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
Phase: PHASE2
Conditions: Acne Vulgaris
Sponsor: AOBiome LLC

 
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CLINICAL TRIAL PROTOCOL 
Phase IIb/III 
 
COMPOUND: 
B244 
 
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 
STUDY NUMBER: 
AOB2016-01 
 
VERSION DATE: October 3, 2016 
PROTOCOL NUMBER: AVB244-002 
 
Sponsor: 
AOBiome LLC 
One Broadway 
14th Floor 
Cambridge, MA 02142-1100 
 
 
 
 
 
NCT02832063

 
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Any and all information presented in this document shall be treated as confidential and shall remain the 
exclusive property of AOBiome LLC (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 LLC (or the concerned affiliated company). 
 
 

 
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Revision Chronology 
Original 
April 15, 2016 
Amendment 1 
July 22, 2016 
Amendment 2 
October 3, 2016 
 
 
 

10-26-2016

 
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INVESTIGATOR AGREEMENT 
I have read this protocol and agree: 
● To conduct the study as outlined herein, in accordance with Good Clinical Practices 
(GCPs), the Declaration of Helsinki and complying with the obligations and 
requirements of Clinical Investigators and all other requirements listed in 21 CFR 
part 312 and according to the study procedures provided by AOBiome LLC and local 
regulations. 
● Not to implement any changes to the protocol without prior agreement from the 
Sponsor and prior review and written approval from the IRB or IEC, except as would 
be necessary to eliminate an immediate hazard to study participant (s), or for 
administrative aspects of the study. 
● To ensure that all persons assisting me with the study are adequately informed about 
the investigational product(s) and of their study-related duties as described in the 
protocol. 
● To completely inform all participants in this study concerning the pertinent details 
and purpose of the study prior to their agreement to participate in the study in 
accordance with GCP and regulatory authority requirements. 
● To be responsible for maintaining each participant’s consent form in a secure study 
file and providing each participant with a signed copy of the consent form. 
● That I am thoroughly familiar with the appropriate use of the investigational 
product(s), as described in the protocol, and any additional information provided to 
me by, or on behalf of AOBiome LLC. 
 
Investigator Printed Name:  
 
 
 
 
 
 
 
 
Signature: 
 
 
 
 
 
Date:   
 
 
 
 
 
 
 

 
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Statement of Compliance 
 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as 
required by the following: 
● United States (US) Code of Federal Regulations (CFR) applicable to clinical 
studies (45 CFR Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
● ICH E6; 62 Federal Register 25691 (1997) 
 
All key personnel (all individuals responsible for the design and conduct of this study) 
have completed Good Clinical Practice Training. 
 

 
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CLINICAL TRIAL SUMMARY 
COMPOUND: B244  
 
 
 
STUDY NUMBER: AVB244-002 
TITLE: 
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 
INVESTIGATOR/TRIAL 
LOCATION: 
Science 37 as a decentralized clinical trial site enrolling 
study participants from within the United States 
STUDY OBJECTIVES: 
Primary Objectives:  
• To evaluate the safety and tolerability of B244 
in participants with acne vulgaris. 
• To assess the efficacy of B244 in participants 
with acne vulgaris from baseline to week 12 
(end of treatment) by: 
o Reduction in inflammatory and non-
inflammatory lesion count 
o IGA success 
 
Secondary Objective:  
• To evaluate the efficacy of B244 in comparison 
to vehicle in participants with acne vulgaris 
from baseline to weeks 2, 4, 8, and 16 
o Reduction in inflammatory and non-
inflammatory lesion counts 
o IGA success  
 
• Improvement in patient reported quality of life 
score using the Skindex-16 questionnaire in 
participants with acne vulgaris from baseline to 
weeks 2, 4, 8, 12, and 16 
 
Exploratory Objective:  
• To evaluate facial skin microbiota in 
participants with acne vulgaris at baseline and 
weeks 2, 4, 8, 12, and 16 in B244-treated 

 
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participants compared to vehicle 
STUDY DESIGN: 
Phase IIb/III, randomized, double blinded,   
decentralized clinical trial evaluating the safety, 
tolerability, and efficacy of B244 compared to vehicle 
in the treatment of acne vulgaris 
INCLUSION CRITERIA: 
1. Males and females age 18 or above 
2. Clinical diagnosis of mild to moderate facial 
acne vulgaris defined as: 
a. ≥5 inflammatory lesions, and; 
b. ≥10 non-inflammatory lesions, and; 
c. IGA 2-3 
3. Willing to refrain from using any treatments, 
other than the investigational product, for acne 
present on the face once consented. This 
includes the use of antibiotics for the treatment 
of acne. Topical acne treatments that do not 
have significant or measurable systemic 
absorption (e.g., benzoyl peroxide, salicylic 
acid) are allowed for treatment of non-facial 
acne  
4. Willing and able to provide informed consent 
and to comply with the study protocol 
 
EXCLUSION CRITERIA: 
 
 
1. Pregnant and/or lactating females 
2. Continuous or planned use of tanning booths or 
excessive sun exposure, as determined by the 
Investigator.  
3. Active cystic acne or acne conglobata, acne 
fulminans, and secondary acne 
4. Two or more active nodular lesions 
5. Treatment with over-the-counter topical 
medications for the treatment of acne vulgaris 
including benzoyl peroxide, topical anti-
inflammatory medications, corticosteroids, α-
hydroxy/glycolic acid, or topical probiotics 
including commercially available product 

 
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AO+Mist on the face within 7 days prior to 
baseline 
6. Treatment with systemic corticosteroids within 
28 days prior to baseline 
7. Treatment with systemic antibiotics or systemic 
anti-acne drugs within 7 days prior to baseline 
8. Prescription topical retinoid use on the face 
within 7 days of baseline (e.g., tretinoin, 
tazarotene, adapalene) 
9. Commencement of new hormonal therapy or 
dose change to hormonal therapy within 90 days 
prior to baseline. Dose and frequency of use of 
any hormonal therapy started more than 90 days 
prior to baseline must remain unchanged 
throughout the study. Hormonal therapies 
include, but are not limited to, estrogenic and 
progestational agents such as birth control pills 
10. Use of androgen receptor blockers (such as 
spironolactone or flutamide) in the 7 days prior 
to randomization 
11. Oral retinoid use (e.g., isotretinoin) within 180 
days prior to baseline or vitamin A supplements 
greater than 10,000 units/day within 180 days of 
baseline 
12. Cosmetic facial procedures (chemical or laser 
peel, microdermabrasion, etc.) within the 28 
days of the first dose or during the study 
13. History of sensitivity to any of the study 
medications, or components thereof or a history 
of drug or other allergy that, in the opinion of 
the Investigator or Medical Monitor, 
contraindicates their participation 
14. Any other condition(s) that the study 
Investigator feels would indicate that 
participation would not be in the best interest of 
the participant 
15. The participant has been previously randomized 
in this study 
16. The participant has received an investigational 

 
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product within 30 days or 5 half-lives, 
whichever is longer prior to randomization 
DOSE REGIMEN: 
Participants will apply 4 pumps of spray to the face 
BID for 12 weeks 
ASSESSMENT SCHEDULE: 
Study assessments will occur at Screening, Baseline, 
and weeks 2, 4, 8, 12, and 16 
STATISTICAL 
CONSIDERATIONS: 
1. Modified Intent to treat analysis 
2. Fisher’s exact test will be used for efficacy 
parameters 
DURATION OF STUDY: 
The estimated total study duration is 12 months. All 
participants will be followed from consent date until the 
end of study visit at week 16. 
 
 
 

 
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Male and Female participants with a diagnosis  
of facial acne vulgaris 
Randomized 
Double Blind 
Vehicle 
Clinical Acne Assessments at: 
Baseline 
Weeks 2, 4, 8, 12 and 16 
End of Study visit for safety assessment at: 
Week 16 
 
B244 
Treatment 
 
STUDY SCHEMA 
 
 
 
 
 

 
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Table of Contents 
1. 
INTRODUCTION ................................................................................................. 16 
1.1. 
Background ............................................................................................................ 16 
1.2. 
Rationale for using decentralized clinical trial model .............................................. 17 
2. 
Risk Assessment ................................................................................................ 19 
3. 
STUDY OBJECTIVES ............................................................................................ 20 
3.1. 
Primary Objectives ................................................................................................. 20 
3.2. 
Secondary Objectives ............................................................................................. 20 
3.3. 
Exploratory Objective ............................................................................................ 21 
4. 
ENDPOINTS ........................................................................................................ 21 
4.1. 
Safety & tolerability: .............................................................................................. 21 
4.2. 
Efficacy: ................................................................................................................. 21 
4.3. 
Exploratory: ........................................................................................................... 21 
5. 
STUDY DESIGN ................................................................................................... 21 
5.1. 
Consenting & Screening Period .............................................................................. 22 
5.2. 
Treatment Period ................................................................................................... 23 
5.3. 
Study Withdrawal and Withdrawal From Investigational Product and Stopping 
Criteria 23 
5.3.1. 
Protocol-Defined Stopping Criteria ..................................................................... 24 
5.4. 
Screen Failures ....................................................................................................... 24 
5.5. 
Early Termination .................................................................................................. 25 
6. 
SELECTION OF STUDY PARTICIPANTS ................................................................. 25 
6.1. 
Number of Participants Planned ............................................................................ 25 
6.2. 
Inclusion Criteria .................................................................................................... 25 
6.3. 
Exclusion Criteria ................................................................................................... 26 
7. 
LIFESTYLE RESTRICTIONS .................................................................................... 27 
8. 
CONTRACEPTION REQUIREMENTS ..................................................................... 27 
9. 
STUDY TREATMENT ........................................................................................... 28 
9.1. 
Investigational Product .......................................................................................... 28 
9.2. 
Modalities of administration .................................................................................. 29 
9.3. 
Description of Blinding Method ............................................................................. 29 

 
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9.4. 
Treatment Assignments: ........................................................................................ 30 
9.5. 
Treatment Compliance ........................................................................................... 30 
9.6. 
Treatment of Investigational Product Overdose ..................................................... 30 
9.7. 
Product Accountability ........................................................................................... 30 
9.8. 
Unblinding Procedures ........................................................................................... 31 
9.9. 
Retrieval and Destruction of Investigational Product ............................................. 31 
10. 
CONCOMITANT MEDICATIONS ....................................................................... 32 
10.1. 
Permitted Medications .......................................................................................... 32 
10.2. 
Prohibited Medications .......................................................................................... 32 
11. 
STUDY PROCEDURES ...................................................................................... 32 
11.1. 
Informed Consent Procedures ................................................................................ 32 
11.2. 
Study Assessments ................................................................................................. 32 
11.3. 
Demographics ........................................................................................................ 32 
11.4. 
Medical History ...................................................................................................... 32 
11.5. 
Pregnancy Testing .................................................................................................. 33 
11.6. 
Skin Swab Samples ................................................................................................. 33 
11.7. 
Safety Assessments ................................................................................................ 33 
11.8. 
Compliance ............................................................................................................ 33 
11.9. 
Pregnancy Reporting .............................................................................................. 33 
12. 
EFFICACY ASSESSMENTS ................................................................................. 34 
12.1. 
Lesion Counts ......................................................................................................... 34 
12.2. 
Investigator Global Assessment ............................................................................. 34 
12.3. 
Skindex-16 ............................................................................................................. 35 
12.4. 
Photography .......................................................................................................... 35 
12.5. 
Study Completion .................................................................................................. 36 
13. 
STATISTICAL CONSIDERATIONS ...................................................................... 36 
13.1. 
Sample Size ............................................................................................................ 36 
13.2. 
Populations for Analysis ......................................................................................... 36 
13.3. 
Data Analysis ......................................................................................................... 36 
13.3.1. 
Demographic and Safety Data ............................................................................. 36 
13.3.2. 
Safety Analyses .................................................................................................... 36 
13.3.3. 
Efficacy Analyses ................................................................................................. 37 
14. 
ADVERSE EVENTS (AE) AND SERIOUS ADVERSE EVENTS (SAE) ......................... 37 
14.1. 
Definition of an AE ................................................................................................. 37 
14.2. 
Definition of a SAE ................................................................................................. 38 
14.3. 
Time Period, Frequency, and Method of Detecting AEs and SAEs ........................... 39 

 
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14.4. 
Recording of AEs and SAEs ..................................................................................... 39 
14.5. 
Evaluating AEs and SAEs ......................................................................................... 40 
1.1.1. 
Severity Rating .................................................................................................... 40 
1.1.2. 
Relationship to Investigational product (IP) ........................................................ 40 
1.1.3. 
Follow-Up of AEs and SAEs .................................................................................. 41 
14.6. 
Pregnancy .............................................................................................................. 41 
14.7. 
Prompt Reporting of SAEs to the Sponsor .............................................................. 41 
15. 
ETHICAL AND REGULATORY STANDARDS ....................................................... 42 
15.1. 
Ethical Conduct of Study ........................................................................................ 42 
15.2. 
Laws and Regulations ............................................................................................. 43 
15.3. 
Informed Consent .................................................................................................. 43 
15.4. 
Institutional Review Board/Independent Ethics Committee (IRB/EC) ..................... 44 
15.5. 
Clinical Monitoring/Record Keeping ....................................................................... 44 
16. 
ADMINISTRATIVE RULES ................................................................................ 45 
16.1. 
Curriculum Vitae .................................................................................................... 45 
16.2. 
Study Documentation ............................................................................................ 45 
16.3. 
Archiving of Study Documentation ......................................................................... 46 
16.4. 
Monitoring and Quality Assurance ......................................................................... 46 
16.5. 
Internal Safety Review Committee ......................................................................... 46 
17. 
PUBLICATIONS ............................................................................................... 46 
18. 
PROTOCOL ADHERENCE ................................................................................. 47 
19. 
CLINICAL TRIAL PROTOCOL AMENDMENTS ..................................................... 47 
20. 
REFERENCES ................................................................................................... 48 
21. 
Appendix A - Time and Events Tables ............................................................. 50 
21.1.1. 
Pregnancy Test2 ................................................................................................... 50 
 
 
 
 
 
 

 
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
AE 
Adverse Event 
AMO 
Ammonia Monooxygenase 
AOB 
Ammonia Oxidizing Bacteria 
BID 
Twice-Daily 
CRF 
Case Report Form 
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 
NORA 
Network Oriented Research Assistant 
QoL 
Quality of Life 
SAE 
Serious Adverse Event 
SPM 
Study Procedures Manual 
 
 
 
 
 
 
 

 
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1. INTRODUCTION 
1.1. 
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 needs.1 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 
oxidation.2 
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 
Staphylcoccus 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 

 
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bacteria, anti-inflammatory activity, and inhibition of lipogenesis by insulin-stimulated 
immortal sebocytes.4,5  
B244 has been developed as a topical application of a natural source of AOB and 
NO/NOx to the human skin. The active ingredient in B244 is available to consumers as a 
cosmetic product.  
Acne vulgaris is a common disease that continues to affect many men and women of all 
ethnicities. Most people have had acne at some point of their life with 15-20% of people 
experiencing moderate to severe acne.6 Prevalence differs from study to study but has 
been estimated as high as 45 million people within the United States alone and accounts 
for 20% of all dermatology visits.7 Although development of effective acne treatments 
has come a long way, much of our understanding of the pathogenesis of acne at the 
cellular level is still lacking.8  
To date, two cosmetic studies have been completed. In the cosmetic studies, a total of 24 
participants applied the cosmetic product to the scalp and 83 participants applied the 
product to their face. In these studies, there were no reports of drug-related serious 
adverse events (SAEs).  
A phase 1b/2a clinical trial was recently completed 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 
have been no attributable drug related SAEs reported.  
1.2. 
Rationale for using decentralized clinical trial model 
For medical science to progress, technology must advance to bridge the gap between 
research and the real-world patient application of medical discovery. For example, 
researchers are beginning to have a better understanding of the diverse human 
microbiome and how derangements might contribute to diseases such as acne. But to 
decipher the complexity of this relationship across growing and diverse populations, new 
technology and methodology is needed to connect with patients. Keeping pace with the 
advent of electronic medical records, social networking, and smartphones, new 
applications of technology will allow patients to interact with physicians and researchers 
more regularly and directly from their home in a way that will lead to better treatment 
and understanding of the diseases that afflict us.  
 
The concept of using mobile smartphone technology in medicine and research is not new. 
Even with regard to acne, research instruments such as telemedicine, mobile electronic 
devices, and the internet have been used. Specifically, this technology has been used to 
assist with compliance, medical education, physician-patient interactions, medical access, 

 
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and evaluation of treatment efficacy in many areas including dermatology. Indeed, 
because of prior challenges associated with unbiased scoring of acne clinical trials, the 
FDA guidelines for registered trials in acne strongly suggest that a digital photographic 
record of all patient visits be made available as the gold standard for FDA audit of 
clinical outcomes. 
Similarly, telemedicine and photographic technology has long been used in dermatology 
to bridge the access gap and to open patient-physician interactions. A limitation in 
telemedicine and the use of store-and-forward technology has been in the user’s skill 
when capturing photos to be evaluated by qualified healthcare professionals. Bergman 
and colleagues examined the feasibility of patient mediated photographic assessment by 
comparing photos taken by trained staff to those taken by patients.10 Photos were then 
evaluated using total inflammatory lesion count, the Burke and Cunliffe Leeds acne 
assessment technique, and the Investigator Global Assessment. The study showed strong 
inter-rater reliability for inflammatory lesion counts, Leeds assessments, and Investigator 
global assessments.11 The study demonstrated that photographs taken by participants were 
reliable and effective to score acne lesion counts and severity.  
Grading disease severity (e.g. acne severity scoring) is of utmost importance in guiding 
and monitoring the success of treatment. Instruments used to score disease often neglect 
to account for the impact of disease on livelihood. In addition, patients tend to score their 
acne severity higher than physicians.12 Interestingly, there is no gold standard for scoring 
acne severity despite extensive acne research over the years and many attempts to create 
a uniform scoring system. The pleomorphic nature of acne makes it difficult to create a 
simple unifying grading system, yet there remains a demand for a homogenized scoring 
system by which acne studies can be compared.13 The FDA has issued guidelines for 
acne trials that include a recommendation to keep digital photographic records of each 
visit so outside investigators or auditors could verify the scoring if needed. 
Many different studies have employed one or more scoring methods or have created their 
own scoring rubric. There are two main variations of acne scoring-lesion counting and 
global assessment. There has been debate regarding the reliability of lesion counting 
compared to global assessment; important considerations include the ease and 
reproducibility of each scoring instrument. In a study done by Lucky et al, lesion 
counting showed high inter-rater reliability (.81 to .97).14 The reliability was highest in 
mild cases of acne and decreased with increased number of lesions. Global assessments 
of acne such as the GEA have been shown to have inter-rater reliability as high as .80-
.84.15 The US FDA, American Academy of Dermatology (AAD) and European 
Medicines Agency (EMA) recommend a global approach.13,15 However, FDA guidelines 

 
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also suggest the use of lesion counting in conjunction with global assessment of acne. 
Application of both systems of acne grading has been validated in studies done by Burke 
and Cunliffe.11   
Access issues continue to limit biomedical research and healthcare. Access limits 
connections between patients and physicians and between researchers and needed 
participants. As modern medical practice shifts toward patient-centered care, a push 
toward greater patient engagement must also evolve. A better partnership between 
patients and doctors to improve understanding and management of disease will lead to 
better healthcare. As an example, patients will be able to enter their own data into secure 
electronic health records (EHR). Today, some patients enjoy access to their EHR through 
providers such as Kaiser Permanente and are allowed to procure certain samples for lab 
analysis (e.g. fecal occult blood test) in their home. This very same principle of patient 
empowered medical evaluation can be applied to research and made easy for patients to 
access. Note that physician guidance is still the foundation of medical and investigational 
practice. However, application of patient involvement can be as simple as using a mobile 
device to securely update reliable patient reported outcomes (PROs) within clinical trials. 
Additionally, less than 5% of clinical research participants nationally are minorities.  The 
decentralized clinical trial model will unlock access to minorities and underserved 
populations that are not normally cared for at traditional clinical research sites and thus 
better reflect the national diversity of race and ethnicity in the United States. 
New technology exists to bridge access between clinical research and the need for 
patient-centered care. One such application of technology is NORA (Network Oriented 
Research Assistant). NORA is currently being used successfully in other FDA-registered 
decentralized clinical trials to assess skin and mucosal disease in the home.  NORA is a 
combination technology that includes the functionality of a telemedicine platform, an 
EMR, an EDC, and a mobile data collection tool.  NORA was recently used to compare 
digital photography as a correlate of face-to-face acne scoring but has been used in other 
therapeutic areas as well. This approach will hopefully improve patient outcomes and 
advance the field of medicine by using more real world data and a better diversity of 
patients. 
The purpose of this study is to demonstrate the safety, tolerability and efficacy of B244 
administered over 12 weeks to participants with mild to moderate acne vulgaris relative 
to vehicle.  
2. RISK ASSESSMENT 
Please refer to Investigator Brochure for in depth risk assessment.  

 
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None of the potential or identified risks seen to date in participants dosed with B244 
preclude further clinical development. Mitigation strategies have been implemented to 
promptly identify and appropriately address these risks in order to protect participant 
safety and to better characterize the safety profile of the drug. Careful safety monitoring 
should also identify any emerging safety issues in a timely fashion. There is a paucity of 
probiotics with novel mechanisms of action available to treat acne vulgaris, given the 
potential unmet medical need for treatment of acne and the risk mitigation strategies in 
place, the benefit to risk profile of B244 for the treatment of acne vulgaris is favorable 
and warrants further study. 
 
Eligibility criteria, monitoring of clinical parameters, and stopping criteria have been 
chosen to mitigate identified and potential risks in this study and to better characterize the 
safety and tolerability of B244. Refer to Appendix A, Time and Events Table for the 
timing of all clinical assessments. Further, the study design in place will allow in stream 
review of the efficacy, safety, and tolerability data for B244 during the study. 
 
 
3. STUDY OBJECTIVES 
3.1. 
Primary Objectives 
1. To evaluate the safety and tolerability of B244 in participants with acne vulgaris 
2. To assess the efficacy of B244 in participants with mild to moderate acne vulgaris 
from baseline to week 12 (end of treatment) by: 
i) Reduction in inflammatory and non-inflammatory lesion count  
ii) IGA success  
 
3.2. 
Secondary Objectives 
1. Improvement in patient reported quality of life score using the Skindex-16 
questionnaire in participants with acne vulgaris from baseline to weeks 2, 4, 8, 12 
and 16. 
2. To evaluate the efficacy of B244 in participants with mild to moderate acne 
vulgaris from baseline to weeks 2, 4, 8, and 16: 
i) Reduction in inflammatory and non-inflammatory lesion count  
ii) IGA success  
 

 
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3.3. 
Exploratory Objective 
1. To evaluate facial skin microbiota in participants with acne vulgaris at baseline, 
week 4, week 8, week 12, and week 16 in B244-treated participants compared to 
vehicle from: 
i) Skin swabs will be taken from forehead, nose, both cheeks and chin  
All participants (vehicle and B244) will undergo skin swabs and testing.  
4. ENDPOINTS 
4.1. 
Safety & tolerability: 
1. Safety and tolerability endpoints will consist of all adverse events reporting 
during the study duration. 
4.2. 
Efficacy: 
1. Number of Inflammatory Lesions and Non-Inflammatory Lesions at baseline and 
weeks 2, 4, 8, 12, and 16. 
2. Investigator Global Assessment (IGA) at baseline and weeks 2, 4, 8, 12, and 16. 
3. Skindex 16 at baseline and weeks 2, 4, 8, 12, and 16. 
4.3. 
Exploratory: 
1. Microbial content, microbiota composition, and B244 presence/absence at 
baseline, weeks 4, 8, 12, and 16. 
 
5. STUDY DESIGN 
 
● This is a randomized, double blinded, Phase IIb/III decentralized study of B244 to 
determine safety and efficacy in participants with mild to moderate acne vulgaris 
● Randomization will be 1:1 so that 186 participants will be treated with B244 and 
186 participants will be treated with vehicle 
● All B244 randomized participants will be treated at the dose of 4x109 cfu/ml 
(mid-dose of the Phase Ib/IIa trial) 
● FDA Guidance efficacy endpoints will be assessed and compared between 
treatment groups 
● Screening will occur at Study Week -3 (Days -21 to 0) 

 
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● Randomization will occur during the baseline period for the study (Days -7 to 0). 
● Clinical acne assessments (IGA, lesion counts) will be made at Baseline; Study 
Weeks 2, 4, 8, 12, and 16. 
● 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. 
 
5.1. 
Consenting & Screening Period 
Electronic 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 virtually, using 
an electronic consent (eConsent) form that the participant will view on his/her home 
computer or tablet. The eConsent form will be identical in content to a paper consent 
form, but will be viewed on a computer or tablet. Participants will receive a link via   to 
access the eConsent web portal. Participants without access to a computer or tablet with 
which to complete the informed consent process will be shipped a tablet to complete the 
eConsent form from the web portal. Site personnel will organize the return of the tablet 
upon completion of the informed consent process.  
 
After informed consent has been obtained, to determine participant eligibility for 
enrollment in the study, screening assessments will be performed within 3 weeks (-28 
days to -8) prior to the Baseline visit (-7 days to 1), randomization and first dose of the 
investigational product (IP).  Participants will be shipped an iPhone pre-loaded, after 
consent, with NORA for use during their participation in the study.  All screening 
evaluations must be completed and reviewed to confirm that participants meet all 
eligibility criteria before randomization. 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. 
 
Re-screening refers to repeating the entire screening process if a participant has not yet 
met all the eligibility criteria within 14 days of the original screening visit. As acne is a 
very dynamic disease, potential participants will be allowed to be re-screened up to three 
times. Each participant must be re-consented before re-screening occurs. 
 

 
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5.2. 
Treatment Period 
Once it has been determined that the participant meets all inclusion criteria and no 
exclusion criteria, they will be enrolled into the trial and randomized. Eligible 
participants will perform baseline assessments after randomization.  All assessments and 
the relative timings are listed in the Time and Events Table (Appendix A).   
Participants will apply the investigational product (IP) to the face BID, once in the 
morning and once in the evening for 12 weeks.  
Participants will be assessed for safety and efficacy endpoints throughout the study at the 
times indicated in the Time & Events Table (Appendix A). The last assessment is at 
Week 16. 
 
5.3. 
Study Withdrawal and Withdrawal From Investigational Product and 
Stopping Criteria  
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 
● 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 participant is to be withdrawn).  
 
Reasons for withdrawal from investigational product (IP) may include the following: 
• Adverse event 
• Protocol deviations at the discretion of the Sponsor 
• Termination of the study by AOBiome 
• Investigator discretion 
• Participant reached protocol-defined stopping criteria 
 
The reason for participant study withdrawal or withdrawal from IP 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. 
 

 
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Any subject who develops Grade 2 or higher adverse events [according to the common 
toxicity criteria for adverse events (CTCAE) version 3] should be discontinued from 
treatment, be continued in the trial, and followed until resolution of the adverse events. 
 
 
Participants who are prematurely withdrawn from IP will remain in the study. If a 
participant is prematurely withdrawn from IP, the Investigator must make every recorded 
effort to ensure the participant attends all visits as scheduled in Appendix A.  
 
5.3.1. 
Protocol-Defined Stopping Criteria  
The number, severity, type, nature and pattern of all clinical and non-clinical findings 
will be considered before making a decision to stop dosing of subsequent participants in 
the study.  Based on these results and the nature of the AE, a decision may be made to 
adjust participant(s) to the original dose. 
Treatment with the investigational product (IP) will be stopped and the participant will be 
withdrawn from the study if any of the following occurs during the study: 
● Hypersensitivity reactions considered possibly related to the investigational 
product (IP) or vehicle that may include allergic (or immediate) and delayed 
responses 
● Severe, localized inflammatory reactions and infections on the face whose signs 
and symptoms may include pain, pruritus, erythema, and edema 
● Bacteremia or sepsis consistent with clinical signs, symptoms, and laboratory 
evaluations 
● Serious Adverse Events (SAEs) considered possibly related to the investigational 
product (IP) as assessed by the Investigator 
● The Investigator considers that the participant is being exposed to unacceptable, 
medical risk(s) for any reason while participating in this study 
The study will be terminated for the following reasons: 
If one or more participants experience a possibly drug-related SAE or a possibly drug-
related significant non-SAE, that in the opinion of the study physician, Investigator or 
AOBiome Medical Monitor or designee warrants discontinuation of further dosing. 
5.4. 
Screen Failures 
Data for screen and baseline failures will be collected in source documentation at the site 

 
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but will not be transmitted to AOBiome. 
 
5.5. 
Early Termination 
Participants who have discontinued the study early will be evaluated by the Investigator 
at approximately 7 ± 1 days after last dose of IP for an Unscheduled Visit.  See the list of 
assessments to be performed at the Unscheduled 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.  
 
6. SELECTION OF STUDY PARTICIPANTS 
6.1. 
Number of Participants Planned 
It is estimated that approximately 372 participants will be enrolled & randomized in order 
to provide a maximum of 322 eligible participants completing the 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 females age 18 or older 
2. Clinical diagnosis of mild to moderate facial acne vulgaris defined as: 
a. ≥5 inflammatory lesions, and; 
b. ≥10 non-inflammatory lesions, and; 
c. IGA 2-3 
3. Willing to refrain from using any treatments, other than the investigational 
product, for acne present on the face. This includes the use of antibiotics for the 
treatment of acne. Topical acne treatments that do not have significant or 
measurable systemic absorption (e.g., benzoyl peroxide, salicylic acid) are 
allowed for treatment of non-facial acne. 
4. Willing and able to provide informed consent and to comply with the study 
protocol. 

 
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6.3. 
Exclusion Criteria 
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. Pregnant and/or lactating females  
2. Continuous or planned use of tanning booths or excessive sun exposure, in the 
opinion of the Investigator. 
3. Active cystic acne or acne conglobata, acne fulminans, and secondary acne 
4. Two or more active nodular lesions 
5. Treatment with over-the-counter topical medications for the treatment of acne 
vulgaris including benzoyl peroxide, topical anti-inflammatory medications, 
corticosteroids, α-hydroxy/glycolic acid, or topical probiotics including 
commercially available product AO+Mist on the face within 7 days prior to 
baseline. 
6. Treatment with systemic corticosteroids within 28 days prior to baseline. 
7. Treatment with systemic antibiotics or systemic anti-acne drugs within 7 days 
prior to baseline. 
8. Prescription topical retinoid use on the face within 7 days of baseline (e.g., 
tretinoin, tazarotene, adapalene). 
9. Commencement of new hormonal therapy or dose change to hormonal therapy 
within 90 days prior to baseline. Dose and frequency of use of any hormonal 
therapy started more than 90 days prior to baseline must remain unchanged 
throughout the study. Hormonal therapies include, but are not limited to, 
estrogenic and progestational agents such as birth control pills. 
10. Use of androgen receptor blockers (such as spironolactone or flutamide) in the 7 
days prior to randomization. 
11. Oral retinoid use (e.g., isotretinoin) within 180 days prior to baseline or vitamin A 
supplements greater than 10,000 units/day within 180 days of baseline. 
12. Cosmetic facial procedures (chemical or laser peel, microdermabrasion, etc.) 
within the 28 days of the first dose or during the study. 

 
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13. History of sensitivity to any of the study medications, or components thereof or a 
history of drug or other allergy that, in the opinion of the Investigator or Medical 
Monitor, contraindicates their participation. 
14. Any condition that the study Investigator feels would indicate that participation 
would not be in the best interest of the participant. 
15. The participant has been previously randomized in this study. 
16. The participant has received an investigational product within 30 days or 5 half-
lives, whichever is longer prior to randomization. 
7. LIFESTYLE RESTRICTIONS 
1. Use of anti-aging and anti-acne products on the face will be prohibited during the 
study. 
2. All makeup must be removed before each photography session.  No other topical 
products should be applied to the face until all photographs have been taken. 
3. Participants will be discouraged from facial washing for at least 2 hours AFTER 
each application of investigational product.  Participants may bathe or shower 
with soap and water BEFORE each application. 
4. Participants will be encouraged to avoid prolonged periods of sun exposure and 
discouraged from the use of tanning beds during the study.  Extra care should be 
taken to wear protective clothing, including sunglasses, and avoid sun exposure 
from 10 AM to 2 PM. 
5. 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.  
8. CONTRACEPTION REQUIREMENTS 
 
Effective contraception is required for all women physiologically capable of becoming 
pregnant during study participation.  
 
Effective contraception methods include:  
• 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.  
• Use of oral, injected or implanted hormonal methods of contraception or 
placement of an IUD or IUS or other forms of hormonal contraception that 

 
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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) 
 
In case of use of oral contraception women should have been stable on the same pill for a 
minimum of 90 days before the baseline visit. 
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 let their female partners know of 
their participation in a research study of a drug, and that the effects of the drug on an 
unborn baby 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.  
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. 
9. STUDY TREATMENT 
9.1. 
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. 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 the 
protocol. 
 
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 

 
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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 bottle. Discrepancies are to be reconciled or resolved.  
 
 
 
 
 
 
 
 
 
Product name: 
B244, 30ml/bottle 
Vehicle, 30ml/bottle 
Dosage form: 
B244 suspension 
Vehicle solution 
Unit dose strength: 
4x109 cfu/ml 
50nM Na2HPO4-2mM 
MgCl2 (pH 7.6) 
Route/administration/duration: Topical application BID 
for 12 weeks 
Topical application BID 
for 12 weeks 
Dosing instruction: 
4 pumps of spray to 
saturate the entire face 
applied BID. 
Applications should 
occur in the morning and 
at night for 12 weeks. 
4 pumps of spray to 
saturate the entire face 
applied BID. 
Applications should 
occur in the morning and 
at night for 12 weeks. 
Physical description: 
Odorless, cloudy, light 
pink suspension 
Odorless, clear, and 
colorless suspension 
Manufacturer/source of 
procurement: 
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 metered 
opaque bottles.  
9.2. 
Modalities of administration  
4 pumps of spray to saturate the entire face must be applied twice a day. Applications 
should occur in the morning and at night for the duration of the study. Participants will be 
instructed to close their eyes and mouth during the application.  
 
9.3. 
Description of Blinding Method 
This study will be double-blinded: neither Investigator(s), nor study participants, nor 

 
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those involved in the conduct of the trial (including sponsor staff) will be aware of the 
treatment the participants are receiving. 
 
 
9.4. 
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 vehicle or 
B244 prior to the initiation of the trial. Randomization will be centrally-based and 
performed using an appropriate IxRS (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.  
    
9.5. 
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 mobile application 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. 
 
 
9.6. 
Treatment of Investigational Product Overdose 
The sponsor does not recommend specific treatment for an overdose. Washing with soap 
and water will remove the product. The Investigator will use clinical judgment to treat 
any overdose.  
 
9.7. 
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 

 
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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 the 
designated refrigerator between 2-8oC. . 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.  
 
9.8. 
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. Investigators have direct access to the 
participant’s individual study treatment. 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. 
 
9.9. 
Retrieval and Destruction of Investigational Product 
All partially used or unused treatments will be returned to the site as shipped by study 
participants. A detailed treatment log of the returned IP shall be established.  
The site will not destroy unused IP unless the Sponsor provides written authorization to 
the contrary.  
 

 
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10. CONCOMITANT MEDICATIONS 
10.1. 
Permitted Medications 
At study entry, participants must not be using any medication to treat acne vulgaris.  
Topical acne treatments that do not have significant or measurable systemic absorption 
(e.g., benzoyl peroxide, salicylic acid) are allowed for treatment of non-facial acne.  
 
Any concomitant medication to treat adverse events will be recorded in the Concomitant 
Medication section of the eCRF. 
 
10.2. 
Prohibited Medications 
Participants will be asked to stop taking medications for treatment of facial acne vulgaris 
from 7 days prior to randomization during the washout period and throughout the study 
until the final follow-up visit.   
11. STUDY PROCEDURES 
 
11.1. 
Informed Consent Procedures 
Eligible participants may only be included in the study after providing virtual electronic 
consent using the IRB-approved informed consent. 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 CRF. 
 
11.2. 
Study Assessments 
Study activities will take place according to the Time and Events table (Appendix A). 
11.3. 
Demographics  
All participant demographic and baseline characteristic data will be collected.  
11.4. 
Medical History 
Relevant medical history/current medical conditions data will be collected from all 
participants.  

 
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11.5. 
Pregnancy Testing 
A urine pregnancy test will be performed for women physiologically capable of 
becoming pregnant prior to administration of the first dose of IP.  
 
11.6. 
Skin Swab Samples 
All participants will self-collect a total of 5 skin swabs sampling the forehead, both 
cheeks, nose and chin at Baseline, Week 4, 8, 12 and Week 16. Participants will collect 
swabs at Baseline and then one swab at Week 4, 8, 12, 16. Patients will be instructed to 
collects samples before washing or applying product on their face. Patients will be 
instructed to send samples back to the study site using the self-addressed stamped 
envelope.  
Swab samples may be subjected to DNA sequencing. While this is not our intention to 
study participants genome, participants sequence may be included as part of this analysis. 
Samples may be stored in the freezer until future research. We will keep the specimen 
indefinitely, or until analyzed.  If we complete our research and no longer need to keep 
the specimens, we will destroy them. Specimens will be coded and no identifiable 
participant information will be used.  
 
11.7. 
Safety Assessments 
Participants will report adverse events immediately to the Investigator and study 
personnel. Participants will also be solicited for adverse events at each scheduled visit. 
Limited vital signs may be measured at home intermittently and recorded in the study 
mobile application by the participants.  
 
11.8. 
Compliance 
Participants will be supplied with scales and will be asked to weigh each spray bottle 
provided before the first use and after 7 days of use. Weight will be recorded in the study 
mobile application. 
11.9. 
Pregnancy Reporting 
Any pregnancy will be reported by study participants during their study participation. 

 
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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.  
 
12. EFFICACY ASSESSMENTS 
12.1. 
Lesion Counts 
Using standardized and validated facial photography modules built into the study mobile 
platform, participants will perform self-photography and photographs will be 
automatically uploaded the mobile platform.  Acne lesion counts will be performed at the 
times indicated in the Time and Events Table (Appendix A).  
Lesion counting will be performed using the photos taken by the participant and stored 
inside the study mobile platform by trained personnel only and every effort should be 
made that counting and clinical evaluation is done by the same evaluator for a given 
participant. The counting is to be done by lesion type and by counting area. The face will 
be separated into five areas: the forehead, the right cheek, the left cheek, the chin and the 
nose. Lesions under the jaw line or behind the hairline (including eyebrows) will not be 
included in the counts (Rizer 2001).The evaluator will be recorded in the eCRF. 
NOTE: Papules, pustules, cysts and nodules are classified as inflammatory acne lesions 
while open and closed comedones are classified as non-inflammatory lesions. 
 
12.2. 
Investigator Global Assessment 
IGA will be performed at the times indicated in the Time and Events Table (Appendix 
A). The Investigator will assess improvement of acne based on the 5-point severity scale 
summarized below based on examination of the standardized photography digital images 
of the face.  
Table 1 
IGA Severity Scale 
Grade 
Description 
0 
Clear skin with no inflammatory or non-inflammatory lesions 
1 
Almost clear; rare non-inflammatory lesions with no more than one small 
inflammatory lesion 

 
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2 
Mild severity; greater than Grade 1; some non-inflammatory lesions with 
no more than a few inflammatory lesions (papules/pustules only, no 
nodular lesions) 
3 
Moderate severity; greater than Grade 2; up to many non-inflammatory 
lesions and may have some inflammatory lesions, but no more than one 
small nodular lesion 
4* 
Severe; greater than Grade 3; up to many non-inflammatory and 
inflammatory lesions, but no more than a few nodular lesions 
* Participants who worsen beyond Grade 4 should be described. 
 
12.3. 
Skindex-16 
Skindex-16 instrument will be administered at the times indicated in the Time and Events 
Table (Appendix A). The participant will answer a questionnaire examining the 
relationship between the patient’s skin health and quality of life (Appendix B). The 
questionnaire will be completed by the participant within the study mobile application as 
an electronic patient reported outcome (ePRO). 
 
12.4. 
Photography 
In order to participate in the trial, all participants will be asked to provide photos at 
Baseline, Weeks 2, 4, 8, 12 and 16. Digital photos will be taken using standardized 
procedures by study participants. Participants will use a guided facial photography 
module on an iPhone to take at least 5 standardized digital photos of their own face using 
validated standards. An online instructional video will demonstrate standard 
recommended photography techniques.  
Each participant will take a total of 5 face images (full face, forehead, right side, left side, 
and chin).  Investigators or their designee will confirm the photographic quality. 
Photo collection is required for participation in the trial. The photographs will be used for 
research purposes only and will not be used for marketing or advertising purposes.   
Photo collection and processing instructions will be available in a separate manual.  
 

 
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12.5. 
Study Completion 
A completed participant is one who has completed all study visits. The Week 16 study 
visit is defined as the participant’s last visit. 
 
13. STATISTICAL CONSIDERATIONS 
13.1. 
Sample Size 
Primary efficacy power calculations assume a >25% difference between B244 treatment 
and vehicle and a 15% dropout.  As per FDA statistical guidance for co-primary 
endpoints, each of the endpoints comprising the co-primary endpoint will be tested at an 
alpha level of p<0.05.  In order to achieve 90% power with a 5% Type I error rate, a total 
of 372 participants is required to be enrolled in the trial.   
 
13.2. 
Populations for Analysis 
Safety Population: includes all randomized participants who receive one dose of 
investigational product (IP). 
mITT Population:  includes all randomized participants who receive one dose of 
investigational product (IP)  
 
13.3. 
Data Analysis 
The analyses will be conducted on all participant data when the trial ends. Details of the 
planned analysis will be presented in the trial Statistical Analysis Plan. 
 
13.3.1. 
Demographic and Safety Data 
Demographics and baseline characteristics will be listed and summarized descriptively by 
cohort and treatment. Participant disposition and incidence of AEs will also be 
summarized overall. 
13.3.2. 
Safety Analyses 
Safety analyses will be performed on data from all participants in the Safety Population.  
AEs, and other safety measures will be listed and summarized.   

 
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13.3.3. 
Efficacy Analyses 
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 mITT Population. 
IGA Success is defined as “Clear” (Grade 0) or “Almost clear” (Grade 1) at the pre-
specified primary time points. For participants whose baseline score is Grade 2, the 
clinically meaningful criterion for IGA success is achieving a score of Grade 0 at the pre-
specified primary time point because of limitations inherent to an ordinal scale. 
Lesion counts will be enumerated as the number of inflammatory and non-inflammatory 
lesions separately.  
Participant reported outcomes will be obtained through the Skindex-16 questionnaire.  
Details of the data analysis will be defined in the statistical analysis plan. 
 
14. 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. 
 
14.1. 
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. 

 
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● 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). 
 
14.2. 
Definition of a SAE 
A serious adverse event is any untoward medical occurrence that, at any dose: 
(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, or 
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 

 
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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. 
14.3. 
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. 
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 14.4 (“Recording of AEs and 
SAEs”). 
14.4. 
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. 

 
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14.5. 
Evaluating AEs and SAEs 
1.1.1. 
Severity Rating 
The severity of an adverse event (AE and SAE) is to be scored according to the following 
scale: 
Mild 
Awareness of sign or symptom, 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 14.2 
“Definition of a SAE”. 
1.1.2. 
Relationship to Investigational product (IP)  
SAEs will be classified as “not related” or “related” (including unknown).  
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 

 
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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. 
1.1.3. 
Follow-Up of AEs and SAEs 
After the initial AE/SAE report, the Investigator is required to proactively follow each 
participant and provide further information for clinically important AEs to the Sponsor’s 
medical monitor or designee on the participant’s condition within 24 hours as described 
in Section 14.7. 
New or updated information will be recorded on the “SAE” Report Form.  The updated 
SAE Report Form should be sent to Sponsor’s medical monitor or designee within 24 
hours as described in Section 14.7. 
14.6. 
Pregnancy 
Any pregnancy that occurs in a female participating in the study must be reported to the 
Sponsor within 3 working days 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 
possibly related or 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. 
14.7. 
Prompt Reporting of SAEs to the Sponsor 
Any SAE occurring in a participant receiving treatment or any SAE that the Investigator 
becomes aware of post-treatment during the follow up period, must be reported by the 
Investigator to the medical monitor or designee within 24 hours even if the SAE does 
not appear to be drug-related. This should be done by telephone and by sending an email 
(high importance) of the Adverse Event form plus other related information.  
Additionally, it may be necessary for the Sponsor to directly communicate with the 
Investigator if additional information is required.   

 
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During both business and non-business hours, the telephone numbers and email addresses 
listed below should be used to notify the Sponsor 
. 
 
Primary Contact 
AOBiome Reportable Events Hotline 
24 Hour Phone: 617-475-1605 
Email: lweiss@aobiome.com  
Call medical monitor to email a scanned report 
 
A Serious Adverse Event Report Form must be completed, scanned and forwarded via 
email to the medical monitor or designee at the number listed above within 24 hours of 
becoming aware of the event.  
All additional follow-up evaluations must be reported to the medical monitor or designee.  
Such data should be sent to the Sponsor within 10 calendar days.  All SAEs will be 
followed until the Investigator and Sponsor agree the event is satisfactorily resolved. 
The Sponsor will be responsible for completing the safety report and for notifying the 
relevant authorities of any SAE as outlined in the International Conference on 
Harmonization (ICH) Guidelines and per local regulatory requirements.  The Investigator 
will also ensure that the appropriate ethics committee is notified of the SAE. 
15. ETHICAL AND REGULATORY STANDARDS 
15.1. 
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 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 

 
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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. 
15.2. 
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.  
15.3. 
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 considered a benefit.  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 virtual electronic consent to take part in the 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.  

 
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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). 
15.4. 
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 AOBiome, in writing, 
of the approval to continue the study.   
 
15.5. 
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 
digitally 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. 

 
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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 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. 
16. ADMINISTRATIVE RULES 
16.1. 
Curriculum Vitae 
An updated, signed, and dated copy of the curriculum vitae limited to the experience, 
qualification and training for each Investigator and/or Sub-Investigator(s) will be 
provided to the Sponsor prior to the beginning of the Clinical Trial.  
16.2. 
Study Documentation 
The Investigator is required to prepare and maintain adequate and accurate case histories 
(i.e., source documents and/or Medical Record Supplement) designed to record all 
observations and other data pertinent to the study for each study participant.  This 
includes accurate documentation of accountability of study medications.  The medical 
records must contain adequate information to allow for verification of participant identity 
throughout the study.   
eCRFs will be completed for each participant who is enrolled (randomized) in the study.  
A participant screening/enrollment log, noting reasons for screen failure where 
applicable, will be maintained for all participants who are consented. 
All information recorded on the eCRFs for this study are considered to be the 
participant’s source documentation. The source documents may include scanned copies 
of records from a hospital and/or the physician’s chart, X-rays, or laboratory test 
documentation.  
The original eCRFs for each participant may be periodically checked against the 
participant’s source documents at the study site by the site monitor.  Instances of missing 
or unclear data will be discussed with appropriate site personnel for resolution.  A quality 
assurance audit will be performed on the eCRF database. 
 

 
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16.3. 
Archiving of Study Documentation 
The Investigator shall retain records for two (2) years following the date a marketing 
application is approved for the indication pertaining to this clinical study; or, if the drug 
is planned to be terminated or if a Regulatory application is not planned to be progressed, 
until two (2) years after the investigation is discontinued and the Food and Drug 
Administration (FDA) or a competent regulatory authority is notified. 
The Sponsor will inform the Investigator, in writing, as to when these documents no 
longer need to be maintained. 
16.4. 
Monitoring and Quality Assurance 
During the course of the study, a monitor will have access to all source documents, 
photographs, and eCRFs. The monitor will be able to remotely monitor the study at any 
time and review protocol compliance and CRFs, assess drug accountability and ensure 
that the study is being conducted according to pertinent regulatory requirements.  The 
review of the participants’ original medical records will be performed in a manner to 
ensure that participant confidentiality is maintained. 
16.5. 
Internal Safety Review Committee 
An internal safety review committee will be set up to protect the ethical and safety 
interests of participants and to protect the scientific validity of the study. Adhoc safety 
interim analyses might be performed by an independent statistician if the safety review 
committee identifies suspicious safety signals during its routine blinded safety review. 
The details for the analysis plan will be documented in the trial’s Statistical Analysis 
Plan. 
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 

 
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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.   
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 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. 
 
 

 
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20. REFERENCES 
 
1. Arp DJ, Sayavedra-Soto LA, Hommes NG (2002) Molecular biology and 
biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch Microbiol 
178: 250-255. 
2. Chandran K, Stein LY, Klotz MG, van Loosdrecht MCM (2011) Nitrous oxide 
production by lithotrophic ammonia-oxidizing bacteria and implications for 
engineered nitrogen-removal systems. Biochem Soc Trans 39: 1832-1837. 
3. Williams HC, Dellavalle RP, Garner S. Acne vulgaris. The Lancet. 2012;  
379(9813): 361-372. doi:10.1016/S0140-6736(11)60321-8. 
4. Weller, R., Price, R. J., Ormerod, A. D., Benjamin, N., & Leifert, C. (2001). 
Antimicrobial effect of acidified nitrite on dermatophyte fungi, Candida and 
bacterial skin pathogens. Journal of Applied Microbiology, 90(4), 648-652. 
5. Rico, J., Quiring, J., Hollenbach, S., Enloe, C., & Stasko, N. (2014, August). 
Phase 2 study of efficacy and safety of SB204 in the treatment of acne vulgaris. 
In Journal Of Investigative Dermatology (Vol. 134, No. 8, pp. S8-S8). 75 Varick 
St, 9th Floor, New York, NY 10013-1917 USA: Nature Publishing Group. 
6. Park C, Kim G, Patel I, Chang J, Tan X. Improving adherence to acne treatment: 
the emerging role of application software. Clin Cosmet Investig Dermatol. 2014; 
7: 65–72. doi: 10.2147/CCID.S46051. 
7. Fitz-Gibbon S, Tomida S, Chiu Bor-Han et. al. Propionibacterium acnes strain 
populations in the human skin microbiome associated with acne. J Invest 
Dermatol. 2013; 133(9): 2152-2160. doi: 10.1038/jid.2013.21. 
8. Yentzer BA, Wood AA, Sagransky MJ, et al. An Internet-Based Survey and 
Improvement of Acne Treatment Outcomes. Arch Dermatol. 2011;147(10):1223-
1224. doi:10.1001/archdermatol.2011.277. 
9. Weiss J, Shavin J, Davis M. Improving Patient Satisfaction and Acne Severity in 
Patients With Mild to Moderate Acne: The BEST Study. Cutis. 2003; 71(2): 3-4. 
Available at: 
http://www.ecardiologynews.com/fileadmin/qhi_archive/ArticlePDF/CT/07102S0
03.pdf. Accessed December 4, 2014. 
10. Bergman H, Tsai KY,  Seo SJ, Kvedar JC, Watson AJ. Remote Assessment of 
Acne: The Use of Acne Grading Tools to Evaluate Digital Skin Images. Telemed 
J E Health. 2009;15(5): 426-30. doi: 10.1089/tmj.2008.0128. 
11. Burke BM, Cunliffe WJ. The assessment of acne vulgaris - the Leeds technique. 
Br J Dermatol. 1984; 111(1): 83-92. doi: 10.1111/j.1365-2133.1984.tb04020.x. 
12. Medansky RS, Handler RM, Medansky DL. Self-evaluation of acne and emotion: 
A pilot study. Psychosomatics. 1981; 22(5) 379-383. doi: 10.1016/S0033-
3182(81)73507-2.  
13. Pochi PE, Shalita AR, Strauss JS. Report of the Consensus Conference on Acne 
Classification. Washington, D.C., March 24 and 25, 1990. J Am Acad Dermatol. 
1991; 24(3): 495-500. doi: http://dx.doi.org/10.1016/S0190-9622(08)80076-X. 

 
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14. Lucky AW, Barber BL, Girman CJ, Williams J, Ratterman J, Waldstreicher J. A 
multirater validation study to assess the reliability of acne lesion counting. J Am 
Dermatol. 1996; 35(4): 559-569. doi: 10.1016/S0190-9622(96)90680-5.  
15. Dréno B, Poli F, Pawin H, et al. Development and evaluation of a Global Acne 
Severity Scale (GEA Scale) suitable for France and Europe. J Eur Acad Dermatol 
Venereol. 2011; 25(1): 43-48. doi: 10.1111/j.1468-3083.2010.03685.x.  
16. Gupta MA, Johnson AM, Gupta AK. The Development of an Acne Quality of 
Life Scale: Reliability, Validity, and Relation to Subjective Acne Severity in Mild 
to Moderate Acne Vulgaris. Acta Derm Venereol. 1998; 78(6): 451-456. doi: 
10.1080/000155598442773 
17. Rizer, RL, Mills, OH, Trookman, NS. (2001) The assessment of acne: a re-
evaluation of grading strategies. Scientific Poster, Annual Meeting of the Am. 
Acad. Dermatol. 
 
 

 
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21. APPENDIX A - TIME AND EVENTS TABLES 
 
1: Informed consent must be provided by all patients before any screening procedures are performed. 
2: Only performed for women of childbearing potential, prior to first dose of Investigational Product. 
3: If study participant has an unscheduled visit to end study participation, these study procedures will be conducted. 
Visit name (Day) 
Screening 
(Day 0) 
Baseline 
(Day 1) 
Week 2 
(Day 14) 
Week 4 
(Day 28) 
Week 8 
(Day 56) 
Week 12 
(Day 84) 
Week 16 
(Day 112) 
Unscheduled 
Visit3 
Visit Window, in Days 
-28 to  -8 
-7 to 1 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
 
Informed Consent1 
X 
 
 
 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical History / Current Medical 
Conditions 
X 
 
 
 
 
 
 
X 
21.1.1. 
Pregnancy Test2 
X 
(X) 
 
 
 
 
 
 
Microbiota swabs 
 
X 
 
X 
X 
X 
X 
X2 
Skindex-16 
 
X 
X 
X 
X 
X 
X 
X2 
Investigational Product  
Administration 
 
X 
X 
X 
X 
X 
 
 
Photography 
 
X 
X 
X 
X 
X 
X 
X2 
IGA 
 
X 
X 
X 
X 
X 
X 
X2 
Lesion Count 
 
X 
X 
X 
X 
X 
X 
X2 
Blood Pressure 
 
X 
X 
X 
X 
X 
X 
X2 
Concomitant Medications4 
As required 
Adverse Events / Serious Adverse 
Events 
As required 

 
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4: A thorough review of any concomitant medications (including medication name, dose, unit, frequency, and route) should be performed at every visit.