A Phase II, Randomized, Double-Blind, Vehicle Controlled Study of the Efficacy, Safety, and Tolerability of B244 Topical Spray for the Treatment of Pruritus in Adults With a History of Atopic Dermatitis
Phase: PHASE2
Conditions: Atopic Dermatitis, Pruritus
Sponsor: AOBiome LLC

Protocol PRB244-01 Amendment 3 28-FEB-2020 
 
 
CLINICAL STUDY PROTOCOL 
 
 
 
TITLE:  
A Phase II, Randomized, Double-Blind, Vehicle Controlled Study 
of the Efficacy, Safety, and Tolerability of B244 Topical Spray for 
the Treatment of Pruritus in Adults with a History of Atopic 
Dermatitis 
 
IND Number:  
 
17485 
 
Protocol Number: 
 
PRB244-01 
 
Protocol Version/Date: 
Amendment 3 / 28-FEB-2020 
 
Development Phase:  
Phase 2 
 
Sponsor: 
 
 
AOBiome Therapeutics 
125 Cambridgepark Drive 
Cambridge, MA 02140 
USA 
 
 
 
Any and all information presented in this document shall be treated as confidential and shall 
remain the exclusive property of AOBiome Therapeutics (or any of its affiliated companies). The 
use of such confidential information must be restricted to the recipient for the agreed purpose 
and must not be disclosed, published or otherwise communicated to any unauthorized persons, 
for any reasons, in any form whatsoever without the prior written consent of AOBiome 
Therapeutics (or the concerned affiliated company).

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 2  
Revision Chronology 
 
 
Original 
September 25, 2019 
Amendment 1 
November 07, 2019 
Amendment 2 
January 06, 2020 
Amendment 3 
February 28, 2020 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 3  
 
SIGNATURE PAGE FOR INVESTIGATOR(S) 
 
 
TITLE:  
A Phase II, Randomized, Double-Blind, Vehicle Controlled Study 
of the Efficacy, Safety, and Tolerability of B244 Topical Spray for 
the Treatment of Pruritus in Adults with a History of Atopic 
Dermatitis 
 
IND Number:  
 
17485 
 
Protocol Number: 
 
PRB244-01 
 
Protocol Version/Date: 
Amendment 3 / 28-FEB-2020 
 
Development Phase:  
Phase 2 
 
Sponsor: 
 
 
AOBiome Therapeutics 
125 Cambridgepark Drive 
Cambridge, MA 02140 
USA 
 
 
I have read the protocol and agree to conduct this study in accordance with the protocol, all 
relevant laws and regulations in force at the time, International Conference on Harmonisation 
Guidelines for Good Clinical Practices, and the Declaration of Helsinki.  
 
 
 
 
 
 
 
 
 
Principal Investigator’s printed name  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator’s signature  
 
 
 
 
Date (DD-MMM-YYYY)  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 5  
PROTOCOL SYNOPSIS 
 
Title 
A Phase II, Randomized, Double-Blind, Vehicle Controlled Study of the 
Efficacy, Safety, and Tolerability of B244 Topical Spray for the Treatment 
of Pruritus in Adults with a History of Atopic Dermatitis 
Protocol Number 
PRB244-01 
Sponsor 
AOBiome Therapeutics 
Development Phase 
II 
Study Objectives 
Primary objective:  
● To assess the efficacy of B244 in the treatment of pruritus in adults 
with a history of atopic dermatitis.  
Secondary objectives:  
 
● To assess the safety and tolerability of B244 in adults with a history of 
atopic dermatitis.  
Study Design 
This is a double-blind, randomized, vehicle-controlled study to assess the 
efficacy, safety, and tolerability of 2 doses of B244 for the treatment of 
pruritus in adults with a history of atopic dermatitis. Subjects who meet the 
study entry criteria will be randomized in a 1:1:1 ratio to receive twice daily 
topical doses of B244 at 1x1010 cells/ml (O.D. 5.0), 4 x1010 cells/ml (O.D. 
20.0) or vehicle (control) for 4 weeks. The total duration of the study will be 
approximately 11 weeks. The study will be conducted at approximately 50 
study sites.  
At Screening and Baseline, all subjects must have atopic dermatitis, which 
involves a minimum of 10% and a maximum of 40% body surface area, score 
of ≥ 7 points on the NRS scale and static IGA of 2-3. 
The total duration of the study will be approximately 11 weeks. Participants 
will report for a Screening visit and if all inclusion/exclusion criteria are met, 
subjects will go through a two-week washout phase before reporting for a 
Baseline visit. At the baseline visit, eligible subjects will be randomly assigned 
to receive B244 1x1010 cells/ml, B244 4x1010 cells/ml or vehicle. Subjects will 
come in for Week 2 and Week 4 visits. After completion of the 4-week 
treatment period, all subjects will enter a 4-week follow-up period. Participants 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 6  
who have discontinued the study early will be evaluated by the Investigator at 
the Early Termination Visit within 7 days after their last dose of study drug. 
 
The primary efficacy endpoint will be assessed after Week 4 of treatment.  
Number of patients 
planned for randomization 
Approximately 576 subjects (192 per treatment group) will be enrolled and 
randomized in the study.   
Study Population 
Male and female adults 18 to 65 years of age with pruritus and a history of 
atopic dermatitis.  
Inclusion Criteria: 
 
 
1. Male and female subjects 18 to 65 years of age. 
2. Pruritus of at least 4 weeks duration prior to the initial Screening visit 
and during the 2 week washout period. 
a. Subjects using stable doses of oral H1 antihistamines at the 
initial Screening visit must be willing to continue these at the 
same doses and frequencies throughout the study inclusive of 
the follow-up period.  
3. Worst Itch Numeric Rating Scale (WI-NRS) score ≥ 7 in the 24-hour 
period prior to the initial Screening as well as Baseline visits.  
4. Average weekly WI-NRS score ≥6 for each week of the washout 
period, as recorded in the eDiary. 
5. A history of atopic dermatitis for greater than 12 months consistent 
with a diagnosis of atopic dermatitis, as defined by the 2014 American 
Academy of Dermatology (AAD) Guidelines of Care for the 
Management of Atopic Dermatitis.1  
a. Subjects using bland emollients at the initial Screening visit 
will be allowed to continue to use their emollient of choice at 
the same dose and frequency throughout the study. 
b. Subjects using low- to mid-potency topical corticosteroids at 
the initial Screening visit will be allowed to use their topical 
corticosteroid of choice at the same dose and frequency no 
more than 7 days per month throughout the study as rescue 
medication. 
6. A minimum of 10% and not more than 40% of the subjects' BSA 
affected by atopic dermatitis (affected is defined by physical 
examination findings: erythema, edema, scaling, lichenification, 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 7  
excoriation, with the excoriation serving as the physical examination 
correlate of pruritus) at Screening and Baseline. 
a. Subjects’ BSA can include face and body OR body alone BUT 
NOT face alone. 
7. An Investigator Global Assessment (IGA) score of 2-3 at Screening 
and Baseline. 
8. Willing and able to complete once-daily eDiary entries within a 
consistent timeframe for the duration of the study and have ≥80% 
eDiary compliance rate during the washout period. 
9. Judged to be in good health in the investigator’s opinion.  
Exclusion Criteria: 
 
1. Clearly defined etiology for pruritus other than atopic dermatitis. These 
include but are not limited to urticaria, psoriasis or other non-atopic 
dermatologic conditions, hepatic or renal disease, psychogenic pruritus, 
drug reaction, untreated hyperthyroidism, parasite presence and 
presence of acute infection either systemically or in the AD lesions.  
2. Presence of any acute condition which may risk inducing an atopic 
dermatitis flare during the course of the study, such as impetigo or 
active herpes simplex infection.  
3. Treatment with systemic corticosteroids within 4 weeks prior to 
randomization.  
4. Treatment with Class III or higher potency topical corticosteroids or 
any topical anti-pruritic therapies (other than stable doses of low- or 
mid-potency topical corticosteroids or bland emollients) within 4 
weeks prior to randomization.  
5. Treatment with systemic therapies with recognized anti-pruritic (e.g. 
tricyclic antidepressants, sedatives, tranquilizers, gabapentin, marijuana 
or other cannabinoids, opioid receptor agonists/antagonists) or pruritic 
(e.g. opioids, angiotensin-converting enzyme inhibitors, cocaine, 
antimalarials) properties within 4 weeks prior to randomization.  
a. Stable doses of H1 antihistamines will be permitted. Subjects 
must be willing to continue these at the same doses and 
frequencies throughout the study inclusive of the follow-up 
period.  
6. Any clinically significant changes in type, dose, or frequency of bland 
emollients, low- or mid-potency corticosteroids, and/or oral H1 
antihistamines throughout the study from screening to follow-up.  
7. Treatment with systemic immunosuppressive/ immunomodulatory 
therapies within 4 weeks prior to randomization (including but not 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 8  
limited to phosphodiesterase-4 inhibitors, cyclosporine, 
mycophenolate-mofetil, methotrexate, azathioprine, interferon-gamma, 
or phototherapy).  
8. Treatment with biologic therapies within 12 weeks or 5 half-lives prior 
to randomization, whichever is longer.  
9. Use of an indoor tanning facility within 4 weeks prior to 
randomization.  
10. Treatment with any investigational therapy within 4 weeks prior to 
randomization.  
11. Allergen immunotherapy within 6 months prior to randomization.  
12. Prior use of AO+ Mist. 
13. History of malignancy within 5 years prior to randomization, with the 
exception of completely treated and non-metastatic basal cell 
carcinoma or squamous cell carcinoma of the skin.  
14. History of a major psychiatric condition (including major depressive 
disorder, bipolar disorder, or schizophrenia), suicidal ideation, or 
suicide attempt.  
15. Known active hepatitis infection.  
16. Known history of human immunodeficiency virus (HIV) infection.  
17. Presence of any medical condition or disability that, in the 
investigator’s opinion, could interfere with the assessment of safety or 
efficacy in this trial or compromise the safety of the subject.  
18. Currently pregnant or breastfeeding, or male subject with a pregnant or 
breastfeeding partner.  
19. Females of childbearing potential who are unable or unwilling to 
practice highly effective contraception (pregnancy prevention); fertile 
males who are unable or unwilling to use condoms with female 
partners of childbearing potential.  
Study Drug 
1) B244 1x1010 cells/ml (O.D. 5.0) 
2) B244 4x1010 cells/ml (O.D. 20.0) 
3) Vehicle 
Dose Regimen 
Subjects will apply 10 pumps of IP per application to all affected areas twice-
a-day (i.e. 10 pumps in the morning and 10 pumps again at night) for 4 Weeks. 
 
Efficacy Endpoints 
The key primary efficacy endpoint is as follows:  
● Mean change in WI-NRS from baseline to Week 4 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 9  
Additional secondary efficacy endpoints include the following:  
● Proportion of subjects with ≥4 point improvement in WI-NRS from 
baseline to Week 4 
● Proportion of subjects with any improvement in WI-NRS from baseline 
to Week 4 
● Mean change in AI-NRS from baseline to Week 4 
● Proportion of subjects with ≥4 point improvement in AI-NRS from 
baseline to Week 4 
● Proportion of subjects with any improvement in AI-NRS from baseline 
to Week 4 
● Mean change in WI-NRS from baseline to Week 2 
● Proportion of subjects with ≥4 point improvement in WI-NRS from 
baseline to Week 2 
● Mean change in POEM from baseline to Week 4 
● Mean change in 5-D Pruritus Scale from baseline to Week 4 
Exploratory endpoints include the following:  
● Mean change in IGA from baseline to Week 4 
● Mean change in EASI from baseline to Week 4 
Safety Endpoints 
Safety and tolerability endpoints include the following:  
● Incidence of treatment-emergent adverse events (TEAEs) and serious 
adverse events (SAEs) 
● Changes in vital signs and clinical laboratory parameters following 
study drug exposure 
● Changes in local skin tolerability following application of study drug 
Assessment Schedule: 
All subjects will attend a screening visit not more than 21 days prior to 
Baseline (Day 0). Subjects will be required to return to the clinic at Baseline, 
Day 14 (Week 2) and Day 28 (Week 4). All subjects will be asked to attend a 
Week 8 follow-up visit 4 weeks (28 days (±3) days) after the last dose of study 
medication. 
Statistical Considerations: Sample Size: 
Approximately 576 subjects may be enrolled to account for 16.7% drop out 
rate prior to completing the study.  
A total of 160 evaluable subjects per group (480 total) are required to achieve 
at least 80% to detect a pairwise difference of 0.65 in mean WI-NRS change 
from baseline to Week 4 between one of two active doses of B244 and vehicle 
control when assuming a standard deviation of 2.5 and applying a Dunnett 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 10  
Testing Method at a one-sided familywise error rate of 0.10. 
Populations:  
● ITT population:  All subjects who are enrolled and apply at least 1 dose 
of study treatment will be included in the Intent to Treat (ITT) 
population.  The ITT population will be the primary population for 
safety and efficacy assessments. 
● Per Protocol (PP) population:  All subjects in the ITT population 
without any protocol deviations that may have an impact on the 
efficacy assessments, who complete their Week 4 visit, and who 
administer at least 50% of investigational product (IP), will be included 
in the Per Protocol (PP) population. 
● Safety Population:  All subjects treated with at least one dose of IP.  
Efficacy Analysis:  
Efficacy analysis will be performed on the ITT population with some 
supportive analysis on the PP population.  Regression models will include all 
eligible subjects for the respective analysis, from the 3 treatment arms.   
Hypothesis tests for the primary efficacy endpoint will be performed using a 
Dunnett Testing Method, applying pairwise comparisons of each respective 
B244 dose group to vehicle using a one-sided familywise error rate of 0.10.  
No additional adjustments will be made for multiple testing.   
Additional supportive analyses may be performed combining the B244 dose 
groups in comparison against vehicle and comparison of the B244 groups to 
one another.   
The frequency and rate of NRS responders at Week 4 (≥4 point change from 
baseline in NRS) will be reported and compared between treatment groups 
using a logistic regression model. 
Continuous measures of change from baseline to Week 1, Week 2, Week 3, 
Week 4, Week 8 for AI-NRS and WI-NRS; and change from baseline to Week 
2, Week 4, Week 8 for POEM and 5-D Pruritus Scale will be analyzed using 
mixed models with repeated measures (MMRM) to account for within-subject 
variability over time.  Comparisons at each time point will be performed 
within the longitudinal model. 
Safety Analysis:  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 11  
Analysis of safety measures will be performed on the Safety Population, 
including all treated subjects. 
 
The incidence of all adverse events (AEs) and treatment-related AEs will be 
tabulated by treatment received. These AEs will be classified by system organ 
class and preferred term using the Medical Dictionary for Regulatory 
Activities (MedDRA). The incidence and severity of changes in local 
tolerability and adverse reactions will be collected as a solicited AE of special 
interest. 
 
For incidence reporting, if a subject reported more than one AE that was coded 
to the same system organ class or preferred term, the subject will be counted 
only once for that specific system organ class or preferred term. An overview 
of AEs, which includes subject incidence of AEs, treatment-related AEs, AEs 
by severity, SAEs, deaths, and AEs leading to discontinuation, will be 
presented.  
 
Summaries of IP exposure, clinical laboratory measures, vital signs, rescue 
medications, and concomitant medications will be presented. 
Study Sites 
Approximately 50 study sites  
Expected Duration of 
Subject’s Participation 
Approximately 11 weeks: Up to 3 weeks of screening, 4 weeks of treatment, 
and a follow-up period of 4 weeks.  
 
This study will be conducted in accordance with the Guidelines of Good Clinical Practices 
(GCPs).  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 12  
Table of Contents 
1 
STUDY SCHEMA ................................................................................................................ 17 
2 
INTRODUCTION ................................................................................................................ 18 
2.1 
Background ...............................................................................................................................18 
2.2 
Clinical Experience ...................................................................................................................18 
2.3 
Safety Profile .............................................................................................................................20 
2.4 
Rationale for the Study ............................................................................................................20 
3 
STUDY OBJECTIVES ........................................................................................................ 21 
3.1 
Primary Objective ....................................................................................................................21 
3.2 
Secondary Objectives ...............................................................................................................21 
4 
ENDPOINTS ........................................................................................................................ 21 
4.1 
Efficacy ......................................................................................................................................21 
4.2 
Safety & Tolerability ................................................................................................................22 
5 
STUDY DESIGN ................................................................................................................. 22 
6 
SELECTION OF STUDY PARTICIPANTS ...................................................................... 23 
6.1 
Number of Participants Planned .............................................................................................23 
6.2 
Inclusion Criteria .....................................................................................................................23 
6.3 
Exclusion Criteria .....................................................................................................................23 
7 
PARTICIPANT ENROLLMENT ....................................................................................... 25 
7.1 
Consenting Participants ...........................................................................................................25 
7.2 
Screening for Eligibility ...........................................................................................................25 
7.3 
Study Withdrawal and Withdrawal from Investigational Product and Stopping Criteria
 
25 
7.4 
Early Termination ....................................................................................................................26 
7.5 
Lifestyle Considerations ...........................................................................................................27 
7.5.1 
Use of Bland Emollients During the Study ...........................................................................................27 
8 
STUDY TREATMENT ........................................................................................................ 27 
8.1 
Investigational Product ............................................................................................................27 
8.2 
Dose Changes ............................................................................................................................28 
8.3 
Storage conditions ....................................................................................................................28 
8.4 
Description of Blinding Method ..............................................................................................29 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 13  
8.5 
Treatment Assignments: ..........................................................................................................29 
8.6 
Treatment Compliance ............................................................................................................29 
8.7 
Treatment Application .............................................................................................................30 
8.8 
Treatment of Investigational Product Overdose ...................................................................31 
8.9 
Study Drug Discontinuation ....................................................................................................31 
8.10 
Product Accountability ............................................................................................................32 
8.11 
Unblinding Procedures ............................................................................................................32 
8.12 
Retrieval and Destruction of Investigational Product ..........................................................32 
8.13 
Concomitant and Excluded Therapies ...................................................................................32 
8.13.1 
Permitted Medications .....................................................................................................................33 
8.13.2 
Excluded Therapies ..........................................................................................................................34 
8.14 
Rescue Medications ..................................................................................................................34 
8.14.1 
Rescue Medication Permitted During the Study ..............................................................................34 
8.14.2 
Use of Excluded Therapies During the Treatment Period ...............................................................35 
8.15 
Handling of Investigational Product .......................................................................................36 
9 
CONTRACEPTION REQUIREMENTS ............................................................................ 36 
10 
STUDY PROCEDURES .................................................................................................. 37 
10.1 
Pre-screening procedures ........................................................................................................37 
10.2 
Informed Consent Procedures ................................................................................................37 
10.3 
Study Assessments ....................................................................................................................37 
10.4 
Inclusion procedures ................................................................................................................37 
10.5 
Timing of patient’s visits to the clinic .....................................................................................38 
10.6 
Description by type of visit ......................................................................................................38 
10.6.1 
Screening Visit (-21 to -14) .............................................................................................................38 
10.6.2 
Washout Phase (-14 to -1) ................................................................................................................38 
10.6.3 
Study Day 0-Baseline visit (-1 to 0) .................................................................................................39 
10.6.4 
Day 5- Phone call to patient .............................................................................................................39 
10.6.5 
Day 14 and Day 28 study visits .......................................................................................................40 
10.6.6 
Day 56-End of Study Visit ...............................................................................................................40 
10.6.7 
Unscheduled/Unanticipated Study visit ...........................................................................................40 
10.6.8 
Early Termination Visit ...................................................................................................................41 
11 
STUDY ASSESSMENTS ................................................................................................. 41 
11.1.1 
Vital Signs .............................................................................................................................41 
11.1.2 
Physical Examinations .........................................................................................................41 
11.1.3 
Laboratory Assessments ......................................................................................................42 
11.2 
Efficacy Assessments ................................................................................................................42 
11.2.2 
Patient Oriented Eczema Measure ...................................................................................................43 
11.2.3 
Itch Numeric Rating Scale ..................................................................................................43 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 14  
11.2.4 
Investigator Global Assessment Score (IGA) ....................................................................44 
11.2.5 
5-D Pruritus Scale ................................................................................................................44 
11.3 
eDiary and Efficacy Assessments ............................................................................................44 
11.4 
Patient Reported Local Tolerability .......................................................................................45 
11.5 
Pregnancy Reporting ...............................................................................................................45 
11.6 
Study Completion .....................................................................................................................45 
11.7 
Subject Withdrawal Criteria ...................................................................................................45 
12 
STATISTICAL CONSIDERATIONS ............................................................................. 46 
12.1 
Sample Size ...............................................................................................................................46 
12.2 
Populations for Analysis ..........................................................................................................46 
12.3 
Data Analysis ............................................................................................................................46 
12.3.1 
Disposition .......................................................................................................................................47 
12.3.2 
Demographic and Baseline ..............................................................................................................47 
12.4 
Safety Analyses .........................................................................................................................47 
12.4.1 
Definitions ........................................................................................................................................47 
12.4.2 
Adverse Events ................................................................................................................................47 
12.4.3 
Deaths and Serious Adverse Events .................................................................................................48 
12.4.4 
Adverse Events leading to treatment discontinuation ......................................................................48 
12.5 
Efficacy Analyses ......................................................................................................................48 
12.6 
Handling of dropouts or missing data ....................................................................................49 
12.7 
Clinical Trial Protocol deviations ...........................................................................................49 
13 
ADVERSE EVENTS (AE) AND SERIOUS ADVERSE EVENTS (SAE) .................... 49 
13.1 
Definition of an AE ...................................................................................................................49 
13.2 
Definition of a SAE ...................................................................................................................49 
13.3 
Time-period, Frequency, and Method of Detecting AEs and SAEs ....................................50 
13.4 
Recording of AEs and SAEs ....................................................................................................51 
13.5 
Evaluating AEs and SAEs .......................................................................................................51 
13.5.1 
Severity Rating .................................................................................................................................51 
13.5.2 
Relationship to Investigational product (IP) ....................................................................................51 
13.6 
Pregnancy ..................................................................................................................................52 
13.7 
Prompt Reporting of SAEs to the Sponsor ............................................................................52 
14 
ETHICAL AND REGULATORY STANDARDS ........................................................... 53 
14.1 
Ethical Conduct of Study .........................................................................................................53 
14.2 
Laws and Regulations ..............................................................................................................54 
14.3 
Informed Consent .....................................................................................................................54 
14.4 
Institutional Review Board/Independent Ethics Committee (IRB/EC) ..............................55 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 15  
14.5 
Clinical Monitoring/Record Keeping .....................................................................................55 
15 
ADMINISTRATIVE RULES .......................................................................................... 56 
15.1 
Curriculum Vitae .....................................................................................................................56 
15.2 
Archiving of Study Documentation ........................................................................................56 
15.3 
Internal Safety Review Committee .........................................................................................56 
16 
STUDY MONITORING .................................................................................................. 56 
16.1 
Responsibilities of the Investigator(s) .....................................................................................56 
16.2 
Responsibilities of the Sponsor ................................................................................................57 
16.3 
Source document requirements ..............................................................................................57 
16.4 
Use and completion of Case Report Forms (CRFs) and additional requests .....................57 
17 
PUBLICATIONS ............................................................................................................. 58 
18 
PROTOCOL ADHERENCE ........................................................................................... 59 
19 
CLINICAL TRIAL PROTOCOL AMENDMENTS ....................................................... 59 
20 
CONFIDENTIALITY ...................................................................................................... 59 
21 
PROPERTY RIGHTS ...................................................................................................... 59 
22 
DATA PROTECTION ..................................................................................................... 60 
23 
SPONSOR AUDITS AND INSPECTIONS BY REGULATORY AGENCIES ............. 60 
24 
PREMATURE DISCONTINUATION OF THE STUDY OR PREMATURE CLOSE-
OUT OF A SITE .......................................................................................................................... 61 
24.1 
Decided by the Sponsor in the following cases: .....................................................................61 
24.2 
Decided by the Investigator .....................................................................................................61 
25 
CLINICAL TRIAL PROTOCOL AMENDMENTS ....................................................... 61 
26 
REFERENCES ................................................................................................................ 62 
APPENDIX A: SCHEDULE OF EVENTS ................................................................................ 63 
APPENDIX B: EXCLUDED MEDICATIONS/THERAPY ...................................................... 68 
APPENDIX C: EASI SCORE ..................................................................................................... 70 
APPENDIX D: PATIENT ORIENTED ECZEMA MEASURE (POEM) ................................ 78 
APPENDIX E: IGA ..................................................................................................................... 80 
APPENDIX F: 5-D PRURITUS SCALE .................................................................................... 81 
APPENDIX G: PATIENT REPORTED LOCAL TOLERABILITY ......................................... 82 
 
 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 16  
 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
AD 
Atopic Dermatitis 
AE 
Adverse Event 
AI 
Average Itch 
AMO 
Ammonia Monooxygenase 
AOB 
Ammonia Oxidizing Bacteria 
BID 
Twice-Daily 
CRF 
Case Report Form 
EASI 
Eczema Area and Severity Index 
E/T 
Early Termination 
FDA 
Food and Drug Administration 
HAO 
NH2OH oxidoreductase 
HbsAg 
Hepatitis B Virus Surface Antigen 
HCV Ab 
Hepatitis C Virus Antibody 
HIV 
Human Immunodeficiency Virus 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IGA 
Investigator Global Assessment 
IP 
Investigational Product 
IRB 
Institutional Review Board 
NH2OH 
Hydroxylamine 
NH3 
Ammonia 
NO 
Nitric oxide 
NO2- 
Nitrite 
NRS 
Itch Numeric Rating Scale 
PCP 
Primary Care Physician 
POEM 
Patient Oriented Eczema Measure 
SAE 
Serious Adverse Event 
SPM 
Study Procedures Manual 
VAS 
Visual Analog Scale  
WI 
Worst Itch 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 17  
1 
STUDY SCHEMA 
 
Consent 
 
 
Screening Visit 
(V1) 
 
 
Meet eligibility 
criteria? 
Screening Period 
 
 
 
Baseline Visit 
(V2) 
 
Randomization: 
1x1010cfu/ml 
4x1010cfu/ml 
vehicle 
 
ET Visit 
 
Screen Fail 
Day 14 
Visit (V3) 
 
Day 28 
Visit (V4) 
 
Yes 
Yes 
Yes 
No 
F/U Visit 
(Day 56 
Visit, V5)  
Washout Phase 
-14 to -1 
Meet 
eligibility 
criteria? 
Meet eligibility 
criteria? 
No 
Meet eligibility 
criteria? 
No 
Stop study before 
Day 56 
Day 5 Call  
 
 
No 
Yes 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 18  
2 
INTRODUCTION 
2.1 
Background 
Ammonia oxidizing bacteria (AOB) are essential for the initial step in environmental nitrification 
processes, specifically the oxidation of ammonia (NH3) to nitrite (NO2-). Nitrosomonas are 
Gram-negative chemolithoautotrophic betaproteobacteria that obtain energy solely from NH3 
oxidation, while fixing CO2 for their carbon needs2. Oxidation of NH3 proceeds in two steps 
(Figure 1) leading to sequential generation of hydroxylamine (NH2OH) and NO2- that require 
two enzyme complexes: the membrane-bound ammonia monooxygenase (AMO) comprised of 
subunits AmoA, AmoB and AmoC; and the periplasmic NH2OH oxidoreductase (HAO). In 
addition to high NO2- levels, NH3 oxidation leads to nitric oxide (NO) and N2O production 
through two independent pathways downstream of NH2OH production: nitrifier denitrification 
and NH2OH oxidation3. 
 
Figure 1 
Nitrifier Denitrification Pathway 
 
B244 is a purified a strain of Nitrosomonas eutropha originally isolated from soil samples. 
Sequencing of the B244 genome revealed a distinct genetic profile from that of other published 
Nitrosomonas strains and AOB genomes. Based on in vitro co-culture studies, B244 was able to 
reduce survival of pathogenic bacteria. Nitrite generation from ammonia concurrently with 
medium acidification by B244 led to strong antibacterial effects and a marked reduction (~100-
fold) in viable counts of methicillin-resistant Staphylococcus aureus and Pseudomonas 
aeruginosa, two pathogens frequently isolated from infected skin and wound sites. In contrast, 
control cultures with B244 in the absence of ammonium or with heat-killed B244 supplemented 
with ammonium, had no antibacterial effects. The unique metabolic and antimicrobial activity of 
Nitrosomonas, in combination with their lack of virulence render these bacteria as attractive 
candidates for topical delivery of nitrite and nitric oxide on human skin with potential to improve 
health in both normal and abnormal skin conditions or wound sites. NO-releasing drugs or NO 
donors have also shown activity against Propionibacterium acnes and other pathogenic bacteria, 
anti-inflammatory activity, and inhibition of lipogenesis by insulin-stimulated immortal 
sebocytes4-6.  
 
2.2 
Clinical Experience 
B244 is being developed as a ‘live topical’ to provide a natural source of AOB and NO/NO2 to the 
human skin.   

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 19  
AOBiome’s first Phase 1b/2a clinical trial titled, “A Double Blind, Vehicle-Controlled, Single 
Center, Randomized, Sequential, Ascending 14-Day Multiple Dose Study in Subjects with Acne 
Vulgaris to Evaluate the Safety, Tolerability and Preliminary Efficacy of B244 Delivered as a 
Topical Spray” was completed in 2016 where 36 participants with clinical diagnosis of facial acne 
vulgaris were randomized to receive ascending doses of investigational product (IP) over 14 days. 
Safety analyses have been completed and there has been no attributable drug related SAEs 
reported.  In addition, a Phase 2b/3 clinical trial titled, “A Randomized, Double Blinded, Phase 
IIb/III, Decentralized Study of B244 Delivered as a Topical Spray to Determine Safety and Efficacy 
in Participants with Mild to Moderate Acne Vulgaris in 372 patients with clinical diagnosis of 
facial acne” has been completed.  B244 was safe and well tolerated with no attributable drug 
related SAEs.  Efficacy was supported by statistically significant 2-point reduction in IGA with 
B244 and a trend in the reduction of the number of inflammatory lesions compared to vehicle.  
 
A study titled, “A Prospective, Vehicle Controlled, Double Blind, Multicenter, Randomized, Phase 
II Study of B244 Delivered as a Topical Spray to Determine Safety and Efficacy in Subjects with 
Mild to Moderate Atopic Dermatitis” has recently been completed.  A total of 122 subjects were 
enrolled and randomized in a 1:1 ratio to receive B244 or vehicle in the study. Overall, there were 
no unexpected safety signals observed following treatment with B244. A total of 30 (25%) subjects 
experienced at least 1 treatment-emergent adverse events (TEAE) during the study, including 16 
(26%) subjects in the B244 treatment group and 14 (23%) subjects in the vehicle group.  
 
AOBiome completed a Phase Ib study in pediatric and adolescent subjects aged 2-17 years of age. 
A study titled, “An Open-label, Multicenter, Phase Ib Study of B244 Delivered as a Topical Spray 
to Assess Safety in Pediatric Subjects aged 2 to 17 years with Atopic Dermatitis” enrolled a total 
of 28 subjects across 3 cohorts.  Overall, there were no unexpected safety signals observed 
following treatment with B244 in pediatric subjects with atopic dermatitis. The B244 spray was 
well tolerated and not associated with any increased pain or redness at the application site. 
 
“A Vehicle-Controlled, Double-Blind, Randomized Phase II Study of B244 Delivered as a Topical 
Spray to Determine Safety and Efficacy in Subjects with Mild to Moderate Rosacea” has been 
completed with a total of 140 subjects randomized. Generally, there were no unexpected safety 
signals observed following treatment with B244. Overall, 31 (22.1) subjects had experienced at 
least one treatment emergent adverse event of which 19 (26.0%) subjects in B244 spray group and 
12 (17.9%) in the Vehicle group. 
 
In addition, other topical development programs with B244 include hypertension.  A study titled, 
“A Prospective, Controlled, Double Blinded, Multicenter, Randomized, Vehicle Controlled, Phase 
II Study of B244 Delivered as a Topical Spray to Determine Safety and Efficacy in Subjects with 
Elevated Blood Pressure” has been completed.  Safety data indicated that B244 was safe and well 
tolerated.  Overall, 16% of subjects experienced at least 1 TEAE during the study with comparable 
incidence in the B244 and Vehicle groups.  Overall, there were no unexpected safety signals 
observed following treatment with B244. 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 20  
Moreover, intranasal application of B244 is being developed for the treatment of allergic rhinitis 
and migraines.  A rat toxicology study performed by intranasal administration of B244 twice daily 
for 28 days found that B244 was safe and well tolerated in rats at levels of up to the maximum 
dose 8x109 cell/mL.  A study titled, “A Prospective, Controlled, Double Blinded, Single Center, 
Randomized, 3 arm, Phase 1b/2a Study to Assess the Safety, Tolerability, and Preliminary Efficacy 
of B244 Delivered as an Intranasal Spray in Healthy Volunteers and Subjects with Seasonal 
Allergic Rhinitis” has been completed.  In addition, a study titled “A Prospective, Randomized, 
Vehicle-Controlled, Double-Blind, Phase 2 Study to Assess the Safety, Tolerability, and Efficacy 
of B244 Delivered as an Intranasal Spray for Preventive Treatment in Subjects with Episodic 
Migraine” is in progress. 
 
2.3 
Safety Profile 
To date, there have been no reported infections or health risks associated with topical application 
or ingestion of Nitrosomonas species.  The absence of any illnesses attributed to these bacteria 
despite our widespread exposure indicates that they pose a minimal health risk, if any at all.  
Infection or tissue damage by Nitrosomonas is unlikely, because the sequenced genomes of several 
Nitrosomonas and other AOB lack genes encoding cytotoxins, or other known bacterial virulence 
factors.  Further, AOB are slow growing, as compared to most heterotrophic bacteria, with 
optimum doubling times of 8 hours or higher.  In particular, Nitrosomonas growth is rate limited 
by the availability of ammonia requiring the oxidation of 27 moles NH3/mole CO2 fixed.  Due to 
their dependence on ammonia for their growth, the numbers of Nitrosomonas on the skin will be 
necessarily limited and naturally regulated by the amount of ammonia produced in sweat.  This 
would ensure that the amount of nitrite and NO generated would be relatively low, without any 
adverse effects. 
  
2.4 
Rationale for the Study 
Eczema or Atopic dermatitis is an inflammatory skin condition that affects nearly 20% of 
children and between 2-10% of adults7. Disease prevalence has steadily grown in the last 30 
years, resulting in a growing field of atopic dermatitis research8.   
Pruritus is a common morbidity associated with Atopic Dermatitis, and its management remains 
a challenge for physicians. Pruritus is defined as an urge or unpleasant sensation that produces 
the urge to scratch9. Modern treatment no longer consists of antihistamines alone. Anti-
inflammatory, antiseptic, as well as antipruritic therapies to address the epidermal barrier as well 
as immunomodulation or infection are now used in clinical practice9.  Mild pruritus may be 
controlled with topical therapies, while moderate to severe pruritus requires combination 
treatment, consisting of antipruritic and immunosuppressive drugs, phototherapy, and topical 
compounds9. 
 
While the complete pathophysiology of pruritus remains unclear, histamines are one of the most 
potent mediators in AD related pruritus. The itch cycle exacerbates damage to the epidermal 
barrier leading to water loss and dryness, thereby creating a hospitable environment for skin 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 21  
pathogens, which cause infections and flare up symptoms. Staphylococcus aureus is consistently 
found in eczematous skin lesions in patients with AD. Correlation between the severity of the 
disease and presence of Staphylococcus aureus has been well established and it has been shown 
that the presence of bacteria is an important factor in skin aggravation10. The goal of therapy for 
AD is to restore the epidermal barrier function and reduce skin inflammation, thereby alleviating 
symptoms and intensity of AD associated pruritus. However, systemic antibiotic use is 
controversial.  
B244 has been developed as a topical application of a natural source of AOB and NO/NOx to the 
human skin.  We hypothesize that application of AOB to eczematous skin may 
reduce Staphylococcus aureus skin load and normalize the inflammatory response by reducing 
Th2 activation. 
The primary purpose of this study is to evaluate the efficacy and safety of B244 in the treatment 
of pruritus in adults with a history of atopic dermatitis. 
3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
● To assess the efficacy of B244 in the treatment of pruritus in adults with a history of 
atopic dermatitis.  
3.2 
Secondary Objectives 
● To assess the safety and tolerability of B244 in adults with a history of atopic dermatitis. 
4 
ENDPOINTS 
4.1 
Efficacy 
4.1.1 Primary 
● Mean change in WI-NRS from baseline to Week 4 
4.1.2 Secondary 
● Proportion of patients with ≥4 point improvement in WI-NRS from baseline to Week 4 
● Proportion of subjects with any improvement in WI-NRS from baseline to Week 4 
● Mean change in AI-NRS from baseline to Week 4 
● Proportion of subjects with ≥4 point improvement in AI-NRS from baseline to Week 4 
● Proportion of subjects with any improvement in AI-NRS from baseline to Week 4 
● Mean change in WI-NRS from baseline to Week 2 
● Proportion of subjects with ≥4 point improvement in WI-NRS from baseline to Week 2 
● Mean change in POEM from baseline to Week 4 
● Mean change in 5-D Pruritus Scale from baseline to Week 4 
4.1.3 Exploratory 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 22  
● Mean change in IGA from baseline to Week 4 
● Mean change in EASI from baseline to Week 4 
4.2 
Safety & Tolerability 
Safety and tolerability endpoints include the following:  
● Incidence of TEAEs and SAEs 
● Changes in vital signs and clinical laboratory parameters following study drug exposure 
● Changes in local skin tolerability following application of study drug 
5 
STUDY DESIGN 
● This is a Prospective, Vehicle Controlled, Double-Blind, Multicenter, Randomized Phase 
II Trial, comparing the effect of twice daily B244 applications for 4 weeks vs vehicle 
applications on treatment of mild to moderate pruritus associated with atopic dermatitis. 
● Approximately 576 subjects may be enrolled. 
● The total duration of the study will be approximately 11 weeks. Participants will report 
for a Screening visit and if all inclusion/exclusion criteria are met, subjects will go 
through a two-week washout phase (see Section 10.6.2) before reporting for a Baseline 
visit.   
● After screening and baseline, participants will be randomized to one of two doses of 
B244 or vehicle application for 4 weeks.  
● Randomization will be 1:1:1 so that an equal number of patients will be treated in each 
Arm of the study. 
● All B244 randomized subjects will be treated at the dose of 1x1010 cells/ml (O.D. 5.0) or 
4x1010 cells/ml (O.D. 20.0). 
● Subjects must be willing and able to complete diary within a consistent time frame on a 
daily basis and to comply with restrictions on allowable therapies for the duration of the 
study.  
● All subjects will attend a screening visit not more than 21 days prior to Baseline (Day 0). 
Subjects will be required to return to the clinic at Baseline, Day 14 (Week 2) and Day 28 
(Week 4) visits. All subjects will be asked to attend a Week 8 follow-up visit 4 weeks (28 
(±3) days) after the last dose of study medication. 
● Subjects will apply a total of 10 pumps of IP per application across all affected 
areas twice-a-day (i.e. 10 pumps in the morning and 10 pumps again at night) for 4 
weeks. 
● Safety evaluations will consist of review of participant’s medical history at screening and 
on-going assessment of adverse events reported throughout the study duration. 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 23  
6 
SELECTION OF STUDY PARTICIPANTS 
6.1 
Number of Participants Planned 
Approximately 576 subjects (192 per treatment group) with pruritus and a history of atopic 
dermatitis will be randomized into this study.  
6.2 
Inclusion Criteria 
Deviations from inclusion criteria are not allowed because they can potentially jeopardize 
the scientific integrity of the study, regulatory acceptability or participant safety. Therefore, 
adherence to the criteria as specified in the protocol is essential. 
 
Participants eligible for enrollment in the study must meet all the following criteria: 
 
1. Male and female subjects 18 to 65 years of age. 
2. Pruritus of at least 4 weeks duration prior to the initial Screening visit and during the 2 
week washout period. 
a. Subjects using stable doses of oral H1 antihistamines at the initial Screening visit 
must be willing to continue these at the same doses and frequencies throughout 
the study inclusive of the follow-up period.  
3. Worst Itch Numeric Rating Scale (WI-NRS) score ≥ 7 in the 24-hour period prior to the 
initial Screening as well as Baseline visits.  
4. Average weekly WI-NRS score ≥6 for each week of the washout period, as recorded in 
the eDiary. 
5. A history of atopic dermatitis for greater than 12 months consistent with a diagnosis of 
atopic dermatitis, as defined by the 2014 American Academy of Dermatology (AAD) 
Guidelines of Care for the Management of Atopic Dermatitis1.  
a. Subjects using bland emollients at the initial Screening visit will be allowed to 
continue to use their emollient of choice at a similar dose and frequency 
throughout the study, if used. 
b. Subjects using low- to mid-potency topical corticosteroids at the initial Screening 
visit will be allowed to use their topical corticosteroid of choice at the same dose 
and frequency no more than 7 days per month throughout the study as rescue 
medication. 
6. A minimum of 10% and not more than 40% of the subjects' BSA affected by atopic 
dermatitis (affected is defined by physical examination findings: erythema, edema, 
scaling, lichenification, excoriation, with the excoriation serving as the physical 
examination correlate of pruritus) at Screening and Baseline. 
a. Subjects’ BSA can include face and body OR body alone BUT NOT face alone. 
7. An Investigator Global Assessment (IGA) score of 2-3 at Screening and Baseline. 
8. Willing and able to complete once-daily eDiary entries within a consistent timeframe for 
the duration of the study and have ≥80% eDiary compliance rate during the washout 
period.  
9. Judged to be in good health in the investigator’s opinion. 
6.3 
Exclusion Criteria 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 24  
Deviations from exclusion criteria are not allowed because they can potentially jeopardize the 
scientific integrity of the study, regulatory acceptability or participant safety. Therefore, 
adherence to the criteria as specified in the protocol is essential. 
 
Participants will be excluded from the study if any of the following criteria are met: 
1. Clearly defined etiology for pruritus other than atopic dermatitis. These include but are 
not limited to urticaria, psoriasis or other non-atopic dermatologic conditions, hepatic or 
renal disease, psychogenic pruritus, drug reaction, untreated hyperthyroidism, parasite 
presence and presence of acute infection either systemically or in the AD lesions.  
2. Presence of any acute condition which may risk inducing an atopic dermatitis flare during 
the course of the study, such as impetigo or active herpes simplex infection.  
3. Treatment with systemic corticosteroids within 4 weeks prior to randomization.  
4. Treatment with Class III or higher potency topical corticosteroids or any topical anti-
pruritic therapies (other than stable doses of low- or mid-potency topical corticosteroids 
or bland emollients) within 4 weeks prior to randomization.  
5. Treatment with systemic therapies with recognized anti-pruritic (e.g. tricyclic 
antidepressants, sedatives, tranquilizers, gabapentin, marijuana or other cannabinoids, 
opioid receptor agonists/antagonists) or pruritic (e.g. opioids, angiotensin-converting 
enzyme inhibitors, cocaine, antimalarials) properties within 4 weeks prior to 
randomization.  
a. Stable doses of H1 antihistamines will be permitted. Subjects must be willing to 
continue these at the same doses and frequencies throughout the study inclusive of 
the follow-up period.  
6. Any clinically significant changes in type, dose, or frequency of bland emollients, low- or 
mid-potency corticosteroids, and/or oral H1 antihistamines throughout the study from 
screening to follow-up. 
7. Treatment with systemic immunosuppressive/ immunomodulatory therapies within 4 
weeks prior to randomization (including but not limited to phosphodiesterase-4 
inhibitors, cyclosporine, mycophenolate-mofetil, methotrexate, azathioprine, interferon-
gamma, or phototherapy).  
8. Treatment with biologic therapies within 12 weeks or 5 half-lives prior to randomization, 
whichever is longer.  
9. Use of an indoor tanning facility within 4 weeks prior to randomization.  
10. Treatment with any investigational therapy within 4 weeks prior to randomization.  
11. Allergen immunotherapy within 6 months prior to randomization.  
12. Prior use of AO+ Mist. 
13. History of malignancy within 5 years prior to randomization, with the exception of 
completely treated and non-metastatic basal cell carcinoma or squamous cell carcinoma 
of the skin.  
14. History of a major psychiatric condition (including major depressive disorder, bipolar 
disorder, or schizophrenia), suicidal ideation, or suicide attempt.  
15. Known active hepatitis infection.  
16. Known history of human immunodeficiency virus (HIV) infection.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 25  
17. Presence of any medical condition or disability that, in the investigator’s opinion, could 
interfere with the assessment of safety or efficacy in this trial or compromise the safety of 
the subject.  
18. Currently pregnant or breastfeeding, or male subject with a pregnant or breastfeeding 
partner.  
19. Females of childbearing potential who are unable or unwilling to practice highly effective 
contraception (pregnancy prevention); fertile males who are unable or unwilling to use 
condoms with female partners of childbearing potential.  
 
7 
PARTICIPANT ENROLLMENT 
 
7.1 
Consenting Participants 
Informed consent for participation in the study must be obtained before performing any study-
specific procedures.  
 
Informed Consent Forms for enrolled participants and for participants who are not subsequently 
enrolled will be maintained at the study site in secure study files. Consent will be obtained by 
trained research study staff trained in taking informed consent. The study will be explained with 
the opportunity for the participant to ask questions. If a participant wishes to enter the study, a 
consent form will be completed and signed. 
 
7.2 
Screening for Eligibility 
After informed consent has been obtained, to determine participant eligibility for enrollment in 
the study, screening assessments will be performed within 1 week (-21 to -14 days) prior to 
starting the Washout period (-14 to -1). All screening evaluations must be completed and 
reviewed to confirm that participants meet all eligibility criteria before Washout period and 
subsequent randomization on Day 0. Subjects will be asked to undergo a 2 week (-14 to -1) 
washout period prior to the Baseline procedure. During the screening, washout, treatment and 
follow-up periods, subjects will be asked to stop utilizing bleach or vinegar as a pruritus or 
atopic dermatitis therapy, as well as any other treatments described in the exclusion criteria and 
the Excluded Medications/Therapy (Appendix B). 
 
All screening assessments are listed in the Time and Events Table (Appendix A).  A participant 
must meet all inclusion criteria, and none of the exclusion criteria, to be enrolled and randomized 
in this study. The Investigator and team will maintain a screening log to record details of all 
persons screened and to confirm eligibility or record reasons for screening failure, as applicable. 
 
7.3 
Study Withdrawal and Withdrawal from Investigational Product and Stopping 
Criteria  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 26  
A participant may withdraw from the study at any time at his/her own request, or may be 
withdrawn at any time at the discretion of the Investigator. 
Reasons for withdrawal (participants who refuse to complete any remaining study visits) or 
discontinuation (participants who prematurely stop the application) at any time during the study 
may include, but are not limited, to the following: 
 
● For safety reasons, either at the discretion of the Investigator or at the participant’s 
request 
● For protocol violations at the discretion of AOBiome 
● Withdrawal of consent by the Subject 
● Lost to follow-up 
● Due to use of concomitant therapy that could interfere with the results of the study (the 
Investigator will report all such information through the CRF and decide, in accordance 
with AOBiome, whether the participant is to be withdrawn).  
 
The reason for participant study withdrawal will be recorded in the electronic Case Report Form 
(eCRF). Data from participants withdrawing from the study will be considered evaluable up to 
the point at which they are withdrawn using the same criteria for evaluability as for participants 
who complete the study. 
 
Study stopping rules will be implemented to stop the study for safety review in the event that: 
• 1 subject reports death, or 
• >2 subjects report an SAE, or 
• >4 subjects experience grade 3 AEs of a similar type, or 
• >6 subjects experience grade 2 AEs of a similar type  
when the reported SAEs or AEs are considered possibly, probably, definitely or related to the 
investigational product. 
  
Atopic Dermatitis is an active disease with known seasonal and personal histories of flares and 
exacerbations of the patient’s underlying disease state. This includes personal histories of 
worsening itch and itch-scratch cycles, which can lead to localized infection and localized 
cellulitis. A subject’s known and collected clinical history should be considered by the principal 
investigator when determining relationship of event to the investigational product. 
 
7.4 
Early Termination 
Early termination from the study may occur due to loss to follow-up or withdrawal of consent by 
the subject. Participants who have discontinued the study early will be evaluated by the 
Investigator at the Early Termination Visit.  See the list of assessments to be performed at the 
Early Termination Visit in the Time and Events Table (Appendix A). Participants with ongoing 
AEs or SAEs believed to be possibly related to investigational product (IP) will continue to be 
followed until resolution or for 30 days as warranted by the nature of the AE.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 27  
7.5 
Lifestyle Considerations 
There are no dietary or activity related restrictions for this study. 
7.5.1 
Use of Bland Emollients During the Study 
 
Subjects will be allowed to use their usual choice of bland emollients (e.g., lotion, moisturizer) 
from screening to follow-up, on an as needed basis, using specific time of use guidelines in 
relation to study medication application.  Subjects will be allowed to use their emollient of 
choice at a similar dose and frequency throughout the study if used.  Any clinically significant 
changes in type, dose, or frequency of bland emollients will not be allowed during the study.  A 
detailed guideline will be shared with the sites and subjects to ensure consistency in study 
medication treatment application and relation to body washing, emollient use, and other chemical 
exposure to skin. 
 
Subjects will be encouraged not to use sunscreen on the treated lesions during participation in the 
study. Sunscreen should be used minimally or used sparingly while in the active treatment phase 
(Baseline to Week 4) of the study.  Alternatively, sunscreen should be washed off prior to study 
medication use. 
 
Subjects who do not have atopic dermatitis on their face may apply makeup as needed. However, 
those subjects who have atopic dermatitis on their face and require to use makeup will be advised 
to use it minimally. 
 
8 
STUDY TREATMENT 
8.1 
Investigational Product 
Under normal conditions of handling and administration, IP is not expected to pose 
significant safety risks to site staff. A Material Safety Data Sheet describing the 
occupational hazards and recommended handling precautions will be provided to site 
staff if required by local laws or will otherwise be available from the Sponsor upon request. 
 
Investigational product must be stored in a secure area under the appropriate physical 
conditions for the product (2-8oC). Access to and administration of the IP will be limited to the 
Investigator and authorized site staff. Investigational product must be dispensed or 
administered only to participants enrolled in the study and in accordance with this protocol (see 
Section 8.6). 
 
The Investigator is responsible for study treatment accountability, reconciliation, and record 
maintenance. The Investigator or designated site personnel must maintain study treatment 
accountability records throughout the course of the study. The responsible person(s) will 
document the amount of study treatment received from and returned to the sponsor and the 
amount administered to participants. The required accountability unit for this study will be the 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 28  
bottle. Discrepancies are to be reconciled or resolved.  
 
 
Product name: 
B244, 30ml/bottle 
B244, 30ml/bottle 
Vehicle, 30ml/bottle 
Dosage form: 
B244 suspension 
B244 suspension 
Vehicle solution 
Unit dose strength: 
1 x 1010 cells/ml 
(O.D. 5.0) 
4x1010 cells/ml 
(O.D. 20.0) 
50nM Na2HPO4-
2mM MgCl2 (pH 7.6) 
Route/administration/duration: 
Topical application 
BID for 28 days 
Topical application 
BID for 28 days 
Topical application 
BID for 28 days 
Dosing instructions: 
Subjects will apply 
a total of 10 
pumps of IP per 
application to all 
affected 
areas twice-a-
day (i.e., 10 pumps 
in the morning and 
10 pumps again at 
night). 
 
Applications 
should occur in the 
morning and at 
night for 4 weeks. 
Subjects will apply 
a total of 10 
pumps of IP per 
application to all 
affected 
areas twice-a-
day (i.e., 10 pumps 
in the morning and 
10 pumps again at 
night). 
 
Applications 
should occur in the 
morning and at 
night for 4 weeks. 
Subjects will apply a 
total of 10 pumps of 
IP per application 
to all affected 
areas twice-a-
day (i.e., 10 pumps in 
the morning and 10 
pumps again at 
night). 
 
Applications should 
occur in the morning 
and at night for 4 
weeks. 
Physical description: 
Odorless, cloudy, 
light pink 
suspension 
Odorless, cloudy, 
light pink 
suspension 
Odorless, clear, and 
colorless suspension 
Manufacturer/source of 
procurement: 
AOBiome, LLC 
AOBiome, LLC 
AOBiome, LLC 
 
The contents of the label will be in accordance with all applicable regulatory requirements. B244 
and matching vehicle will be packaged in identical 30 ml white bottles.  
 
8.2 
Dose Changes  
No dose changes are anticipated. 
 
8.3 
Storage conditions  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 29  
All investigational drug supplies in the study will be stored in a secure, refrigerated (2-8°C) safe 
place, under the responsibility of the Investigator or other authorized individual.  
 
8.4 
Description of Blinding Method 
This study will be double-blinded: neither Investigator(s), nor study participants, nor those 
involved in the conduct of the trial (including sponsor staff) will be aware of the treatment the 
participants are receiving.  The appearance of B244 and vehicle dispensing containers will be 
identical. 
 
8.5 
Treatment Assignments: 
This is a double-blind study. Participants will be assigned to study treatment in accordance with 
the randomization schedule generated for the allocation of B244 or vehicle prior to the initiation 
of the trial. Randomization will be centrally-based and performed using an appropriate IWRS (an 
automated randomization system).  
 
Each participant scheduled to receive investigational product (IP) will receive a randomization 
number at the time of randomization. The randomization number will be used to identify the 
study medication kit assigned to the participant and indicate the treatment to be administered to 
that participant.  
    
8.6 
Treatment Compliance 
Study treatment must be dispensed or administered according to procedures described 
herein. Only participants enrolled in the study may receive study treatment. Only authorized site 
personnel may supply study treatment. Participants will record use of the study medication 
utilizing the study diary at the time of use each day. Participants will review study medication 
compliance with the Investigator or designee. Any missed doses, timing, and reason for missed 
dose will be recorded in the eCRF. There should be no doubling of doses to make up for missed 
doses. If a dose is missed, the next dose of study medication should be taken as scheduled. 
 
Each site participating in the trial will be instructed to assess subject’s compliance by weighing 
the investigational product at the Baseline visit, Week 2 visit, and Week 4 visit and followed 
every time study medication is dispensed and returned per instructions below. Sites will be 
provided scales, which will be calibrated prior to each use. Study personnel will be instructed to 
record measurements into the eCRF. 
 
All weight measurements will be performed without the safety cap.  Subjects will be dispensed 2 
bottles upon randomization, and each bottle will be labeled 1 and 2 with a permanent marker at 
the site.  Unused Bottle 1 will be vortexed, primed, and weighed without the cap by the site per 
treatment application instructions and dispensed to the subject who will mix and apply the first 
dose at the site under study staff supervision.  Bottle 2 will also be vortexed, primed, and 
weighed at the site at the time of dispense without the cap, the cap inserted back on the bottle, 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 30  
and the bottle returned to its carton unused. After the first treatment application of Bottle 1, 
subjects will take home both bottles where they will place Bottle 1 on the counter at room 
temperature for in-home application twice a day until empty.   Bottle 2 will be stored in the 
refrigerator until Bottle 1 is empty, at which point Bottle 2 will be taken out of the refrigerator, 
mixed per treatment application instructions, and used.  Should Bottle 1 run out during a 
treatment application, the subject will count the number of pumps that were made with Bottle 1 
and complete the remaining number of pumps needed to achieve a total of 10 pumps using Bottle 
2.   
 
At the Week 2 visit, subjects will return any empty bottle (likely Bottle 1 since each bottle 
should last approximately 10 days and the subject should be on Bottle 2 at the time of visit) to 
the site for weight measurement (without the safety cap) to confirm usage.  At this visit, a third 
bottle of the same treatment group will be labeled as Bottle 3 and will be vortexed, primed, and 
weighed at the site at the time of dispense without the cap, the cap inserted back on the bottle, 
and the bottle returned to its carton unused.  The subject will be dispensed Bottle 3 to take home 
and store in the refrigerator until Bottle 2 is empty, at which point Bottle 3 is taken out of the 
refrigerator and used on the counter until the Week 4 visit.  All empty bottles returned to the site 
will be collected and stored by the site in ambient temperature until return to the drug depot. If 
the subject is still on Bottle 1 at the Week 2 visit, the site will confirm daily dosing application 
and retrain the subject on the appropriate use of IP application, and the subject will continue 
using Bottle 1 until empty, at which point Bottle 2 is taken out of the refrigerator and used on the 
counter until Bottle 2 is empty, after which Bottle 3 is taken out of the refrigerator and used on 
the counter until the Week 4 visit.  Any bottle currently in use at the time of the Week 2 visit 
should not be returned to the site.   
 
At the Week 4 visit, all used and unused bottles will be returned to the site for weight 
measurements and storage at the site in ambient temperature until return to the drug depot.   
 
8.7 
Treatment Application 
Subjects will receive study drug for application throughout the study. Subjects will be instructed 
in the use of the spray bottle and asked to self-administer the Investigational Product following a 
detailed instruction on treatment application that will be shared with the sites and subjects to 
ensure consistency in treatment application and relation to body washing, emollient use, and 
other chemical exposure to skin.  Key guidelines include: 
 
● The subject should wash his/her hands before applying study treatment. 
● Subjects will apply a total of 10 pumps of IP per application to all affected. areas twice-a-
day (i.e., 10 pumps in the morning and 10 pumps again at night). 
● The subject will apply the IP to the affected area twice daily (approximately 12 hours apart) 
for 28 consecutive days. 
● Subject should mix the bottle thoroughly per instructions provided before each application 
and then saturate the application area well, holding the bottle approximately 6 inches away 
from the skin. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 31  
● Subjects will be asked to let the product air dry after each application. 
● Subjects will follow instructions provided for body washing, emollient use, and other 
chemical exposure to skin in relation to timing of IP application. 
● The spray bottle in use may be stored at ambient temperature until used up, while additional 
unused bottles will be stored in the refrigerator until use.  DO NOT FREEZE. 
● The subject will apply the IP to the worst and largest lesions first and then apply the IP to the 
smaller lesions. If after covering all lesions, the total number of pumps per application has 
not been exhausted, subject should go back and cover the worst lesions with the remaining 
number of pumps. However, the total number of 10 pumps allowed should not be exceeded 
per application.  
● Subjects will be asked not to expose the treatment kit to conditions which are unnatural or 
harmful to the product, such as excessive heat (temperatures over 77°F [25°C] and freezing 
temperatures below 32°F [ 0°C]). Subjects may travel with their study medication but should 
not leave it in a hot car, outside in the cold temperatures, etc. Subjects will also be asked not 
to tamper or cause damage to the IP bottle. 
8.8 
Treatment of Investigational Product Overdose 
The sponsor does not recommend specific treatment for an overdose. Washing with conventional 
cleanser and water will remove the product. The Investigator will use clinical judgment to treat 
any overdose.  
 
8.9 
Study Drug Discontinuation  
Subjects will be discontinued from study drug treatment in the following events:  
● The subject experiences a Grade 2 or higher treatment emergent AE that is assessed as 
likely related to study drug  
● The subject receives treatment with an excluded therapy or has a clinically significant 
change in dose or frequency of allowed adjunctive therapies during the treatment period 
● The female subject becomes pregnant or female partner of a male subject becomes 
pregnant or is breastfeeding  
Discontinuation from study drug treatment may also occur for any of the following reasons:  
● Subject decision to discontinue study drug treatment, or subject decision to withdraw 
consent from the study  
● Any medical condition that may jeopardize the subject’s safety if study drug is continued, 
in the investigator’s and/or Sponsor’s opinion  
● Discontinuation is deemed to be in the best interest of the subject, in the investigator’s 
and/or Sponsor’s opinion  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 32  
Subjects who discontinue treatment with study drug prior to completing the treatment period will 
have an ETD visit within 7 days after their last dose of study drug in addition to a follow-up 
visit.  
8.10 Product Accountability 
In accordance with federal and local regulatory requirements, the Investigator and designated site 
personnel must document the amount of investigational product dispensed to study participants, 
the amount returned by study participants, and amount received and returned to the sponsor, 
when applicable. Product accountability records must be maintained throughout the course of the 
trial. Any quality issue noticed with the receipt or use of an IP (deficient IP in condition, 
appearance, pertaining documentation, labeling, expiry date, etc.) should be promptly reported to 
the Sponsor, who will initiate a complaint procedure. All investigational product must be stored 
in a secure locked room with access limited to the Investigator and designated site personnel. 
Study product is to be stored in a refrigerator between 2-8°C. Maintenance of a temperature log 
is required. Under no circumstances will the Investigator allow IP to be used other than as 
directed by this Clinical Trial Protocol, or dispose of IP in any other manner.  
 
8.11 Unblinding Procedures 
The Investigator may unblind a participant’s treatment assignment only in the case of emergency 
or in the event of a serious medical condition when knowledge of the study treatment is essential 
for the appropriate clinical management or welfare of the participant, as determined by the 
Investigator. It is preferred (but not required) that the Investigator first contacts the Medical 
Monitor to discuss options before unblinding the participant’s treatment assignment. The 
Investigator must notify the Sponsor as soon as possible when a participant’s treatment 
assignment is unblinded without revealing the treatment assignment of the unblinded participant 
unless that information is deemed important for the safety of participants currently in the study. 
The date and reason for the unblinding must be documented in the participant’s study record.  
The Medical Monitor may unblind the treatment assignment for any participant with an SAE. If 
the SAE requires that an expedited regulatory report be sent to one or more regulatory agencies, 
a copy of the report, identifying the participant’s treatment assignment, may be sent to clinical 
Investigators in accordance with local regulations and/or sponsor policy. 
 
8.12 Retrieval and Destruction of Investigational Product 
All partially used or unused treatments will be returned to the site as brought by study 
participants. A detailed IP log of the returned IP shall be established.  
The site will not destroy unused IP unless the Sponsor provides written authorization to the 
contrary. All used and unused bottles will be shipped to the authorized drug depot at the end of 
the study.  
8.13 Concomitant and Excluded Therapies  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 33  
Any medication or vaccine (including over-the-counter or prescription medicines, vitamins, 
and/or herbal supplements) that the subject is receiving within 30 days prior to screening and 
through the final study visit will be recorded on the appropriate CRF) along with: 
 
● Reason for use. 
● Dates of administration including start and end dates. 
● Dosage information including dose and frequency. 
 
Any medications started during the study (including “as needed” medications) will be recorded 
in the concomitant medication list as soon as the Investigational Site will become aware of the 
medication being added. 
 
Details of any medication taken by the subject outside of the study center will be reviewed by the 
Investigator or designee on each study center visit. The Medical Monitor should be contacted if 
there are any questions regarding concomitant or prior therapy. 
 
A list of excluded medications/therapy is provided in Appendix B. 
 
Previous treatment of atopic dermatitis must be recorded irrespective of the term it was given. 
Acetaminophen is permitted for use any time during the study. Other concomitant medication 
may be considered on a case-by-case basis by the Investigator in consultation with the Medical 
Monitor. 
Concomitant therapies include any therapies (including over-the-counter medications) used by a 
subject from initiation of study drug treatment through the follow-up period. A record of all 
medications used will be maintained for each subject throughout the study. Reported information 
will include a description of the type of drug, treatment period, dosing regimen, the route of 
administration, and drug indication.  
8.13.1 Permitted Medications 
Subjects using oral contraceptives, hormone-replacement therapy, or other maintenance therapies 
that are not Excluded Therapies (Appendix B) may continue their use during the study.   
 
Stable doses of the following therapies for pruritus and/or atopic dermatitis will be allowed as 
adjunctive therapies during the study:  
● Oral H1 antihistamines 
● Bland emollients 
A detailed guideline will be shared with the sites and subjects to ensure consistency in study 
medication treatment application and relation to body washing, emollient use, and other chemical 
exposure to skin. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 34  
Low- or mid-potency topical corticosteroids will be allowed per subjects’ usual medication, 
frequency, and dose throughout the study from screening to washout, treatment, and follow-up 
periods as rescue medication up to 7 days per month. 
 
 
Subjects using any of these adjunctive therapies should maintain their regimen without clinically 
significant changes in type, dose or frequency throughout the screening, treatment, and follow-up 
periods.  
All participants will be screened for concomitant medications prior to inclusion into the study.  
Any concomitant medication to treat adverse events will be recorded in the Concomitant 
Medication section of the eCRF. 
 
8.13.2 Excluded Therapies 
Therapies and medications that are excluded from the initial Screening visit through the follow-
up period are listed in Appendix B.  
8.14 Rescue Medications 
8.14.1 Rescue Medication Permitted During the Study 
Over the course of the study, the Investigator will monitor and evaluate subject’s condition and 
determine whether rescue therapy may be necessary.  The use of medications to treat a subject’s 
atopic dermatitis during the study will be permitted if a medical professional determines that it 
becomes medically necessary.  Such rescue treatment should not exceed 7 days per month for the 
duration of the trial (screening, washout, treatment, and follow-up), e.g., no more than 3 days 
total during the 2 week washout period, no more than 7 days total during the 4 week treatment 
period, and no more than 7 days total during the 4 week follow-up period.   If a subject requires 
treatment for more than 7 days per month or requires systemic therapy, they may be discontinued 
from the study.  Medications permitted are listed in Table 8.1. 
In the event of an atopic dermatitis or pruritus exacerbation in between visits, subjects should 
contact study staff as soon as they are able. Subjects should seek approval on the use of rescue 
medications.  In the event that the subject did not notify the study staff, every effort should be 
made by the subject to contact the PI or study staff as soon as feasible. The details of the 
medication should be recorded in the eDiary and transferred to the CRF.  Details recorded should 
include medication name, date of administration, dosage, and number of applications. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 35  
Table 8.1 
Permitted Rescue Medication 
Class 4 – Mid-strength  
Clocortolone pivalate (0.1%) 
Cloderm® Cream 
Mometasone furoate (0.1%) 
Elocon® Cream 
Triamcinolone acetonide (0.1%) 
Aristocort® A Cream, Kenalog® Ointment 
Betamethasone valerate (0.1%) 
Valisone Ointment 
Fluocinolone acetonide (0.025%) 
Synalar® Ointment 
Class 5 – Lower Mid-strength 
Fluticasone propionate (0.05%) 
Cutivate® Cream/Cutivate Lotion 
Prednicarbate (0.1%) 
Dermatop® Cream 
Hydrocortisone probutate (0.1%) 
Pandel® Cream 
Triamcinolone acetonide (0.1%) 
Aristocort A Cream, Kenalog Lotion 
Fluocinolone acetonide (0.025%) 
Synalar Cream 
Class 6 – Mild 
Alclometasone dipropionate (0.05%) 
Aclovate® Cream/Ointment 
Desonide (0.05%) 
Verdeso™ Foam, Desonate Gel™ 
Triamcinolone acetonide (0.025%) 
Aristocort A Cream, Kenalog Lotion 
Hydrocortisone butyrate (0.1%) 
Locoid Cream/Ointment 
Fluocinolone acetonide (0.01%) 
Derma-Smoothe/FS® Scalp Oil, Synalar Topical 
Solution 
Class 7 – Least potent 
Hydrocortisone (2%/2.5%) 
Nutracort® Lotion, Synacort Cream 
Hydrocortisone (0.5 to 0.1%) 
Cortaid® Cream/Spray/Ointment and many other over-
the-counter products 
 
 
8.14.2 Use of Excluded Therapies During the Treatment Period  
Subjects who use an excluded therapy during the treatment period for treatment of atopic 
dermatitis or pruritus outside of the rescue medication allowance will be discontinued from study 
drug treatment, with the following exception:  
● Subjects who require short treatment for indications other than atopic dermatitis or 
pruritus (up to a total of 3 days over the treatment period) with sedatives, tranquilizers, 
opioids, and/or topical therapies will not be required to discontinue from study drug 
treatment.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 36  
Use of any excluded therapies will be recorded for subjects who receive them.  
8.15 Handling of Investigational Product 
 
Subjects will receive study IP in 30 ml white bottles. Each bottle will be anticipated for use over 
10 days and brought to all study appointments. The IP bottle can be kept at room temperature 
while in use by the subject.  
 
Subjects will be asked not to subject the treatment kit to conditions which are unnatural or 
harmful to the product, such as excessive heat (temperatures over 77°F (25°C)) and freezing 
temperatures (below 32°F (0°C)). Subjects may travel with their study medication but should not 
leave it in a hot car, outside in the cold temperatures, etc. Subjects will also be asked not to 
tamper or cause damage to IP. 
 
9 
CONTRACEPTION REQUIREMENTS 
 
Effective contraception is required for all women physiologically capable of becoming pregnant 
during study participation. Women of child-bearing potential must agree to use an acceptable 
form of contraception for up to 2 weeks after the study completion as detailed. Women of 
childbearing potential are defined as any female who has experienced menarche and who is NOT 
permanently sterile (e.g., by tubal ligation) or postmenopausal. Postmenopausal is defined as 12 
consecutive months with no menses without an alternative medical cause. 
 
Effective contraception methods include:  
● Use of oral, injected or implanted hormonal methods of contraception or placement of 
an IUD or IUS or other forms of hormonal contraception that have comparable 
efficacy, for example hormone vaginal ring or transdermal hormone contraception  
● Use of barrier methods (i.e., condom, diaphragm) used with a spermicide (i.e., foam, 
cream, or gel that kills sperm) 
● Total abstinence (when this is in line with the preferred and usual lifestyle of the 
study participant).  
 
Periodic abstinence (i.e., calendar, ovulation, symptothermal, postovulation methods) and 
withdrawal are not acceptable methods of contraception.  
 
Male participants of the study who are having sexual intercourse with a woman who can become 
pregnant must use an acceptable form of birth control while participating in the study. 
Additionally, male participants are expected to inform their female partners of their participation 
in a research study of an investigational drug, the effects of which on a fetus and on a pregnant 
woman are unknown. Male participants will also be expected to provide their female partners 
with the contraception requirements information previously described and the study doctor’s 
contact information for questions.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 37  
Payment for all aspects of obstetrical care, child-or related care will be the study participant’s 
responsibility.  
 
In case of pregnancy, Investigational Product should be discontinued and the Sponsor should be 
informed immediately. Follow-up of the pregnancy will be mandatory until the outcome is 
available. 
10 STUDY PROCEDURES 
10.1 Pre-screening procedures  
Study subjects will be recruited from among participating hospitals, clinics, and diagnostic 
centers or from general population under the responsibility of a participating Investigator. Prior 
to initiation of the recruitment phase, participating Investigators will identify a pool of potential 
study subjects. Each of these centers will identify potentially eligible patients in advance, by 
either reviewing past medical records and diagnoses, screening in clinics, referral from other 
physicians, or other sources of recruitment, to identify those aged 18 to 65 with clinical diagnosis 
of mild or moderate Atopic Dermatitis. Medical records from patient’s dermatologist or primary 
care physician to confirm the diagnosis are optional. Verbal confirmation of the diagnosis 
present for > 12 months is sufficient to fulfill this criterion.  
 
10.2 Informed Consent Procedures 
 
Eligible participants may only be included in the study after providing a consent using the IRB-
approved informed consent form. Informed consent must be obtained before conducting any 
study-specific procedures (i.e., all of the procedures described in the protocol). The process of 
obtaining informed consent should be documented in the participant’s source documents. The 
date of signing of informed consent (and withdrawal, if later withdrawn) should be documented 
in the eCRF. 
 
10.3 Study Assessments 
 
Study activities will take place according to the Time and Events table (Appendix A). 
 
Demographics and a complete medical history will be recorded during the Screening period. The 
medical history will include a complete review of all current diseases and their respective 
durations and treatments. 
 
10.4 Inclusion procedures  
 
Once all inclusion/exclusion criteria are fulfilled, the patient becomes eligible for randomization 
and inclusion into the treatment period. Treatment allocation will be performed as stated above 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 38  
in Section 8.5. Study medication will be delivered as stated in Section 8.6. Patients will be 
counseled on product application and eDiary completion.  
 
10.5 Timing of patient’s visits to the clinic 
Patients will be asked to report to the clinic for their scheduled appointments. If a subject is 
unable to schedule an appointment within the required time frame, study staff will be asked to 
reschedule the patient to a day when they are able to come in within predetermined time frame.  
 
10.6 Description by type of visit 
10.6.1 Screening Visit (-21 to -14) – Visit 1 
● informed consent completed and signed  
● inclusion and exclusion criteria  
● demographic data  
● medical/surgical history 
● concomitant medications 
● physical exam 
● vital signs 
● blood for clinical chemistry, serology and hematology 
● urine pregnancy test (for women of childbearing potential) 
● NRS assessment 
● POEM questionnaire 
● Pruritus 5-D questionnaire 
● IGA scoring assessment 
● EASI scoring assessment 
● study counseling 
● start eDiary 
● start AE monitoring 
10.6.2 Washout Phase (-14 to -1) 
For those subjects who meet the eligibility criteria at the Screening visit and is determined by the 
Principal Investigator to be eligible for the study after the safety labs testing results are available, 
subjects will be notified by telephone to start the Washout period phase.  Subjects will be 
allowed to use their regular emollients if needed but will be asked to stop using bleach or vinegar 
baths. 
Stable doses of the following therapies for pruritus and/or atopic dermatitis will be allowed as 
adjunctive therapies during the study:  
● Oral H1 antihistamines 
● Bland emollients 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 39  
A detailed guideline will be shared with the sites and subjects to ensure consistency in study 
medication treatment application and relation to body washing, emollient use, and other chemical 
exposure to skin. 
Low- or mid-potency topical corticosteroids will be allowed per subjects’ usual medication, 
frequency, and dose during the washout period but no more than 7 days per month as rescue 
medication. 
 
 
Subjects using any of these adjunctive therapies should maintain their regimen without clinically 
significant changes in type, dose or frequency throughout the screening, treatment, and follow-up 
periods.  
Subjects will be prohibited from using Excluded Medications/Therapy listed in Appendix B. 
● phone call to subjects 
● study counseling 
 
10.6.3 Study Day 0-Baseline visit (0) – Visit 2 
● inclusion and exclusion criteria  
● concomitant medications 
● medical history 
● vital signs 
● urine pregnancy test (for women of childbearing potential) 
● NRS assessment 
● POEM questionnaire 
● Pruritus 5-D questionnaire 
● IGA scoring assessment 
● EASI scoring assessment 
● allocation of a randomized treatment kit number via IWRS 
● delivery of the corresponding Investigational Product  
● obtain study medication weight for Investigational Product compliance 
● first application of Investigational Product (under medical supervision)  
● study counseling 
● eDiary review 
● recording of AEs if any 
10.6.4 Day 5- Phone call to patient 
The Day 5 visit is a telephone visit that occurs 5 days (±2 days) after the Baseline visit. Study 
staff will discuss IP application with the subject, answer any questions and counsel the subject on 
any other matters related to the study.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 40  
● phone call to subjects 
● application of study drug 
● study counseling 
 
10.6.5 Day 14 and Day 28 study visits – Visit 3 and Visit 4 
● concomitant medications 
● vital signs 
● blood for clinical chemistry and hematology (Day 28) 
● urine pregnancy test (for women of childbearing potential) (Day 28) 
● NRS assessment 
● POEM questionnaire 
● Pruritus 5-D questionnaire 
● IGA scoring assessment 
● EASI scoring assessment 
● obtain study medication weight for Investigational Product compliance 
● dispense investigational product to patient (Day 14) 
● collect study medication 
● application of study drug 
● eDiary 
● study counseling 
● recording of AEs if any 
 
10.6.6 Day 56-End of Study Visit – Visit 5 
● concomitant medications  
● vital signs 
● physical exam 
● urine pregnancy test (for women of childbearing potential) 
● NRS assessment 
● POEM questionnaire 
● Pruritus 5-D questionnaire 
● IGA scoring assessment 
● EASI scoring assessment 
● eDiary 
● recording of AEs if any 
 
10.6.7  Unscheduled/Unanticipated Study visit 
If an event arises that requires patient to come in to the research center, subjects should be 
scheduled for the Unscheduled visit and assessments are performed based on investigator 
discretion. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 41  
 
  
10.6.8 Early Termination Visit 
Every attempt should be made to complete all visits during the defined window periods. Subjects 
who do not complete all required study visits and withdraw from the study before the Day 56 
final visit will be asked to complete the Early Termination Visit. 
 
During the visit, the following will be obtained: 
 
● concomitant medications  
● vital signs 
● physical exam 
● urine pregnancy test (for women of childbearing potential) 
● NRS assessment 
● POEM questionnaire 
● Pruritus 5-D questionnaire 
● IGA scoring assessment 
● EASI scoring assessment 
● blood for clinical chemistry and hematology (if visit occurs before Day 28) 
● eDiary 
● collect study medication (if visit occurs before Day 28) 
● obtain study medication weight for Investigational Product compliance 
● recording of AEs if any 
11 STUDY ASSESSMENTS 
11.1 Safety Assessments 
11.1.1 Vital Signs  
Vital signs will include measurements of heart rate, sitting blood pressure, respiration rate, and 
temperature. Vital signs will be assessed as outlined in Appendix A and at unscheduled study 
visits when clinically indicated. The subjects’ height and weight will be measured as outlined in 
Appendix A.  
11.1.2 Physical Examinations  
The physical examination will be performed at Screening, Day 56, and at any Unanticipated visit 
should one occur as outlined in Appendix A. The physical examination includes an assessment 
of general appearance and a review of systems (dermatologic, head, eyes, ears, nose, 
mouth/throat/neck, thyroid, lymph nodes, respiratory, cardiovascular, gastrointestinal, 
extremities, musculoskeletal, and neurologic systems).  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 42  
11.1.3 Laboratory Assessments 
Blood samples should be taken using standard venipuncture techniques. Blood sampling will be 
performed according to the site SOPs. 
 
The following laboratory variables will be determined as outlined below: 
 
The following routine clinical chemistry, hematology and Lipid Panel will be performed 
according to Time and Events table (Appendix A): Albumin, Alkaline Phosphatase, ALT, AST, 
Total Bilirubin, BUN, BUN:Creatinine ratio, Calcium, Chloride, Creatinine, eGFR, Glucose, 
Potassium, Sodium, Uric Acid, Total Protein, Bicarbonate 
 
Lipid Panel: HDL Cholesterol, LDL cholesterol, Total Cholesterol, Triglycerides, VLDL 
Cholesterol, LDL/HDL Cholesterol Ratio, Non-HDL Cholesterol. 
 
Urinalysis: Color, clarity, pH, specific gravity, bilirubin, glucose, ketones, leukocytes, nitrite, 
blood, protein, urobilinogen, microscopic analysis.  
  
 
 
 
 
 
 
Hematology: WBC, RBC, Hemoglobin, Hematocrit, Platelets, WBC Differential (Neutrophils, 
Lymphocytes, Monocytes, Eosinophils, Basophils) 
 
Serology will only be done at Screening: HIV Ab, HCV Ab, and HBsAg. 
 
 
Pregnancy testing: All females of childbearing potential will have a local urine pregnancy test 
performed at specified visits.  
 
Patients will be asked to fast for at least 8 hours before all blood tests are done. An exception 
will be made on the laboratory testing for the screening visit such that either fasting or non-
fasting blood samples can be collected.  This is to avoid requesting participants to fast prior to 
the informed consent.   
 
The total blood volume collected for clinical labs for Screening visit will be approximately 20 ml 
of whole blood. Volume collected for subsequent visits would be approximately 10 ml of whole 
blood.  
 
Any value outside the normal range will be flagged for the attention of the Investigator or 
designee at the site. The Investigator or designee will indicate whether or not the value is of 
clinical significance. If the result of the clinical chemistry test from the samples taken during the 
screening phase is indicated as clinically significant, the study subject will NOT be allowed into 
the study.  
 
11.2 Efficacy Assessments 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 43  
Efficacy endpoints will be descriptively summarized and will include the number of 
observations, mean, median, standard deviation, minimum, and maximum of scores/values at all 
applicable time points and for all treatments in the ITT Population. 
11.2.1 EASI Assessment 
An EASI score is used to measure the extent (area) and severity of atopic eczema (Eczema Area 
and Severity Index). The score will be calculated at the times indicated in the Time and Events 
Table (Appendix A). The investigator will assess improvement of eczema based on intensity and 
severity of the disease. EASI score sheet can be found in Appendix C. 
 
The severity strata for the EASI are as follows:  
0 = clear 
0·1-1·0 = almost clear 
1·1-7·0 = mild 
7·1-21·0 = moderate 
21·1-50·0 = severe 
50·1-72·0 = very severe  
 
11.2.2 Patient Oriented Eczema Measure 
The POEM is a tool developed by the University of Nottingham, United Kingdom, for 
monitoring atopic dermatitis severity (Appendix D). The subject will complete the questionnaire 
at each of the assessment timepoints as outlined in Appendix A. 
Each of the 7 questions in the POEM questionnaire carries equal weight and is scored from 
0 to 4: 
● No days = 0. 
● 1 to 2 days = 1. 
● 3 to 4 days = 2. 
● 5 to 6 days = 3. 
● Every day = 4. 
Scores are then added to yield a total score of 0 to 28; higher scores mean the greater the severity 
of atopic dermatitis. 
11.2.3 Itch Numeric Rating Scale 
The Itch NRS is a validated, self-reported instrument for measurement of itch intensity. It uses a 
24-hour recall period and asks subjects to rate the intensity of their itch on an 11-point scale 
ranging from 0 (no itch) to 10 (worst itch imaginable). Higher scores indicate greater itch 
intensity. In this study, both worst itch intensity (WI-NRS) and average itch intensity (AI-NRS) 
during a 24-hour recall period will be captured. Subjects will record their Itch NRS scores once 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 44  
daily via eDiary throughout the screening, treatment, and follow-up periods, as outlined in 
Appendix A.  
The question for WI-NRS would be, “Please rate the itching severity due to your atopic 
dermatitis by circling the number that best describes your worst level of itching in the past 24 
hours.” 
 
The question for AI-NRS would be, “Please rate the itching severity due to your atopic 
dermatitis by circling the number that best describes your average level of itching in the past 24 
hours.” 
 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
0 = No itching  
 
 
 
 
 
 
10 = Worst itch imaginable 
 
11.2.4 Investigator Global Assessment Score (IGA) 
IGA will be performed at the times indicated (Screening, Baseline, Day 14, Day 28, and Day 56) 
in the Time and Events Table of the Protocol (Appendix A) as static IGA. The Investigator will 
assess improvement of Atopic Dermatitis based on the 5-point severity scale summarized below 
and in Appendix E.  
 
 
 
11.2.5 5-D Pruritus Scale 
The 5-D Pruritus Scale is a validated, multi-dimensional measure of itching that assesses the five 
domains of degree, duration, direction, disability and distribution (Appendix F). Subjects rate 
their symptoms over the preceding 2-week period on a 1 to 5 scale, with 5 being the most 
affected. Subjects will complete the 5-D Pruritus Scale during study visits as outlined in 
Appendix A.  
11.3 eDiary and Efficacy Assessments 
All patient reported outcomes and clinical outcome assessments (ePRO/eCOA) will be obtained 
electronically using either a smartphone application or a tablet.  Participants will be provided 
with eDiary at screening to initiate daily diary entries from screening to follow-up and will 
include responses to WI-NRS, AI-NRS, daily dose confirmation (during the treatment period), 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 45  
rescue medication (if any), and local skin tolerability (Day 1 to Day 7 of treatment).  In addition, 
participants’ usual adjunctive therapies (e.g., bland emollient, oral H-1 antihistamine) may be 
collected using eDiary or CRF at screening and optionally throughout the study.  POEM and 5-D 
Pruritus Scale will be reported by the participants at the designated site visits using a site 
provided tablet.  IGA and EASI will also be assessed at the designated site visits by the clinician. 
11.4 Patient Reported Local Tolerability 
Solicited local adverse reactions (e.g., itching, new rash, pain, tenderness, stinging, skin color 
change, etc.) and severity from subjects will be collected during the first week of treatment using 
eDiary with the questions outlined in Appendix G to inform local skin tolerability.  
11.5 Pregnancy Reporting 
Any pregnancy will be reported by study participants during their study participation. 
Participants who report pregnancy or lactation during the review of inclusion/exclusion criteria 
prior to randomization will not be enrolled in the trial. In case of pregnancy, Investigational 
Product should be discontinued and the Sponsor informed. Follow-up of the pregnancy will be 
mandatory until the outcome is available.  
11.6 Study Completion 
A completed participant is one who has completed all study visits. Day 56 study visit is defined 
as the participant’s last visit. 
11.7 Subject Withdrawal Criteria 
A subject may withdraw from the study at any time at his/her own request, or may be withdrawn 
at any time at the discretion of the Investigator for safety, behavioral, or administrative reasons.  
Reasons for withdrawal (subjects who refuse to return for any remaining study visits) or 
discontinuation (subjects who prematurely stop the application) at any time during the study may 
include, but are not limited, to the following: 
 
● For safety reasons, either at the discretion of the Investigator or at the subject’s request 
● For protocol violations at the discretion of AOBiome 
● Due to concomitant therapy that could interfere with the results of the study (the 
Investigator will report all such information on the CRFs and decide, in accordance with 
AOBiome, whether the subject is to be withdrawn). 
  
All premature discontinuations and their causes must be carefully documented by the 
Investigator on the CRF or if needed on the AE form.   
 
If, for any reason, a subject is withdrawn before completing the final visit, the reason for 
termination will be entered on the CRF.  All data gathered on the subject prior to termination will 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 46  
be made available to AOBiome. Subjects not completing the entire study should be fully 
evaluated when possible.  The appropriate CRFs should be completed. 
 
If the subject chooses to withdraw before completing the study, the subject should notify the 
study coordinator who will instruct the subject on completion of assessments for End of Study 
(EOS) visit.  For subjects who refuse to complete the assessments for their early termination, 
every attempt must be made to check on their status, using any mode of communication such as 
telephone, email, fax, or text. 
 
12 STATISTICAL CONSIDERATIONS 
12.1 Sample Size 
Approximately 576 subjects may be enrolled to account for 16.7% drop out rate prior to 
completing the study.  A total of 160 evaluable subjects per group (480 total) are required to 
achieve at least 80% power to detect a pairwise difference of 0.65 in mean WI-NRS change from 
baseline to Week 4 between one of two active doses of B244 and vehicle control when assuming 
a standard deviation of 2.5 and applying a Dunnett Testing Method at a one-sided familywise 
error rate of 0.10.  
12.2 Populations for Analysis 
Intent to Treat (ITT): All randomized participants who apply at least 1 dose of study medication. 
Subjects will be grouped as randomized.   
Per Protocol: All subjects in the ITT population without any major protocol deviations that may 
have an impact on the efficacy assessments, who complete their Week 4 visit, and who 
administer at least 50% of investigational product (IP). Subjects will be grouped as treated. 
Safety: All subjects who apply at least 1 dose of study medication. Subjects will be grouped as 
treated. 
12.3 Data Analysis 
A separate statistical analysis plan will be developed. All data collected will be documented 
using summary tables, figures, and/or patient data listings. For continuous variables, descriptive 
statistics (number (n), mean, median, standard deviation, minimum and maximum) will be 
presented. For categorical variables, frequencies and percentages will be presented. As 
appropriate, a 95% CI will be presented. Graphical displays will be presented, as appropriate.  
Data will be presented by treatment group and overall. 
Descriptive statistics for each treatment group will be provided for clinical laboratory values 
(e.g., hematology, serum chemistry, and urinalysis) and vital signs data, presented as both actual 
values and changes from baseline relative to each on-study evaluation. Abnormal clinical 
laboratory values will be listed.  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 47  
12.3.1 Disposition 
A tabulation of the disposition of subjects will be presented, including the number enrolled, the 
number randomized, the number treated, and the reasons for study discontinuation. Summaries 
of the number in each analysis set will be presented. Entry criteria violations and protocol 
deviations will be listed. 
12.3.2 Demographic and Baseline 
Demographic and baseline characteristic data summarization will be performed in order to 
descriptively assess the comparability of treatment groups. Data to be tabulated will include age, 
race, ethnicity, height, weight, and BMI, as well as baseline characteristics related to medical 
history. 
12.4 Safety Analyses 
12.4.1 Definitions  
All adverse events recorded during the study will be coded according to MedDRA.  
12.4.2 Adverse Events  
All adverse events (AEs) recorded during the study through the date of randomization through 28 
days after the last dose of study drug will be analyzed.  
 
Adverse events will be summarized by treatment group using the Medical Dictionary for 
Regulatory Activities (MedDRA) system organ class and preferred term. Separate tabulations 
will be produced for all treatment-emergent AEs (TEAEs), treatment-related AEs (those 
considered by the Investigator as at least possibly drug related), SAEs, discontinuations due to 
AEs, and AEs ≥ Grade 3 severity. By-subject listings will be provided for any deaths, SAEs, and 
AEs leading to discontinuation. 
 
AE’s will be summarized using incidence rates. Therefore, each subject will only contribute once 
for a given adverse event SOC or PT.  
 
Any treatment emergent AEs related to local safety (e.g., erythema, edema, induration, 
vesiculation, etc.) will be collected by the investigator during scheduled clinic visits.  In addition, 
solicited local adverse reactions (e.g., itching, new rash, pain, tenderness, stinging, skin color 
change, etc.) and severity from subjects will be collected during the first week of treatment using 
eDiary to inform local skin tolerability. 
A summary of the incidence of any adverse event, SAE, and adverse events leading to 
discontinuation will be presented.  Summaries will display, by treatment group, the incidence of 
patients with events, the frequency of patients with events within each primary system organ 
class and by preferred terms. For each preferred term and each system organ class a patient will 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 48  
be counted only once. For summaries on severe or drug-related AE, for a given patient, the 
highest severity or relationship for a specific preferred term will be considered.  
12.4.3 Deaths and Serious Adverse Events  
Serious adverse events and events leading to death will be summarized overall and by primary 
system organ class and preferred term.  
12.4.4 Adverse Events leading to treatment discontinuation  
Adverse events leading to treatment discontinuation will be summarized overall and by primary 
system organ class and preferred term.  
12.5 Efficacy Analyses  
All efficacy analyses will be performed on the ITT population and PP population. Regression 
models will include all eligible subjects for the respective analysis, from the 3 treatment arms.   
Hypothesis tests for the primary efficacy endpoint will be performed using a Dunnett Testing 
Method, applying pairwise comparisons of each respective B244 dose group to vehicle using a 
one-sided familywise error rate of 0.10.  No additional adjustments will be made for multiple 
testing.  As such, p-values from analysis of secondary and exploratory efficacy analyses must be 
interpreted in an exploratory fashion. 
The primary endpoint of mean change from baseline to Week 4 in WI-NRS will be analyzed 
using analysis of covariance (ANCOVA) models. 
 
In addition, continuous efficacy endpoints will be summarized using descriptive statistics at 
Baseline, Week 2, Week 4 and Week 8 for actual values and change-from-baseline values. The 
difference in treatment groups in change-from-baseline values at post-baseline visits will be 
analyzed using a mixed model with repeated measures to account for within subject variability 
and including visit (Baseline vs. Week 2, Week 4, Week 8), treatment group, visit-by-treatment 
interaction, and baseline value as explanatory variables. 
 
Categorical variables will be summarized using descriptive statistics and analyzed using a 
logistic regression model at each respective timepoints.  Generalized estimating equations to 
account for repeated measures and within-subject variability may also be applied.  
Supportive analyses may be performed combining the B244 dose groups in comparison against 
vehicle and comparison of the B244 groups to one another.   
 
 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 49  
12.6 Handling of dropouts or missing data  
All available data will be analyzed.  The details for any imputations for missing data will be 
documented in the trial’s Statistical Analysis Plan.  Subjects who dropout after enrollment but 
prior to randomization will be replaced.  
 
12.7 Clinical Trial Protocol deviations  
At minimum, the following deviations will be summarized on the ITT patient population:  
 
● Inclusion or exclusion criteria not satisfied.  
● Deviations related to the Investigational Product administration  
● Not permitted concomitant medications.  
13 ADVERSE EVENTS (AE) AND SERIOUS ADVERSE EVENTS (SAE) 
The Investigator and study staff are responsible for detecting and recording AEs and SAEs 
during scheduled safety evaluations and whenever such information is brought to their attention. 
This section of the protocol provides definitions and detailed procedures to be followed. 
 
13.1 Definition of an AE 
An AE is any untoward medical occurrence in a study participant which is temporally associated 
with the use of a medicinal product, regardless of its potential relationship to the medicinal 
product.  An AE, therefore, can be any unfavorable or unintended sign, including an abnormal 
symptom, or disease (new or exacerbated), whether or not related to the investigational product 
(IP).   
 
Examples of an AE include: 
● Exacerbation of a pre-existing condition including either an increase in frequency and/or 
intensity of the condition. 
● New conditions detected or diagnosed after investigational product administration even 
though it may have been present prior to the start of the study. 
● Signs, symptoms of a drug interaction. 
● Signs, symptoms of a suspected overdose of either investigational product or a concurrent 
medication (overdose per se should not be reported as an AE/SAE). 
 
AEs may include pre- or post-treatment events that occur as a result of protocol-mandated 
procedures (i.e., modification of participant’s previous therapeutic regimen). 
 
13.2 Definition of a SAE 
A serious adverse event is any untoward medical occurrence that, at any dose: 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 50  
a) results in death. 
b) is life-threatening. 
NOTE:  The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event.  It does not refer to an event, which 
hypothetically might have caused death, if it were more severe. 
 
c) requires hospitalization or prolongation of existing hospitalization. 
NOTE:  In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation and/or 
treatment that would not have been appropriate in the physician’s office or out -patient setting.  
Complications that occur during hospitalization are AEs.  If a complication prolongs 
hospitalization or fulfills any other serious criteria, the event is serious.  When in doubt as to 
whether “hospitalization” occurred or was necessary, the AE should be considered serious. 
Routine hospitalizations or elective surgeries are generally not regarded as SAEs.  
 
d) results in disability/incapacity 
NOTE:  The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.  This definition is not intended to include experiences of relatively minor 
medical significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and 
accidental trauma (e.g. sprained ankle) which may interfere or prevent everyday life functions 
but do not constitute a substantial disruption. 
 
e) is a congenital anomaly/birth defect 
f) Medical or scientific judgment should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
participant or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition.  These should also be considered serious.  
Examples of such events are invasive or malignant cancers, intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions 
that do not result in hospitalization, or development of drug dependency or drug abuse. 
13.3 Time-period, Frequency, and Method of Detecting AEs and SAEs 
 
All AEs occurring after administration of the first dose of study medication and on or before the 
final assessment must be reported as AEs.  All AEs must be recorded irrespective of whether 
they are considered drug-related. 
 
At each assessment in the period defined above, AEs will be evaluated by the Investigator and 
recorded. 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 51  
Any AEs already documented at a previous assessment and designated as ongoing, should be 
reviewed at subsequent assessments as necessary.  If these have resolved, this should be 
documented.  Changes in intensity or frequency of AEs should be recorded as separate events 
(i.e., a new record started). 
 
The recording of AEs and SAEs are described in Section 13.4 (“Recording of AEs and SAEs”). 
 
13.4 Recording of AEs and SAEs 
All clinical events, including either observed or volunteered problems, complaints or symptoms 
are to be recorded on the Adverse Events page(s) of the CRF.  The need to capture this 
information is not dependent upon whether the clinical event is associated with study treatment. 
Adverse clinical events resulting from concurrent illnesses or reactions to concurrent 
medications are also to be recorded.  In order to avoid vague, ambiguous, or colloquial 
expressions, the AE should be recorded in standard medical terminology rather than the 
participant’s own words. 
 
Each adverse clinical event is to be evaluated for duration, intensity, and whether the event may 
be associated with the investigational product (IP) or other causes.  Start and stop dates, 
relationship to investigational product (IP), medical management, and alternative causality of 
event must be recorded in the Adverse Events section of the CRF.  AEs believed to be possibly 
related to investigational product (IP) must be followed until resolution. 
 
13.5 Evaluating AEs and SAEs 
 
13.5.1 Severity Rating 
The severity of an adverse event (AE and SAE) is to be scored according to the following scale: 
Mild 
Awareness of symptoms, but easily tolerated 
Moderate 
Discomfort enough to cause interference with usual 
activity 
Severe 
Incapacitating with inability to work or perform usual 
activity 
An AE that is assessed as severe should not be confused with a SAE.  An event is defined as 
‘serious’ when it meets one of the pre-defined outcomes as described in Section 13.2 “Definition 
of a SAE”. 
 
13.5.2 Relationship to Investigational product (IP)  
SAEs will be classified as “not related” or “related” (including unknown).  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 52  
For AEs, the relationship to study treatment is to be assessed according to the following 
definitions: 
Definitely not related: The AE is definitely not related to the drug.  This designation 
should be reserved for those events which occur prior to study treatment or for those 
events which cannot be even remotely related to study participation (e.g., injuries 
sustained in an automobile accident). 
 
Unlikely related: There is no reasonable association between the study treatment and the 
suspected event and the event could have been produced by the participant 's clinical state 
or other modes of therapy administered to the participant. 
 
Possibly related:  The suspected adverse event may or may not follow a reasonable 
temporal sequence from study treatment administration but seems to be the type of 
reaction that cannot be dismissed as unlikely.  The event could have been produced or 
mimicked by the participant 's clinical state or by other modes of therapy concomitantly 
administered to the participant. 
 
Probably related: The suspected adverse event follows a reasonable temporal sequence 
from study treatment administration, abates upon discontinuation of the treatment, and 
cannot be reasonably explained by the known characteristics of the participant 's clinical 
state. 
 
Definitely related: This designation should be reserved for those events which have no 
uncertainty in their relationship to treatment administration. 
 
13.6 Pregnancy 
 
Any pregnancy that occurs in a female participating in the study must be reported to the Safety 
Team or to a designated Safety email or fax number provided by the Safety Team within 24 
hours of learning of the pregnancy. Follow-up must occur to determine the outcome of the 
pregnancy (including premature termination) and the status of mother and child.  Pregnancy 
complications and elective terminations for medical reasons must be reported as AEs or SAEs.  
Spontaneous abortions must be reported as an SAE. 
 
Any SAE occurring in association with a pregnancy and considered by the Investigator as related 
to the investigational product must be promptly reported to the Sponsor, even if the event 
occurred after the participant completed the study. 
 
The Investigator must attempt to collect pregnancy information on any female partners of male 
participants who become pregnant while the male participant is enrolled in the study. Pregnancy 
information must be reported to the Sponsor as described above. 
 
13.7 Prompt Reporting of SAEs to the Sponsor 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 53  
 
In the case of a Serious Adverse Event the Investigator must immediately: 
 
● SEND (within 1 working day) the signed and dated SAE Report Form to the Safety Team 
or to a designated Safety email or fax number provided by the Safety Team;  
● ATTACH a photocopy of all examinations carried out and the dates on which these 
examinations were performed. Care should be taken to ensure that the patient's identity is 
protected and the patient's identifiers in the Clinical Trial are properly documented on all 
copies of source documents provided to the Sponsor. For laboratory results, include the 
laboratory normal ranges;  
● Follow-up of any Serious Adverse Event that is fatal or life threatening should be 
provided within one additional calendar week. The treatment code will be unblinded for 
reporting of Serious Adverse Events that are unexpected and determined to be related to 
the use of the Investigational Product.  
Follow-up  
● The Investigator should take all appropriate measures to ensure the safety of the patients, 
including referral to a specialist if indicated. Notably the Investigator should follow up 
the outcome of any adverse events (clinical signs, laboratory values or other, etc.) until 
the return to normal or stabilization of the patient's condition;  
● In the case of any Serious Adverse Event, the patient must be followed up until clinical 
recovery is complete and laboratory results have returned to normal, or until progression 
has been stabilized. This implies that follow-up may continue after the patient has left the 
Clinical Trial and that additional investigations may be requested by the Safety Team;  
● In case of any Serious Adverse Event brought to the attention of the Investigator at any 
time after cessation of Investigational Product and considered by the Investigator to be 
related to the Investigational Product, this should be reported to the Safety Team.  
 
 
AOBiome Reportable Events Hotline 
Email: safety@aobiome.com 
 
 
14 ETHICAL AND REGULATORY STANDARDS 
14.1 Ethical Conduct of Study 
This clinical trial was designed and shall be implemented, executed, and reported in accordance 
with the ICH Harmonized Tripartite Guidelines for Good Clinical Practice, with applicable local 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 54  
regulations (including US Code of Federal Regulations Title 21), and with the ethical principles 
laid down in the Declaration of Helsinki. 
 
The Investigator(s) should conduct the study in accordance with this protocol, the Declaration of 
Helsinki and ICH GCP guidelines and FDA regulations.  The Investigator(s) and the Sponsor 
will sign the protocol and study contract, to confirm agreement.  The Investigator(s) will not 
implement any amendment (deviation or changes of the protocol) without agreement by the 
Sponsor and IRB approval, except where necessary to eliminate immediate hazard(s) to study 
participants, or when change(s) involve only logistical or administrative aspects of the study. 
Records that may reveal the identities of participants must be well protected, with consideration 
given to confidentiality and the right to privacy of participants. 
14.2 Laws and Regulations 
This Clinical Trial will be conducted in compliance with all international laws and regulations, 
and national laws and regulations of the countries in which the clinical trial is performed, as well 
as any applicable guidelines. The trial will be registered and updated on www.clintrials.gov and 
on other sites, as deemed appropriate.  
14.3 Informed Consent 
Each participant must be provided with a statement that the investigation involves research and 
that the IRB has approved solicitation of participants to participate; a fair explanation of the 
procedures to be followed and their purposes, including identification of any procedures which 
are experimental; a description in lay language of any possible side effects; a description of any 
attendant discomforts and risks reasonably to be expected; a description of any benefits 
reasonably to be expected; a disclosure of any appropriate alternative procedures that might be 
advantageous for the participant; an offer to answer any inquiries concerning the procedures, and 
instruction that the person is free to withdraw consent and discontinue participation in the project 
or activity at any time without prejudice to the participant.  Payment to research participants for 
taking part in the study is based on time and inconvenience.   All information concerning 
payment, including the schedule of payments, must be set forth in the informed consent, 
including a disclosure that the Investigator is being paid to perform the stated research. 
 
A participant must give consent to take part in the study.  Participants below the age of majority 
in the municipality must give written assent to participate in this study.  This consent must be 
dated and retained by the Principal Investigator as part of the study records.  A downloadable 
digital copy shall be given to the person signing the form.  The informed consent process must be 
documented in the participant’s source documents. 
 
The Investigator agrees that the Sponsor, its employees or agents will have the right from time to 
time during the course of this study to audit and review pertinent medical records relating to this 
clinical trial.  A statement will be obtained from each person participating in the study permitting 
the release of his/her medical records as necessary for inspection by authorized personnel of the 
Sponsor, FDA, and the staff managing the clinical study.  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 55  
The release of medical records and the review of the contents will be in compliance with the 
Health Insurance Portability and Accountability Act of 1996 (HIPAA). 
 
14.4 Institutional Review Board/Independent Ethics Committee (IRB/EC) 
 
The protocol and informed consent form and the electronic version of the consent for this study 
must be approved by the IRB.  A copy of the Letter of Approval from the Board, which contains 
specific identification of the documents approved, must be received by the Sponsor prior to 
shipment of drug supplies to the Principal Investigator. 
 
All changes to the protocol, as well as a change of Investigator, must also be approved by the 
IRB and documentation of this approval provided to the study monitor.  Records of the IRB’s 
review and approval of all documents pertaining to the study must be kept on file by the 
Principal Investigator and are subject to FDA inspection at any time.  IRB renewal for approval 
is required each year.  The Investigator is to inform AOBiome, in writing, of the approval to 
continue the study.   
 
14.5 Clinical Monitoring/Record Keeping 
There shall be no alterations in the protocol design without the written consent and approval of 
the Sponsor and the approval of the IRB, except in the case that participants are at immediate 
risk without immediate implementation of such alterations.  In the aforementioned situation, the 
site should notify the Sponsor and IRB of the deviation as soon as possible, and should seek the 
written consent and approval of the Sponsor and the approval of the IRB. 
 
All results of this trial must be recorded on eCRFs.  Each participant who has been randomized 
must have a completed eCRF.  Reasons for termination must be stated in the early termination 
section.  Study participants are not to be identified by name on eCRFs, but rather by coded 
identifiers and participant initials.   
 
The study monitor will verify the accuracy of the data by reviewing pertinent source documents 
such as office records or hospital charts of the participants. 
 
Study records include eCRFs, signed FDA Form 1572, original reports of test results, and signed 
electronic informed consent forms.  IRB approval letters and other documents pertaining to the 
conduct of the study are to be kept on file by the Investigator.   If the study files are assigned to 
someone else or removed to another location, the Investigator is to notify the study monitor or 
Sponsor in writing of the change.  All study records are subject to FDA inspection at any time. 
All information supplied to the Investigator by the Sponsor before, during, and after the study is 
confidential.  Such information is to be used solely in connection with the clinical study.  The 
study protocol, IB, and any other pertinent study related materials or records provided are to be 
maintained in a confidential manner, reviewed carefully with attention to admonitions and 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 56  
returned to the Sponsor upon request.  No part of these materials may be reproduced or 
transmitted in any form without prior written permission from the Sponsor. 
15  ADMINISTRATIVE RULES 
15.1 Curriculum Vitae 
An updated, signed, and dated copy of the curriculum vitae with the experience, qualifications 
and training for each Investigator and/or Sub-Investigator(s) will be provided to the Sponsor 
prior to the beginning of the Clinical Trial.  
15.2 Archiving of Study Documentation 
The Investigator must maintain confidentiality for all study documentation, and take measures to 
prevent accidental or premature destruction of these documents.  
 
It is recommended that the Investigator retain the study documents at least fifteen (15) years after 
the completion or discontinuation of the Clinical Trial. However, applicable regulatory 
requirements should be taken into account in the event that a longer period is required.  
 
The Investigator must notify the Sponsor prior to destroying any study essential documents 
within the fifteen (15) year period following the Clinical Trial completion or discontinuation.  
If the Investigator's personal situation is such that archiving can no longer be ensured by him/her, 
the Investigator shall inform the Sponsor and the relevant records shall be transferred to a 
mutually agreed upon designee.  
 
15.3 Internal Safety Review Committee 
An internal safety review committee may be set up as needed to protect the ethical and safety 
interests of participants and to protect the scientific validity of the study. Ad-hoc safety interim 
analyses might be performed by an independent statistician if the safety review committee 
identifies potential safety signals during its routine blinded safety review. The details for the 
analysis plan may be documented in the trial’s Statistical Analysis Plan. 
  
16 STUDY MONITORING 
16.1 Responsibilities of the Investigator(s)  
The Investigator(s) undertake(s) to perform the Clinical Trial in accordance with this Clinical 
Trial Protocol, ICH guidelines for Good Clinical Practice and the applicable regulatory 
requirements.  
 
The Investigator is required to ensure compliance with all procedures required by the Clinical 
Trial Protocol and by study procedures provided by the Sponsor (including security rules). The 
Investigator agrees to provide reliable data and all information requested by the Clinical Trial 
Protocol (with the help of the Case Report Form [CRF], Discrepancy Resolution Form [DRF] or 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 57  
other appropriate instrument) in an accurate and legible manner according to the instructions 
provided and to ensure direct access to source documents to Sponsor representatives. If any 
particular circuits have to be defined (e.g., e-CRF, Fax), particular attention should be paid to the 
confidentiality of the patient's data to be transferred. The Investigator may appoint such other 
individuals as he/she may deem appropriate as Sub-Investigators to assist in the conduct of the 
Clinical Trial in accordance with the Clinical Trial Protocol. All Sub-Investigators shall be 
timely appointed and listed. The Sub-Investigators will be supervised by and under the 
responsibility of the Investigator. The Investigator will provide them with a Clinical Trial 
Protocol and all necessary information.  
 
16.2 Responsibilities of the Sponsor  
 
The Sponsor of this Clinical Trial is responsible to Health Authorities for taking all reasonable 
steps to ensure the proper conduct of the Clinical Trial Protocol with regard to ethics, Clinical 
Trial Protocol compliance, integrity and validity of the data recorded on the Case Report Forms. 
Thus, the main duty of the Monitoring Team is to help the Investigator and the Sponsor maintain 
a high level of ethical, scientific, technical and regulatory quality in all aspects of the Clinical 
Trial. At regular intervals during the Clinical Trial, the site will be contacted, through monitoring 
visits, letters or telephone calls, by a representative of the Monitoring Team to review study 
progress, Investigator and patient compliance with Clinical Trial Protocol requirements, and any 
emergent problems. During these monitoring visits, the following but not exhaustive list of 
points will be scrutinized with the Investigator: patient informed consent, patient recruitment and 
follow-up, Serious Adverse Event documentation and reporting, outcome events documentation 
and reporting, Investigational Product allocation, patient compliance with the Investigational 
Product regimen, Investigational Product accountability, concomitant therapy use and quality of 
data.  
 
16.3 Source document requirements  
According to the ICH guidelines for Good Clinical Practice, the Monitoring Team must check 
the Case Report Form entries against the source documents. The Informed Consent Form will 
include a statement by which the patient allows the Sponsor’s duly authorized personnel, the 
Ethics Committee (IRB/EC), and the regulatory authorities to have direct access to source data 
which supports the data on the Case Report Forms (e.g., patient's medical file, appointment 
books, original laboratory records, etc.). These personnel, bound by professional secrecy, must 
keep confidential all personal identity or personal medical information (according to 
confidentiality rules).  
 
16.4 Use and completion of Case Report Forms (CRFs) and additional requests  
The sponsor or CRO will be responsible for activities associated with the data management 
of this study. This will include setting up a relevant database and data transfer mechanisms, 
along with appropriate validation of data and resolution of queries. Data generated within this 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 58  
clinical study will be handled according to the relevant SOPs of the data management and 
biostatistics departments of sponsor or CRO. 
  
For Electronic Data Capture (EDC): 
Study sites will enter data directly into an EDC system by completing the eCRF via a secure 
internet connection.  Data entered into the eCRF must be verifiable against source documents 
at the study site. Any changes to the data entered into the EDC system will be recorded in an 
automated, secure audit trail and is Food and Drug Administration (FDA) Code of Federal 
Regulations (CFR) Title 21 Part 11 compliant. 
  
Data entered into the eCRF will be validated as defined in the Data Validation Specifications 
(DVS). Validation includes, but is not limited to, validity checks (for example, missing data, 
range checks) and consistency checks (logical checks between variables) to ensure that study 
data are accurately reported. Additionally, CRO Data Management will perform aggregate data 
review as defined in the DVS to ensure that the data are complete, consistent and reasonable. The 
electronic edit checks will run continually throughout the course of the study and queries 
reviewed by CRO personnel to assure validity as compared to source records. Manual queries 
may also be entered into EDC by Monitoring or Data Management personnel to address 
identified discrepancies. 
  
Medical conditions/procedures will be coded using MedDRA and prior and concomitant 
medications will be coded using WHODrug. 
  
At the conclusion of the study, each site will be provided with their subject CRFs in Portable 
Document Format (PDF) for archival.  The CRF PDFs will contain subject data, audit trail 
information, queries including responses, and comments. 
 
17 PUBLICATIONS 
All data generated from this study are the property of AOBiome LLC and shall be held in strict 
confidence along with all information furnished by AOBiome.  Independent analyses and/or 
publication of these data by the Investigator or any member of his/her staff is not permitted 
without prior written consent of AOBiome.   
 
Any formal presentation or publication of data from this trial will be considered as a joint 
publication by the Investigator(s) and appropriate Sponsor personnel.  Authorship will be 
determined by mutual agreement. Written permission to the Investigator will be contingent on 
the review by the Sponsor of the methodology and statistical analysis and any publication or 
presentation will provide for nondisclosure of AOBiome confidential or proprietary information.  
In all cases, the parties agree to submit all manuscripts or abstracts to all other parties at least 30 
days prior to submission.  This will enable all parties to protect proprietary information and to 
provide comments based on information that may not yet be available to other parties.   

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 59  
18 PROTOCOL ADHERENCE 
This protocol defines the study objectives, the study procedures and the data to be collected on 
study participants. Additional assessments required to ensure the safety of participants should be 
administered as deemed necessary on a case by case basis. Under no circumstances is an 
Investigator allowed to collect additional data or conduct any additional procedures for any 
research related purpose involving any investigational drugs under the protocol. 
Investigators ascertain they will apply due diligence to avoid protocol deviations. If an 
Investigator feels a protocol deviation would improve the conduct of the study this must be 
considered a protocol amendment, and unless such an amendment is agreed upon by the sponsor 
and approved by the IRB/IEC and health authorities, where required, it cannot be implemented. 
19 CLINICAL TRIAL PROTOCOL AMENDMENTS 
Any protocol amendments will be added as stand-alone documents.  In addition, any and all 
revisions dictated by the amendments will be made in the protocol.  Each time a protocol is 
amended, a new amended version date will be added to the cover page. 
 
The Investigator should not implement any deviation from, or changes of the Clinical Trial 
Protocol without agreement by the sponsor and prior review and documented approval/favorable 
opinion from the IRB/EC of an amendment. The Investigator is expected to take any immediate 
action required for the safety of any participant included in this study, even if this action 
represents a deviation from the protocol. In such cases, the reporting requirements identified in 
Section 13 must be followed and the Study Lead. 
20 CONFIDENTIALITY  
All information disclosed or provided by the Sponsor (or any company/institution acting on their 
behalf), or produced during the Clinical Trial, including, but not limited to, the Clinical Trial 
Protocol, the CRFs, the Investigator's Brochure and the results obtained during the course of the 
Clinical Trial, is confidential. The Investigator or any person under his/her authority agrees to 
undertake to keep confidential and not to disclose the information to any third party without the 
prior written approval of the Sponsor. However, the submission of this Clinical Trial Protocol 
and other necessary documentation to the Ethics Committee (IRB/EC) is expressly permitted, the 
IRB/EC members having the same obligation of confidentiality. The Sub-Investigators shall be 
bound by the same obligation as the Investigator. The Investigator shall inform the Sub-
Investigators of the confidential nature of the Clinical Trial. The Investigator and the Sub-
Investigators shall use the information solely for the purposes of the Clinical Trial, to the 
exclusion of any use for their own or for a third party's account. 
 
21 PROPERTY RIGHTS  
All information, documents and Investigational Product provided by the Sponsor or its designee 
are and remain the sole property of the Sponsor. The Investigator shall not mention any 
information or the Product in any application for a patent or for any other intellectual property 
rights. All the results, data, documents and inventions, which arise directly or indirectly from the 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 60  
Clinical Trial in any form, shall be the immediate and exclusive property of the Sponsor. The 
complete verified database will be shared with the Operations Committee, which shall have full 
access to all data. The Sponsor may use or exploit all the results at its own discretion, without 
any limitation to its property right (territory, field, continuance). The Sponsor shall be under no 
obligation to patent, develop, market or otherwise use the results of the Clinical Trial. As the 
case may be, the Investigator and/or the Sub-Investigators shall provide all assistance required by 
the Sponsor, at the Sponsor's expense, for obtaining and defending any patent, including 
signature of legal documents.  
 
22 DATA PROTECTION  
The patient's personal data and Investigator's personal data which may be included in the 
Sponsor database shall be treated in compliance with all applicable laws and regulations;  
 
When archiving or processing personal data pertaining to the Investigator and/or to the patients, 
the Sponsor shall take all appropriate measures to safeguard and prevent access to this data by 
any unauthorized third party.  
23 SPONSOR AUDITS AND INSPECTIONS BY REGULATORY AGENCIES  
For the purpose of ensuring compliance with the Clinical Trial Protocol, Good Clinical Practice 
and applicable regulatory requirements, the Investigator should permit auditing by or on behalf 
of the Sponsor and inspection by applicable regulatory authorities.  
 
The Investigator agrees to allow the auditors/inspectors to have direct access to his/her study 
records for review, being understood that these personnel are bound by professional secrecy, and 
as such will not disclose any personal identity or personal medical information.  
 
The Investigator will make every effort to help with the performance of the audits and 
inspections, giving access to all necessary facilities, data, and documents. As soon as the 
Investigator is notified of a future inspection by the authorities, he will inform the Sponsor and 
authorize the Sponsor to participate in this inspection.  
 
The confidentiality of the data verified and the protection of the patients should be respected 
during these inspections.  
 
Any result and information arising from the inspections by the regulatory authorities will be 
immediately communicated by the Investigator to the Sponsor.  
 
The Investigator shall take appropriate measures required by the Sponsor to take corrective 
actions for all problems found during the audit or inspections.  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 61  
24 PREMATURE DISCONTINUATION OF THE STUDY OR PREMATURE CLOSE-
OUT OF A SITE  
 
24.1 Decided by the Sponsor in the following cases: 
1. In the event the results of the Clinical Trial do not appear to be scientifically convincing 
to the Sponsor;  
2. If the aim of the Clinical Trial has become outdated or is no longer of interest;  
3. If the information on the product leads to doubt as to the benefit/risk ratio;  
4. If the Investigator has received from the Sponsor all Investigational Product, means and 
information necessary to perform the Clinical Trial and has not included any patient after 
a reasonable period of time mutually agreed upon;  
5. In the event of breach by the Investigator of a fundamental obligation under this 
agreement, including but not limited to breach of the Clinical Trial Protocol, breach of 
the applicable laws and regulations or breach of the ICH guidelines for GCP;  
6. If the total number of patients are included earlier than expected; In any case the Sponsor 
will notify the Investigator of its decision by written notice.  
 
24.2 Decided by the Investigator  
The Investigator must notify (30 days' prior notice) the Sponsor of his/her decision and give the 
reason in writing. In all cases (decided by the sponsor or by the Investigator), the appropriate 
Ethics Committee(s) (IRB/EC) and Health Authorities should be informed.  
 
25 CLINICAL TRIAL PROTOCOL AMENDMENTS  
All appendices attached hereto and referred to herein are made part of this Clinical Trial 
Protocol. The Investigator should not implement any deviation from, or changes of the Clinical 
Trial Protocol without agreement by the sponsor and prior review and documented 
approval/favorable opinion from the IRB/EC of an amendment, except where necessary to 
eliminate an immediate hazard(s) to clinical trial patients, or when the change(s) involves only 
logistical or administrative aspects of the trial. Any change agreed upon will be recorded in 
writing, the written amendment will be signed by the Investigator and by the Sponsor and the 
signed amendment will be filed with this Clinical Trial Protocol. Any amendment to the Clinical 
Trial Protocol requires written approval/favorable opinion by the IRB/EC prior to its 
implementation, unless there are overriding safety reasons. In some instances, an amendment 
may require a change to the Informed Consent Form. The Investigator must receive an IRB/EC 
approval/favorable opinion concerning the revised Informed Consent Form prior to 
implementation of the change. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 62  
26 REFERENCES 
1. Eichenfield LF, Tom WL, Chamlin SL, et al. Guidelines of care for the management of 
atopic dermatitis: Section 1: Diagnosis and assessment of atopic dermatitis. J Am Acad 
Dermatol. 2014; 70(2):338-351. 
2. Arp DJ, Sayavedra-Soto LA, Hommes NG. Molecular biology and biochemistry of 
ammonia oxidation by Nitrosomonas europaea. Arch Microbiol 2002; 178: 250-255. 
3. Chandran K, Stein LY, Klotz MG, van Loosdrecht MCM. Nitrous oxide production by 
lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-
removal systems. Biochem Soc Trans 2011; 39: 1832-1837. 
4. Williams HC, Dellavalle RP, Garner S. Acne vulgaris. The Lancet 2012; 379(9813): 361-
372. doi:10.1016/S0140-6736(11)60321-8. 
5. Weller R, Price RJ, Ormerod AD, Benjamin N, & Leifert C. Antimicrobial effect of 
acidified nitrite on dermatophyte fungi, Candida and bacterial skin pathogens. Journal of 
Applied Microbiology 2001; 90(4): 648-652. 
6. Rico J, Quiring J, Hollenbach S, Enloe C, & Stasko N. Phase 2 study of efficacy and 
safety of SB204 in the treatment of acne vulgaris. J Invest Dermatology 2014; 134(8): S8. 
7. Williams H, Robertson C, Stewart A, et al. Worldwide variations in the prevalence of 
symptoms of atopic eczema in the international study of asthma and allergies in 
childhood. J Allergy Clin Immunol 1999; 103: 125–138. 
8. Diepgen, TL. Is the prevalence of atopic dermatitis increasing? In: Williams HC 
ed. Atopic Dermatitis: the Epidemiology, Causes and Prevention of Atopic Eczema. 2000 
Cambridge: Cambridge University Press. 
9. Roekevisch E, Spuls PI, Kuester D, Limpens J, Schmitt J. Efficacy and safety of systemic 
treatments for moderate-to-severe atopic dermatitis: a systematic review. J Allergy Clin 
Immunol 2014; 133(2):429-38. 
10. Kobayashi T, Glatz M, Horiuchi K, Kawasaki H, Akiyama H, Kaplan DH, Kong 
HH, Amagai M, Nagao K. Dysbiosis and Staphylococcus aureus Colonization Drives 
Inflammation in Atopic Dermatitis. Immunity 2015; 42(4):756-66. 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 63  
APPENDIX A: SCHEDULE OF EVENTS 
 
Visit Name 
Screening 
 
Washout 
phase 
Baseline 
Day 0 
Day 5 
Day 14 
(Week 2) 
Day 28 
(Week 4) 
 Day 56  
(Week 8) 
 
Early 
Termination 
Visit 
 
 
Comments/Clarification 
Visit Number 
V1 
 
V2 
 
V3 
V4 
V5 
 
 
Visit Window in 
days 
-21 to -14 
-14 to -1 
0 
+/-2 
+/-2 
+/-2 
+/-3 
 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
Informed consent will 
occur prior to any protocol-
mandated procedures, 
including the stopping of 
any excluded therapies.  
Inclusion / 
Exclusion 
Criteria 
Х 
 
X 
 
 
 
 
 
 
Demographics 
Х 
 
 
 
 
 
 
 
 
Medical History 
Х 
 
X 
 
 
 
 
 
 
Concomitant 
Medications 
Х 
 
X 
 
Х 
Х 
X 
X 
 
Physical Exam 
X 
 
 
 
 
 
X 
X 
 
Vital Signs 
X 
 
X 
 
X 
X 
X 
X 
Height and weight will be 
assessed at screening; BP, 
pulse, respiration rate, and 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 64  
temperature will be 
assessed at each visit. 
Smoking status will be 
recorded at Screening 
 eDiary  
X 
X 
X 
X 
X 
X 
X 
X 
Subjects will be provided 
eDiary to be completed at 
home daily and in-clinic. 
At home eDiary will 
include WI-NRS, AI-NRS, 
rescue medication, dose 
administration, and local 
skin tolerability.  In clinic 
eDiary will include POEM 
and 5-D Pruritus Scale.  
eDiary will be reviewed at 
the baseline visit to 
determine eligibility. 
POEM 
X 
 
X 
 
X 
X 
X 
X 
Patient reported assessment 
by ePRO 
5-D Pruritus 
Scale 
X 
 
X 
 
X 
X 
X 
X 
Patient reported assessment 
by ePRO 
IGA 
X 
 
X 
 
X 
X 
X 
X 
Clinician assessment by 
eCOA 
EASI 
X 
 
X 
 
X 
X 
X 
X 
Clinician assessment by 
eCOA 
Itch Numeric 
Rating Scale 
(NRS) 
X 
X 
X 
X 
X 
X 
X 
X 
Patient reported assessment 
by ePRO 
Clinical Labs 
X 
 
 
 
 
X 
 
X  
Patients should fast for at 
least 8 hours before the test 
but optional at screening. 
Blood and urine for clinical 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 65  
(if visit 
occurs before 
Day 28) 
chemistry will be shipped 
to the central lab for 
processing. Chemistry, 
Hematology, Lipid Panel, 
and Urinalysis will be done 
at Screening and Day 28. 
Serology will only be done 
at Screening. Kits and lab 
manuals will be provided 
by a central lab. 
Urine pregnancy 
test for WOCBP 
X 
 
X 
 
 
X 
X 
X 
 
IWRS 
 
 
X 
 
 
 
 
 
 
Investigational 
Product 
compliance 
 
 
X 
 
Х 
Х 
 
X 
Weights of dispensed and 
collected bottles to be 
recorded following 
instructions provided to 
sites. 
Dispense study 
drug 
 
 
X 
 
X 
 
 
 
 
Application of 
study drug 
 
 
X 
X 
X 
X 
 
 
First application of study 
drug will occur in the 
office under the 
supervision of the study 
staff.  
Phone call to 
subjects 
 
X 
 
X 
 
 
 
 
Call to initiate washout 
phase will be placed once 
results of screening clinical 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 66  
labs are finalized. Call on 
Day 5 will be made to 
counsel subjects on the use 
of IP and eDiary 
recordings, as well as 
answer any questions.  
Collect study 
drug 
 
 
 
 
X 
X 
 
X 
 
Study 
counseling 
X 
X 
X 
X 
X 
X 
 
 
Subjects will be counseled 
at each visit on the 
appropriate use of IP, 
eDiary entries and use.  
AE monitoring 
X 
 
X 
 
Х 
Х 
X 
X 
AE will be monitored from 
screening to follow-up.  
After informed consent, 
but prior to study drug 
administration, only SAEs 
caused by a protocol-
mandated intervention will 
be collected  
 
 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 67  
APPENDIX A continued: 
Schedule of eDiary and Efficacy Assessments  
eDiary (ePRO) is provided to subjects at the screening visit for daily entry at home (e.g., application on 
smartphone). Questionnaires and clinician assessments are recorded via ePRO/eCOA (e.g., tablet) or CRF 
during the site visits per schedule of events.  Additional patient reported information include rescue medication 
use, dose administration confirmation, and adjunctive therapy use. 
Device 
Assessment 
Frequency and Duration of Assessment 
eDiary 
WI-NRS 
Once daily from Screening visit through the Follow-up visit 
eDiary 
AI-NRS 
Once daily from Screening visit through the Follow-up visit 
eDiary 
Local Tolerability 
Once daily from Day 1 to Day 7 of treatment 
eDiary 
POEM 
Patient reported at site visits 
eDiary 
5-D Pruritus Scale 
Patient reported at site visits 
eCOA/CRF 
IGA 
Clinician assessment at site visits 
eCOA/CRF 
EASI 
Clinician assessment at site visits 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 68  
APPENDIX B: EXCLUDED MEDICATIONS/THERAPY 
Excluded medication/therapy is listed below. The use of an excluded medication/therapy is a 
protocol violation and must be recorded in the CRF. 
Within 4 weeks prior to Baseline through the Follow-up period 
● Systemic immunosuppressive/immunomodulating drugs (i.e., methotrexate, cyclosporine, 
etc.). 
● Immunoglobulin or blood products. 
● Systemic corticosteroids (oral, IV, injectable) 
● NK1-R antagonists  
● Class III or higher topical corticosteroids or any topical anti-pruritic therapies (other than 
stable doses of low- or mid-potency topical corticosteroids or bland emollients) 
● Systemic therapies with recognized anti-pruritic (e.g. tricyclic antidepressants, sedatives, 
tranquilizers, gabapentin, marijuana or other cannabinoids, opioid receptor 
agonists/antagonists) or pruritic (e.g. opioids, angiotensin-converting enzyme inhibitors, 
cocaine, antimalarials) properties  
− Stable doses of oral H1 antihistamines will be permitted  
● Systemic immunosuppressive/immunomodulatory therapies (including but not limited to 
PDE4 inhibitors, cyclosporine, mycophenolate-mofetil, methotrexate, azathioprine, 
interferon-gamma, or phototherapy)  
● Allergen immunotherapy 
● Any investigational therapy   
● Strong CYP3A4 inhibitors, such as 
− boceprevir  
− clarithromycin  
− cobicistat  
− conivaptan  
− danoprevir and ritonavir  
− diltiazem  
− elvitegravir and ritonavir  
− regular grapefruit juice consumption  
− idelalisib  
− indinavir and ritonavir  
− itraconazole 
− ketoconazole 
− lopinavir and ritonavir  
− nefazodone  
− nelfinavir  
− paritaprevir and ritonavir and (ombitasvir and/or dasabuvir)  
− posaconazole 
− ritonavir  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 69  
− saquinavir and ritonavir  
− telaprevir  
− tipranavir and ritonavir  
− troleandomycin  
− voriconazole 
Within 2 weeks prior to Baseline through the Follow-up period 
● High-potency topical corticosteroids (Class 1-3). The use of low- to mid-potency topical 
corticosteroids (Class 4-7), inhaled corticosteroids, or intranasal corticosteroids will be 
allowed. 
● Use of crisaborole ointment. 
● Systemic antibiotics. 
● Bleach baths or topical coal tar. 
● Topical calcineurin inhibitor use (e.g., pimecrolimus, tacrolimus). 
● New onset use of systemic antihistamines. 
● UVA or UVB phototherapy 
● Topical and oral antibiotics/antiviral/antifungal/antiseptic agents 
● Topical probiotics 
● Topical antihistamines. 
● The use of intranasal and oral antihistamines will be alloweda. 
a Subjects following stable regimens (≥2 weeks consistent for intranasal and 3 months for oral use before study 
baseline) with systemic antihistamines are permitted to continue use but should not alter the dose or stop the 
regimen while in the study within 1 week prior to Baseline. 
Biologics 
● Cell-depleting agents, including but not limited to rituximab: within 6 months of baseline. 
● Infliximab, adalimumab, golimumab, certolizumab pegol, abatacept, etanercept, anakinra, 
and dupilumab: within 12 weeks of baseline, or 5 half-lives, whichever is longer. 
● Other biologics: within 12 weeks of Baseline, or 5 half-lives (if known), whichever is longer.

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 70  
APPENDIX C: EASI SCORE 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 71  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 72  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 73  
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 74  
 
 
 
 
 
 
 
 
 
 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 75  
 
 
 
 
 
 
 
 
 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 76  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 77  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 78  
APPENDIX D: PATIENT ORIENTED ECZEMA MEASURE (POEM)  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 79  
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 80  
APPENDIX E: IGA 
 
Score 
Category 
Definition 
0 
Clear 
Minor residual discoloration, no erythema or induration/papulation, no 
oozing/crusting 
1 
Almost Clear 
Trace faint pink erythema with almost no induration/papulation and no 
oozing/crusting 
2 
Mild Disease 
Faint pink erythema with mild induration/papulation and no 
oozing/crusting 
3 
Moderate 
Disease 
Pink-red erythema with moderate induration/papulation and there may 
be some oozing/crusting 
4 
Severe Disease 
Deep/bright red erythema with severe induration/papulation with 
oozing/crusting 
 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 81  
APPENDIX F: 5-D PRURITUS SCALE 
 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 82  
 
APPENDIX G: PATIENT REPORTED LOCAL TOLERABILITY 
PATIENT ASSESSMENT OF LOCAL SKIN TOLERABILITY AT APPLICATION SITE(S) 
SUBJECT AGE RANGE: 12 years old and above 
  
INSTRUCTIONS: 
Please examine all of your skin lesions that are treated with B244 topical solution.  Please circle 
one score for each category that best describes the changes you observed in all skin lesions.  For 
each day of the first 7 days of treatment, please examine all skin lesions at approximately the 
same time every day. 
  
DAY OF TREATMENT (1-7): _________________ 
  
TIME OF DAY: _____________AM or PM 
  
SKIN REDNESS AND/OR COLOR CHANGE AT APPLICATION SITE 
Score 
Grade 
Definition 
0 
None 
No new redness or new color change; no new increase in redness 
or new increase in color change 
1 
Mild 
Slight increase in redness or slight increase in color change 
2 
Moderate 
Increase in redness or increase in color change 
3 
Severe 
Intense redness or intense color change 

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 83  
   
ITCHING AT APPLICATION SITE 
Score 
Grade 
Definition 
0 
None 
No new itching or new scratching 
1 
Mild 
Slight increase in itching or slight increase in scratching 
2 
Moderate 
Increase in itching or increase in scratching that is not disturbing 
sleep 
3 
Severe 
Increase in itching or increase in scratching that is disturbing sleep 
  
BURNING AND/OR STINGING AT APPLICATION SITE 
Score 
Grade 
Definition 
0 
None 
No new burning or new stinging 
1 
Mild 
Slight increase in warm or tingling sensation that is not 
bothersome 
2 
Moderate 
Increase in warm or tingling sensation that is bothersome 
3 
Severe 
Hot or stinging sensation that is causing definite discomfort 
  

 
Protocol PRB244-01 
Amendment 3 28-FEB-2020 
 
Page 84  
PAIN AND/OR TENDERNESS AT APPLICATION SITE 
Score 
Grade 
Definition 
0 
None 
No new pain or new tenderness 
1 
Mild 
Slight increase in pain or slight increase in tenderness that is not 
bothersome 
2 
Moderate 
Increase in pain or increase in tenderness that is bothersome 
3 
Severe 
Intense pain or intense tenderness causing definite discomfort or 
disturbing sleep 
  
NEW OR CHANGING RASH AT APPLICATION SITE 
Score 
Grade 
Definition 
0 
None 
No new rash or no change in rash (such as swelling, fullness, or 
blistering) 
1 
Mild 
Slight new rash or slight increase in rash (such as swelling, 
fullness, or blistering) that is not bothersome 
2 
Moderate 
New rash or increase in rash (such as swelling, fullness, or 
blistering) that is bothersome 
3 
Severe 
Significant new rash or significant increase in rash (swelling, 
fullness, or blistering) causing discomfort