A Phase II Randomized, Double Blind, Placebo-controlled, Parallel Group, Multicenter Study to Evaluate the Safety and Efficacy of Repeated Oral Doses of Blautix in Adult Subjects With Irritable Bowel Syndrome (IBS) Subtypes IBS-C and IBS-D
Phase: PHASE2
Conditions: Irritable Bowel Syndrome
Sponsor: 4D pharma plc

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
 
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PROTOCOL 
 
 
 
PROTOCOL NUMBER:  BHT-II-002 
 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE BOWEL 
SYNDROME (IBS) SUBTYPES IBS-C and IBS-D 
 
 
 
 
Sponsor Name and Address: 
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 
Date and Version: 
 
6 February 2018; Version 1.0 
 
 
6 April 2018; Version 1.1 
 
 
19 May 2018: Version 1.2 
 
 
31 May 2018: Version 2.0 
 
 
21 June 2018: Version 2.1 
 
 
13 February 2019: Version 3.0  
 
 
31 March 2020: Version 3.1 
 
EudraCT Number: 
 
2018-001203-36  
IND Number:  
 
 
 
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Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
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TABLE OF CONTENTS 
1 
SYNOPSIS ...................................................................................................................................... 7 
2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ................................. 14 
2.1. 
Background Information .................................................................................................. 14 
2.2. 
Scientific Rationale Test .................................................................................................... 14 
2.2.1 
Non-clinical Studies ................................................................................................ 16 
2.2.2 
Clinical Studies ....................................................................................................... 17 
2.3. 
Potential Risks and Benefits .............................................................................................. 18 
2.3.1 
Potential Risks ........................................................................................................ 18 
2.3.2 
Known Potential Benefits ....................................................................................... 19 
2.3.3 
Risk-Benefit Assessment ........................................................................................ 19 
3 
STUDY OBJECTIVES AND ENDPOINTS .............................................................................. 20 
3.1 
Objectives ........................................................................................................................... 20 
3.2 
Endpoints ............................................................................................................................ 20 
4 
STUDY RATIONALE AND DESIGN ....................................................................................... 22 
4.1 
Rationale ............................................................................................................................. 22 
4.1.1 
Rationale for the Study Design ............................................................................... 22 
4.1.2 
Rationale for the Dosage ......................................................................................... 22 
4.2 
Study Design ....................................................................................................................... 23 
4.2.1 
Overall Study Design .............................................................................................. 23 
4.2.2 
Stopping Criteria ..................................................................................................... 30 
4.2.3 
Independent Data and Safety Monitoring Board (DSMB) ..................................... 30 
5 
POPULATION ............................................................................................................................. 30 
5.1 
Inclusion Criteria ............................................................................................................... 30 
5.2 
Exclusion Criteria .............................................................................................................. 31 
5.3 
Treatment assignment procedures ................................................................................... 32 
5.3.1 
Randomisation ........................................................................................................ 32 
5.3.2 
Blinding Procedures ................................................................................................ 33 
5.3.3 
Subject Numbering ................................................................................................. 33 
5.3.4 
Emergency Unblinding Procedures ........................................................................ 33 
5.4 
Subject withdrawal ............................................................................................................ 33 
5.4.1 
Reasons for Withdrawal .......................................................................................... 33 
5.4.2 
Replacement of Subjects ......................................................................................... 34 
6 
INVESTIGATIONAL PRODUCTS ........................................................................................... 34 
6.1 
Formulation ........................................................................................................................ 34 
6.2 
Dosage, Frequency and duration of Dosing ..................................................................... 34 
6.3 
Packaging and Labelling ................................................................................................... 34 

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6.4 
Storage and Expiry ............................................................................................................ 35 
6.5 
Treatment Compliance ...................................................................................................... 35 
6.6 
Accountability .................................................................................................................... 35 
6.7 
Permitted Dose Adjustments and Interruptions ............................................................. 35 
6.8 
Concomitant Treatment .................................................................................................... 35 
6.9 
Prohibited Medication ....................................................................................................... 35 
7 
STUDY ASSESMENTS ............................................................................................................... 36 
7.1 
Clinical Assessments .......................................................................................................... 36 
7.2 
Laboratory Assessments .................................................................................................... 38 
7.2.1 
Safety Assessments: ................................................................................................ 38 
7.2.2 
Exploratory Assessments ........................................................................................ 39 
7.2.3 
Total Blood Volumes .............................................................................................. 40 
7.2.4 
Laboratory Manual .................................................................................................. 40 
8 
SAFETY DATA COLLECTION, RECORDING AND REPORTING .................................. 40 
8.1 
Responsibilities ................................................................................................................... 40 
8.2 
Definitions ........................................................................................................................... 40 
8.2.1 
Adverse Events ....................................................................................................... 40 
8.2.2 
Serious Adverse Events .......................................................................................... 41 
8.3 
Serious Adverse Event Reporting..................................................................................... 41 
8.3.1 
Pregnancies ............................................................................................................. 42 
8.4 
Clinical Management of Events ........................................................................................ 43 
9 
DATA REVIEW, DATABASE MANAGEMENT AND STUDY DOCUMENTATION ...... 43 
9.1 
Site Monitoring .................................................................................................................. 43 
9.2 
Source Documents.............................................................................................................. 43 
9.3 
Data Collection ................................................................................................................... 44 
9.4 
Database Management and Quality Control ................................................................... 44 
9.5 
Protocol Deviations ............................................................................................................ 45 
9.6 
Study Records Retention ................................................................................................... 45 
10 STATISTICAL CONSIDERATIONS ....................................................................................... 45 
10.1 Populations for Analysis .................................................................................................... 45 
10.2 Sample Size Calculation .................................................................................................... 46 
10.3 Interim Analysis ................................................................................................................. 46 
10.4 Statistical Analysis Plan .................................................................................................... 46 
10.5 Statistical Analysis ............................................................................................................. 46 
10.5.1 Primary Efficacy Analysis ...................................................................................... 46 
10.5.2 Secondary Efficacy Analysis .................................................................................. 47 
10.5.3 Exploratory Efficacy Analysis ................................................................................ 47 
10.5.4 Safety Analysis ....................................................................................................... 47 

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Clinical Study Protocol No: BHT-II-002  
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10.6 Concomitant Medications ................................................................................................. 47 
10.7 Handling of Missing Data .................................................................................................. 47 
10.8 Multiplicity ......................................................................................................................... 48 
11 QUALITY CONTROL AND QUALITY ASSURANCE ......................................................... 48 
12 ETHICS/REGULATORY ........................................................................................................... 48 
12.1 Institutional Review Board (IRB)/ Ethics Committee (EC) ........................................... 48 
12.2 Regulatory .......................................................................................................................... 49 
12.3 Serious Breaches ................................................................................................................ 49 
12.4 Informed Consent .............................................................................................................. 49 
12.5 Future use of stored samples ............................................................................................. 50 
12.6 Subject Confidentiality ...................................................................................................... 50 
12.7 Insurance ............................................................................................................................ 50 
13 PUBLICATION POLICY ........................................................................................................... 50 
14 REFERENCES ............................................................................................................................. 52 
APPENDIX 1:  IBS-QOL .................................................................................................................... 53 
APPENDIX 2:  HOSPITAL ANXIETY AND DEPRESSION SCALE (HADS) ........................... 62 
APPENDIX 3:  BRISTOL STOOL CHART..................................................................................... 64 
APPENDIX 4:  IBS-SSS ...................................................................................................................... 65 
APPENDIX 5:  MODIFIED FFQ ....................................................................................................... 72 
APPENDIX 6:  ROME IV CLASSIFICATION FOR IBS .............................................................. 79 
APPENDIX 7:  PROHIBITED MEDICATIONS ............................................................................. 80 
APPENDIX 8:  SUMMARY OF PROTOCOL CHANGES VERSION 1.1 ................................... 82 
APPENDIX 9:  SUMMARY OF PROTOCOL CHANGES VERSION 1.2 ................................... 94 
APPENDIX 10:  SUMMARY OF PROTOCOL CHANGES VERSION 2.0 ................................. 97 
APPENDIX 11:  SUMMARY OF PROTOCOL CHANGES VERSION 2.1 ............................... 108 
APPENDIX 12:  SUMMARY OF PROTOCOL CHANGES VERSION 3.0 ............................... 112 
APPENDIX 13:  SUMMARY OF PROTOCOL CHANGES VERSION 3.1 ............................... 136 
 
 

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HADS 
Exploratory Endpoints 
 
 
 
Safety Endpoints 
Incidence, nature, severity, relatedness, seriousness, expectedness and outcome of adverse 
events 
Haematology and blood chemistry assessments  
Vital signs 
Study Design: A randomised, double-blind, placebo controlled parallel group study.  
Planned Number of Sites: Approximately 20 sites in North America, UK and Ireland 
Study Population: 500 subjects with a diagnosis of IBS as defined by the Rome IV 
criteria.  Subjects will be classified into Cohorts according to the Rome IV classification of 
IBS subtypes:   
Cohort C: subjects diagnosed with IBS- C  
Cohort D: subjects diagnosed with IBS-D   
Within each cohort, 250 subjects will randomly receive either Blautix™ or matching 
placebo in a 1:1 ratio overall of treated to control subjects.    
Test Product, Dose and Route of Administration: 
Capsules of Blautia hydrogenotrophica (Blautix™) administered orally.   
(DFK FDSVXOH FRQWDLQV [ 9MPN Blautia hydrogenotrophica /capsule and subjects will 
receive two capsules twice daily approximately 30 minutes before food for 8 weeks.  
Reference Therapy, Dose and Route of Administration: 
Matching placebo, administered orally.  
Each placebo capsule will be comparable in size, weight and appearance to the test 
formulation and subjects will receive two capsules twice daily approximately 30 minutes 
before food for 8 weeks.  
Duration of Study: 
Subjects will be screened up to 4 weeks prior to commencement of dosing followed by 
an 8-week treatment period and a 4-week follow-up. In total, subjects will make 5 
visits to the clinic including screening. 
 
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Criteria for Evaluation 
Efficacy: 
Abdominal pain intensity using a numeric pain rating scale 
Subject global assessment of relief 
Stool consistency/frequency 
IBS-QOL 
IBS-SSS 
HADS 
Exploratory: 
 
 
 
 
Safety: 
Safety evaluation will include occurrence of adverse events; clinical laboratory safety tests 
(serum biochemistry and haematology), physical examinations, vital signs (blood pressure, 
pulse rate, respiratory rate and oral temperature), 12-lead ECG parameters and BMI. 
Statistical Analysis: 
All baseline, compliance, disposition, efficacy and safety variables will be listed and 
summarised. 
The standard summary statistics for continuous baseline and outcome variables are: N, 
mean, standard deviation, median, quartiles, minimum and maximum. The standard 
summary statistics for categorical baseline and outcome variables are: count and 
proportion (expressed as percentage). 
The two Cohorts, Cohort C and Cohort D, will be analysed separately except where 
exploratory pooled or combined analyses are conducted across cohorts. 
Primary Efficacy Analysis 
The primary efficacy analysis is the comparison of the proportion of overall responders 
between the Blautix™ and placebo groups using Pearson’s test with Yates’s correction in 
the full analysis set within each Cohort. 
All other analyses of the primary endpoint are secondary analyses. 
 
 
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Clinical Study Protocol No: BHT-II-002  
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
AE 
Adverse event 
BAM 
Bile Acid Malabsorption 
BHT 
Blautia hydrogenotrophica 
BSFS 
Bristol stool form scale 
BMI 
Body mass index 
CFU 
Colony forming units 
CH4 
Methane 
CI 
Confidence interval  
CMC 
Chemistry, manufacturing and controls 
CRF 
Case Report Form 
CO2 
Carbon dioxide 
CSBMs 
Complete spontaneous bowel movements 
DSMB 
Data Safety Monitoring Board  
EC 
Ethics Committee 
ECG 
Electrocardiogram 
eCOA 
Electronic Clinical Outcomes Assessments 
ELISA 
Enzyme linked immunosorbent assay 
FFQ 
Food Frequency Questionnaire 
FODMAP 
Fermentable, Oligo-,Di-,Mono- Saccharide and Polyol 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HADS 
Hospital Anxiety and Depression Scale  
HIV 
Human Immunodeficiency Virus 
H2 
Hydrogen 
H2S 
Hydrogen sulphide 
IBS 
Irritable bowel syndrome 
IBS-C 
Constipation predominant IBS 
IBS-D 
Diarrhoea predominant IBS 
IBS-HMA 
IBS-human microbiota-associated 

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IBS-M 
Mixed bowel pattern IBS 
IBS-QOL 
IBS Quality of Life 
IBS-SSS 
IBS-Symptom Severity Score 
IBS-U 
Unclassified IBS 
ICF 
Informed consent form  
ICH 
International Conference on Harmonization 
IL 
Interleukin 
IMP 
Investigational medicinal product  
IRB 
Institutional Review Board 
ITT 
Intent-to-treat 
IV/WRS 
Interactive Voice/Web recognition system 
Kg 
Kilogram 
LBP 
Live biotherapeutic product 
L 
Litre 
MedDRA 
Medical Dictionary for Regulatory Activities 
MPN 
Most probable number 
NOEL 
No observable effect level 
NOAEL 
No observable adverse effect level 
OD 
Optical density 
PP 
Per protocol  
PCR 
Polymerase Chain Reaction 
PRO 
Patient-reported outcome 
QA 
Quality Assurance 
QC 
Quality Control 
QP 
Qualified Person  
QOL 
Quality of life 
qRT-PCR 
Quantitative real-time polymerase chain reaction 
SAP 
Statistical analysis plan  
NRS 
Numeric Rating Scale 
SAE 
Serious adverse event  

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Clinical Study Protocol No: BHT-II-002  
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SRB 
Sulphate-reducing bacteria 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TAMC 
Total aerobic microbial count 
TNF 
Tumour necrosis factor  
WHO 
World Health Organisation  
 

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2 BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
2.1.Background Information 
Irritable bowel syndrome (IBS) is a chronic, debilitating, gastrointestinal functional disorder with 
an estimated population prevalence in the United States of approximately 12% (Chey, Kurlander, 
and Eswaran 2015) and in Europe between 10-15% (2). IBS is characterised by abdominal pain or 
discomfort in association with alteration in either stool form or frequency. IBS patients can be 
classified as either diarrhoea predominant (IBS-D), constipation predominant (IBS-C), or with a 
mixed bowel pattern (IBS- M). Furthermore, IBS is associated with several comorbidities, 
including somatic pain syndromes (fibromyalgia, chronic fatigue, and chronic pelvic pain), 
gastrointestinal disorders (gastroesophageal reflux and dyspepsia) and psychiatric disorders (i.e., 
depression, anxiety, and somatisation) (Chey, Kurlander, and Eswaran 2015). Therefore, IBS-
related symptoms can be debilitating and lead to a significant reduction in quality of life. 
While the complete pathophysiology of IBS has not been fully elucidated, a disruption of the 
function of the brain-gut axis and an altered intestinal microbiota and/or host immune response are 
thought to be important factors. The risk of developing IBS increases six-fold after acute 
gastrointestinal infection. Furthermore, antibiotics are known to affect the homeostasis of the gut 
microbiome and its usage appears to increase the risk of developing IBS. Several recent studies 
using high throughput sequencing methods have independently shown that the microbiota of IBS 
patients is distinct from that of healthy controls and is characterised by differentially abundant taxa 
and lower global diversity (Jeffery et al. 2012; Tap et al. 2017). 
Studies have demonstrated that treatment with live bacteria may have beneficial effects on IBS 
symptoms by restoring homeostasis in the gut microbiota, normalisation of cytokine blood levels, 
normalising intestinal transit time, decreasing small intestine permeability, and eliminating small 
intestinal overgrowth of fermenting bacteria. This emerging field of research demonstrates the 
feasibility of therapeutic manipulation of the microbiome using live bacteria to improve symptoms 
of IBS. (Shanahan F and Quigley EMM. 2014). 
2.2.Scientific Rationale Test 
Blautix™ is a live biotherapeutic product (LBP) under development by 4D Pharma for the 
treatment of Irritable Bowel Syndrome. The active ingredient of Blautix™ is a strain of the 
bacterium Blautia hydrogenotrophica (B. hydrogenotrophica) (BHT), which is 
 
 for oral administration. B. hydrogenotrophica is a 
Gram-positive anaerobic, non-sporulating coccobacillus bacterium that is a sub-dominant 
commensal in the human gastrointestinal tract. 
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2.2.1.2 
Toxicology 
2.2.2 Clinical Studies  
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2.3.2 Known Potential Benefits  
Although there was no statistically significant clinically relevant effect on symptoms of IBS 
observed in the Phase I study, treatment with Blautix™ demonstrated a trend towards reduction of 
abdominal pain/discomfort/cramps, bloating and diarrhoea by more than 33%. Blautix™ has also 
been shown to exert a positive impact on the microbiota in both pre-clinical models and the phase I 
clinical study. 
2.3.3 Risk-Benefit Assessment 
In this study, there may be some potential benefit of an 8-week treatment of Blautix™ >1x1010 
MPN given twice daily to IBS patients in the alleviation of IBS-related symptoms and associated 
quality of life.  
The risk benefit assessment is considered appropriate to continue clinical development and 
initialise the Phase 2 study in the population described.  
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Clinical Study Protocol No: BHT-II-002  
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IBS-C 
IBS-D 
Primary Endpoint 
Responder Symptom 
Definition 
Abdominal Pain Intensity  
Decrease in weekly average 
of worst abdominal pain in 
the past 24 hours score of at 
least 30% compared with 
baseline  
 
And 
 
Stool Frequency  
Increase of 1 or more 
Complete spontaneous 
bowel movements (CSBM) 
per week compared with 
baseline. 
Abdominal Pain Intensity  
Decrease in weekly average 
of worst abdominal pain in 
past 24 hours score of at 
least 30% compared with 
baseline  
 
and  
 
Stool Consistency (BSS) 
Decrease at least 50% in the 
proportion of days per week 
with at least one stool that 
has a consistency of Type 6 
or 7 compared with baseline. 
 
Secondary Efficacy Endpoints 
x 
Subject global assessment of relief 
x 
Stool consistency/frequency 
x 
IBS-QOL 
x 
IBS-SSS 
x 
HADS 
Exploratory Endpoints 
Safety Endpoints 
x 
Incidence, nature, severity, relatedness, seriousness, expectedness and outcome of adverse 
events 
x 
Haematology and blood chemistry assessments  
x 
Vital signs 
 
 
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4 STUDY RATIONALE AND DESIGN 
 
4.1 Rationale 
4.1.1 Rationale for the Study Design 
This is a randomised, placebo-controlled, parallel group, repeat dose multicentre study in adult 
male and female subjects of 18 to 70 years of age with a confirmed diagnosis of either IBS- C or 
IBS-D. 
An 8-week treatment period of Blautix™ or placebo is considered sufficient to assess any changes 
in the subject assessment of IBS symptoms and the analysis of pharmacodynamic changes on the 
microbiota.  Subject assessment of symptoms by use of rating scales/scores and a quality of life 
questionnaire are considered appropriate. 
Studies of microbiota composition in IBS have indicated that those patients with a normal 
microbiota displayed higher scores for anxiety and depression, whereas IBS patients with an 
abnormal microbiota had on average normal scores for anxiety and depression (ref).  The Hospital 
Anxiety and Depression score (HADS) will be used to determine levels of anxiety and depression 
during the study. 
Use of periodic dietary recall by means of the Food Frequency Questionnaire (FFQ) may be used 
to determine if dietary habits influence exploratory study outcomes.  
4.1.2 Rationale for the Dosage 
Blautia hydrogenotrophica is a commensal bacterium that is naturally present in the human body. 
The bacterium was isolated and purified from human faeces and pre-clinical toxicology did not 
detect any clinical, morphological or laboratory deviations of clinical relevance.  As Blautia 
hydrogenotrophica is a bacterium that naturally occurs in man, it is expected that it does not have 
any genotoxic, carcinogen, or mutagenic potential.  
The dose of B. hydrogentotrophica administered in the study will be two capsules twice a day.  
Each capsule will contain a minimum of 5 x 109 MPN.  Hence the daily dose will be 1010 to 1011 
MPN.  There is no established methodology for calculating human equivalent dose for live 
biotherapeutic products. 
 
 In order to calculate the equivalent human dose, body surface area calculations were 
used. It is known that the ratio of intestinal surface area (possibly the most relevant aspect for live 
biotherapeutic dosing) to total body surface area in rodents is similar to man (Casteleyn et al. 
2010). In order to calculate body surface area, MPN/kg was first calculated, then multiplied by 
published factors (Nair and Jacob, 2016) to convert to MPN/m2. 
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4.2 Study Design 
4.2.1 Overall Study Design 
Subjects who are potentially suitable for the study will be identified from current patient lists at 
appropriate gastroenterology clinics and or by advertisement. Subjects will be approached to 
determine if they are interested and willing to participate. The investigative sites will provide 
interested subjects with a ‘Research Study Information and Informed Consent Form’ and they will 
be booked in for the initial screening visit when consent will be obtained, and the Subjects will be 
asked to sign the Consent Form 
Screening Visit (Up to 4 weeks before dosing starts Day -28 to Day -1)  
Following completion of the study consent form, subjects will be screened against the protocol 
requirements, and a medical history and concomitant medications will be documented, a full 
physical examination will be performed, and bloods will be drawn for haematology, serum 
biochemistry and viral serology.  Vital signs (pulse, blood pressure, respiratory rate and body 
temperature) will be assessed and recorded, and a 12 lead ECG will be taken. Women of child 
bearing potential will have a serum pregnancy test.  Height and weight will be measured, and if 
subjects fulfil all the eligibility criteria at this part of the Screening process they will be provided 
with a Smartphone containing an ‘Electronic Clinical Outcomes Assessment’ (eCOA) App and 
will be provided with training and instructions on how to use the device. At this Visit they will 
complete the IBS-Symptom Severity Score (SSS) on a tablet device during the clinic visit.  
The subject will be asked to take the smart phone home and complete the following assessments 
daily over a 7-day period. These assessments will need to be completed prior to attending the clinic 
for their Baseline Visit 1 
x 
An abdominal pain intensity rating scale daily 
x 
Bowel movements, Stool consistency/frequency daily and laxative use 
x 
If no bowel movements, this should be recorded as zero  
The subject will need to ensure they complete these two assessments at least one day prior to the 
day next clinic visit. As soon as the subjects have completed their assessments and click submit on 
the smartphone, their data will be sent to the hub for review by the investigator and study team. 
The same smartphone will be used throughout the study for all patient reported outcome 
assessments. 
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Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next clinic 
visit. If necessary, a stool sample may be provided up to 2 days after the visit provided it is from 
the night before or from the day it is delivered to the clinic.  
The next clinic visit will be booked with the Subjects prior to leaving the clinic. 
Reasons for screen failure will be documented in the subject’s notes.  Subjects may be re-screened 
once, after the initial screening visit if considered to be appropriate by the Investigator. A waiting 
period of at least 2 weeks is required between screening visits.  
Baseline: Visit 1 (Day 1) 
Subjects will return to the clinical unit on Day 1 (Visit 1; within 28 days of screening visit or re-
screening visit if applicable). The study doctor will review the subject screening data obtained 
from the smartphone eCOA app and the subject will be classified into either IBS-C or IBS-D 
subtypes.  
A brief physical examination, assessment of vital signs and a urine pregnancy test for female 
subjects of child bearing potential, weight and BMI will be conducted and any changes in 
medication and adverse events will be checked.  Blood samples will be taken for measurement of 
inflammatory markers (cytokine levels). A fresh urine sample will be taken to analyse the 
metabolites.  
Final confirmation of eligibility for the study will be documented, signed by the Investigator and 
filed in the subject notes. 
Classification into IBS subtype will be confirmed, and randomisation will take place. Subjects will 
receive either Blautix™ or placebo capsules, with instructions for two (2) capsules to be taken at 
approximately 12 hourly intervals; morning and evening, approximately 30 minutes before meals, 
giving a total of four (4) capsules per day. Subjects will receive instructions about storing the 
capsule packs in the fridge and be asked not to open the capsules. Subjects will be provided with 
enough medication to last until the next clinic visit. 
Subjects will use their smartphone issued at the Screening visit to capture their study related data. 
The IBS-SSS and FFQ questionnaires will be performed at the clinic on a tablet device.  
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Abdominal Pain Score completed daily 
• 
Stool consistency, frequency, spontaneity and laxative use, completed daily  
• 
Subject Global Assessment of Relief completed weekly  
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit or within 24 hours 
prior to the visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of each Clinic visit 

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• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 24 
hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of each Clinic visit 
• 
Review of concomitant medications 
Subjects will be provided with a contact card containing an appropriate contact telephone number 
for the study team and hospital, should they need medical advice. 
Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next clinic 
visit.   
Visit 2 (Week 4)  
Subjects will return after 4 weeks (± 7 days) for a safety check including adverse events and to 
record any changes in concomitant medication. A urine pregnancy test for female subjects of 
childbearing potential. Blood samples will be taken for measurement of inflammatory markers 
(cytokine levels). A fresh urine sample will be taken to analyse the metabolites.  
Subjects will continue to use their smartphones to capture all the patient related outcome data, 
except for IBS-SSS and FFQ questionnaires which will be completed during the clinic visit on a 
tablet device. The data will be reviewed on an ongoing basis as required by the study team and 
sponsor. At this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Score completed daily 
• 
Stools consistency/frequency and use of laxatives daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit or within 24 hours 
prior to the visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 24 
hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit  
• 
Review of concomitant medications 
Subjects will receive further study medication to last until the next clinic visit. Subjects will be 
reminded to take their medication; two (2) capsules to be taken at 12 hourly intervals 
approximately 30 minutes before meals.  Subjects will receive a total of four (4) capsules  per day 
and will be reminded to store the capsules in the fridge and not to open the capsules.  

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Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next clinic 
visit  
End of Treatment (Week 8)  
Subjects will return to the clinic at the end of the 8-week (or up to -7 days) treatment period.   
Subjects will be assessed (vital signs, concomitant medication and adverse events) and a physical 
examination, including weight and BMI and an ECG will be conducted. A urine pregnancy test for 
female subjects of child bearing potential.  Bloods for haematology, serum biochemistry and 
inflammatory markers will be taken.  A fresh urine sample will be taken to analyse the metabolites.  
Subjects will continue to use their smartphones to capture all the patient related outcome data, 
except for IBS-SSS and FFQ questionnaires which will be completed during the clinic visit on a 
table device. The data will be reviewed on an ongoing basis as required by the study team and 
sponsor. At this visit the following information will be reviewed with the subject; 
x 
Information on Adverse Events 
x 
Review of Medication log completed daily  
x 
Abdominal Pain Score completed daily 
x 
Stool consistency/frequency and laxative use daily  
x 
Subject Global Assessment of Relief completed weekly 
x 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit or within 24 hours 
prior to the visit 
x 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
x 
Hospital Anxiety and Depression score (HADS) on Day of Clinic visit or within 24 hours 
prior to the visit 
x 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit 
x 
Review of concomitant medications 
Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next clinic 
visit  
Follow-Up (Week 12-14) 
Subjects will return 4-6 weeks after the last dose of study medication for a safety check and 
clinical update.  All female subjects of child bearing potential will have a urine pregnancy test.  A 
physical examination, weight and vital signs will be measured and recorded, changes in 
concomitant medication and adverse events will be recorded.  Bloods will be drawn for 
haematology, serum biochemistry 
 
 
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Subjects will use their smartphone issued to capture their study related data, except for IBS-SSS 
and FFQ questionnaires which will be completed during the clinic visit on a tablet device. 
• 
Information on Adverse Events 
• 
Abdominal Pain Score completed daily 
• 
Stool consistency/frequency and laxative use, daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit or within 24 hours 
prior to the visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
• 
Hospital Anxiety and Depression score (HADS) on Day of Clinic visit or within 24 hours 
prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit  
• 
Review of concomitant medications 
At this visit the smartphones will be returned from the subjects and given back to the study site 
team. 
 

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4.2.2 Stopping Criteria 
4.2.2.1 
Individual Subjects 
Subjects will be withdrawn from the study on safety grounds to be established by the DSMB and 
as per section 5.4.1. 
4.2.2.2 
Study as a Whole 
The DSMB will establish criteria for stopping the study.  If the criteria are met, a substantial 
amendment will be filed, and the study will be stopped temporarily, and the events investigated to 
determine if they are related to the investigational medicinal product (IMP).  If it is determined that 
the events are related to the IMP, the study will be stopped, and the Health Authorities and ethics 
committees informed. 
4.2.3 Independent Data and Safety Monitoring Board (DSMB) 
An independent DSMB comprising experts in drug safety, biostatistics and other relevant fields 
will be established by the sponsor to assess the progress of the trial, the safety data and efficacy 
endpoints.  Stopping criteria for individual subjects and the study will be established. 
The composition, responsibilities and procedures of the Board will be documented prior to the 
commencement of the study and randomisation of the first subject.  Recommendations of the 
Board and actions taken by the sponsor will be documented.   
5 POPULATION 
500 subjects diagnosed with IBS will be enrolled into the study.  Diagnosis of IBS will be defined 
by the Rome IV criteria and must be met for the last 3 months with symptom onset at least 6 
months prior to diagnosis. 
IBS patients will be screened based on the inclusion and exclusion criteria described in 5.1 below, 
and will be classified into one of the following Cohorts according to the Rome IV classification of 
IBS subtypes. 
Cohort C: 250 subjects diagnosed with IBS-C  
Cohort D: 250 subjects diagnosed with IBS-D 
5.1 
Inclusion Criteria 
All subjects must meet all the following inclusion criteria: 
1. 
Written consent on an Institutional Review Board (IRB)/ Independent Ethics Committee 
(IEC) approved ICF before any study specific evaluation  
2. 
Males and Females between 18 and 70 years of age 
3. 
Body Mass Index (BMI): 18-39 kg/m2 
4. 
Having IBS-C or IBS-D as defined by Rome IV, including Subtype Classification as defined 
per Table 2  
 
Recurrent abdominal pain on average, at least 1 day/week in the last 3 months associated 
with two or more of the following criteria: 
• 
Related to defecation 

 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
  
  
 
 
 
  
  
 
  
 
 
 
 
 
 
 
 
 
  
 
 
 
  
  
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
 
 
  
 
  
 
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
 
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
  
 
 
   
  
 
 
  
 
 
 
  
 
 
  
  
 
 
 
 
 
 
 
 
 
 
  
  
 
  
  
 
 
 
 
  
 
 
 
  
 
 
   
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
  
 
 
 
 
   
 
 
  
  
 
 
 
 
  
  
 
 
 
 
 
 
  
  
 
  
 
  
 
 
 
  
 
 
 
  
 
 
  
  
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
 
 
 
  
 
 
 
  
 
  
 
 
 
 
 
 
 
  
 
 
 
  
 
 
 
  
 
 
 
 
 
  
 
 
 
   
 
 
 
 
 
  

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6. 
Individuals who, in the opinion of the investigator, are poor attendees or unlikely for any 
reason to be able to comply with the study requirements 
7. 
Patient is currently enrolled in or has not yet completed at least 30 days since ending other 
investigational device or drug study(s), or patient is receiving other investigational agent(s) 
8. 
Have an active or recent (within 3 years) malignant disease or any concomitant end-stage 
organ disease. A non-melanoma skin cancer that has been adequately treated with no 
recurrence within 3 months of screening is not excluded.  
9. 
Females who are pregnant or breast feeding  
10. 
Refusal to use acceptable methods of birth control (true abstinence, sterilisation, birth control 
pills, injections or contraceptive implants) for women of child bearing potential while on 
treatment and following completion of 2 menstrual cycles/ months after the last dose of study 
treatment. For Males, a barrier method of birth control from randomisation until the Follow-
Up visit, unless vasectomised 
11. 
Use of antibiotics within 30 days of screening 
12. 
Use of systemic steroids within 30 days of screening  
13. 
Change in dose or introduction of an antipsychotic within the last month 
14. 
Have suffered from an uncontrolled or current major psychiatric disorder 
15. 
Clinically diagnosed Lactose intolerance 
16. 
Clinically diagnosed Coeliac disease 
17. 
Change of diet e.g. FODMAP, gluten-free within last 3 months 
18. 
Those > 50 will be excluded if their diagnosis of IBS is recent (<12 months) and if they have 
not had a sigmoidoscopy or colonoscopy within previous 5 years. 
19. 
Any GI related abdominal surgery other than hernia repair or appendectomy. 
Cholecystectomy more than 6 months previously is not an exclusion  
20. 
Subjects taking prucalopride  
21. 
Known HIV infection, or hepatitis A, B, or C active infection 
22. 
Subjects with abnormal laboratory values at screening deemed by the investigator to be 
clinically significant 
23. 
Subjects who have taken commercially available probiotics within the last month (30 days 
prior to randomisation). See Appendix 7 
24. 
Subjects with known or suspected hereditary fructose intolerance, glucose-galactose 
malabsorption or sucrase-isomaltase insufficiency  
25. 
Subjects taking guanylate cyclase agonists; such as linaclotide and lubiprostone 
 
5.3 Treatment assignment procedures 
 
5.3.1 Randomisation 
This is a double-blind study and subjects will randomly receive either Blautix™ or placebo in a 1:1 
ratio respectively.  At Baseline (Visit 1) , subjects will be classified into either the IBS- C (Cohort 
C) or the IBS-D (Cohort D) cohort of subjects and 250 subjects will be enrolled into each cohort.  
Within each cohort, 125 subjects will randomly receive Blautix™ and 125 subjects will randomly 
receive matching placebo.  Allocation of randomisation numbers will be performed centrally using 
an interactive voice/web response allocation system (IV/WRS). 

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Subjects, 4D pharma Plc, designated CRO and clinical site staff will be blinded to the 
investigational product allocation for each subject. Randomisation data will be kept strictly 
confidential, filed securely by the Sponsor (or designee) and accessible only to authorised persons 
of the Sponsor (or designee), and accessible only to authorised per Sponsor (or designee)’s 
standard operating procedures (SOPs) until the time of unbinding.  
5.3.2 Blinding Procedures 
The blinding will be maintained using placebo capsules comparable in appearance to the Blautix™ 
capsules.  
5.3.3 Subject Numbering 
The investigators must maintain a screening log of all screened subjects and those randomised into 
the study. 
Each subject will be given a unique identification subject number, to facilitate anonymous 
identification within the study. Once assigned to a subject, a subject number will not be re-used. 
5.3.4 Emergency Unblinding Procedures 
The IV/WRS will be used for emergency unblinding of the treatment codes. The treatment codes 
should not be broken except in medical emergencies when the management of the subject requires 
knowledge of the treatment randomisation.  The investigator must sign and date the code break 
form supplied and document the reason for breaking the code.  The Sponsor Medical Monitor 
should be informed as soon as possible and within 24 hours of the event.  
In addition, each subject will be given a card and asked to carry it with them at all times in case of 
an emergency.  The card will include the study number, subject number, Investigational product 
and the name and 24-hour telephone number of the investigative site.  Subjects should be asked to 
return this card at the end of the study.   
The Sponsor’s pharmacovigilance provider may break the code for serious adverse events that are 
unexpected and are suspected to be causally related to the investigational product and potentially 
require expedited reporting to the regulatory authorities.  
5.4 Subject withdrawal 
 
5.4.1 Reasons for Withdrawal  
Subjects have the right to withdraw from the study at any time and for any reason without 
prejudice to future medical care by the investigator or at the institution.  
Reasons for removal from the Protocol-required investigational product(s) or procedural 
assessments might include: 
• 
Withdrawal of informed consent 
• 
Pregnancy 
• 
Investigator’s opinion that it is in the best interests of the subject  

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Subjects are not eligible for the study if they have received antibiotics or systemic corticosteroids 
within 30 days prior to the screening visit.  Subjects on inhaled steroids for asthma may be 
included but oral or infused steroids for asthma are not permitted.  If these medications are 
required during the study, the subject does not need to be withdrawn but details will be recorded in 
the concomitant medication case report forms. 
 
 
7 STUDY ASSESMENTS 
7.1 Clinical Assessments 
Medical History 
A medical history including prior and current medication will be obtained during screening. In 
addition, demographic data will be collected, including gender, race, and ethnic origin.  
Physical Examination 
A full physical examination will be performed by the Investigator at screening and at study visits 
as indicated in Table 1.  Body weight in kilograms (kg) and height in metres (m) will be measured 
and the BMI will be calculated.    
Vital Signs  
Blood pressure, heart rate, respiratory rate and temperature will be taken as indicated in Table 1.  
Blood pressure and heart rate will be measured in the supine position using an automated blood 
pressure monitor.   
ECG 
A 12-lead ECG will be taken as indicated in Table 1.  Subjects will be in a supine position prior to 
recording an ECG. The ECG traces will be reviewed and commented on by the Investigator and 
the traces signed and dated.  
Subject Assessments: 
Refer to Table 1: Schedule of Assessments and Procedures. The assessments will be completed by 
the subject either at home using the smartphone provided or during the clinic visit.  
Abdominal Pain Score  
Subjects will be asked to rate their worst abdominal pain over the past 24 hours using an 11-point 
Numeric Rating Scale (NRS) and a weekly average will be recorded.  This data will be recorded 
daily for a 7-day period up to Baseline (Visit 1) to allow Subject to be classified into Cohorts of 
IBS-C or IBS-D sub-types. Once subjects are randomised they will be asked to record their score 
daily on their smartphone. 
Subjects will respond to the following question: ‘How much abdominal pain have you felt today’ 
on a scale from 0 (none) to 10 (worst possible pain)” using an 11-point NRS ranging from 0 (none) 
to 10 (worst possible pain)?  
 

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Subject Global Assessment of Relief 
A weekly subject response to the question Please consider how you felt this past week in regard to 
your IBS, in particular your overall wellbeing and symptoms of abdominal discomfort, pain and 
altered bowel habit. Compared to the way you usually felt before entering the study, how would 
you rate your relief of symptoms during the past week?.   
Using a 5-point Likert scale with the following response options:  
Completely relieved; considerably relieved; somewhat relieved; unchanged; worse 
Subjects will be asked to record their assessment weekly on their smartphone. 
IBS Quality of Life (QOL) (Appendix 1)  
Subjects will be asked to complete a quality of life questionnaire consisting of 34 questions with a 
five-point response. The individual responses to the 34 items are summed and averaged for a total 
score and then transformed to a 0-100 scale for ease of interpretation with higher scores indicating 
better IBS specific quality of life. Once subjects are randomised they will be asked to record their 
score on their smartphone at each clinic visit or within 24hrs prior to the Clinic Visit. 
Hospital Anxiety and Depression Score (HADS) (Appendix 2) 
Subjects will be asked to complete HADS which is a 14-item scale that generates ordinal data. 
Seven of the items relate to anxiety and 7 relate to depression.  Each item is scored from 0-3 so a 
subject can score 0 to 21 for either anxiety or depression. Once subjects are randomised they will 
be asked to complete this questionnaire on their smartphone at each clinic visit or within 24hrs 
prior to the Clinic Visit. 
Bristol Stool Chart 
Subjects will be asked to record stool frequency and consistency. Stool types will be based on the 
Bristol Stool Chart (Appendix 3). This data will be recorded daily for a 7-day period up to 
Baseline (Visit 1) to allow Subject to be classified into Cohorts of IBS sub-types. Once subjects 
are randomised they will be asked to record their stool frequency daily up to 10 bowel movements 
per day and score each stool type on their smartphone. No bowel movements in any given day will 
also be recorded.   
Stool frequency will be defined as a sum of weekly CSBMs. Stool frequency will be recorded 
using the following question: "How many times did you open your bowels during the past 24 
hours? If you have not had any bowel movements today, please enter 0." Subjects will be reminded 
to rate all of their bowel movements in the Bristol Stool Chart before answering the question. 
CSBMs will be determined by absence of laxative use recorded in concomitant medication 
questions for the same 24 hour period. 
IBS Symptom Severity Score (SSS) (Appendix 4) 
Subjects will be asked to complete a questionnaire on severity of abdominal distension, severity of 
abdominal pain, frequency of abdominal pain, dissatisfaction with bowel habits, and interference 
of IBS symptoms with daily life.  A score ranging from 0 (no symptoms) to 500 (maximum 
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The initial data from this questionnaire will be collected during the Screening visit on a tablet 
device during the clinic visit. This data will be used to classify into Cohorts of IBS sub-types prior 
to randomisation. Once subjects are randomised, they will be asked to record their responses to the 
questionnaire on a tablet device during each clinic visit.  
Food Frequency Questionnaire (FFQ) (Appendix 5)  
Subjects will be asked to complete a questionnaire to obtain frequency information about food and 
beverage consumption over a specified period of time (day, week and month). FFQ completion at 
baseline will be in relation to food (s) consumed over the prior 6 months. At each subsequent visit 
the FFQ shall capture responses to food (s) consumed since last subject visit. Subjects will be 
asked to complete this questionnaire on a tablet device during each Clinic Visit. 
 Concomitant Medications:  
Subjects will be asked to record name, dose and time of administration of any medication, other 
than IMP, taken during the study. This will be reviewed at each visit. 
Dosing Diary: 
Subjects will be asked to confirm their daily study medication intake by indicating "yes" or "no" to 
the following questions on the smartphone provided: "Did you take two of your study medication 
capsules this morning? " in the morning and "Did you take two of your study medication capsules 
this evening?" in the evening. Subjects will be prompted with reminders via this device.  
Diet and Study Restrictions 
Dosing should be twice daily at twelve hourly intervals (approximately 30 minutes before 
breakfast and 30 minutes before evening meal). Subjects with lactose intolerance and Coeliac 
disease and those with a change of diet within the 3 months before screening are excluded. 
Subjects taking commercially available supplements (probiotics formulated as capsules or sachets 
as in Appendix 7) are excluded 1 month (30 days) prior to randomisation and throughout the study 
to follow-up. Probiotic yogurts, yogurt-type drinks and soft drinks e.g. fruit juices are acceptable if 
taken during the month before screening and continued to follow-up. 
7.2 Laboratory Assessments 
A Laboratory Manual will be prepared to document the address and contact numbers for all 
laboratories involved in the study and the collection, processing, shipment, storage and reporting 
procedures for all samples. 
7.2.1 Safety Assessments: 
Refer to Table 1: Schedule of Assessments and Procedures for sample collection timepoints.  
Blood Samples: 
Haematology:  
Haemoglobin (HGB), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin 
(MCH), mean cell haemoglobin concentration (MCHC), red blood cells (RBC), C-reactive Protein 
(CRP), white cell count (WCC) and platelets (PLT).  Differential white cell count will include 

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neutrophils, lymphocytes, eosinophils, basophils and monocytes as absolute values and 
percentages. 
Clinical Chemistry: 
Total protein (TP), albumin (ALB), total bilirubin (BIL-T), alanine transaminase (ALT), aspartate 
transaminase (AST), gamma-glutamyl transferase (GGT), glucose (GLU), sodium (NA), 
potassium (K), bicarbonate (HCO3), creatinine (CREA), urea, calcium (Ca), phosphate (PO4), 
globulin. 
Serology:  
At screening the analyses will also include HIV-1 & HIV-2, Hepatitis B surface antigen (HBsAg), 
a Hepatitis B surface antibody (Anti-HBs),a Hepatitis core antibody test (anti-HBc) and Hepatitis 
C. 
Pregnancy test: 
A serum pregnancy test will be conducted for all female subjects at the screening visit.  
Urine Samples: 
A fresh urine sample will be collected for a pregnancy test in all female subjects at  Baseline (Visit 
1), Visit 2, End of Treatment and Follow-up (dipstick testing). 
7.2.2 Exploratory Assessments  
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7.2.3 Total Blood Volumes  
The total amount of blood taken from each subject during the study will be approximately 150 ml  
7.2.4 Laboratory Manual 
A Laboratory Manual will be prepared to document the address and contact numbers for all 
laboratories involved in the study and the collection, processing, shipment, storage and reporting 
procedures for all samples. 
8 SAFETY DATA COLLECTION, RECORDING AND REPORTING 
8.1 Responsibilities 
The Safety and Medical Management Plan details the roles and responsibilities, Serious Adverse 
event processing, reporting of safety data, SAE reconciliation and Medical monitoring for this 
study.  
8.2 Definitions 
8.2.1 Adverse Events  
An adverse event is any untoward medical occurrence in a subject administered study medication 
and which does not necessarily have to have a causal relationship with this treatment.   Abnormal 
laboratory values or test results constitute adverse events only if they induce clinical signs or 
symptoms, are considered clinically significant, or require therapy. 
The occurrence of adverse events should be sought by non-direct questioning of the subject at 
baseline (pre-dose) Adverse events will be checked at every clinic visit. Adverse events may be 
detected when they are volunteered by the subject or through physical examination, laboratory test, 
or other assessments. All adverse events must be recorded in the CRF with the following 
information: 
1. 
Diagnosis: diagnosis if known, otherwise sign or symptom; 
2. 
Seriousness: whether it constitutes a serious adverse event (SAE) and therefore requires 
reporting immediately (see Section 8.3) 
3. 
Criteria for Adverse Events:  
Mild: Asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated 
Moderate: Moderate; minimal, local or non-invasive intervention indicated; limiting age-
appropriate instrumental ADL (Activities of Daily Living) 
Severe: Severe or medically significant but not immediately life-threatening; hospitalization 
or prolongation of existing hospitalization indicated; limiting self-care ADL (Activities of 
Daily Living) 
4. 
Relatedness: relationship to the study medication will be assessed as reasonable possibility of 
being related or no reasonable possibility of being related;  
5. 
Duration: start and end dates or, if continuing, at final examination; 
6. 
Action: any action taken to manage it (none, dosing interrupted, dosing stopped, other, 
medication); 

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7. 
Outcome: the outcome (completely recovered, recovered with sequelae, condition 
improving, condition still present and unchanged, condition deteriorated, death). 
8.2.2 Serious Adverse Events  
A serious adverse event is defined as an event which: 
• 
results in death  
• 
is life-threatening 
• 
requires hospitalisation or prolongation of hospitalisation 
• 
results in persistent or significant disability or incapacity 
• 
consists of a congenital anomaly or birth defect 
• 
is a medically important event  
All adverse events should be treated appropriately.  Treatment may include one or more of the 
following: no action taken (i.e., further observation only); study medication discontinued due to 
this adverse event; concomitant medication given; non-drug therapy given.  The action taken to 
treat the adverse event or resolve the adverse incident should be recorded on the CRF.  
Once an adverse event is detected, it should be followed until its resolution. The suspected 
relationship to the study medication, the interventions required to treat it and the outcome must be 
recorded in the CRF. 
8.3 Serious Adverse Event Reporting 
A Serious Adverse Event (SAE) Form must be completed for every SAE, regardless of suspected 
causality, occurring after protocol-specified procedures begin after signing the consent form until 4 
weeks after the End of Treatment Visit and must be reported within 24 hours of the investigative 
site becoming aware of the event and ideally within 24 hours of its occurrence.  The Safety and 
Medical Management Plan will provide full details.  
Any SAEs experienced after this 4-week period should only be reported to if the Investigator 
suspects a causal relationship to the study medication.   
Recurrent episodes, complications, or progression of the initial SAE must be reported as follow-up 
to the original episode, regardless of when the event occurs.  This report must be submitted within 
24 hours of the investigator receiving the follow-up information.  An SAE that is considered 
completely unrelated to a previously reported one should be reported separately as a new event. If 
recurrence after resolution of the previous episode occurs if will be reported as a new SAE.    
SAEs should be followed to resolution. Follow-up information should describe whether the event 
has resolved or continues, if and how it was treated, and whether the subject continued or 
withdrew from study participation.  
If the SAE is not previously documented in the investigator’s brochure and is thought to be related 
to the investigational study medication, then this is defined as a serious unexpected suspected 
adverse reaction (SUSAR).  It is the responsibility of the Sponsor to determine whether a reported 
SAE fits the classification of a SUSAR and to inform the Investigator of their decision as soon as 

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possible. It is the responsibility of the Sponsor to determine whether an event requires expedited 
reporting and to notify the Investigators of their decision as soon as possible. 
4D Pharma, or its designee, reports SAE’s and/or SUSARS as required to regulatory authorities, 
investigators/institutions, and IRBs/IECs in compliance with all reporting requirements according 
to local regulations and good clinical practice. 
The investigator is to notify the appropriate IRB/IEC of SAEs occurring at the site and other AE 
reports received from 4D Pharma or it’s designee, in accordance with local procedures and 
statutes.  
8.3.1 Pregnancies 
There is no reason to believe that Blautix™ would be harmful to a mother or baby during 
pregnancy. If a patient becomes pregnant during the study the investigator is to stop dosing with 
study medication immediately.  
A pregnancy is not considered to be an AE or SAE; however, it must be reported to the sponsor 
using the Pregnancy report forms within the same timelines as an SAE. This applies to female 
patients as well as female partners of male patients. A pregnancy should be followed through to 
outcome, wherever possible.  
Subjects who are women and who may be able to have children will be given a serum pregnancy 
test at screening and a urine dipstick test at all subsequent study visits. If the result is positive at 
screening, the subject will not be able to enter the study.  Sexually-active women of child bearing 
potential must use an acceptable form of birth control throughout the study until completion of two 
(2) periods after the last dose to avoid pregnancy for at least one complete menstrual cycle. The 
following methods of birth control are considered acceptable for this study; true abstinence, 
sterilisation, birth control pills, vasectomised partner, injections or contraceptive implants. The 
following birth control pills are recommended:  
• 
Combined (oestrogen and progestogen containing) hormonal contraception associated with 
inhibition of ovulation: 
o 
Oral 
o 
Intravaginal 
o 
Transdermal 
• 
Progestogen-only hormonal contraception associated with inhibition of ovulation: 
o 
Oral 
o 
Injectable 
o 
Implantable 
Female Subjects must not  breast feed an infant during the study. 
Male subjects, able to father children, and sexually active with a female partner who is able to bear 
children, must use a barrier method of birth control (male condom with or without spermicide) 
from the time of randomisation until the Follow-up visit. The study doctor will discuss methods of 

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birth control with the subjects if needed. If a subject becomes pregnant or think they may be 
pregnant during the study, they will be told to stop using the study medication and contact the 
study doctor’s office immediately. They will be asked to withdraw from the study. For Male 
Subjects with a partner who becomes pregnant or thinks she may be pregnant during the study, the 
subjects will be asked to contact the study doctor’s office immediately.  
Refer to the Safety Management and Medical Monitoring Plan for further details.  
8.4 Clinical Management of Events  
If a subject chooses to withdraw or is withdrawn, the Investigator must ensure the safety and well-
being of the subject prior to final discharge from the study.  All adverse events will be evaluated, 
treated and followed up.  Repeated and unscheduled examinations and laboratory assessments 
should be conducted as needed until appropriate and satisfactory resolution of any abnormalities.  
Every effort should be made for all randomised subjects to undergo final follow-up.  In the event 
that a subject fails to return for the study visits and follow-up assessments, every effort should be 
made to determine the reason(s).  
Any pregnancy will be followed up by the investigator to determine outcome for the mother and 
baby. 
9 DATA REVIEW, DATABASE MANAGEMENT AND STUDY DOCUMENTATION 
9.1 Site Monitoring 
On behalf of 4D Pharma Plc contract research organisation (CRO) representative will manage the 
monitoring aspects of this study. Before study initiation, at a site initiation visit, a sponsor 
representative will review the protocol and CRFs with the investigator(s) and their staff.  During 
the study, a sponsor representative (monitor) will visit the site regularly to check the completeness 
of subject records, the accuracy of entries on the CRFs, the adherence to the protocol and to Good 
Clinical Practice, the progress of enrolment, and to ensure that study medication is being stored, 
dispensed, and accounted for according to specifications.  Key study personnel must be available 
to assist during these visits. In addition, 4D Pharma Plc staff may also attend site initiation, 
monitoring and close out visits with CRO personnel.  
The investigator must maintain source documents for each subject in the study and must provide 
the monitor access to all relevant source documents for confirmation of their consistency with the 
CRF entries. Monitoring will require full verification for the presence of informed consent, 
adherence to the inclusion/exclusion criteria, documentation of serious adverse events, and the 
recording of data that will be used for all primary and safety variables.  
Additional checks of the consistency of the source data with the CRFs may be performed 
according to the study-specific monitoring plan.  
9.2 Source Documents  
Source documents are original documents, data (e COA Smartphone data) and records and may 
include medical records, laboratory data, electrocardiograms, pharmacy records, subjects’ diaries 
and the results of any other tests or assessments.  Information on CRFs must be traceable to these 

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source documents in the subject's file.  Data not requiring a source document record will be 
defined before study start and will be recorded directly on the CRFs.  
The Investigator and clinical site will permit trial-related monitoring, audits, EC review, and 
regulatory inspections as requested, and must give direct access to source data/documents, CRFs 
and the Investigator site file.  
Clinical Investigators and authorized study personnel will review and access the electronic eCOA 
data; collected on smartphones and tablets, according to patient activity on site (following 
screening and scheduled clinic visits as indicated in the Protocol, e.g. Screening Visit, Baseline 
Visit 1, Visit 2, End of Treatment and Follow-Up. Discrepancies will be resolved using data 
queries and site response, with the oversight of the monitor. 
9.3 Data Collection 
Data collected from the study must be recorded in case report forms by delegated personnel at the 
Investigator sites.  Diary data will be collected using an app-based electronic Clinical Outcome 
Assessments (eCOA) solution for smartphone. Patients will receive daily reminders through the 
app to go and complete their diaries. As soon as they have completed their diary and click submit, 
their data will be sent to the hub. Patients will use the same devices to complete their other patient-
reported outcome questionnaires when they come into sites. Only personnel delegated the duty 
from the site’s PI, with documentation on the signature delegation log, will collect and enter data.  
CRFs will be reviewed for completeness and accuracy by the monitor and site personnel may be 
asked to make any required corrections or additions, as applicable.  The nature and location of 
source documents for the study will be documented in a specific document.  
Externally collected laboratory data will be received and processed to present a complete dataset.  
9.4 Database Management and Quality Control 
The database includes password protection and encryption and the data management process 
includes quality checks, to identify data that appear inconsistent, incomplete, or inaccurate. Data 
will be reviewed, and queries will be raised by data management in accordance with the study Data 
Management Plan.  
Errors or omissions will be detailed as Data Queries by Data Management and sent to the 
Investigational site for resolution. Quality control of all data in the database will be made prior to 
locking the database and the data set will then be transferred to the statistical programming team 
for creation of Tables, Figures, and Listing for review in accordance with the Statistical Analysis 
Plan. 
Access to the eCOA data will be controlled via the vendor Support Helpdesk. Users must have 
successfully completed the vendor training module, with a pass rate of >80%. Full log of query 
and resolution steps will be maintained within the system, with full audit trail 
Concomitant medications entered into the database will be coded using the WHO Drug Reference 
List, which employs the Anatomical Therapeutic Chemical classification system. Current medical 
conditions and adverse events will be coded using the Medical Dictionary for Regulatory 
Activities (MedDRA) terminology. 

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9.5 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol or GCP.  As a result of 
deviations, corrective actions are to be developed by the site and implemented promptly.  
Exemptions or eligibility criteria waivers for enrolment are not permissible. Any subject enrolled 
who does not meet eligibility criteria will be considered an enrolment deviation and must be 
reported as such.  All protocol deviations, as defined above, must be addressed in study subject 
source documents.  
9.6 Study Records Retention 
The study site will maintain a study file, which should contain, at minimum, the Investigator’s 
brochure, the Protocol and any Amendments, drug accountability records, correspondence with the 
IEC/IRB, 4D Pharma plc and assigned CRO and other study related documents. 
The investigator agrees to keep records and those documents that include (but are not limited to) 
the identification of all participating patients, medical records, study specific source documents, 
source worksheets, all original signed and dated ICFs, copies of all eCRFs, query responses and 
detailed records of drug disposition to enable evaluations or audits from regulatory authorities and 
4D Pharma or its designees.  
Study documents should be retained for a minimum of 25 years after the end of the clinical study. 
These documents should be retained for a longer period, however, if required by local regulations. 
Medical files of subjects should be archived in accordance with national requirements. No records 
will be destroyed without the written consent of the Sponsor, if applicable. It is the responsibility 
of the Sponsor to inform the Investigator when these documents no longer need to be retained 
The Investigator and study staff are responsible for maintaining an Investigator site file containing 
all study related Essential documents as defined by ICH GCP Section 8.  This must be available at 
any time during the study for review by the sponsor monitor and for inspection by the regulatory 
authority.  
10 STATISTICAL CONSIDERATIONS  
10.1 
Populations for Analysis 
The Safety Analysis Set includes all subjects randomised into the study who received at least one 
dose of Blautix™ or Placebo. Subjects will be allocated to the treatment group corresponding to 
the treatment actually received. 
The Safety Analysis Set is the primary analysis set for the safety analyses. 
The Full Analysis Set includes all members of the Safety Analysis Set who were appropriately 
randomised into the Study. Subjects will be allocated to the treatment group corresponding to the 
treatment actually received. 
The Full Analysis Set is the primary analysis set for the efficacy analyses. 
The Efficacy Evaluable Analysis Set includes all members of the Full Analysis Set who completed 
the 8-week treatment and assessment period without major protocol violations. Subjects will be 
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The final determination of the membership of analysis sets will be made at a blinded data review 
meeting convened by the Sponsor. 
10.2 
Sample Size Calculation 
Within each Cohort, 125 subjects per treatment group provide at least 80% power to demonstrate a 
statistically significant difference between the treatment groups at a one-sided alpha of 10% using 
Pearson's test with Yates's correction when the true response rate in Placebo group is 40% and the 
true response rate in the Blautix™ group is 55%.  
The total planned study size is therefore 500 subjects. 
10.3 
Interim Analysis  
An informal interim analysis will be conducted once all recruited patients have completed the 
primary efficacy analysis 8-week treatment period.  This interim analysis is planned to enable 4D 
pharma to expedite the clinical development strategy based on the outcome of the interim analysis. 
Planning for pivotal trials and the EU Paediatric Investigation Plan and the US Paediatric Study 
Plan can be initiated in preparation for Agency meetings and further clinical development.  There 
will be no other consequences of the interim analysis, there is no need to adjust the value of alpha. 
10.4 
Statistical Analysis Plan 
A detailed and comprehensive statistical analysis plan (SAP) describing the planned final analysis 
in detail with tables, figures and listings templates will be developed and finalised before the study 
is unblinded. 
Minor changes to the analyses specified in this section need not trigger a protocol amendment but 
must documented in the Clinical Study Report. 
10.5 
Statistical Analysis 
All baseline, compliance, disposition, efficacy and safety variables will be listed and summarised. 
The standard summary statistics for continuous baseline and outcome variables are: N, mean, 
standard deviation, median, quartiles, minimum and maximum. The standard summary statistics 
for categorical baseline and outcome variables are: count and proportion (expressed as percentage). 
There are 2 cohorts in the study: 
Cohort C: 250 subjects diagnosed with IBS-C  
Cohort D: 250 subjects diagnosed with IBS-D 
The two Cohorts will be analysed separately except there may be exploratory pooled or combined 
analyses.  
10.5.1 Primary Efficacy Analysis 
The primary efficacy analysis is the comparison of the proportion of overall responders between 
the Blautix™ and placebo groups using Pearson's test with Yates's correction in the full analysis 
set within each Cohort. 
All other analyses of the primary endpoint are secondary analyses. 

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evaluable weeks (if the total number of evaluable weeks is four, five or six) or for ≥ 50% of the 
evaluable weeks (if the total number of evaluable weeks is seven or eight). 
10.8 
Multiplicity 
As this is a Phase II study, there will be no formal adjustments for multiplicity.  
11 QUALITY CONTROL AND QUALITY ASSURANCE 
By signing this protocol, the Sponsor and Investigator agree to be responsible for implementing 
and maintaining QC and QA systems where applicable to ensure that the study is conducted, and 
the data is generated, documented and reported in compliance with the protocol, GCP and 
applicable regulations.  
The study may be audited by the Sponsor or its designee.  If such an audit occurs, the Investigator 
must allow access to required subject records and study related documentation.  
Should the Investigator or designee be notified of a regulatory inspection involving this study, they 
should notify the Sponsor immediately.  
12 ETHICS/REGULATORY 
This study will be conducted in compliance with the Protocol: Good Clinical Practices (GCPs) 
including International Conference on Harmonization (ICH) Technical Requirements for 
Registration of Pharmaceuticals for Human Use Guidelines; FDA regulatory requirements; and in 
accordance with the ethical principles of the Declaration of Helsinki. 
The sponsor or designee will submit the study protocol plus all relevant study documents to 
concerned regulatory agencies for approval before the study start. No patient will be admitted to 
the study until appropriate approval of the study protocol has been received.  
Each investigator must complete a Form FDA 1572 or equivalent and provide the completed form 
according to written instructions to the sponsor (or designee). Each investigator must submit to the 
sponsor (or designee) financial disclosure information according to national law and/or local 
requirements.  
US-generated data will be handled in accordance with the Health Information Portability and 
Accountability Act (HIPAA). The trial will be registered at www.clinicaltrials.gov using the 
Protocol Registration system. 
12.1 Institutional Review Board (IRB)/ Ethics Committee (EC) 
Prior to initiation of the study, the Investigator will submit the study protocol, participant 
information sheet, consent form and any other documents that pertain to subject information, to the 
IRB/EC. The Investigator must also submit any other information that may be requested to the 
IRB/EC for review and approval. The Investigator will request that the IRB/EC provide written 
favourable opinion of the study identifying all documents reviewed by name and version. The site 
will keep on file records of all documents and correspondence pertaining to this study. A letter 
confirming the favourable opinion must be forwarded to the Sponsor prior to initiation of this 
study.  

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The Investigator must submit and, where necessary, obtain favourable opinion from the IRB/EC 
for all substantial protocol amendments and changes to the ICF.  The Investigator should notify the 
IRB/EC of serious breaches, urgent safety measures or SUSARs occurring at the site, in 
accordance with local procedures. The Investigator will be responsible for submission of periodic 
progress reports at intervals not to exceed 1 year, or as otherwise specified by the IRB/EC. 
The Investigator will notify the IRB/EC within 90 days of the end of the study or within 15 days of 
early termination.  A reason for early termination will be provided.   
12.2 Regulatory 
The Sponsor will obtain approval of the appropriate regulatory body prior to study initiation. The 
Sponsor will also ensure that the implementation of substantial amendments to the protocol occurs 
only after approval of the relevant regulatory body.  The Sponsor should notify the regulatory body 
of serious breaches, urgent safety measures or SUSARs occurring at the site. 
The Sponsor will notify the regulatory body within 90 days of the end of the study or within 15 
days of early termination.  A reason for early termination will be provided.   
12.3 Serious Breaches  
A serious breach of GCP is defined as a breach which is likely to effect, to a significant degree, the 
safety or mental integrity of the subjects or the scientific value of the study.  Serious breaches must 
be reported by the Investigator to the Sponsor. The Sponsor must notify the MHRA of any serious 
breach within 7 days of becoming aware of the breach. The Investigator should report a serious 
breach to the IRB/EC.  
12.4 Informed Consent  
Informed consent must be obtained in writing for all subjects before any study specific procedure 
is performed. Subjects may be approached to discuss the study to determine if they are interested 
in participating in the study.  Information sheets will be provided, and consent taken when they 
return for the initial study assessment.  The information sheet will clearly explain what the study 
will involve and subjects must have adequate opportunity to ask questions.    
The physician or designee who conducts the informed consent discussion must also sign and date 
the consent form.  A copy of the signed consent document must be given to the subject. The 
original signed consent document will be retained by the Investigator. 
The information sheet and consent forms must contain the minimum elements required by ICH 
GCP and must be submitted for favourable opinion from the IRB/EC.  
If an amendment to the protocol changes the subject participation schedule in scope or activity, or 
if important new information becomes available that may be relevant to the subject’s consent, the 
ICF must be revised with a new version number and submitted to the IRB/EC for review and 
favourable opinion. The revised ICF must be used to obtain consent from a subject currently 
enrolled in the study if he or she is affected by the amendment.   The revised ICF must be used to 
obtain consent from any new subjects who are enrolled into the study after the date of the 
favourable opinion of the protocol amendment.  

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12.5 Future use of stored samples 
Samples will not be labelled with information that directly identifies the subject but will be coded 
with the identification number for the subject. 
Collected samples will be transferred for analysis to the Sponsor, or to other laboratories working 
for the Sponsor. 
Biological samples will be stored for the time established by regulatory requirements or destroyed 
after the final clinical study report has been finalized if storage is not required. There might be a 
new request for these samples to be used for purposes related to the QA of the laboratory tests 
described in this protocol, in which case they will be used for this purpose. This may include the 
assessment of the quality of current tests, the maintenance or improvement of these tests, the 
development of new test methods for the markers described in this protocol, as well as making sure 
that new tests are comparable to previous methods and work reliably. 
If study results suggest that further investigations using stored biological samples are warranted, 
these tests might be carried out on an exploratory basis. In addition, biological samples may be 
used by the Sponsor or their research partners for further research that is not related to the disease 
or the product under study. This testing will be done on pseudonymized samples (meaning that 
subjects will not be identifiable from their biological samples as their identity will have been 
removed and they will be assigned a unique clinical study identification number). Subjects will be 
asked to sign an additional, separate consent form for this optional testing and refusal of consent 
will not affect their possibility of participating in the study.  
During the conduct of the study, an individual subject can choose to withdraw consent to have 
biological specimens stored for future research. 
12.6 Subject Confidentiality 
In order to maintain subject privacy, all CRFs, diary cards, study drug accountability records, 
study reports, and communications will identify the subject only by the assigned subject number. 
The Investigator will grant monitors and auditors from the Sponsor or its designee, and regulatory 
authorities, access to the subject’s original medical records for verification purposes.  
12.7 Insurance 
The Sponsor has established an insurance policy for the anticipated duration of the study, covering 
the subjects with respect to the risks involved in taking part in this study in accordance with this 
protocol. In the case of injury or disability deriving from participation in the study, subjects are 
requested to inform the Investigator or his/her staff responsible for the study at the institution 
without delay 
13 PUBLICATION POLICY  
The Sponsor and Investigator will mutually manage the publication and presentation process. All 
manuscripts, abstracts, or other modes of presentation arising from the results of the study must be 
reviewed and approved in writing by the Sponsor in advance of submission in accordance with the 

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clinical site agreement.  The review is aimed at protecting the Sponsor’s proprietary information 
existing either at the date of the commencement of the study or generated during the study.  
The Sponsor will register the study on a public registry and results database of clinical trials.  
The Sponsor will publish the summary study results in EudraCT within six months following the 
end of the trial.  
The Sponsor and the Investigator will disclose the study results, in the form of a clinical study 
synopsis to the Health Authorities and the EC / IRB respectively, within 1 year of the end of the 
study. The format of this synopsis and that of the clinical study report will comply with the ICH3 
guidelines for the structure and content of a clinical study report.  

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14 
REFERENCES 
(1) 
Bai, T., J. Xia, Y. Jiang, H. Cao, Y. Zhao, L. Zhang, H. Wang, J. Song, and X. Hou. 2017. 
'Comparison of the Rome IV and Rome III criteria for IBS diagnosis: A cross-sectional 
survey', J Gastroenterol Hepatol, 32: 1018-25. 
(2) 
C Casteleyn, A Rekecki1, A Van der Aa, P Simoens and W Van den Broeck. 2010. Surface 
area assessment of the murine intestinal tract as a prerequisite for oral dose translation from 
mouse to man, Laboratory Animals, 44: 176–183. 
(3) 
Chassard, C., M. Dapoigny, K. P. Scott, L. Crouzet, C. Del'homme, P. Marquet, J. C. 
Martin, G. Pickering, D. Ardid, A. Eschalier, C. Dubray, H. J. Flint, and A. Bernalier-
Donadille. 2012. 'Functional dysbiosis within the gut microbiota of patients with 
constipated- irritable bowel syndrome', Aliment Pharmacol Ther, 35: 828-38. 
(4) 
Chey, W. D., J. Kurlander, and S. Eswaran. 2015. 'Irritable bowel syndrome: a clinical 
review', JAMA, 313: 949-58. 
(5) 
Collins, S. M. 2014. 'A role for the gut microbiota in IBS', Nat Rev Gastroenterol Hepatol, 
11: 497-505. 
(6) 
Crouzet, L., E. Gaultier, C. Del'Homme, C. Cartier, E. Delmas, M. Dapoigny, J. 
Fioramonti, and A. Bernalier-Donadille. 2013. 'The hypersensitivity to colonic distension 
of IBS patients can be transferred to rats through their fecal microbiota', 
Neurogastroenterol Motil, 25: e272-82. 
(7) 
Distrutti, E., L. Monaldi, P. Ricci, and S. Fiorucci. 2016. 'Gut microbiota role in irritable 
bowel syndrome: New therapeutic strategies', World J Gastroenterol, 22: 2219-41. 
(8) 
Drossman, D. A., C. B. Morris, Y. Hu, B. B. Toner, N. Diamant, J. Leserman, M. 
Shetzline, C. Dalton, and S. I. Bangdiwala. 2005. 'A prospective assessment of bowel habit 
in irritable bowel syndrome in women: defining an alternator', Gastroenterology, 128: 580-
9. 
(9) 
Enck, P., Q. Aziz, G. Barbara, A. D. Farmer, S. Fukudo, E. A. Mayer, B. Niesler, E. M. 
Quigley, M. Rajilic-Stojanovic, M. Schemann, J. Schwille-Kiuntke, M. Simren, S. Zipfel, 
and R. C. Spiller. 2016. 'Irritable bowel syndrome', Nat Rev Dis Primers, 2: 16014.  
(10) 
Jeffery, I. B., P. W. O'Toole, L. Ohman, M. J. Claesson, J. Deane, E. M. Quigley, and M. 
Simren. 2012. 'An irritable bowel syndrome subtype defined by species-specific alterations 
in faecal microbiota', Gut, 61: 997-1006. 
(11) 
Nair, J. and Jacobs, S. 2016. A simple practice guide for dose conversion between animals 
and human, Journal of Basic and Clinical Pharmacy, 7:27-30. 
(12) 
Tap, J., M. Derrien, H. Tornblom, R. Brazeilles, S. Cools-Portier, J. Dore, S. Storsrud, B. 
Le Neve, L. Ohman, and M. Simren. 2017. 'Identification of an Intestinal Microbiota 
Signature Associated With Severity of Irritable Bowel Syndrome', Gastroenterology, 15 
(13) 
Shanahan F and Quigley EMM. Manipulation of the microbiota for treatment of IBS and 
IBD – challenges and controversies. Gastroenterology 2014;146:1554-63. 

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About how you feel 
 
Please think about your life over the past month (last 30 days) and look at the statements 
below.  Each statement has five different responses.  For each statement, please circle the 
response that best describes your feelings. 
 
Q1.    I feel helpless because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q2.    I am embarrassed by the smell caused by my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q3.    I am bothered by how much time I spend on the toilet. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q4.    I feel vulnerable to other illnesses because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 

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Q5.    I feel fat/bloated because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q6.    I feel like I'm losing control of my life because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q7.    I feel my life is less enjoyable because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q8.    I feel uncomfortable when I talk about my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q9.    I feel depressed about my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 

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5 
EXTREMELY 
 
Q10.    I feel isolated from others because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q11.    I have to watch the amount of food I eat because of my bowel problems. (Please circle 
one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
 
Q12.    Because of my bowel problems, sexual activity is difficult for me. (Please circle one 
number)  
(If not applicable, please circle “NOT AT ALL”) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q13.    I feel angry that I have bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 

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Q14.    I feel like I irritate others because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q15.    I worry that my bowel problems will get worse. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q16.    I feel irritable because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
 
Q17.    I worry that people think I exaggerate my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q18.    I feel I get less done because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 

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5 
A GREAT DEAL 
 
Q19.    I have to avoid stressful situations because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q20.    My bowel problems reduce my sexual desire. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q21.    My bowel problems limit what I can wear. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q22.    I have to avoid strenuous activity because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 59 of 137 
Q23.    I have to watch the kind of food I eat because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q24.    Because of my bowel problems, I have difficulty being around people I do not know 
well. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q25.    I feel sluggish because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q26.    I feel unclean because of my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 60 of 137 
Q27.    Long trips are difficult for me because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q28.    I feel frustrated that I cannot eat when I want because of my bowel problems. (Please 
circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q29.    It is important to be near a toilet because of my bowel problems. (Please circle one 
number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 
Q30.    My life revolves around my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 61 of 137 
Q31.    I worry about losing control of my bowels. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q32.    I fear that I won't be able to have a bowel movement. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q33.    My bowel problems are affecting my closest relationships. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
A GREAT DEAL 
 
Q34.    I feel that no one understands my bowel problems. (Please circle one number) 
 
1 
NOT AT ALL 
2 
SLIGHTLY 
3 
MODERATELY 
4 
QUITE A BIT 
5 
EXTREMELY 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 64 of 137 
APPENDIX 3:  BRISTOL STOOL CHART  
 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 65 of 137 
APPENDIX 4:  IBS-SSS 
 
IBS QUESTIONNAIRE 
Name: 
 G.P. Name: 
 
Address: 
 Address: 
 
 
  
 
 
  
 
 
  
 
Telephone: 
 Telephone: 
 
Date of birth: 
  
Marital status: Single / Married / Divorced / Widowed / Co-Habit 
Occupation: 
 Sex: 
M 
 
F 
 
 
 
INSTRUCTIONS 
This form is designed to enable us to record and monitor the severity of your IBS. It is to be 
expected that your symptoms might vary over time, so please try and answer the questions 
based on how you currently feel (ie over the last ten days or so). All information will be kept 
in strict confidence. 
 
1. For questions where a number of different responses are a possibility please circle the 
response appropriate to you. 
 
2. Some questions will require you to write in an appropriate response. 
 
3. Some questions require you to put a cross on a line which enables us to judge the severity 
of a particular problem. 
 
For example: 
 
How severe was your pain? 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 66 of 137 
Please place your cross () anywhere on the line between 0-100%  in order to indicate as 
accurately as possible the severity of your symptom. 
 
This example shows a severity of approximately 90% 
 
0 % 
100 % 
no pain 
not 
very 
severe 
quite 
severe 
severe 
very severe 
 
1 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 67 of 137 
PART 1: SEVERITY SCORE 
1. a) Do you currently suffer from abdominal (tummy) 
pain? 
YES  
NO 
 
For 
office 
use 
only 
 
 
Circle 
appropriate box 
 
 
b) If yes, how severe is your abdominal (tummy) pain? 
SCORE 
 
 
 
0 % 
100 % 
 
 
 
no pain 
not 
very 
severe 
quite 
severe 
severe 
very severe 
 
 
 
 
 
c) Please enter the number of days that you get the pain in every ten days. 
For example if you enter 4 it means that you get pain 4 out of 10 days. If 
you get pain every day enter 10 
 
 
 
 
 
Number of days with pain 
 
x 10 
 
 
 
 
 
 
2. a) Do 
you 
currently 
suffer 
from 
abdominal 
distension* (bloating, swollen or tight tummy) 
(* women, please ignore distension related to your 
periods) 
YES  
NO 
 
 
 
Circle 
appropriate box 
 
 
 
 
 
 
b) If yes, how severe is your abdominal distension/tightness 
 
 
 
 
0 % 
100 % 
 
 
 
no distension 
not 
very 
severe 
quite 
severe 
severe 
very severe 
 
 
 
 
3. 
How satisfied are you with your bowel habit? 
 
 
 
 
0 % 
100 % 
 
 
 
very happy 
quite happy 
unhappy 
very unhappy 
 
 
 
 
 
 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 68 of 137 
 
4. 
Please indicate with a cross on the line below how much your Irritable 
Bowel Syndrome is affecting or interfering with your life in general. 
 
 
 
 
0 % 
100 % 
 
 
 
not at all 
not much 
quite a lot 
completely 
 
 
 
 
 
IBS SEVERITY SCORE: 
 
 
 
 
2 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 69 of 137 
PART 2: OTHER IBS DATA 
BOWEL HABIT 
 
5. a) What is the most number of times you open your bowels per day/week/month? 
 
 
 
Number of times 
 
per day / week / month 
(Circle appropriate) 
 
 
 
Note: For some people the answer to part a and b could be the same 
 
 
 
b) What is the least number of times you open your bowels per day/week/month? 
 
 
 
Number of times 
 
per day / week / month 
(Circle appropriate) 
 
 
6. In the following questions you may circle more than one answer: 
 
Are your motions ever: 
 
a) normal 
often / occasionally / 
never 
(Circle 
appropriate) 
 
b) hard 
often / occasionally / 
never 
(Circle 
appropriate) 
 
c) very thin (like string) 
often / occasionally / 
never 
(Circle 
appropriate) 
 
d) in small pieces (like rabbit pellets) 
often / occasionally / 
never 
(Circle 
appropriate) 
 
e) mushy (like porridge) 
often / occasionally / 
never 
(Circle 
appropriate) 
 
f) 
watery 
often / occasionally / 
never 
(Circle 
appropriate) 
 
 
7. In the following questions you may circle more than one answer: 
 
Do you ever: 
 
 
Circle 
appropriate box 
 
 
a) pass mucus (or slime or jelly) with your motions 
YES 
 
NO 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 70 of 137 
 
b) pass blood with your motions 
YES 
 
NO 
 
 
c) have to hurry/rush to the toilet to open your bowels 
YES 
 
NO 
 
 
d) strain to open your bowels 
YES 
 
NO 
 
 
e) feel 
you 
haven’t 
emptied 
your 
bowel 
completely 
after you have passed a motion 
YES 
 
NO 
 
 
 
 
3 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 71 of 137 
 
PART 2: Continued 
SITE OF PAIN 
 
Please mark with a cross (x) on the diagram below where you get your pain 
(use more than one x if necessary) 
 
 
RIGHT 
SIDE 
LEFT 
SIDE 
 
 
8.Do you ever: 
 a) notice your stools are more frequent or loose when you get pain 
YES 
 
NO 
 
 
Circle 
appropriate box 
 
 b) notice whether the pain is frequently eased by opening your 
bowels 
YES 
 
NO 
 
 
Circle 
appropriate box 
 
9.In the last year on approximately how many weeks were you: 
 i) 
absent from work due to IBS 
(enter 52 if you have given up completely work because of IBS) 
---------------------   
 ii) at work suffering from IBS 
---------------------   
 
4 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 72 of 137 
APPENDIX 5:  MODIFIED FFQ 
(US/UK Terminology) 
NOTE: FFQ completion at baseline will be in relation to food (s) consumed over the prior 6 
months. At each subsequent visit the FFQ shall capture responses to food (s) consumed since 
last subject visit. 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
1.   Beef roast (roast/steak) 
 
 
2.   Beef: steak 
 
 
3.   Beef: mince, ground beef 
 
 
4.   Beef: stew 
 
 
5.   Beef burger/meat loaf (1 burger) 
 
 
6.   Pork: roast/chops 
 
 
7.   Pork: chops 
 
 
8.   Pork: slices/escalopes 
 
 
9.   Lamb: roast 
 
 
10. Lamb: chops 
 
 
11. Lamb: stew 
 
 
12. Chicken portion or other poultry e.g. 
turkey: roast 
 
 
13. Breaded chicken, chicken nuggets, 
fried chicken, chicken burger 
 
 
14. Bacon 
 
 
15. Ham 
 
 
16. Corned beef, Spam, Luncheon meats, 
processed meats/deli meats 
 
 
17. Sausages, hot dog, chorizo, 
Frankfurters (1 sausage) 
 
 
18. Savoury pies (e.g. meat pie, pork pie, 
steak & kidney pie, sausage rolls) 
 
 
19. Liver, heart, kidney, tongue 
 
 
20. Liver paté 
 
 
21. Fish fried in batter), shrimps e.g. fish 
and chips 
 
 
22. Fish fried in breadcrumbs/cornmeal 
(catfish) 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 73 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
23. Oven baked/grilled fish (in 
breadcrumbs or batter) 
 
 
24. Fish fingers/fish cakes/fish sticks/fish 
sandwich/fish taco 
 
 
25. Other white fish, fresh or frozen (e.g. 
cod, haddock, plaice, sole, snapper, trout, 
tilapia, halibut, coli) 
 
 
26. Oily fish, fresh or canned (e.g. 
mackerel, kippers, tuna, salmon, sardines, 
herring) 
 
 
27. Shellfish (e.g. crab, prawns, mussels) 
 
 
28.   White bread and rolls (including 
ciabatta, panini bread & gluten free) 
 
 
29.   Brown bread/rolls, whole wheat , 
gluten free 
 
 
30.   Whole meal, rye, wholegrain bread/ 
rolls 
 
 
31.   Cream crackers, crackers, cheese 
biscuits, Ritz, saltines 
 
 
32. Tortilla, (corn or flour) 
 
 
 
 
 
 
 
 
 
 
 
33.   Crisp bread, crisp rye bread, e.g. 
Ryvita 
 
 
34. Pancakes, muffins, oatcakes, waffles, 
French toast 
 
 
35.   Hot cereal (Porridge, Ready Brek, 
cream of wheat, oatmeal, grits) 
 
 
36.   All Bran, High fibre, Raisin Bran, 
whole grain, grape nuts, bran cereal, 
Weetabix, Shredded Wheat, bran buds 
 
 
37.   Bran flakes, Bran Buds 
 
 
38.   Cornflakes, Rice Krispies, Cheerios 
 
 
39.   Muesli (e.g. Country Store, Alpen, 
sugar coated) 
 
 
40.   Sugar Coated Cereals (e.g. Frosties, 
Crunchy Nut Cornflakes, Crunchy Sugar 
Coated Muesli, fruit loops, lucky charms, 
fruit pebbles) 
 
 
41.   Boiled, instant or jacket potatoes 
 
 
42.   Mashed potatoes 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 74 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
43.   Chips (fries, French fries) 
 
 
44.   Roast potatoes 
 
 
45.   Potato Salad 
 
 
46.   White Rice 
 
 
47.   Brown Rice 
 
 
48.   White/yellow or green pastas (e.g. 
spaghetti, macaroni, noodles, penne) 
without meat sauce 
 
 
49.   Whole meal/gluten free pasta 
 
 
50. Lasagne (meat based) 
 
 
51. Lasagne (vegetarian) 
 
 
52. Moussaka, Burritos, Tacos, quesadillas 
 
 
53. Pizza 
 
 
54. Macaroni Cheese 
 
 
55.   Cream (tablespoon) 
 
 
56.   Full-fat (whole milk) yoghurt or 
Greek- style Yoghurt (125g carton) 
 
 
57.   Dairy desserts (ice cream - 125g 
carton) 
 
 
58.   Cheddar cheese, Monterey jack, 
Havarti, Munster, Swiss (medium serving) 
 
 
59.   Low-fat cheddar cheese (medium 
serving) 
 
 
60. Cottage Cheese 
 
 
61.   Eggs as boiled, fried, scrambled, 
poached (one) 
 
 
62.   Quiche (medium serving) 
 
 
63. Omelette  
 
 
 
 
 
 
 
 
 
 
 
64.   Light salad cream/dressing or light 
mayonnaise (tablespoon) 
 
 
65.   Salad cream, regular mayonnaise/ 
salad dressing (tablespoon) 
 
 
66. French dressing (tablespoon) 
 
 
67. Other salad dressing e.g. ranch/blue 
cheese 
Other - specify(tablespoon) 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 75 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
68. Butter (teaspoon) 
 
 
69. Lite Butter (reduce fat) e.g. Dawn Lite, 
Connacht Gold (teaspoon) 
 
 
70. Margarine (soft tub) e.g. Sunflower 
margarine, Flora (teaspoon) 
 
 
71. Low-fat/reduced fat margarine e.g. 
low- low (teaspoon) 
 
 
72.Lard or bacon grease 
 
 
 
 
 
 
 
 
 
 
 
73. Cholesterol Lowering Spreads e.g. 
Flora Pro Active, Dairy Gold Heart 
(teaspoon) 
 
 
74. Cream & Vegetable Oil spread e.g. 
Golden Pasture, Kerrymaid, Dairy Gold – 
teaspoon 
 
 
75. Olive oil spread/butter with olive oil 
e.g. Golden Olive (teaspoon) 
 
 
76.   Apples 
 
 
77.   Pears 
 
 
78.   Oranges, satsumas, mandarins 
 
 
79.   Grapefruit 
 
 
80.   Bananas 
 
 
81.   Grapes 
 
 
82.   Melon, water melon, cantaloupe 
 
 
83.   Peaches, plums 
 
 
84.   Apricots 
 
 
85. Strawberries, raspberries, kiwi fruit, 
blue berries 
 
 
86. Tinned/canned fruit, fruit cocktails 
(specify) 
 
 
87. Dried fruit e.g. raisins 
 
 
88. Frozen fruit 
 
 
89.   Carrots 
 
 
90.   Spinach 
 
 
91.   Broccoli, spring greens, kale, chard, 
spinach, collard greens 
 
 
92.   Brussel sprouts 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 76 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
93.   Cabbage 
 
 
94.   Peas 
 
 
95.   Green beans, broad beans, runner 
beans, lima beans, fava beans 
 
 
96.   Courgettes, zucchini, yellow squash 
 
 
97.   Cauliflower 
 
 
98.   Parsnips, turnips 
 
 
99.   Leeks 
 
 
100. Onions 
 
 
101. Garlic 
 
 
102. Mushrooms 
 
 
103. Sweet peppers 
 
 
104. Beansprouts 
 
 
105. Green salad, lettuce 
 
 
106. Cucumber, celery 
 
 
107. Tomatoes 
 
 
108. Sweetcorn, corn, kernels on the cob 
 
 
109. Beetroot, beet 
 
 
110. Coleslaw 
 
 
111. Baked beans 
 
 
112. Dried lentils, beans, peas, pinto, 
black, red, peas 
 
 
113. Tofu, soya meat, TVP 
 
 
114.   Chocolate coated sweet biscuits e.g. 
digestive (one) 
 
 
115.   Plain sweet biscuits e.g. Marietta, 
digestives, rich tea (one) 
 
 
116.   Cakes e.g. fruit, sponge 
 
 
117.   Buns, pastries e.g. croissants, 
doughnuts 
 
 
118.   Fruit pies, tarts, crumbles, cobblers 
 
 
119.   Sponge puddings 
 
 
120.   Milk puddings e.g. rice, custard, 
trifle, flan 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 77 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
121.   Ice cream, choc ices, Frozen 
desserts, ice cream sandwiches, frozen 
popsicles 
 
 
122.   Chocolates, singles or squares 
 
 
123. Sweets, candy, toffees, mints 
 
 
124. Sugar added to tea coffee, cereal 
(teaspoon) 
 
 
125. Sugar substitute e.g. Canderel added 
to tea coffee, cereal (teaspoon) 
 
 
126. Crisps, potato chips, tortilla, Pretzel, 
popcorn or other packet snacks 
 
 
127. Peanuts 
 
 
128. Nuts (other than peanuts) 
 
 
129.   Vegetable soups: homemade/fresh 
(1 bowl) 
 
 
130.   Vegetable soups: tinned/packet (1 
bowl) 
 
 
131.   Meat soups: homemade/fresh (1 
Bowl) 
 
 
132.   Meat/cream soups: 
tinned/canned/packet (1 bowl) 
 
 
133.Chicken and noodle soup 
(hand/homemade) 1 bowl 
 
 
 
 
 
 
 
 
 
 
 
134.   Sauces e.g. white sauce, cheese 
sauce, gravy (1 tablespoon) 
 
 
135.   Tomato based sauces e.g. pasta 
sauces 
 
 
136.Tomato sauce with meat 
 
 
 
 
 
 
 
 
 
 
 
137. Miso, wonton soup 
 
 
 
 
 
 
 
 
 
 
 
138. Curry-type sauces 
 
 
139. Pickles, chutney, ketchup, mustard 
(tablespoon) 
 
 
140. Marmite, Bovril (tablespoon) 
 
 
141. Jam, marmalade, honey, syrup 
(teaspoon) 
 
 
142. Peanut butter (teaspoon) 
 
 
143. Tea (cup) 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 78 of 137 
Food Name 
Never 
or < 
than 
once a 
month 
once a 
month 
2-3 times a 
month 
once a 
week 
2-4 times a 
week 
5 times a 
week 
once a day 
2-4 times a 
day 
5 times a 
day 
> 5 times a 
day 
Comment 
144. Herbal tea (cup) 
 
 
145. Coffee instant (cup) 
 
 
146.   Coffee ground (cup) 
 
 
147.   Coffee, decaffeinated (cup) 
 
 
148.   Coffee whitener e.g. coffee-mate, 
coffee creamer milk (teaspoon) 
 
 
149.   Cocoa, Hot Chocolate (cup) 
 
 
150.   Horlicks, Ovaltine, malt drinks (cup) 
 
 
151.   Wine (glass) 
 
 
152.   Beer, Larger or Cider (half pint) 
 
 
153. Alcopops (low alcoholic/alcohol free 
drinks e.g. Bacardi Breezer 
 
 
154. Whole milk (cup) – cow or goat 
(including milk used in tea, cereal etc) 
 
 
155. Milk substitute (almond, rice, cashew, 
soya) specify 
 
 
 
 
 
 
 
 
 
 
 
156. Semi-skimmed milk/half milk (cup) 
(including milk used in tea, cereal etc) 
 
 
157. Port, Sherry, Vermouth, liqueurs 
(glass) 
 
 
158. Spirits e.g. Gin, Whiskey (single 
measure) 
 
 
159. Low calorie or diet soft fizzy (glass) 
 
 
160. Fizzy Soft drinks e.g. Cocoa Cola 
(glass) 
 
 
161. Pure fruit drinks/fruit juice e.g. 
orange juice (small glass) 
 
 
162. Fruit squash/cordial (small glass) 
 
 
163.   Probiotic drinks/yoghurt e.g. 
Actimel, Yakult, kefir 
 
 
164.   Vitamin supplements (if so please 
include details below) 
 
 
 
 
 

 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
 
 
  
 
  
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
  
 
 
   
  
 
 
  
 
 
 
  
 
  
  
 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 80 of 137 
APPENDIX 7:  PROHIBITED MEDICATIONS 
 
IBS-C Patients: 
Guanylate cyclase agonists - 
Linaclotide 
Lubiprostone 
 
IBS-D Patients: 
5-hydroxytryptamine (serotonin) 3 receptor antagonists 
Alosetron 
Bile Acid sequestrants 
Cholestyramine 
Colesevelam 
Colestipol  
Eluxadoline 
Rifaximin 
 
Probiotics 
Probiotics 
Alflorex (EU) 
Align (North America) 
Align ® (B. longu m infantis 35 624) Capsules 
Align Probiotic Supplement Capsules ((Lactobacillus GG) 
BioGaia L.Reuteri ProTectis Probiotic (Lactobacillus) Capsules 
Bioglan products 
Bio-Kult 
Culturelle Probiotic (Lactobacillus GG)Capsules 
Digestive Care™ (L. plantarum 299v) Capsules 
Human MicroFlora products by Genestra™  
IbSium® (Saccharomyces cerevisiae I-3856) Capsules 
Mutaflor ® (active ingredient Escherichia coli strain Nissle 1917) 
Prescript-Assist (Broad Spectrum Probiotic Prebiotic ) Capsules 
ProBio 7 products 
Proxiflor ® (L. rhamnosus R0011 L.helveticus R0052 )Capsules 
TuZen® (L. plantar um 299v) Capsules 
Udos probiotics 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 81 of 137 
Ultra Probiotic Complex by GNC-  
UltraFlora Intensive Care (L. planta rum 299v) Capsules 
Vitabiotic products 
Yakult (Lactobacillus casei Shirota) packet 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 82 of 137 
APPENDIX 8:  SUMMARY OF PROTOCOL CHANGES VERSION 1.1 
 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Original Version:  
Version 1  
Version Date:  
6 February 2018 
 
Updated Version: 
Version 1.1  
Version Date: 
6 April 2018 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 83 of 137 
A6.1 Section: Change to the header information on all Protocol pages 
Old Text:  
Blautix™, Blautia hydrogenotrophica 
 
February 2018 
New Text:  
Blautix™, Blautia hydrogenotrophica  
Irritable Bowel Syndrome 
Clinical Study Protocol No: BHT-II-002 Version 1.1 
Date: 3rd April 2018 
Reason:  
To amend the Protocol header information from the IND submission format and make clear 
reference to the therapy area and Protocol version and date. This change has been made at the 
request of the Investigators.  
 
A6.2 Section: Page 8 Synopsis  
Old Text:    
Reference Therapy, Dose and Route of Administration: 
Matching placebo, administered orally.  
Each placebo capsule will be comparable in size, weight and appearance to the test formulation 
and subjects will receive one capsule twice daily before food for 8 weeks.  
New Text:  
Reference Therapy, Dose and Route of Administration: 
Matching placebo, administered orally.  
Each placebo capsule will be comparable in size, weight and appearance to the test formulation 
and subjects will receive two capsules twice daily approximately 30 minutes before food for 
8 weeks.  
Reason:  
To amend an administrative error and correct the reference to ‘one capsule twice daily’ the 
daily dose is ‘two capsules twice daily’ and amend the timing of the dosing to be consistent 
with other sections of the Protocol. This administrative change has been made to ensure 
correctness and consistency throughout the protocol.  
 
CCI

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 84 of 137 
A6.3 Section: Page 8 Synopsis  
Old Text:   
Test Product, Dose and Route of Administration: 
Capsules of Blautia hydrogenotrophica (Blautix™) administered orally.   
Each capsule contains ≥5x 109MPN Blautia hydrogenotrophica /capsule and subjects will 
receive two capsules twice daily before food for 8 weeks. 
New Text:  
Test Product, Dose and Route of Administration: 
Capsules of Blautia hydrogenotrophica (Blautix™) administered orally.   
Each capsule contains ≥5x 109MPN Blautia hydrogenotrophica /capsule and subjects will 
receive two capsules twice daily approximately 30 minutes before food for 8 weeks 
Reason:  
To amend an administrative error regarding the timing of the dosing to be consistent with other 
sections of the Protocol. This administrative change has been made to ensure correctness and 
consistency throughout the protocol.  
 
A6.4 Section:  Overall Study Design Section 4.2.1 
Old Text:  
Baseline Visit 1 (Day 1) 
Subjects will use their smartphone issued at the Screening visit to capture their study related 
data; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Abdominal Pain Intensity Numeric Rating Scale (NRS) completed daily 
• 
Stools consistency/frequency completed daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) completed on the Day of each Clinic 
Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of each Clinic visit or within 24 
hrs prior to the visit 
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of each Clinic visit or within 24hours 
prior to the visit  

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 85 of 137 
New Text:  
Subjects will use their smartphone issued at the Screening visit to capture their study related 
data. The IBS-SSS and FFQ questionnaires will be performed at the clinic on a tablet 
device.  
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Abdominal Pain Intensity Numeric Rating Scale (NRS) completed daily 
• 
Stools consistency/frequency completed daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) completed on the Day of each Clinic 
Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of each Clinic visit 
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of each Clinic visit 
Reason:  
Modification of data capture of IBS-SSS and FFQ questionnaires to a tablet device during the 
clinic visit as the smartphone functionality cannot accommodate these assessments due to the 
both the length and complexity. This change has been made at the request of the Investigators.  
 
A6.5 Section:  Overall Study Design Section 4.2.1 
Old Text:  
Visit 2 (Week 4-5)  
Subjects will continue to use their smartphones to capture all the patient related outcome data.  
The data will be reviewed on an ongoing basis as required by the study team and sponsor. At 
this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 86 of 137 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit or within 24 hrs 
prior to the visit 
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit or within 24hours prior 
to the visit  
New Text:  
Subjects will continue to use their smartphones to capture all the study related outcome data, 
except for IBS-SSS and FFQ questionnaires which will be completed during the clinic 
visit. The data will be reviewed on an ongoing basis as required by the study team and sponsor. 
At this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit  
Reason:  
Modification of data capture of IBS-SSS and FFQ questionnaires to a tablet device during the 
clinic visit as the smartphone functionality cannot accommodate these assessments due to the 
both the length and complexity. This change has been made at the request of the Investigators.  
 
A6.6 Section:  Overall Study Design Section 4.2.1 
Old Text:  
End of Treatment (Week 8-9)  
Subjects will continue to use their smartphones to capture all the study related outcome data.  
The data will be reviewed on an ongoing basis as required by the study team and sponsor. At 
this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 87 of 137 
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit or within 24 hrs 
prior to the visit 
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit or within 24hours prior 
to the visit  
New Text:  
Subjects will continue to use their smartphones to capture all the patient related outcome data, 
except for IBS-SSS and FFQ questionnaires which will be completed during the clinic 
visit. The data will be reviewed on an ongoing basis as required by the study team and sponsor. 
At this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit  
Reason:  
Modification of data capture of IBS-SSS and FFQ questionnaires to a tablet device during the 
clinic visit as the smartphone functionality cannot accommodate these assessments due to the 
both the length and complexity. This change has been made at the request of the Investigators.  
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 88 of 137 
A6.7 Section:  Overall Study Design Section 4.2.1 
Old Text:  
Follow-Up (Week 12-14)  
Subjects will continue to use their smartphones to capture all the study related outcome data.  
The data will be reviewed on an ongoing basis as required by the study team and sponsor. At 
this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit or within 24 hrs 
prior to the visit 
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 
• 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit or within 24hours prior 
to the visit  
New Text:  
Subjects will continue to use their smartphones to capture all the study related outcome data, 
except for IBS-SSS and FFQ questionnaires which will be completed during the clinic 
visit. The data will be reviewed on an ongoing basis as required by the study team and sponsor. 
At this visit the following information will be reviewed with the subject; 
• 
Information on Adverse Events 
• 
Review of Medication log completed daily  
• 
Recording times of taking their medication completed daily 
• 
Abdominal Pain Intensity NRS completed daily 
• 
Stools consistency/frequency daily  
• 
Subject Global Assessment of Relief completed weekly 
• 
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
• 
IBS Symptom Severity Score (IBS-SSS) on the Day of Clinic visit  
• 
Hospital Anxiety and Depression score (HADS) on Day of each Clinic visit or within 
24 hours prior to the visit 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 89 of 137 
x 
Food Frequency Questionnaire (FFQ) on the Day of Clinic visit  
Reason:  
Modification of data capture of IBS-SSS and FFQ questionnaires to a tablet device during the 
clinic visit as the smartphone functionality cannot accommodate these assessments due to the 
both the length and complexity. This administrative change has been made to ensure 
correctness and consistency throughout the protocol.  
 
A6.8 Section:  INVESTIGATIONAL PRODUCTS: Formulation Section 6.1  
Old Text:  
 
New Text:  
   
Reason:  
 
 
A6.9 Section:  Dosage, Frequency and Duration of dosing Section 1.2 
Old Text:  
Blautix™ or placebo capsules will be administered twice daily at 12 hourly intervals before 
food for a duration of 8 weeks. 
 
 Subjects will be supplied with enough treatment supply at the Baseline 
Visit to last until the next scheduled Clinic Visit and then at subsequent visits.   
New Text:  
Blautix™ or placebo capsules will be administered twice daily at 12 hourly intervals 
approximately 30 minutes before food for a duration of 8 weeks. 
 
 Subjects will be supplied with enough 
treatment supply at the Baseline Visit to last until the next scheduled Clinic Visit and then at 
subsequent visits.   
CCI
CCI
CCI
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Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 90 of 137 
Reason:  
Correction of an administrative error and consistency with other sections of the Protocol. This 
administrative change has been made to ensure correctness and consistency throughout the 
protocol.  
 
A6.10 Section: Clinical Assessments Section 7.1 
Old Text:  
Food Frequency Questionnaire (FFQ) (Appendix 5)  
Subjects will be asked to complete a questionnaire to obtain frequency information about food 
and beverage consumption over a specified period of time (day, week and month). Once 
subjects are randomised they will be asked to complete this questionnaire on their smartphone 
at each clinic visit or within 24hrs prior to the Clinic Visit. 
New Text:  
Food Frequency Questionnaire (FFQ) (Appendix 5)  
Subjects will be asked to complete a questionnaire to obtain frequency information about food 
and beverage consumption over a specified period of time (day, week and month). Once 
subjects are randomised they will be asked to complete this questionnaire on a tablet device 
during their clinic visit. 
Reason:  
Modification of data capture of FFQ to a tablet device during the clinic visit as the smartphone 
functionality cannot accommodate this assessment. This change has been made at the request 
of the Investigators.  
 
A6.11 Section: Clinical Assessments Section 7.1 
Old Text:  
IBS Symptom Severity Score (SSS) (Appendix 4) 
Subjects will be asked to complete a questionnaire on severity of abdominal distension, 
severity of abdominal pain, frequency of abdominal pain, dissatisfaction with bowel habits, and 
interference of IBS symptoms with daily life.  A score ranging from 0 (no symptoms) to 500 
(maximum severity) can be achieved as mild (75-175), moderate (176-300) or severe (>300) 
IBS symptoms.  
The initial data from this score will be collected during the Screening visit on the smartphone 
provided to the subject. This data will be used to classify into Cohorts of IBS sub-types prior to 
randomisation. Once subjects are randomised they will be asked to record their score on their 
smart phone at each clinic visit or within 24hrs prior to the Clinic Visit. 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 91 of 137 
New Text:  
IBS Symptom Severity Score (SSS) (Appendix 4) 
Subjects will be asked to complete a questionnaire on severity of abdominal distension, 
severity of abdominal pain, frequency of abdominal pain, dissatisfaction with bowel habits, and 
interference of IBS symptoms with daily life.  A score ranging from 0 (no symptoms) to 500 
(maximum severity) can be achieved as mild (75-175), moderate (176-300) or severe (>300) 
IBS symptoms.  
The initial data from this score will be collected during the Screening visit on a tablet device 
during the clinic visit.  This data will be used to classify into Cohorts of IBS sub-types prior 
to randomisation. Once subjects are randomised they will be asked to record their score 
during each clinic visit on a tablet device.   
Reason:  
Modification of data capture of IBS-SSS to a tablet device during the clinic visit as the 
smartphone functionality cannot accommodate this assessment. This change has been made at 
the request of the Investigators.  
 
A6.12 Section: Safety Assessments Section 7.2.1  
Old Text:  
Haematology:  
Haemoglobin (HGB), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin 
(MCH), mean cell haemoglobin concentration (MCHC), red blood cells (RBC), erythrocyte 
sedimentation rate (ESR), white cell count (WCC) and platelets (PLT).  Differential white 
cell count will include neutrophils, lymphocytes, eosinophils, basophils and monocytes as 
absolute values and percentages. 
New Text:  
Haematology:  
Haemoglobin (HGB), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin 
(MCH), mean cell haemoglobin concentration (MCHC), red blood cells (RBC), C-reactive 
Protein (CRP), white cell count (WCC) and platelets (PLT).  Differential white cell count will 
include neutrophils, lymphocytes, eosinophils, basophils and monocytes as absolute values and 
percentages. 
Reason:  
Removal of ESR and replacement with CRP test within the Hematology panel as this is a more 
accurate measurement for investigator review. In addition, CRP can be analyzed at the central 
lab, ESR would need to be measurement locally. The Protocol does not allow for local 
laboratory assessment, all safety assessments are to be carried out by the central laboratory. 
This change has been made at the request of the Investigators.  
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 92 of 137 
A6.13 Section: Safety Assessments Section 7.2.1 
Old Text:  
Serology: At screening the analyses will also include HIV-1 & HIV-2, hepatitis B and hepatitis 
C.    
New Text:  
Serology: At screening the analyses will also include HIV-1 & HIV-2, Hepatitis B surface 
antigen (HBsAg), a Hepatitis B surface antibody (Anti-HBs) and a Hepatitis core 
antibody test (anti-HBc)  
Reason:  
Additional testing for Hepatitis B surface antigen, a Hepatitis B surface antibody and a 
Hepatitis core antibody test has been added for all subject at screening. This should avoid 
inclusion of subjects with previous or ongoing HBV infection, a positive result will exclude a 
subject unless the Hepatitis B surface antibody is the sole positive result (indicating subject's 
immunization). This change has been made at the request of the Investigators. 
 
A6.14 Section: Safety Assessments Section 7.2.1 
Old Text:  
Fasting serum will be required for bile acid analysis where 7a-hydroxy-4-cholesterol-3-one 
(C4) will be assayed using LC-MS/MS as an alternative to the SeHCAT test where it is 
unavailable.  
New Text:  
Removal of this test from the planned per Protocol assessments.  
Reason:  
Fasting serum is required for this test and it is not feasible for sites to obtain a fasting sample 
from patients enrolled in the study. In addition, this assessment is for an Exploratory Endpoint 
assessment and the decision was made to remove the test from the Protocol. This change has 
been made at the request of the Investigators.  
 
A6.15 Section: Serious Adverse Event Reporting Section 8.3  
Old Text:  
It is the responsibility of the Sponsor to report fatal or life-threatening SUSARs to the MHRA 
as soon as possible but no later than 7 calendar days after they first become aware of the 
reaction. The Investigator is required to notify the ethics committee (EC) of any fatal or life-
threatening SUSARs as soon as possible but no later than 7 calendar days after they first 
become aware of the reaction.  Any additional relevant information should be sent within 8 
days of the report. 
The Sponsor is responsible for reporting other (non-fatal, non-life-threatening) SUSARS to the 
Health Authorities as soon as possible but no later than 15 calendar days after they first become 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 93 of 137 
aware of the reaction.  The Investigator is required to notify the EC of any non-fatal or non-
life-threatening SUSARs as soon as possible but no later than 15 calendar days after they first 
become aware of the reaction. 
The Sponsor is required to inform the Health Authorities, the Investigator and EC within 3 
calendar days of an urgent safety issue.  
New Text:  
4D Pharma, or it’s designee, reports SAE’s and/or SUSARS as required to regulatory 
authorities, investigators/institutions, and IRBs/IECs in compliance with all reporting 
requirements according to local regulations and good clinical practice. 
The investigator is to notify the appropriate IRB/IEC of SAEs occurring at the site and 
other AE reports received from 4D Pharma or it’s designee, in accordance with local 
procedures and statutes.  
Reason:  
The original text referred to the MHRA specifically and as this is a global study, the change 
was made to apply to a generic statement to comply with all agency and local requirements in 
all countries conducting the trial. This administrative change has been made to ensure 
correctness and consistency throughout the protocol. 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 94 of 137 
APPENDIX 9:  SUMMARY OF PROTOCOL CHANGES VERSION 1.2 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Original Version:  
 
Version 1  
Version Date:  
 
6 February 2018 
 
Updated Version: 
 
Version 1.1  
Version Date: 
 
6 April 2018 
 
Updated Version as per MHRA response:  
Version 1.2 
 
 
19 May 2018 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 95 of 137 
Section: Study Design Section 1.2  
Old Text:  
Screening Visit  
Sexually-active men or women must use an accepted form of birth control throughout the study 
until completion of two (2) periods after the last dose to avoid pregnancy for at least one 
complete menstrual cycle). The following methods of birth control are considered highly 
effective and acceptable for this study; true abstinence, sterilization, birth control pills, 
vasectomised partner, injections or contraceptive implants. For male subjects, able to father 
children, and sexually active with a female partner who is able to bear children, must use a 
barrier method of birth control (male condom with or without spermicide). Subjects should not 
donate sperm or have unprotected intercourse with a female who is pregnant or is breast-
feeding from the time of informed consent until the Follow-up visit. 
New Text:  
Remove text relating to birth control from screening section as the text is repeated in Section 
8.3.1 Pregnancies.  
 
Section 8.3.1 Pregnancies  
Old Text:  
Subjects who are women and who may be able to have children, will be given a pregnancy test 
at screening and all subsequent study visits. If the result is positive at screening, the subject 
will not be able to enter the study.  Sexually-active men and women must use an accepted form 
of birth control throughout the study until completion of two (2) periods after the last dose to 
avoid pregnancy for at least one complete menstrual cycle). The following methods of birth 
control are considered highly effective and acceptable for this study; true abstinence, 
sterilization, birth control pills, vasectomised partner, injections or contraceptive implants. 
Male subjects, able to father children, and sexually active with a female partner who is able to 
bear children, must use a barrier method of birth control (male condom with or without 
spermicide) together with a highly effective method of birth control used by your female 
partners. Subjects should not donate sperm or have unprotected intercourse with a female who 
is pregnant or is breast-feeding from the time of informed consent until the Follow-up visit. 
Female Subjects must not be breast feed an infant during the study. 
New Text: 
Subjects who are women and who may be able to have children will be given a serum 
pregnancy test at screening and a urine dipstick test at all subsequent study visits. If the result 
is positive at screening, the subject will not be able to enter the study.  Sexually-active women 
must use an acceptable form of birth control throughout the study until completion of two (2) 
periods after the last dose to avoid pregnancy for at least one complete menstrual cycle. The 
following methods of birth control are considered acceptable for this study; true abstinence, 
sterilization, birth control pills, vasectomised partner, injections or contraceptive implants. 
Female Subjects must not be breast feed an infant during the study. 

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Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 96 of 137 
Male subjects, able to father children, and sexually active with a female partner who is able to 
bear children, must use a barrier method of birth control (male condom with or without 
spermicide) from the time of randomisation until the Follow-up visit.  
Reason:  
Revisions made in response to MHRA requests during review of the clinical trial application.  
 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 97 of 137 
APPENDIX 10:  SUMMARY OF PROTOCOL CHANGES VERSION 2.0 
 
 
 
Summary of Protocol Changes 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Previous Date and Version: 6 February 2018; Version 1.0 
 
6 April 2018; Version 1.1 
 
19 May 2018: Version 1.2 
 
Updated Current Version: 31 May 2018: Version 2.0 
 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 

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Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 98 of 137 
Summary of Protocol Changes 
 
Section: 3.2 Endpoints – Primary Efficacy Endpoint  
Old Text:  
IBS-D 
Abdominal Pain Intensity  
Decrease in weekly average 
of worst abdominal pain in 
past 24 hours score of at 
least 30% compared with 
baseline  
 
and  
 
Stool Consistency (BSS) 
Decrease at least 50% in the 
number of days per week 
with at least one stool that 
has a consistency of Type 6 
or 7 compared with baseline. 
 
New Text:  
IBS-D 
Abdominal Pain Intensity  
Decrease in weekly average 
of worst abdominal pain in 
past 24 hours score of at 
least 30% compared with 
baseline  
 
and  
 
Stool Consistency (BSS) 
Decrease at least 50% in the 
proportion of days per week 
with at least one stool that 
has a consistency of Type 6 
or 7 compared with baseline. 
 

Blautix™, Blautia hydrogenotrophica  (Irritable Bowel Syndrome) 
Clinical Study Protocol No: BHT-II-002  
Date and Version: 31-Mar-20 Version 3.1 
 
 
 
Page 99 of 137 
Reason: A subject may only have recorded the worst stool on four, five or six days of the week 
(see Section 10.7: Handling of Missing Data).  A reduction in the number of days with Type 6 
or 7 consistency recorded may simply be due to data being available for fewer days.  This 
difficulty is avoided by expressing the decrease as a proportion. This change has been made at 
the request of the Investigators. 
 
Section: 4.2 Study Design 
Screening Visit  
Old Text:  
At this visit they will complete the IBS-Symptom Severity Score on this device.  
New Text:  
At this visit they will complete the IBS-Symptom Severity Score on this on a tablet device 
during the clinic visit.  
Reason:  
Modification of data capture of IBS-SSS questionnaire to a tablet device during the clinic visit 
as the smartphone functionality cannot accommodate these assessments due to the both the 
length and complexity. This change has been made at the request of the Investigators. 
Screening/Baseline (Visit 1)/ Visit 2 (Week 4-5)/ End of Treatment (Week 8-9) 
Old Text:  
Subjects will be provided with a stool collection kit to take home. They will be provided with 
instructions and asked to provide a stool sample within 48 hours prior to or on the morning of 
Baseline (Visit 1) 
New Text:  
Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next 
clinic visit  
Reason:  
Stool samples will need to be frozen at -80 degrees as soon as is possible to avoid significant 
degradation we are requesting that subjects provide samples as close to the timing of their 
clinic visit as possible. This change has been made at the request of the Investigators. 
Baseline (Visit 1)/ Visit 2 (Week 4-5)/ End of Treatment (Week 8-9)/Follow-Up week 12-
14) 
Old Text:   
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit 
New Text:  
IBS Quality of Life questionnaire (IBS QOL) on the Day of Clinic Visit or within 24 hours 
prior to the visit 

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Reason:  
Consistency throughout the Protocol and provide more flexibility for completion of this 
assessment. This change has been made at the request of the Investigators. 
 
Section: 10.1: Population for Analysis  
Old Text:  
The Safety Analysis Set includes all subjects randomised into the study who received at least 
one dose of Blautix™ or Placebo. 
The Safety Analysis Set is the primary analysis set for the safety analyses. 
The Full Analysis Set includes all members of the Safety Analysis Set who were appropriately 
randomised into the Study. 
The Full Analysis Set is the primary analysis set for the efficacy analyses. 
The Efficacy Evaluable Analysis Set includes all members of the Full Analysis Set who 
completed the 8-week treatment and assessment period without major protocol violations. 
New Text:  
The Safety Analysis Set includes all subjects randomised into the study who received at least 
one dose of Blautix™ or Placebo.  Subjects will be allocated to the treatment group 
corresponding to the treatment actually received. 
The Safety Analysis Set is the primary analysis set for the safety analyses. 
The Full Analysis Set includes all members of the Safety Analysis Set who were appropriately 
randomised into the Study.  Subjects will be allocated to the treatment group corresponding 
to the treatment actually received. 
The Full Analysis Set is the primary analysis set for the efficacy analyses. 
The Efficacy Evaluable Analysis Set includes all members of the Full Analysis Set who 
completed the 8-week treatment and assessment period without major protocol violations.  
Subjects will be allocated to the treatment group corresponding to the treatment actually 
received. 
Reason:  
Text has been added to clarify that subjects will be analysed according to the treatment actually 
received, as is appropriate for a Phase II randomised study. Change made at the request of 
FDA.  
 
Section: 10.7 Handling of Missing Data  
Old Text:   
Subjects who do not provide enough data to enable assessment of whether or not that subject is 
a responder will be deemed a non-responder. 

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New Text:  
Subjects who do not provide enough data to enable assessment of whether or not that subject is 
a responder will be deemed a non-responder. 
An 'evaluable week' is one in which there are at least four twenty-four hour (4 day) 
records of abdominal pain score and at least four daily records of number of complete 
spontaneous bowel movements (IBS-C subjects) or of highest Bristol stool chart (IBS-D 
subjects). 
If a subject has not provided four evaluable weeks of data, then they are deemed to be a 
non-responder. 
If a subject has provided at least four evaluable weeks of data then they are deemed to 
be a responder if they have reported an improvement in their weekly symptoms for ≥ 
60% of the evaluable weeks (if the total number of evaluable weeks is four, five or six) 
or for ≥ 50% of the evaluable weeks (if the total number of evaluable weeks is seven or 
eight). 
Reason:  
Text has been added to set out the rules (i) for determining whether sufficient data is available 
to confirm a response and (ii) for determining whether or not a response has occurred if there is 
incomplete but sufficient data. Change made at the request of FDA.  
 
Section: 5.2 Exclusion Criteria  
Exclusion Old Text: 
Number 10.  
Refusal to use highly effective methods of birth control (true abstinence, sterilisation, 
birth control pills, injections or contraceptive implants) for fertile patients (females and 
males) while on treatment and following completion of 2 periods months after the last 
dose of study treatment  
Exclusion New Text:  
Number 10 
Refusal to use acceptable methods of birth control (true abstinence, sterilisation, birth 
control pills, injections or contraceptive implants) for fertile patients (females) while on 
treatment and following completion of 2 menstrual cycles/months after the last dose of 
study treatment. For Males, a barrier method of birth control from randomisation until 
the Follow-Up visit 
Reason: Changes have been made to the classification of birth control during this study 
following MHRA requested changes. Further changes have been made to the Exclusion criteria 
to align with these revisions in Section 8.3.1 Pregnancies.  
 

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Exclusion Old Text:  
Number 15:  
Lactose intolerance  
Number 16: 
Coeliac disease  
Exclusion New Text  
Number 15. Clinically diagnosed Lactose intolerance 
Number 16. Clinically diagnosed Coeliac disease  
Reason:  
To provide further guidance to the investigators that these exclusion points are referring to a 
clinical diagnosis and therefore clear medical records to confirm. This change has been made at 
the request of the Investigators. 
 
Old Text  
Exclusion Number 18  
Those > 55 will be excluded if their diagnosis of IBS is recent (<12 months) and if they 
have not had a sigmoidoscopy or colonoscopy within previous 5 years. 
New Text: 
Exclusion Number 18 
Number 18: Those > 50 will be excluded if their diagnosis of IBS is recent (<12 
months) and if they have not had a sigmoidoscopy or colonoscopy within previous 
5 years. 
Reason:  
Reduction to the upper age limit from >55 to >50 to comply with the American standard as 
opposed to the European standard.  This change has been made at the request of the 
Investigators. 
 
New Exclusion Criteria 
Old Text:  
Addition of new exclusion criteria. No old text.  
New Text:  
Exclusion Number 22 
22. Subjects with abnormal laboratory values at screening deemed by the investigator to be 
clinically significant 

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Reason:  
Addition of a new Exclusion following a recommendation from the FDA.  
 
New Exclusion Criteria  
Old Text:  
Addition of new exclusion criterion. No old text.  
New Text: 
Exclusion Number 23 
23. Subjects who have taken commercially available probiotics within the last month (30 days 
prior to randomisation) 
Reason:  
Following FDA recommendation to revise the protocol and the eligibility criteria such that 
subjects are not consuming over the counter probiotics from 1 month (30 days) before 
administration of the investigational product through the end of the study. There is a concern 
may impact the biological action.  
 
New Text: 
Exclusion Number 24 
24.  Subjects with known or suspected hereditary fructose intolerance, glucose-galactose 
malabsorption or sucrase-isomaltose insufficiency  
Reason:  
 
Section: 7.1 Clinical Assessments 
Old Text:  
Food Frequency Questionnaire (FFQ) (Appendix 5)  
Subjects will be asked to complete a questionnaire to obtain frequency information about food 
and beverage consumption over a specified period of time (day, week and month). Once 
subjects are randomised they will be asked to complete this questionnaire on a tablet device 
during their Clinic Visit. 
CCI

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New Text:  
Food Frequency Questionnaire (FFQ) (Appendix 5)  
Subjects will be asked to complete a questionnaire to obtain frequency information about food 
and beverage consumption over a specified period of time (day, week and month). FFQ 
completion at baseline will be in relation to food (s) consumed over the prior 6 months. At each 
subsequent visit the FFQ shall capture responses to food (s) consumed since last subject visit. 
Subjects will be asked to complete this questionnaire on a tablet device during each Clinic 
Visit. 
Reason:  
Further clarification has been added to the Protocol regarding the length of time the subject 
will need to review their food frequency and consistency throughout the Protocol regarding 
completion of this questionnaire via a tablet device. This change has been made at the request 
of the Investigators. 
 
Old Text:  
Diet and Study Restrictions 
Dosing should be twice daily at twelve hourly intervals (approximately 30 minutes before 
breakfast and 30 minutes before evening meal).  Subjects with lactose intolerance and Coeliac 
disease and those with a change of diet within the 3 months before screening are excluded. 
Supplements (pre-, pro-biotics) are allowed provided they are recorded by the subject in the 
FFQ at Visit 1 and subsequent visits.  
New Text:  
Diet and Study Restrictions 
Dosing should be twice daily at twelve hourly intervals (approximately 30 minutes before 
breakfast and 30 minutes before evening meal).  Subjects with lactose intolerance and Coeliac 
disease and those with a change of diet within the 3 months before screening are excluded. 
Subject taking commercially available supplements (probiotics) are excluded 1 month (30 
days) prior to randomisation and throughout the study to follow-up.  
Reason:  
Following FDA recommendation to revise the protocol and the eligibility criteria such that 
subjects are not consuming over the counter probiotics from 1 month (30 days) before 
administration of the investigational product through the end of the study. There is a concern 
may impact the biological action.  
 

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Section: 7.2.1 Safety Assessments  
Old Text:  
Serology:  
At screening the analyses will also include HIV-1 & HIV-2, Hepatitis B surface antigen 
(HBsAg), a Hepatitis B surface antibody (Anti-HBs) and a Hepatitis core antibody test (anti-
HBc)  
New Text: 
Serology:  
At screening the analyses will also include HIV-1 & HIV-2, Hepatitis B surface antigen 
(HBsAg), a Hepatitis B surface antibody (Anti-HBs) and a Hepatitis core antibody test (anti-
HBc) and Hepatitis C 
Reason:  
Hepatitis C was removed in error during the changes to the Version 1.1. This administrative 
change has been made to ensure correctness and consistency throughout the protocol. 
 
Section: 8.2.1 Adverse Events  
Old Text:  
3. Severity; severity grade (mild, Moderate, Severe)  
New Text:  
3.  
Mild 
Asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated 
Moderate 
Moderate; minimal, local or non-invasive intervention indicated; limiting age-appropriate 
instrumental ADL (Activities of Daily Living) 
Severe 
Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of existing hospitalization indicated; limiting self-care ADL (Activities of Daily 
Living) 
Reason:  
The FDA requested definition of the terms; mild, moderate and severe within the Protocol. The 
revised terminology which is in accordance with CTCAE (Common Terminology Criteria for 
Adverse Events): 
 

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Section: 7.1 Clinical Assessments  
Old Text:  
Stool Frequency and Consistency Score   
Subjects will be asked to record stool frequency and consistency   Stool types will be based on 
the Bristol Stool Chart (Appendix 3). This data will be recorded for a 7-day period up to 
Baseline (Visit 1) to allow Subject to be classified into Cohorts of IBS sub-types. Once 
subjects are randomised they will be asked to record their score daily on their smartphone. 
New Text: 
Bristol Stool Frequency and Consistency Score 
Subjects will be asked to record stool frequency and consistency. Stool types will be based on 
the Bristol Stool Chart (Appendix 3). This data will be recorded daily for a 7-day period up to 
Baseline (Visit 1) to allow Subject to be classified into Cohorts of IBS sub-types. Once 
subjects are randomised they will be asked to record their stool frequency daily up to 10 bowel 
movements per day and score each stool type on their smartphone. No bowel movements in 
any given day will also be recorded.   
Reason: 
To ensure data required to meet the primary endpoint is sufficient we will require subject to 
record up to 10 bowel movements per day. This change has been made at the request of the 
Investigators. 
 
Section: 13. PUBLICATION POLICY 
Old Text: 
PUBLICATION POLICY  
The Sponsor and Investigator will mutually manage the publication and presentation process. 
All manuscripts, abstracts, or other modes of presentation arising from the results of the study 
must be reviewed and approved in writing by the Sponsor in advance of submission. The 
review is aimed at protecting the Sponsor’s proprietary information existing either at the date 
of the commencement of the study or generated during the study.  
New Text: 
PUBLICATION POLICY  
The Sponsor and Investigator will mutually manage the publication and presentation process. 
All manuscripts, abstracts, or other modes of presentation arising from the results of the study 
must be reviewed and approved in writing by the Sponsor in advance of submission in 
accordance with the clinical site agreement. The review is aimed at protecting the Sponsor’s 
proprietary information existing either at the date of the commencement of the study or 
generated during the study.  
Reason: 

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To ensure consistency with the terms of the Clinical Site Agreement. This change has been 
made at the request of the Investigators. 
 
Appendix 5 Modified FFQ  
Updates have been made to the questionnaire relating to certain food groups. US and UK 
terminology have been added to form a single column for ease of use and consistency for all 
countries in this global clinical trial.  
Reason:  
This administrative change has been made to ensure correctness and consistency throughout 
the protocol. 
 

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APPENDIX 11:  SUMMARY OF PROTOCOL CHANGES VERSION 2.1 
 
 
 
Summary of Protocol Changes 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Previous Date and Version: 6 February 2018; Version 1.0 
 
6 April 2018; Version 1.1 
 
19 May 2018: Version 1.2 
 
31 May 2018: Version 2.0 
 
Updated Current Version: 21 June 2018: Version 2.1 
 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 

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Summary of Protocol Changes 
 
Section: 8.3.1 Pregnancy  
Old text:  
Subjects who are women and who may be able to have children will be given a serum 
pregnancy test at screening and a urine dipstick test at all subsequent study visits. If the result 
is positive at screening, the subject will not be able to enter the study.  Sexually-active women 
must use an acceptable form of birth control throughout the study until completion of two (2) 
periods after the last dose to avoid pregnancy for at least one complete menstrual cycle. The 
following methods of birth control are considered acceptable for this study; true abstinence, 
sterilisation, birth control pills, vasectomised partner, injections or contraceptive implants. 
Female Subjects must not be breast feed an infant during the study. 
 
New Text:  
Subjects who are women and who may be able to have children will be given a serum 
pregnancy test at screening and a urine dipstick test at all subsequent study visits. If the result 
is positive at screening, the subject will not be able to enter the study.  Sexually-active women 
must use an acceptable form of birth control throughout the study until completion of two (2) 
periods after the last dose to avoid pregnancy for at least one complete menstrual cycle. The 
following methods of birth control are considered acceptable for this study; true abstinence, 
sterilisation, birth control pills, vasectomised partner, injections or contraceptive implants. The 
following birth control pills are recommended:  
• 
Combined (estrogen and progestogen containing) hormonal contraception 
associated with inhibition of ovulation: 
o 
Oral 
o 
Intravaginal 
o 
Transdermal 
• 
Progestogen-only hormonal contraception associated with inhibition of ovulation: 
o 
Oral 
o 
Injectable 
o 
Implantable 
Female Subjects must not be breast feed an infant during the study. 
 
Reason:  
Information on the specific types of birth control pills has been added at the request of the UK 
Medicines and Healthcare products Regulatory Agency (MHRA) during review of the clinical 
trial application.  

 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
   
 
  
  
 
 
 
 
 
 
 
 
   
 
  
  
 
 
 
 
 
 
  
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
   
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
   
 
    
 
  
 
 
 
 
 
  
 
 
  
 
 
  
  
   
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
  
 
 
  
 
 

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6 or Type 7. Subject must have fewer than 3 
CSBMs within a one week period (7 days) 
Type 6 or Type 7 bowel movements on at least 
four days within a one week period (7 days).  
 
Reason:  
Inclusion Number 4 and Subtype Classifications (Table 2) have been updated in response to 
FDA recommendations to further pre-define the sub-type population groups 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX 12:  SUMMARY OF PROTOCOL CHANGES VERSION 3.0 
 
 
 
Summary of Protocol Changes 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Previous Date and Version: 6 February 2018; Version 1.0 
 
6 April 2018; Version 1.1 
 
19 May 2018: Version 1.2 
 
31 May 2018: Version 2.0 
                                                21 June 2018: Version 2.1 
 
Updated Current Version: 13 February 2019: Version 3.0 
 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 
 

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The subject will need to ensure they complete these two assessments at least one day prior to 
the next clinic visit. 
Reason: 
For clarity and to include laxative use. This change has been made at the request of the 
Investigators.  
Old Text:  
Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be asked to provide a stool sample from the night before or on the morning of the next 
clinic visit.  
New Text:     
Subjects will be provided with a kit with instructions for the collection of a stool sample.  They 
will be  asked to provide a stool sample from the night before or on the morning of the next 
clinic visit. If necessary, a stool sample may be provided up to 2 days after the visit 
provided it is from the night before or from the day it is delivered to the clinic.  
Reason:  
To provide additional time for subjects to provide a stool sample. This change has been made 
at the request of the Investigators.  
Old Text:  
N/A 
New Text:     
Reasons for screen failure will be documented in the subject’s notes.  Subjects may be re-
screened once, after the initial screening visit if considered to be appropriate by the 
Investigator. A waiting period of at least 2 weeks is required between screening visits.  
Reason:  
To provide guidance for screen failure and re-screening.  This change has been made at the 
request of the Investigators.  
 
Baseline: Visit 1 (Day 1) 
Old Text:  
Subjects will return to the clinical unit on Day 1 (Visit 1; within 28 days of screening visit) 
New Text:     
Subjects will return to the clinical unit on Day 1 (Visit 1; within 28 days of screening visit or 
re-screening visit if applicable)) 
Reason:  
To confirm time interval between a re-screen and Visit 1.  This change has been made at the 
request of the Investigators.  

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Old Text:  
A brief physical examination, assessment of vital signs and a urine pregnancy test for female 
subjects, height, weight and BMI …… 
New Text:     
A brief physical examination, assessment of vital signs and a urine pregnancy test for female 
subjects of child bearing potential, weight and BMI …… 
Reason:  
To clarify that a pregnancy test ifs required only in female subjects of child bearing potential 
and to remove measurement of height as this will be measured at the screening visit only. 
These changes have also been made to the Follow-up Visit.  
Old Text:  
N/A 
New Text:     
Final confirmation of eligibility for the study will be documented, signed by the 
Investigator and filed in the subject notes.  
Reason:  
To confirm the procedure for final confirmation of eligibility at baseline prior to 
randomisation. This change has been made at the request of the Investigators.  
Old Text:  
…two (2) capsules to be taken at 12 hourly intervals, approximately 30 minutes before meals.  
Total of four (4) capsules per day.  
New Text:    
…two (2) capsules to be taken at approximately 12 hourly intervals, morning and evening 
approximately 30 minutes before meals giving a total of four (4) capsules per day.   
Reason:  
To clarify dosage instructions.  This change has been made at the request of the Investigators.  

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Old Text:  
 
Subjects will use their smartphone issued at the screening visit to capture their study related 
data  
• Abdominal Pain completed daily 
• Stools consistency, frequency completed daily 
New Text:    
Subjects will use their smartphone issued at the screening visit to capture their patient related 
data  
• Abdominal Pain Score completed daily 
• Stools consistency/frequency, spontaneity and laxative use completed daily 
• Review of concomitant medications 
Reason:  
For consistency throughout protocol and addition of concomitant medication review and 
laxative use. These changes have also been made for Visit 2, End of treatment and Follow-
up.  
 
Visit 2 (Week 4-5) 
Old Text: 
Visit 2 (Week 4-5) 
Subjects will return after 4-5 weeks  
New Text:    
Visit 2 (Week 4)  
Subjects will return after 4 weeks (± 7 days) 

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Reason:  
To provide a window around the visit date. This change has been made at the request of the 
Investigators. 
Old Text: 
Four (4) capsules total per day and to store in the fridge and not to open the capsules.  
New Text: 
Subjects will receive a total of four (4) capsules per day and will be reminded to store the 
capsules in the fridge and not to open the capsules.  
Reason: 
To correct grammatical errors for clarity. 
 
End of Treatment (Week 8-9) 
Old Text: 
End of Treatment (Week 8-9) 
Subjects will return to the clinic at the end of the 8 week treatment period.   
New Text:  
End of Treatment (Week 8)    
Subjects will return to the clinic at the end of the 8 week (or up to ± 7 days) treatment period.  
Reason:  
To provide a window around the visit date. This change has been made at the request of the 
Investigators. 
 
Follow-Up (Week 12-14) 
Old Text: 
Subjects will return 4-6 weeks after the last dose of study medication 
New Text:     
Subjects will return after 4-6 weeks (± 7 days) after the last dose of study medication.  
Reason:  
To provide a window around the visit date. This change has been made at the request of the 
Investigators. 
 
Table 1: Schedule of Assessments  
Old Text: 

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Visit 2: Week 4-5  
New Text:     
Visit 2: Week 4 (±7 days)  
Reason:  
This change has been made for clarity at the request of the Investigators  
Old Text: 
End of Treatment Week 8-9 
New Text:     
End of Treatment Week 8 (±7 days) 
Reason:  
This change has been made for clarity at the request of the Investigators  
Old Text: 
Abdominal Pain Intensity NRS score 
New Text:     
Abdominal Pain score  
Reason:  
For consistency throughout protocol 
Old Text: 
Weight/Height/BMI 
New Text:  
Weight/Height/BMI 
Footer: e   Weight only to be measured and BMI to be calculated, after the screening visit   
Reason:  
To clarify that height is measured only at screening  
Footer Pregnancy testc 
Old Text: 

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c  Pregnancy Test (Urine) : Serum at screening and Urine Dipstick test for all other visits. 
Fertile male patients and female patients of childbearing potential are to continue using 
highly effective  (refer to section 4.2.1) contraception for two (2) periods after the last 
dose of study medication to avoid pregnancy for one complete menstrual cycle. 
New Text: 
  c Pregnancy Test : Serum at screening and Urine Dipstick test for all other visits. Female 
patients of childbearing potential are to continue using acceptable (refer to section 4.2.1) 
contraception for two (2) menstrual cycles after the last dose of study medication.  Male 
subjects who are not vasectomised must use a barrier method of birth control from 
randomisation until the follow-up visit.  
Reason:  
To provide consistency with other sections in the protocol. This change has been made at the 
request of the Investigators. Changes have also been made to section 7.2.1 below.  
 
Section 5.1 Inclusion Criteria  
Footer Table 2  
Old Text: 
 N/A 
New Text:    
1 CSBM defined as a bowel movement that is both complete and spontaneous. Bowel movements 
where laxative use is recorded in the concomitant medication questions on the same calendar day 
by IBS-C subjects will not be counted as CSBMs when subtyping for randomisation.   
Reason:  
To provide a definition and clarification for CSBMs. This change was made at the request of 
the Investigators.  
Inclusion Criteria 4  
Old Text: 
Have a moderate or severe IBS symptom severity score as defined by IBS-SSS. 
New Text:     
Have a moderate or severe IBS symptom severity score: >175 at the screening visit as defined 
by IBS-SSS. A tolerance of -10% (≥ an IBS-SSS score of 157.5) will be allowable at the 
Baseline (Visit 1). 
Reason:  
This change was made for clarity at the request of the Investigators.  
 
Section 5.2 Exclusion Criteria  
  

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Exclusion 8 
Old Text: 
Have a malignant disease or any concomitant end-stage organ disease.  
New Text:     
8. Have an active or recent (within 3 years) malignant disease or any concomitant 
end-stage organ disease. A non-melanoma skin cancer that has been adequately 
treated with no recurrence within 3 months of screening is not excluded.  
Reason:  
To provide clarification concerning malignant disease. This change has been made at the 
request of the Investigators. 
Exclusion 10  
Old Text: 
Refusal to use acceptable methods of birth control (true abstinence, sterilisation, birth control 
pills, injections or contraceptive implants) for fertile patients while on treatment and following 
completion of 2 menstrual cycles/months after the last dose of study treatment.  For Males, a 
barrier method of birth control from randomisation until the Follow-Up visit 
New Text:     
Refusal to use acceptable methods of birth control (true abstinence, sterilisation, birth control 
pills, injections or contraceptive implants) for women of childbearing potential  while on 
treatment and following completion of 2 menstrual cycles/months after the last dose of study 
treatment.  For Males, a barrier method of birth control from randomisation until the Follow-
Up visit, unless vasectomised.  
Reason:  
To provide consistency with other sections in the protocol. This change has been made at the 
request of the Investigators. 
Exclusion 11 
Old Text: 
Use of antibiotics within 1 month of screening 
New Text:     
Use of antibiotics within 30 days of screening  
Reason:  
To provide consistency with other sections in the protocol. This change has been made at the 
request of the Investigators. 
Exclusion 12 
Old Text: 
Use of systemic steroids within the last month  

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New Text:     
Use of systemic steroids within 30 days of screening  
Reason:  
To provide consistency with other sections in the protocol. This change has been made at the 
request of the Investigators. 
Exclusion 14 
Old Text: 
Have suffered from a major psychiatric disorder.  
New Text:     
Have suffered from an uncontrolled or current major psychiatric disorder.  
Reason:  
To provide clarification. This change has been made at the request of the Investigators. 
Exclusion 19 
Old Text: 
Any abdominal surgery other than hernia repair or appendectomy 
New Text:     
Any GI related abdominal surgery other than hernia repair or appendectomy. 
Cholecystectomy more than 6 months previously is not an exclusion.  
Reason:  
To provide clarification. This change has been made at the request of the Investigators 
Exclusion 20 
Old Text: 
Other investigational procedures while participating in this study are excluded. 
New Text:     
N/A Text deleted. 
Reason:  
This text has been deleted to avoid ambiguity and repetition. This change has been made at the 
request of the Investigators. 
Exclusion 20 
New Text:     
The deleted exclusion has been replaced with: 
Subjects taking prucalopride 
 

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Exclusion 23  
Old Text: 
Subjects who have taken commercially available probiotics within the last month (30 days 
prior to randomization. 
New Text:     
Subjects who have taken commercially available probiotics within the last month (30 days 
prior to randomization). See Appendix 7 
Reason:  
To reference the Appendix 
 
Exclusion 24 
Old Text: 
Subjects with sucrose-isomaltose insufficiency 
New Text:   
Subjects with sucrose-isomaltase insufficiency 
Reason:  
To correct a spelling error 
 
Exclusion 25 
Old Text: 
N/A 
New Text:   
Subjects taking guanylate cyclase agonists: such as linaclotide and lubiprostone 
Reason:  
To add a new exclusion criterion 
 
Section 5 Randomisation 
Old Text: 
Subjects, 4D Pharma Plc, designated CRO and other Clinical site staff  
New Text:   
Subjects, 4D pharma PLC, designated CRO and clinical site staff 
Reason:  
For clarification 

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Section 6 Investigational Products  
6.1 Formulation 
Old Text: 
New Text:     
Reason:  
This change has been made for consistency with the formulation used in this study. 
 
6.2 Dosage, Frequency and duration of dosing  
Old Text: 
capsules will be administered twice daily at 12 hourly intervals 
New Text:     
capsules will be administered twice daily at approximately 12 hourly intervals morning and 
evening 
Reason:  
This change has been made for consistency throughout the protocol.  
 
Section 6.8 Concomitant Treatment  
Old Text: 
Any concomitant medication taken within 30 days prior to the first dose of study medication 
should be recorded in the case report forms.  
CCI
CCI

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New Text:     
Any concomitant medication taken within 30 days prior to the first dose of study medication 
and up to the follow-up visit should be recorded in the case report forms.  
Reason:  
To clarify the period for recording of concomitant medication.  
 
Section 6.9 Prohibited Medication  
Old Text: 
N/A 
New Text:     
Probiotics such as yogurts, yogurt-type drinks and other soft drinks e.g. fruit juices may 
be taken during the study.  Probiotic supplements formulated as capsules or sachets are 
prohibited (see Appendix 7).  Acceptable probiotics taken during the month before 
screening should be continued through the study period and to follow-up to ensure 
consistency. 
Prucalopride is a prohibited medication and should not be taken during the study.  
IBS-C subjects should not take guanylate cyclase agonists (such as linaclotide) and 
lubiprostone.  IBS-D subjects should not take bile acid sequestrants (such as 
cholestyramine, colestipol, colesevelam), 5-hydroxytryptamine (serotonin) 3 receptor 
antagonists (such as alosetron), rifaximin and eluxadoline (see Appendix 7). 
Subjects are not eligible for the study if they have received antibiotics or systemic 
corticosteroids within 30 days prior to the screening visit date.  Subjects on inhaled 
steroids for asthma may be included but oral or infused steroids for asthma are not 
permitted.  If these medications are required during the study, the subject does not need 
to be withdrawn but details will be recorded in the concomitant medication case report 
forms. 
Reason:  
A section on prohibited medication has been added for clarity. This change has been made at 
the request of the Investigators 
 
Section 7.1 Clinical Assessments  
Abdominal Pain Score 
Old Text: 
Abdominal Pain Intensity NRS Score  
Subjects will be asked to rate their worst abdominal pain over the past 24 hours using an 11-
point (NRS) and a weekly average will be recorded.  

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New Text: 
Abdominal Pain Score  
Subjects will be asked to rate their worst abdominal pain over the past 24 hours using an 11-
point Numeric Rating Scale (NRS) and a weekly average will be recorded.  
Reason 
This change has been made for clarity at the request of the Investigators.  
 
Subject Global Assessment of Relief 
Old Text: 
A weekly subject response to the question ‘How have you felt during the past week in regard to  
your IBS, particularly overall well being and symptoms of abdominal comfort, pain and altered 
bowel habit’.  
New Text: 
A weekly response to the question ‘Please consider how you felt this past week in regard to 
your IBS, in particular your overall wellbeing and symptoms of abdominal discomfort, 
pain and altered bowel habit.  Compared to the way you usually felt before entering the 
study, how would you rate your relief of symptoms during the past week?’ 
Reason: 
This change has been made at the request of the Investigators.  
 
IBS Quality of Life 
Old Text: 
N/A 
New Text: 
The individual responses to the 34 items are summed and averaged for a total score and 
then transformed to a 0-100 scale for ease of interpretation with higher scores indicating 
better IBS specific quality of life 
Reason 
This change has been made at the request of the Investigators. 
Bristol Stool Chart 
Old Text: 
Bristol Stool Frequency and Consistency Score  
New Text: 
Bristol Stool Chart  

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Stool frequency will be defined as a sum of weekly CSBMs.  Stool frequency will be 
recorded using the following question: “How many times did you open your bowels 
during the past 24 hours? If you have not had any bowel movements today, please enter 
0.” Subjects will be reminded to rate all of their bowel movements in the Bristol Stool 
Chart before answering the question. CSBMs will be determined by absence of laxative 
use recorded in concomitant medication questions for the same calendar day.  
Reason 
This change has been made at the request of the Investigators.  
 
IBS Symptom Severity Score 
Old Text: 
A score ranging from 0 (no symptoms) to 500 (maximum severity) can be achieved as mild, 
moderate or severe (>300) IBS symptoms. 
The initial data from this score will be collected during the Screening visit on a tablet device 
during the clinic visit. This data will be used to classify into Cohorts of IBS sub-types prior to 
randomisation. Once subjects are randomised, they will be asked to record their score during 
each visit on a tablet device.  
New Text: 
A score ranging from 0 (no symptoms) to 500 (maximum severity) can be achieved as mild 
(74-174), moderate (175-299) or severe (>300) IBS symptoms. 
The initial data from this questionnaire will be collected during the Screening visit on a tablet 
device during the clinic visit. This data will be used to classify into Cohorts of IBS sub-types 
prior to randomisation. Once subjects are randomised, they will be asked to record their 
responses to the questionnaire on a tablet device during each clinic visit.  
Reason 
This change has been made at the request of the Investigators.  
 
Food Frequency Questionnaire 
Old Text: 
N/A 
New Text: 
• Concomitant Medications 
Subjects will be asked to record name, dose and time of administration of any 
medication, other than IMP, taken during the study. This will be reviewed at each visit.  
Reason 
This change has been made at the request of the Investigators.  

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Dosing Diary 
 Old Text: 
Subjects will be asked to complete times that the medication was taken on their smartphone 
and will be prompted with reminders via this device.  
New Text: 
Subject will be asked to confirm their daily study medication intake by indicating “yes” or 
“no” to the following questions on the smartphone provided: “Did you take two of your 
study medication capsules this morning?” in the morning and “Did you take two of your 
study medication capsules this evening?” in the evening. Subjects will be prompted with 
reminders via this device.  
Reason 
This change has been made for clarity at the request of the Investigators 
 
Diet and Study Restrictions 
Old Text: 
Dosing should be twice daily at twelve hourly intervals (approximately 30 minutes before 
breakfast and 30 minutes before evening meal). Subjects with lactose intolerance and Coeliac 
disease and those with a change of diet within the 3 months before screening are excluded. 
Subject taking commercially available supplements (probiotics) are excluded 1 month (30 
days) prior to randomisation and throughout the study to follow-up. 
New Text:    
Dosing should be twice daily at twelve hourly intervals (approximately 30 minutes before 
breakfast and 30 minutes before evening meal). Subjects with lactose intolerance and Coeliac 
disease and those with a change of diet within the 3 months before screening are excluded. 
Subject taking commercially available supplements (probiotics formulated as capsules or 
sachets as in Appendix 7) are excluded 1 month (30 days) prior to randomisation and 
throughout the study to follow-up.  Probiotic yogurts, yogurt-type drinks and soft drinks 
e.g.fruit juices are acceptable if taken during the month before screening and continued 
to follow-up. 
Reason:  
Clarification of probiotic use and for consistency with section 6.9, prohibited medications. This 
change has been made at the request of the Investigators 
 
Section 7.2.1 Safety Assessments 
Blood Samples: 
Old Text: 

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 Sexually-active women must use an acceptable form of birth control throughout the study  
New Text:    
 Sexually-active women of child bearing potential must use an acceptable form of birth 
control throughout the study  
Reason: 
This change has been made for consistency throughout the protocol.  
Old Text: 
Female Subjects must not be breast feed an infant during the study.  
New Text:    
Female Subjects must not breast feed an infant during the study.  
Reason: 
To correct a grammatical error.   This change has been made at the request of the Investigators. 
 
Section 9.2 Source Documents 
Old Text: 
N/A 
New Text:    
Clinical Investigators and authorized study personnel will review and access the 
electronic eCOA data; collected on smartphones and tablets, according to patient activity 
on site (following screening and scheduled clinic visits as scheduled in the Protocol, 
e.g.Screening Visit, Baseline Visit, Visit 2, End of Treatment, and Follow-Up.  
Discrepancies will be resolved using data queries and site response, with the oversight of 
the monitor.  
Reason:  
To add the procedure for review of eCOA data. 
 
Section 9.4 Database Management and Quality Control 
Old Text: 
N/A 
New Text : 
Access to the eCOA data will be controlled via the vendor Support Helpdesk.  Users must 
have successfully completed the vendor training module, with a pass-rate of >80%.  Full 
log of query and resolution steps will be maintained within the system, with full audit 
trail. 
Reason:  

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New Text : 
Subjects who do not provide enough data to enable assessment of whether that subject is a 
responder will be deemed a non-responder. 
An ‘evaluable week’ is one in which there are at least four twenty-four hour (4 day) records of 
abdominal pain score and at least four daily records of the number of complete spontaneous 
bowel movements and  stool chart. 
Reason:  
This change has been made at the request of the Investigators 
Old Text: 
Partial dates will be imputed in a standard manner. 
There will be no other imputation for missing data.  
New Text : 
N/A Old Text deleted 
Reason:  
This change has been made at the request of the Investigators 
 
Section 12.4 Informed Consent  
Old Text: 
Subjects may be approached to discuss the study determine if they are interested in 
participating in the study. 
New Text: 
Subjects may be approached to discuss the study to determine if they are interested in 
participating in the study.  
Reason: 
To correct a grammatical error. 
Old Text: 
The physician who conducts the informed consent discussion must also sign and date the 
consent form. 
New Text: 
The physician or designee who conducts the informed consent discussion  
Reason: 
To allow the informed consent discussion to be delegated to a non- physician.  This change has 
been made at the request of the Investigators.  
 

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Section 12.5 Future use of stored samples  
Old Text: 
N/A 
New Text: 
Samples will not be labelled with information that directly identifies the subject but will 
be coded with the identification number for the subject. 
Collected samples will be transferred for analysis to the Sponsor, or to other laboratories 
working for the Sponsor. 
Biological samples will be stored for the time established by regulatory requirements or 
destroyed after the final clinical study report has been finalized if storage is not required. 
There might be a new request for these samples to be used for purposes related to the QA 
of the laboratory tests described in this protocol, in which case they will be used for this 
purpose. This may include the assessment of the quality of current tests, the maintenance 
or improvement of these tests, the development of new test methods for the markers 
described in this protocol, as well as making sure that new tests are comparable to 
previous methods and work reliably. 
If study results suggest that further investigations using stored biological samples are 
warranted, these tests might be carried out on an exploratory basis. In addition, 
biological samples may be used by the Sponsor or their research partners for further 
research that is not related to the disease or the product under study. This testing will be 
done on pseudonymized samples (meaning that subjects will not be identifiable from their 
biological samples as their identity will have been removed and they will be assigned a 
unique clinical study identification number). Subjects will be asked to sign an additional, 
separate consent form for this optional testing and refusal of consent will not affect their 
possibility of participating in the study.  
During the conduct of the study, an individual subject can choose to withdraw consent to 
have biological specimens stored for future research. 
Reason: 
To provide procedures for the future use of stores samples.  
Section 12.7 Insurance 
Old Text: 
N/A 
New Text:    
The Sponsor has established an insurance policy for the anticipated duration of the study, 
covering the subjects with respect to the risks involved in taking part in this study in 
accordance with this protocol. In the case of injury or disability deriving from 

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participation in the study, subjects are requested to inform the Investigator or his/her 
staff responsible for the study at the institution without delay 
Reason: 
To add insurance details.  
 
Section 12.6 Study Discontinuation and Closure 
Old Text: 
Section deleted  
New Text:    
N/A Section deleted 
 
Appendices 
Appendix 6: Rome IV classification for IBS 
Old Text: 
N/A  
New Text:    
Appendix 6: Rome IV Classification for IBS has been added.  
Reason:  
This change has been made at the request of the Investigators.  
Appendix 7 Prohibited Probiotics 
Old Text: 
N/A 
New Text: 
Appendix 7 Prohibited Probiotics has been added 
Reason: 
This change has been made at the request of the Investigators.  
Appendices 6, 7, 8  and 9 have been re-numbered as Appendices 8, 9, 10 and 11 
respectively.   
 
 

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APPENDIX 13:  SUMMARY OF PROTOCOL CHANGES VERSION 3.1 
 
 
 
Summary of Protocol Changes 
 
 
PROTOCOL 
 
PROTOCOL NUMBER: BHT-II-002 
A PHASE 2 RANDOMISED, DOUBLE BLIND, PLACEBO-CONTROLLED, PARALLEL 
GROUP, MULTICENTRE STUDY TO EVALUATE THE SAFETY AND EFFICACY OF 
REPEATED ORAL DOSES OF Blautix™ IN ADULT SUBJECTS WITH IRRITABLE 
BOWEL SYNDROME (IBS) SUBTYPES IBS-C and IBS-D  
 
 
 
Previous Date and Version: 6 February 2018; Version 1.0 
 
6 April 2018; Version 1.1 
 
19 May 2018: Version 1.2 
 
31 May 2018: Version 2.0 
                                                21 June 2018: Version 2.1 
 
13 February 2019: Version 3.0 
 
Updated Current Version: 31 March 2020: Version 3.1 
 
 
 
Sponsor Name and Address:  
4D Pharma Plc 
9 Bond Court 
Leeds, U.K 
LS1 2JZ 
 

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Section: 10.3 Interim Analysis 
Old Text:    
An informal interim analysis following randomisation of approximately 250 Subjects for 
futility will take place under the auspices of the DSMB.  As there will be no provision of 
stopping the study for efficacy and as – other than stopping for futility – there will be no other 
consequences of the interim analysis, there is no need to adjust the value of alpha. 
New Text:  
An informal interim analysis will be conducted once all recruited patients have completed the 
primary efficacy analysis 8-week treatment period.  This interim analysis is planned to enable 
4D pharma to expedite the clinical development strategy based on the outcome of the interim 
analysis. Planning for pivotal trials and the EU Paediatric Investigation Plan and the US 
Paediatric Study Plan can be initiated in preparation for Agency meetings and further clinical 
development.  There will be no other consequences of the interim analysis, there is no need to 
adjust the value of alpha. 
 
Reason:  
As there were no SUSAR submissions, it was deemed that there was no need to call a DSMB 
after the randomisation of 250 subjects and there was no need to stop the trial for futility. 
Although the DSMB was not called, this is not considered a withdrawal of the DSMB as no 
SUSARs have been raised during the study, to this point.  
As all patients have now been randomised and have completed the assessments which will be 
used in the primary efficacy analysis, 4D is including an interim analysis for efficacy.  This 
change in the interim analysis is not considered to be a change of study design which is likely 
to have a significant impact on primary or major secondary statistical analysis or the 
risk/benefit assessment.