A Prospective, Controlled, Double Blind, Multi-Center, Randomized, 3 Arm, Phase 1b/2a Study to Assess the Safety, Tolerability, and Preliminary Efficacy of B244 Delivered as an Intranasal Spray in Healthy Volunteers and Subjects With Seasonal Allergic Rhinitis
Phase: PHASE1
Conditions: Allergic Rhinitis, Allergic Rhinitis Due to Grass Pollen, Healthy Volunteers
Sponsor: AOBiome LLC

CONFIDENTIAL 
 
 
 
CLINICAL TRIAL PROTOCOL 
Phase 1b/2a 
 
COMPOUND: 
B244 
 
A Prospective, Controlled, Double Blind, Multi-Center, Randomized, 3 arm, Phase 
1b/2a Study to Assess the Safety, Tolerability, and Preliminary Efficacy of B244 
Delivered as an Intranasal Spray in Healthy Volunteers and Subjects with Seasonal 
Allergic Rhinitis 
 
STUDY NUMBER: ARB244-001 
VERSION DATE: August 29, 2018 
 
Sponsor: 
AOBiome LLC 
One Broadway 
14th Floor 
Cambridge, MA 02142-1100 
 
 
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)
ARB244-001 Amendment 6 (29Aug2018). AOBiome, LLC. 
1 
NCT03290248

CONFIDENTIAL 
 
Revision Chronology 
 
 
Original 
August 15, 2016 
Amendment 1 
June 5, 2017 
Amendment 2 
July 31, 2017 
Amendment 3 
September 21, 2017 
Amendment 4 
November 27, 2017 
Amendment 5 
April 9, 2018 
Amendment 6 
August 29, 2018 
 
 
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CONFIDENTIAL 
 
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 SYNOPSIS 
COMPOUND: B244  
 
 
 
STUDY NUMBER: ARB244-001 
TITLE: 
A Prospective, Controlled, Double Blind, 
Multi-Center, Randomized, 3 arm, Phase 1b/2a 
Study to Assess the Safety, Tolerability, and 
Preliminary Efficacy of B244 Delivered as an 
Intranasal Spray in Healthy Volunteers and 
Subjects with Seasonal Allergic Rhinitis 
INVESTIGATIONAL PRODUCT 
B244 
STUDY ARMS 
B244 (140ul per nostril; OD 0.5; 1x109 
cells/ml) twice-a-day for 14 days 
B244 (140ul per nostril; OD 2.0; 4x109 
cells/ml) twice-a-day for 14 days 
Vehicle (140ul per nostril) twice-a-day for 14 
days 
PURPOSE: 
To assess the safety and tolerability of B244 
relative to Vehicle in healthy volunteers and to 
evaluate the preliminary efficacy of B244 
relative to Vehicle in subjects with seasonal 
allergic rhinitis. 
OBJECTIVES: 
Primary Objective 
x To assess the safety and tolerability of 
B244 relative to vehicle in healthy 
volunteers during 14 days of treatment 
and up to 28 days of follow-up.   
 
Secondary Objective(s) 
x To evaluate the efficacy of B244 
relative to vehicle in treating Total 
Nasal Symptom Score (TNSS) in 
subjects with seasonal allergic rhinitis 
evaluated in a nasal allergen challenge 
model as a prophylaxis therapy after 14 
days of treatment. 
x To evaluate the safety and tolerability 
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of B244 relative to vehicle in subjects 
with allergic rhinitis after 14 days of 
treatment.  
x To evaluate the efficacy of B244 
relative to vehicle in treating individual 
nasal symptom scores in subjects with 
allergic rhinitis evaluated in a nasal 
allergen challenge model as a 
prophylaxis therapy after 14 days of 
treatment. 
 
Exploratory Objective  
x To test the hypothesis that B244 
inhibits airway inflammation driven by 
nasal allergen challenge. The outcome 
measures will be changes in Peak Nasal 
Inspiratory Flow, nasal inflammation 
score using otoscope, intranasal nitric 
oxide levels and cytokine concentration 
from nasal cavity pre- and post- nasal 
allergen challenge in a prophylaxis 
treatment setting. 
 
ENDPOINTS: 
Primary 
x 
Safety and tolerability will be assessed 
by reporting of AEs, physical 
examination and vital signs (blood 
pressure, heart rate, respiratory rate) 
during 14 days of treatment and up to 
28 days of follow-up (Part 1). 
 
Secondary 
x 
Change in TNSS from baseline to 14 
days of prophylaxis treatment. 
x 
Change in subjective nasal symptom 
scores of nasal congestion, rhinorrhea, 
nasal itching, and sneezing from 
baseline to 14 days of prophylaxis 
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treatment. 
x 
Nasal symptom-free response rate 
within each individual symptom scores 
and percentage of subjects with a 25% 
or 50% reduction in TNSS after 14 
days of prophylaxis treatment. 
x 
Safety and tolerability will be assessed 
in subjects with allergic rhinitis by 
reporting of AEs, physical examination 
and vital signs (blood pressure, heart 
rate, respiratory rate) during 14 days of 
treatment (Part 2). 
 
Exploratory 
x 
Change in Peak Nasal Inspiratory Flow 
(PNIF), nasal inflammation score using 
otoscope, intranasal nitric oxide levels, 
and cytokine concentration in nasal 
cavity and blood from baseline to 14 
days of B244 application and during 
prophylaxis treatment setting. 
 
STUDY DESIGN: 
This is a Prospective, Controlled, Double 
Blinded, Multi-Center, Randomized, 3 Arm, 
Parallel Assignment, Phase 1b/2a Study to 
assess the safety, tolerability, and preliminary 
efficacy of B244 delivered as an intranasal 
spray in healthy volunteers and subjects with 
seasonal allergic rhinitis. This will be a 2-part 
study.  In Part 1, safety and tolerability will be 
evaluated during 14 days of study treatment 
twice-a-day followed by 4 weeks of follow-up 
in healthy volunteers.  In Part 2, preliminary 
efficacy will be evaluated in subjects with a 
history of seasonal allergic rhinitis outside of 
the local pollen season. 
 
Part 1 (safety and tolerability evaluation in 
healthy volunteers) will enroll 24 subjects.  Up 
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to 12 subjects per dose cohort (n=8 active, n=4 
vehicle) will be enrolled, with a target of 20 
subjects completed (10 subjects per dose 
cohort; n=7 active, n=3 vehicle) assuming a 
16.7% drop out rate.   
Safety and tolerability will be assessed by 
reporting of AEs, physical examination and 
vital signs (blood pressure, heart rate, 
respiratory rate) during 14 days of treatment 
and up to 28 days of follow-up. 
 
Subjects will be asked to report for a screening 
visit and if all inclusion/exclusion criteria are 
met, subjects will be asked to report for a 
baseline visit and randomization. Subjects will 
then be trained on the use of the nasal spray, 
and have their first dose of treatment on site. 
Subjects will remain onsite for observation for 
1 hour post dosing. Subjects will then be 
dispensed study medication for 14 days of at 
home dosing. While at home, subjects will 
dose twice daily (am and pm), and record their 
dosing, any occurrence of an AE, and Total 
Nasal Symptom Score (TNSS) using a study 
diary. Subjects will be asked to visit the site at 
Day 14 (Week 2) and at follow-up visits at Day 
21 (Week 3) and Day 42 (Week 6) for safety 
assessments and will additionally obtain Peak 
Nasal Inspiratory Flow (PNIF), intranasal 
fluid, intranasal nitric oxide (nNO) and nasal 
inflammation score. 
 
During the 2 week safety phase (Part 1), a 
continuous Ambulatory Blood Pressure 
Monitoring (ABPM) device will monitor blood 
pressure of each subject for 24 hours at 
baseline and Day 14.  ABPM would be worn 
for 24 hours prior to the baseline visit and 
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again for 24 hours prior to the Day 14 visit. 
 
For Part 1, study will be paused for safety 
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adverse events considered possibly, probably, 
or definitely related to the investigational 
product.  In addition, a 180 day follow up 
contact for collecting serious adverse events 
and new onset medical conditions will be 
provided. 
 
An internal safety committee meeting will 
review the 2 week Part 1 safety data and, if 
there are no safety concerns, the study will 
proceed to Part 2 to evaluate preliminary 
efficacy in subjects with a history of seasonal 
allergic rhinitis (SAR)   
 
Part 2 (preliminary efficacy evaluation in 
subjects with a history of SAR to ragweed 
pollen) will enroll 42 subjects.  Up to 14 
subjects per arm (1x109 cells/ml; 4x109 
cells/ml or vehicle) will be enrolled, with a 
target of 12 subjects per arm for a total of 36 
subjects completed, assuming a 10-15% drop-
out rate.   
Safety and tolerability will be assessed in 
subjects with allergic rhinitis by reporting of 
AEs, physical examination and vital signs 
(blood pressure, heart rate, respiratory rate) 
during 14 days of treatment. 
 
A preliminary efficacy assessment will be 
performed in response to nasal allergen 
challenge (NAC) in a prophylaxis treatment 
setting.  
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Subjects will be asked to report for a screening 
visit (Visit 1) and if all inclusion/exclusion 
criteria are met, subjects will be asked to report 
for a NAC titration (ragweed pollen nasal 
allergen titration) visit to administer a single 
provocative challenge dose for each subject 
(Visit 2).  Subjects will then return 14 days 
after the washout period for basic health 
checks and baseline assessments (intranasal 
fluid, TNSS, PNIF, intranasal nitric oxide, and 
have a nasal inflammation score) followed by 
confirmatory NAC (NAC#1) (Visit 3). During 
this time subjects will use a study diary to 
complete TNSS at -30 (30 minutes prior to 
NAC), 0 (baseline prior to NAC), 15, 30, 45, 
60, 90, and 120 minutes. PNIF at -30, 0, 15, 
30, 60, and 120 minutes will also be obtained.  
TNSS is the sum of four individual symptom 
scores (running nose, congestion, itchy nose, 
and sneezing) rated on a four point scale (0-3).  
Subjects are required to meet the minimum 
qualifying TNSS score of a change of 6/12 
from their baseline score, on at least 2 diary 
cards. Subjects will also provide samples for 
intranasal fluid at 0, 20, and 60 minutes, 
measure intranasal nitric oxide (nNO) and have 
a nasal inflammation score assessed by the 
investigator using a nasal otoscope at 0 and 
120 minutes, in addition to blood collection at 
120 min for biomarkers.  Subjects meeting the 
requirement will be randomized to one of the 
three treatment arms.  Randomization will be 
1:1:1 so that equal number of subjects will be 
treated in each arm of the study. Subjects will 
then be trained on the use of the nasal spray, 
and have their first dose of treatment on site 
(self-dosing). Subjects will remain onsite for 
observation for 1 hour post dosing. Subjects 
will then be dispensed study medication for 14 
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days of at home dosing. While at home, 
subjects will dose twice daily (am and pm), 
and record their dosing, and any occurrence of 
an AE and TNSS using a study diary.  
 
Subjects will return to the clinic for the last 
dose following the 14 days of at home dosing 
for their final visit (Visit 4).  During this time 
subjects will again have their baseline 
assessments and be exposed to ragweed pollen 
challenge (NAC#2), and will use a study diary 
to complete TNSS at -30, 0, 15, 30, 45, 60, 90, 
and 120 minutes and PNIF at -30, 0, 15, 30, 60, 
and 120 minutes.  Prior to and 20 and 60 
minutes after NAC #2, subjects will 
additionally provide samples for intranasal 
fluid, and have nNO measured and nasal 
inflammation score assessed by the 
investigator using a nasal otoscope at 0 and 
120 min, in addition to blood collection at 120 
min for biomarkers.  
 
For Part 2, study will be paused for safety 
review using the same stopping criteria as in 
Part 1. 
 
Rescue medications will not be allowed during 
the study. 
STUDY POPULATION: 
 
Normal healthy volunteers and subjects with a 
history of seasonal allergic rhinitis to ragweed 
pollen are eligible for enrollment.  
Patients must be willing to refrain from using 
any treatments, investigational products, 
including herbal medicines while participating 
in this study.   
MAIN INCLUSION/EXCLUSION 
CRITERIA: 
Inclusion Criteria 
x Males and Females, 18 to 65 years of 
age (committed to consistent use of an 
acceptable method of birth control). 
x In good general health as determined by 
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a thorough medical history and physical 
examination, and vital signs. 
x Nonsmoker or ex-smoker (stopped >1 
year prior to study entry). 
x Subjects willing and able to provide 
written informed consent. 
x Is willing and able to comply with the 
requirements of the protocol and must 
be available for the full duration of the 
study. 
x For Part 1, subjects asymptomatic from 
any seasonal or perennial allergens. 
x For Part 1, elevated systolic blood 
pressure greater than 120 but less than 
160 and never been on antihypertensive 
medications or have been off any 
hypertensive treatment for a period of 
12 weeks or longer. 
x For Part 2, subjects with a well-
documented history of seasonal allergic 
rhinitis (specifically ragweed pollen) 
with documentation of sensitivity by 
positive skin testing and/or IgE testing 
to the relevant allergens 12 months 
prior to enrollment that correlate with 
clinical history. 
x For Part 2, a ragweed positive skin 
prick test with a wheal diameter at least 
5 mm larger than the negative control 
and/or a ragweed specific IgE greater or 
equal to 0.7 kU/L. 
x For Part 2, subjects with confounding 
allergies, or sensitization to cat 
epithelia, dog epithelia, 
Dermatophagoides farinae, or 
Dermatophagoides pteronyssinus or 
prevalent and relevant seasonal 
allergens as per the skin prick test may 
be included if sensitization is not 
clinically relevant (ie. subject is non-
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symptomatic and/or can avoid the 
allergen during the study) at the 
discretion of the Investigator. 
x For Part 2, subjects with a baseline 
TNSS  at Visit 3 and a minimum 
qualifying TNSS score of a change of 
6/12 from their baseline score after 
NAC #1 on at least 2 diary cards. 
x For Part 2, subjects’ average post 
diluents nasal congestion score must be 
1 at admission for each study visit. 
 
Exclusion Criteria 
Patients who meet any of these criteria are not 
eligible for enrollment as study participants: 
x Pregnancy or breast-feeding 
x Female of childbearing potential not 
using adequate contraceptive measures 
x Smoking within the past year or during 
the protocol. 
x Systemic corticosteroid or other 
immunosuppressive medications use in 
the previous three months or during the 
protocol. 
x Intranasal corticosteroid use in the 
previous month or during the protocol. 
x Intranasal antihistamine or cromolyn 
use in the previous week or during the 
study. 
x Allergen immunotherapy during 
previous 12 months or during the 
protocol. 
x Omalizumab use in previous 12 months 
or during the protocol. 
x Systemic antihistamine or leukotriene 
modifying medication use in the 
previous week or during the protocol. 
x Use of antibiotics, NSAIDS, 
antihypertensives, beta-blockers, 
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photosensitizing medications, or 
vitamin D supplements during study.  
Antihypertensive and beta-blocker 
exclusion applies to Part 1 only. 
x Use of any intranasally administered 
over-the-counter product or nasal 
irrigation (e.g., neti pot) during study. 
x Inability to give informed consent. 
x Persistent asthma or any medical 
condition that in the opinion of the 
investigator may compromise the 
subject’s ability to safely participate in 
the study.  
x Subjects with any significant clinical 
abnormalities which may interfere with 
study participation. 
x Prior use of AO+ Mist 
x Subjects with immunodeficiencies, 
nasal lesions, nasal polyps, or sinus 
infections. 
x Use of an investigational drug within 
30 days before screening Visit 1 
DOSE REGIMEN: 
In Part 1, after screening and recruitment, 
participants will be randomized to one of the 
following arms of the study for 2 weeks of 
treatment and 4 weeks of follow-up: 
1. B244 (140ul per nostril; OD 0.5; 1x109 
cells/ml) twice-a-day for 14 days,  
2. B244 (140ul per nostril; OD 2.0; 4x109 
cells/ml) twice-a-day for 14 days 
3. Vehicle (140ul per nostril) twice-a-day for 
14 days 
In Part 2, subjects with a history of SAR to 
ragweed pollen will be enrolled to one of the 
following arms: 
1. B244 (140ul per nostril; OD 0.5; 1x109 
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cells/ml) twice-a-day for 14 days, 
2. B244 (140ul per nostril; OD 2.0; 4x109 
cells/ml) twice-a-day for 14 days, 
3. Vehicle (140ul per nostril) twice-a-day for 
14 days, 
 
Subjects will undergo nasal allergen challenge, 
followed by one-time treatment in clinic on 
Day 0, followed by twice-a-day in-home 
treatment for 14 days, followed by one 
additional treatment in clinic on Day 14 prior 
to second nasal allergen challenge. 
 
ASSESSMENT SCHEDULE: 
In Part 1, study assessments will occur at 
Baseline and Week 2 (Day 14) visit.  Follow-
up visit will be at Week 3 (Day 21) and Week 
6 (Day 42) after treatment to perform safety 
assessments and clinical examination of the 
nasal mucosa, sinuses, and upper airway.   
In Part 2, study assessments will occur at NAC 
titration visit (Day 0), NAC#1 visit (Day 14) 
and NAC#2 visit (Day 28).  Preliminary 
efficacy assessments will be after nasal 
allergen challenge in a prophylaxis treatment 
setting. 
STATISTICAL CONSIDERATIONS: 
Intent to treat analysis 
INVESTIGATIONAL DRUG AND 
PLACEBO: 
Investigational drug refers to B244. 
Investigational Product/treatment refers to 
either B244 or Vehicle.  
PLANNED DURATION PER SUBJECT: 
Up to 9 weeks 
DURATION OF STUDY: 
First subject first visit: Sep 2017 
Study readout: Mar 2018 
 
 
 
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Part 1:  
Normal Healthy Volunteers 
Cohort 1: 
Active B244 1x: Vehicle  
(2:1) 14 days BID 
Treatment and Study Assessments at: 
Baseline, Treatment (Day 14) 
Cohort 2: 
Active B244 4x: Vehicle  
(2:1) 14 days BID 
1 
STUDY SCHEMA 
 
 
 
Screening visit 
Baseline visit 
Randomize
Follow up visits Day 21 and Day 42 
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Vehicle (14 
days BID) 
B244 1x (14 
days BID) 
B244 4x (14 
days BID) 
NAC #2 
Part 2: 
Male and Female subjects with a history of seasonal allergic rhinitis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study assessments at baseline (Day 14) and 2 weeks (Day 28)  
Nasal allergen challenge (NAC) (Titration) 
NAC #1 
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ABPM 
Ambulatory Blood Pressure Monitoring 
AE 
Adverse Event 
AMO 
Ammonia Monooxygenase 
AOB 
Ammonia Oxidizing Bacteria 
BID 
Twice-Daily 
CBC 
Complete Blood Count 
CRF 
Case Report Form 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
ECG 
Electrocardiogram 
E/T 
Early Termination 
FDA 
Food and Drug Administration 
HAO 
NH2OH oxidoreductase 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IP 
Investigational Product 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
MedDRA 
Medical Dictionary for Regulatory Activities 
NAC 
Nasal Allergen Challenge 
NH2OH 
Hydroxylamine 
NH3 
Ammonia 
NO 
Nitric Oxide 
NO2- 
Nitrite 
PNIF 
Peak Nasal Inspiratory Flow 
PT 
Preferred Term 
SAE 
Serious Adverse Event 
SPM 
Study Procedures Manual 
SOC 
System Organ Class 
TEAE 
Treatment Emergent Adverse Event 
TNSS 
Total Nasal Symptom Score 
WOCBP 
Women of Child Bearing Potential 
 
 
 
 
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Table of Contents 
1 
STUDY SCHEMA ............................................................................................................ 17 
2 
BACKGROUND AND RATIONALE .................................................................................... 24 
2.1 
Background ....................................................................................................................... 24 
3 
OBJECTIVES .................................................................................................................. 26 
3.1 
Primary Objective.............................................................................................................. 26 
3.2 
Secondary Objective(s) ...................................................................................................... 26 
3.3 
Exploratory ....................................................................................................................... 26 
4 
ENDPOINTS .................................................................................................................. 26 
4.1 
Primary ............................................................................................................................. 26 
4.2 
Secondary ......................................................................................................................... 27 
4.3 
Exploratory ....................................................................................................................... 27 
5 
STUDY DESIGN .............................................................................................................. 27 
6 
SELECTION OF STUDY PARTICIPANTS ............................................................................. 30 
6.1 
Study Population ............................................................................................................... 30 
6.2 
Number of Participants Planned ........................................................................................ 30 
6.3 
Inclusion Criteria ............................................................................................................... 30 
6.4 
Exclusion Criteria ............................................................................................................... 31 
7 
PARTICIPANT ENROLLMENT .......................................................................................... 32 
7.1 
Consenting Participants ..................................................................................................... 32 
7.2 
Screening for Eligibility ...................................................................................................... 32 
7.3 
Study Withdrawal and Withdrawal from Investigational Product and Stopping Criteria ...... 33 
7.4 
Screen Failures .................................................................................................................. 34 
7.5 
Early Termination .............................................................................................................. 34 
8 
STUDY TREATMENT ...................................................................................................... 34 
8.1 
Investigational Product...................................................................................................... 34 
8.2 
Dose Changes .................................................................................................................... 35 
8.3 
Storage Conditions ............................................................................................................ 35 
8.4 
Description of Blinding Method ......................................................................................... 36 
8.5 
Treatment Assignments: .................................................................................................... 36 
8.6 
Treatment Compliance ...................................................................................................... 36 
8.7 
Treatment Application ...................................................................................................... 36 
8.8 
Treatment of Investigational Product Overdose ................................................................. 38 
8.9 
Product Accountability ...................................................................................................... 38 
8.10 
Unblinding Procedures ...................................................................................................... 38 
8.11 
Retrieval and Destruction of Investigational Product .......................................................... 39 
8.12 
Permitted Medications ...................................................................................................... 39 
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8.13 
Prohibited Medications ..................................................................................................... 39 
8.14 
Rescue Medications .......................................................................................................... 39 
8.15 
Handling of Investigational Product ................................................................................... 39 
9 
CONTRACEPTION REQUIREMENTS ................................................................................ 40 
10 
STUDY PROCEDURES ................................................................................................. 41 
10.1 
Screening .......................................................................................................................... 41 
10.2 
Informed Consent Procedures ........................................................................................... 41 
10.3 
Study Assessments ............................................................................................................ 41 
10.4 
Inclusion Procedures ......................................................................................................... 42 
10.5 
Description by Type of Visit – Part 1 ................................................................................... 42 
10.5.1 
Screening Visit (Visit 1; Study Day -1) (-21 to 0 Days Visit Window) ............................. 43 
10.5.2 
Baseline ABPM Placement (-3 to 0 Days Visit Window) .................................................. 43 
10.5.3 
Baseline Visit (Visit 2; Study Day 0) (-3 to 1 Days Visit Window) ................................... 43 
10.5.4 
Baseline Repeat ABPM Placement (if Necessary) (-3 to 1 Days Visit Window) ............... 44 
10.5.5 
Week 2 ABPM Placement (±3 Days Visit Window) .......................................................... 44 
10.5.6 
Week 2 Visit (Visit 3; Study Day 14) (±3 Days Visit Window) ......................................... 44 
10.5.7 
Week 2 Repeat ABPM Placement (if Necessary) (±3 Days Visit Window) ...................... 45 
10.5.8 
Follow-up #1 Visit (Visit 4; Study Day 21) (±3 Days Visit Window) ............................... 45 
10.5.9 
Follow-up #2 Visit (Visit 5; Study Day 42) (±3 Days Visit Window) ............................... 46 
10.6 
Description by Type of Visit – Part 2 ................................................................................... 46 
10.6.1 
Screening Visit (Visit 1; Study Day -1) (-21 to 0 Days Visit Window) ............................. 46 
10.6.2 
NAC Titration Visit (Visit 2; Study Day 0) (±3 Days Visit Window)................................ 47 
10.6.3 
NAC #1 Visit (Visit 3; Study Day 14) (±3 Days Visit Window) ....................................... 47 
10.6.4 
NAC #2 Visit (Visit 4; Study Day 28) (±3 Days Visit Window) ....................................... 47 
10.6.5 
Unscheduled/Unanticipated Study Visit ............................................................................. 48 
11 
METHODS OF ASSESSMENTS ..................................................................................... 49 
11.1 
Blood Pressure Measurement ............................................................................................ 49 
11.2 
Physical Examinations ....................................................................................................... 49 
11.3 
Allergen Skin Test (Skin Prick Test) ..................................................................................... 49 
11.4 
Blood Collection for IgE ..................................................................................................... 49 
11.5 
Ambulatory Blood Pressure Monitoring (ABPM) ................................................................ 50 
11.5.1 
ABPM Monitoring Schedule .............................................................................................. 50 
11.5.2 
Office Visit .......................................................................................................................... 50 
11.5.3 
ABPM Test ......................................................................................................................... 51 
11.5.4 
ABPM Technical Success/Failure Criteria ......................................................................... 51 
11.5.5 
Repeat ABPM ..................................................................................................................... 51 
11.6 
Nasal Inspection ................................................................................................................ 51 
11.7 
Nasal Allergen Challenge (NAC) ......................................................................................... 51 
11.8 
12-Lead ECG ...................................................................................................................... 53 
11.9 
Laboratory Assessments .................................................................................................... 54 
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11.10 
Urine Pregnancy Test ..................................................................................................... 54 
11.11 
Nasal Fluid Samples for Biomarkers................................................................................ 54 
11.12 
Blood Collection for Biomarkers ..................................................................................... 55 
11.13 
Peak Nasal Inspiratory Flow (PNIF) ................................................................................. 56 
11.14 
Intranasal Nitric Oxide ................................................................................................... 56 
11.15 
Nasal Inflammation Score .............................................................................................. 56 
11.16 
Total Nasal Symptom Score (TNSS) ................................................................................. 57 
11.17 
Study Diary .................................................................................................................... 58 
11.18 
Sample Shipment .......................................................................................................... 58 
12 
SAFETY ASSESSMENTS ............................................................................................... 58 
12.1 
Compliance ....................................................................................................................... 58 
12.2 
Safety Monitoring ............................................................................................................. 59 
12.3 
Pregnancy Reporting ......................................................................................................... 59 
12.4 
Study Completion .............................................................................................................. 60 
12.5 
Internal Safety Committee ................................................................................................. 60 
12.6 
Subject Withdrawal Criteria ............................................................................................... 60 
13 
EFFICACY ASSESSMENTS ............................................................................................ 61 
14 
STATISTICAL CONSIDERATIONS .................................................................................. 61 
14.1 
Sample Size ....................................................................................................................... 61 
14.2 
Populations for Analysis .................................................................................................... 61 
14.3 
Data Analysis .................................................................................................................... 62 
14.3.1 
Disposition .......................................................................................................................... 62 
14.3.2 
Demographic and Baseline ................................................................................................. 62 
14.4 
Safety Analyses ................................................................................................................. 62 
14.4.1 
Definitions ........................................................................................................................... 63 
14.4.2 
Adverse Events ................................................................................................................... 63 
14.4.3 
Deaths and Serious Adverse Events .................................................................................... 63 
14.4.4 
Adverse Events Leading to Treatment Discontinuation ..................................................... 63 
14.5 
Efficacy Analyses ............................................................................................................... 64 
14.5.1 
Handling of Dropouts or Missing Data ............................................................................... 64 
14.6 
Clinical Trial Protocol Deviations ........................................................................................ 64 
15 
ADVERSE EVENTS (AE) AND SERIOUS ADVERSE EVENTS (SAE) .................................... 64 
15.1 
Definition of an AE ............................................................................................................ 64 
15.2 
Definition of a SAE ............................................................................................................ 65 
15.3 
Time Period, Frequency, and Method of Detecting AEs and SAEs ........................................ 65 
15.4 
Recording of AEs and SAEs ................................................................................................. 66 
15.5 
Evaluating AEs and SAEs .................................................................................................... 66 
15.5.1 
Severity Rating .................................................................................................................... 66 
15.5.2 
Relationship to Investigational Product (IP) ....................................................................... 67 
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15.6 
Pregnancy ......................................................................................................................... 67 
15.7 
Prompt Reporting of SAEs to the Sponsor .......................................................................... 68 
16 
ETHICAL AND REGULATORY STANDARDS ................................................................... 69 
16.1 
Ethical Conduct of Study .................................................................................................... 69 
16.2 
Laws and Regulations ........................................................................................................ 69 
16.3 
Informed Consent ............................................................................................................. 69 
16.4 
Institutional Review Board/Independent Ethics Committee (IRB/EC) .................................. 70 
16.5 
Clinical Monitoring/Record Keeping ................................................................................... 70 
17 
ADMINISTRATIVE RULES ............................................................................................ 71 
17.1 
Curriculum Vitae ............................................................................................................... 71 
17.2 
Archiving of Study Documentation..................................................................................... 71 
17.3 
Internal Safety Review Committee ..................................................................................... 72 
18 
STUDY MONITORING ................................................................................................. 72 
18.1 
Responsibilities of the Investigator(s) ................................................................................ 72 
18.2 
Responsibilities of the Sponsor .......................................................................................... 72 
18.3 
Source Document Requirements ........................................................................................ 73 
18.4 
Use and Completion of Case Report Forms (CRFs) and Additional Requests ........................ 73 
19 
PUBLICATIONS .......................................................................................................... 74 
20 
PROTOCOL ADHERENCE............................................................................................. 74 
21 
CONFIDENTIALITY ...................................................................................................... 74 
22 
PROPERTY RIGHTS ..................................................................................................... 75 
23 
DATA PROTECTION .................................................................................................... 75 
24 
SPONSOR AUDITS AND INSPECTIONS BY REGULATORY AGENCIES .............................. 75 
25 
PREMATURE DISCONTINUATION OF THE STUDY OR PREMATURE CLOSE-OUT OF A SITE
 
76 
25.1 
Decided by the Sponsor in the Following Cases: ................................................................. 76 
25.2 
Decided by the Investigator ............................................................................................... 76 
26 
CLINICAL TRIAL PROTOCOL AMENDMENTS ................................................................ 77 
27 
APPENDIX A-Schedule of Events ................................................................................ 77 
28 
REFERENCES .............................................................................................................. 82 
 
 
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2 
BACKGROUND AND RATIONALE 
2.1 
Background 
Allergic rhinitis (AR) is an upper airway inflammatory disease, characterized by symptoms of 
rhinorrhea, sneezing, and nasal congestion.  Although AR is often considered a benign condition, 
it greatly impacts the quality of life of affected individuals, with economic costs estimated at 
$5.3 billion per year in the USA (1,2).  World-wide prevalence of AR is estimated at 9% to 42% 
(3,4).  A variety of treatment approaches exist, including anti-histamines, corticosteroids, and 
immunotherapies, but have associated side effects and variable responses that warrant alternative 
options that exhibit improved safety and efficacy profiles.  None of the current anti-allergic and 
anti-inflammatory interventions is designed to address a microbiome based approach to restore 
colonization of a human commensal bacteria to the nasal microbiome to regulate its anti-
inflammatory and anti-infective properties.  Intranasal administration of ammonia oxidizing 
bacteria maybe beneficial for the treatment of a variety of local and systemic indications through 
its anti-inflammatory and anti-infective properties.  The objective of this study is to evaluate the 
safety of intranasally delivered ammonia oxidizing bacteria (AOB) in healthy volunteers and 
subsequently to evaluate preliminary efficacy of AOB as a prophylaxis treatment in response to a 
ragweed pollen nasal allergen challenge (NAC) in subjects with a history of seasonal allergic 
rhinitis (SAR).    
Ammonia oxidizing bacteria 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 (5). 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 (6).  
 
Figure 1 
Nitrifier Denitrification Pathway 
 
 
 
 
 
 
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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 bacteria, 
anti-inflammatory activity, and inhibition of lipogenesis by insulin-stimulated immortal 
sebocytes (7-9). 
To date, there have been no reported infections or health risks associated with topical application 
or ingestion of Nitrosomonas species.  The absence of any illnesses attributed to these bacteria 
despite our widespread exposure indicates that they pose a minimal health risk, if any at all.  
Infection or tissue damage by Nitrosomonas is unlikely, because the sequenced genomes of 
several Nitrosomonas and other AOB lack genes encoding cytotoxins, or other known bacterial 
virulence factors.  Further, AOB are slow growing, as compared to most heterotrophic bacteria, 
with optimum doubling times of 8 hours or higher.  In particular, Nitrosomonas growth is rate 
limited by the availability of ammonia requiring the oxidation of 27 moles NH3/mole CO2 fixed.  
Due to their dependence on ammonia for their growth, the numbers of Nitrosomonas on the skin 
will be necessarily limited and naturally regulated by the amount of ammonia produced in sweat. 
This would ensure that the amount of nitrite and NO generated would be relatively low, without 
any adverse effects. 
Currently, Nitrosomonas eutropha D23 in buffer at a concentration of 1x109 cells/mL is 
available as a cosmetic product for topical application of a natural source of AOB and NO/NO2 
to improve the appearance of human skin. AOBiome plans to study Nitrosomonas eutropha D23 
diluted in storage solution (50 mM Na2HPO4 and 2 mM MgCl2, pH 7.6) at defined doses to be 
provided as an intranasal spray (B244) for therapeutic applications.  
B244 is being developed under IND #16487 as a ‘live topical’ to provide a natural source of 
AOB and NO/NO2 to the human skin.  Under IND #16487, a phase 1b/2a clinical trial was 
completed  in 2016 where 36 participants with clinical diagnosis of facial acne vulgaris were 
randomized to receive ascending doses of investigational product (IP) over 14 days. Safety 
analyses have been completed and there have been no attributable drug related SAEs reported.  
In addition, a phase 2b/3 clinical trial in 372 patients with clinical diagnosis of facial acne is 
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currently ongoing. To date, there have been no attributable drug related SAEs reported in this 
trial.  
The proposed clinical study is designed to evaluate the safety of intranasally delivered AOB in 
healthy volunteers and subsequently to evaluate preliminary efficacy of AOB as a prophylaxis 
treatment in response to a ragweed pollen nasal allergen challenge in subjects with a history of 
seasonal allergic rhinitis. 
 
3 
OBJECTIVES 
3.1 
Primary Objective 
x To assess the safety and tolerability of B244 relative to vehicle in healthy volunteers 
during 14 days of treatment and up to 28 days of follow-up.  Safety and tolerability will 
be assessed by reporting of AEs, physical examination and vital signs (blood pressure, 
heart rate, respiratory rate).   
 
3.2 
Secondary Objective(s) 
x To evaluate the efficacy of B244 relative to vehicle in treating Total Nasal Symptom 
Score (TNSS) in subjects with seasonal allergic rhinitis evaluated in a nasal allergen 
challenge model as a prophylaxis therapy after 14 days of treatment.  
x To evaluate the safety and tolerability of B244 relative to vehicle in subjects with allergic 
rhinitis after 14 days of treatment. Safety and tolerability will be assessed by reporting of 
AEs, physical examination and vital signs (blood pressure, heart rate, respiratory rate). 
x To evaluate the efficacy of B244 relative to vehicle in treating individual nasal symptom 
scores in subjects with allergic rhinitis evaluated in a nasal allergen challenge model as a 
prophylaxis therapy after 14 days of treatment. 
 
3.3 
Exploratory 
x To test the hypothesis that B244 inhibits airway inflammation driven by nasal allergen 
challenge. The outcome measures will be changes in Peak Nasal Inspiratory Flow, nasal 
inflammation score using otoscope, intranasal nitric oxide levels and cytokine 
concentration from nasal cavity pre- and post- nasal allergen challenge in a prophylaxis 
treatment setting. 
 
4 
ENDPOINTS 
4.1 
Primary 
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x Safety and tolerability will be assessed by reporting of AEs, physical examination and 
vital signs (blood pressure, heart rate, respiratory rate) during 14 days of treatment and up 
to 28 days of follow-up (Part 1). 
 
4.2 
Secondary 
x 
Change in TNSS from baseline to 14 days of prophylaxis treatment. 
x 
Change in subjective nasal symptom scores of nasal congestion, rhinorrhea, nasal itching, 
and sneezing from baseline to 14 days of prophylaxis treatment. 
x 
Nasal symptom-free response rate within each individual symptom scores and percentage 
of subjects with a 25% or 50% reduction in TNSS after 14 days of prophylaxis treatment. 
x 
Safety and tolerability will be assessed in subjects with allergic rhinitis by reporting of 
AEs, physical examination and vital signs (blood pressure, heart rate, respiratory rate) 
during 14 days of treatment (Part 2). 
4.3 
Exploratory 
x 
Change in Peak Nasal Inspiratory Flow (PNIF), nasal inflammation score using otoscope, 
intranasal nitric oxide levels, and cytokine concentration in nasal cavity and blood from 
baseline to 14 days of B244 application and during prophylaxis treatment setting. 
5 
STUDY DESIGN 
x The purpose of this study is to assess the safety and tolerability of B244 relative to 
Vehicle in healthy volunteers and to evaluate the preliminary efficacy of B244 relative to 
Vehicle in subjects with seasonal allergic rhinitis. 
x This is a Prospective, Controlled, Double Blinded, Multi-Center, Randomized, 3 Arm, 
Parallel Assignment, Phase 1b/2a Study to assess the safety, tolerability, and preliminary 
efficacy of B244 delivered as an intranasal spray in healthy volunteers and subjects with 
seasonal allergic rhinitis. This will be a 2-part study.   
o In Part 1, safety and tolerability will be evaluated during 14 days of study 
treatment twice-a-day followed by 4 weeks of follow-up in healthy volunteers.   
o In Part 2, preliminary efficacy will be evaluated in subjects with a history of 
seasonal allergic rhinitis outside of the local pollen season. 
x Subjects will be recruited to participate and randomized 1:1:1 to the following 
interventions: 
1. B244 (140ul per nostril; OD 0.5; 1x109 cells/ml) twice-a-day for 14 days  
2. B244 (140ul per nostril; OD 2.0; 4x109 cells/ml) twice-a-day for 14 days 
3. Vehicle (140ul per nostril) twice-a-day for 14 days 
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x Part 1 (safety and tolerability evaluation in healthy volunteers) will enroll 24 subjects.  
Up to 12 subjects per dose cohort (n=8 active, n=4 vehicle) will be enrolled, with a target 
of 20 subjects completed (10 subjects per dose cohort; n=7 active, n=3 vehicle) assuming 
a 16.7% drop out rate. 
x Safety and tolerability will be assessed by reporting of AEs, physical examination and 
vital signs (blood pressure, heart rate, respiratory rate) during 14 days of treatment and up 
to 28 days of follow-up. 
x Subjects will be asked to report for a screening visit and if all inclusion/exclusion criteria 
are met, subjects will be asked to report for a baseline visit and randomization. Subjects 
will then be trained on the use of the nasal spray, and have their first dose of treatment on 
site. Subjects will remain onsite for observation for 1 hour post dosing. Subjects will then 
be dispensed study medication for 14 days of at home dosing. While at home, subjects 
will dose twice daily (am and pm), and record their dosing, any occurrence of an AE, and 
Total Nasal Symptom Score (TNSS) using a study diary. Subjects will be asked to visit 
the site at Day 14 (Week 2) and at follow-up visits at Day 21 (Week 3) and Day 42 
(Week 6) for safety assessments and will additionally record Peak Nasal Inspiratory Flow 
(PNIF), intranasal fluid, intranasal nitric oxide (nNO) and nasal inflammation score. 
x During the 2 week safety phase (Part 1), a continuous Ambulatory Blood Pressure 
Monitoring (ABPM) device will monitor blood pressure of each subject for 24 hours at 
baseline and Day 14.  ABPM would be worn for 24 hours prior to the baseline visit and 
again for 24 hours prior to the Day 14 visit. 
x Safety monitoring will include infectious complications related to B244 (by symptoms 
and/or testing; pulmonary, nasal, neurological), local and systemic AEs related to 
intranasal route during treatment and at follow-up.  Local AEs include runny nose, nasal 
congestion, sneezing, nasal itching, palate itching, anosmia, nasal ulceration, nasal 
bleeding, sore throat, cough, Bell’s palsy, other neurologic complications, fevers, chills, 
headache, muscle aches, decreased appetite, nausea, vomiting, and rash. 
x Sponsor will distinguish between immediate (onset of reaction is during the first 30 
minutes after administration) and delayed (onset of action is after the first 30 minutes of 
administration) effects for safety reporting of adverse events. 
x For Part 1, study will be paused for safety review LQWKHHYHQWWKDWVXEM
VHULRXVDGYHUVHHYHQWJUDGHVHYHUHDGYHUVH
adverse events considered possibly, probably, or definitely related to the investigational 
product.  In addition, a 180 day follow up contact for collecting serious adverse events 
and new onset medical conditions will be provided. 
x An internal safety committee meeting will review the 2 week Part 1 safety data and, if 
there are no safety concerns, the study will proceed to Part 2 to evaluate preliminary 
efficacy in subjects with a history of seasonal allergic rhinitis (SAR). 
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x Part 2 (preliminary efficacy evaluation in subjects with a history of SAR to ragweed 
pollen) will enroll 42 subjects.  Up to 14 subjects per arm (1x109 cells/ml; 4x109 cells/ml 
or vehicle) will be enrolled, with a target of 12 subjects per arm for a total of 36 subjects 
completed, assuming a 10-15% drop-out rate. 
x Safety and tolerability will be assessed in subjects with allergic rhinitis by reporting of 
AEs, physical examination and vital signs (blood pressure, heart rate, respiratory rate) 
during 14 days of treatment. 
x A preliminary efficacy assessment will be performed in response to nasal allergen 
challenge (NAC) in a prophylaxis treatment setting. 
x Subjects will be asked to report for a screening visit (Visit 1) and if all 
inclusion/exclusion criteria are met, subjects will be asked to report for a NAC titration 
(ragweed pollen nasal allergen titration) visit to administer a single provocative challenge 
dose for each subject (Visit 2).  Subjects will then return 14 days after the washout period 
for basic health checks and baseline assessments (intranasal fluid, TNSS, PNIF, intranasal 
nitric oxide, and have a nasal inflammation score) followed by confirmatory NAC 
(NAC#1) (Visit 3). During this time subjects will use a study diary to complete TNSS at -
30 (30 minutes prior to NAC), 0 (baseline prior to NAC), 15, 30, 45, 60, 90, and 120 
minutes. PNIF at -30, 0, 15, 30, 60, and 120 minutes will also be obtained.  TNSS is the 
sum of four individual symptom scores (running nose, congestion, itchy nose, and 
sneezing) rated on a four point scale (0-3).  Subjects are required to meet the minimum 
qualifying TNSS score of a change of 6/12 from their baseline score, on at least 2 diary 
cards. Subjects will also provide samples for intranasal fluid at 0, 20, and 60 minutes, 
measure intranasal nitric oxide (nNO) and have a nasal inflammation score assessed by 
the investigator using a nasal otoscope at 0 and 120 minutes, in addition to blood 
collection at 120 min for biomarkers.  Subjects meeting the requirement will be 
randomized to one of the three treatment arms.  Randomization will be 1:1:1 so that equal 
number of subjects will be treated in each arm of the study. Subjects will then be trained 
on the use of the nasal spray, and have their first dose of treatment on site (self-dosing). 
Subjects will remain onsite for observation for 1 hour post dosing. Subjects will then be 
dispensed study medication for 14 days of at home dosing. While at home, subjects will 
dose twice daily (am and pm), and record their dosing, and any occurrence of an AE and 
TNSS using a study diary. 
x Subjects will return to the clinic for the last dose following the 14 days of at home dosing 
for their final visit (Visit 4).  During this time subjects will again have their baseline 
assessments and be exposed to ragweed pollen challenge (NAC#2), and will use a study 
diary to complete TNSS at -30, 0, 15, 30, 45, 60, 90, and 120 minutes and PNIF at -30, 0, 
15, 30, 60, and 120 minutes.  Prior to and 20 and 120 minutes after NAC #2, subjects will 
additionally provide samples for intranasal fluid, and have nNO measured and have a 
nasal inflammation score assessed by the investigator using a nasal otoscope at 0 and 120 
min, in addition to blood collection at 120 min for biomarkers. 
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x For Part 2, study will be paused for safety review using the same stopping criteria as in 
Part 1. 
x Rescue medications will not be allowed during the study. 
6 
SELECTION OF STUDY PARTICIPANTS 
6.1 
Study Population 
Normal healthy volunteers and subjects with a history of seasonal allergic rhinitis to ragweed 
pollen are eligible for enrollment.  Patients must be willing to refrain from using any treatments, 
investigational products, including herbal medicines while participating in this study. 
6.2 
Number of Participants Planned 
Part 1 (safety and tolerability evaluation in healthy volunteers) will enroll 24 subjects.  Up to 12 
subjects per dose cohort (n=8 active, n=4 vehicle) will be enrolled, with a target of 20 subjects 
completed (10 subjects per dose cohort; n=7 active, n=3 vehicle) assuming a 16.7% drop out 
rate. 
 
Part 2 (preliminary efficacy evaluation in subjects with a history of SAR to ragweed pollen) will 
enroll 42 subjects.  Up to 14 subjects per arm (1x109 cells/ml; 4x109 cells/ml or vehicle) will be 
enrolled, with a target of 12 subjects per arm for a total of 36 subjects completed, assuming a 10-
15% drop-out rate. 
 
6.3 
Inclusion Criteria 
x Males and Females, 18 to 65 years of age (committed to consistent use of an acceptable 
method of birth control as described in Section 10). 
x In good general health as determined by a thorough medical history and physical 
examination, and vital signs. 
x Nonsmoker or ex-smoker (stopped >1 year prior to study entry). 
x Subjects willing and able to provide written informed consent. 
x Is willing and able to comply with the requirements of the protocol and must be available 
for the full duration of the study. 
x For Part 1, subjects asymptomatic from any seasonal or perennial allergens. 
x For Part 1, elevated systolic blood pressure greater than 120 but less than 160 and never 
been on antihypertensive medications or have been off any hypertensive treatment for a 
period of 12 weeks or longer. 
x For Part 2, subjects with a well-documented history of seasonal allergic rhinitis 
(specifically ragweed pollen) with documentation of sensitivity by positive skin testing 
and/or IgE testing to the relevant allergens 12 months prior to enrollment that correlate 
with clinical history. 
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x For Part 2, a ragweed positive skin prick test with a wheal diameter at least 5 mm larger 
than the negative control and/or a ragweed specific IgE greater or equal to 0.7 kU/L. 
x For Part 2, subjects with confounding allergies, or sensitization to cat epithelia, dog 
epithelia, Dermatophagoides farinae, or Dermatophagoides pteronyssinus or prevalent 
and relevant seasonal allergens as per the skin prick test may be included if sensitization 
is not clinically relevant (ie. subject is non-symptomatic and/or can avoid the allergen 
during the study) at the discretion of the Investigator. 
x For Part 2, subjects with a baseline TNSS  at Visit 3 and a minimum qualifying 
TNSS score of a change of 6/12 from their baseline score after NAC #1 on at least 2 diary 
cards. 
x For Part 2, subjects’ average post diluents nasal congestion score must be  1 at 
admission for each study visit. 
 
6.4 
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 
 
Patients who meet any of these criteria are not eligible for enrollment as study participants: 
x Pregnancy or breast-feeding 
x Female of childbearing potential not using adequate contraceptive measures. 
x Smoking within the past year or during the protocol. 
x Systemic corticosteroid or other immunosuppressive medications use in the previous 
three months or during the protocol.* 
x Intranasal corticosteroid use in the previous month or during the protocol.* 
x Intranasal antihistamine or cromolyn use in the previous week or during the study.* 
x Allergen immunotherapy during previous 12 months or during the protocol.* 
x Omalizumab use in previous 12 months or during the protocol.* 
x Systemic antihistamine or leukotriene modifying medication use in the previous week or 
during the protocol.* 
x Use of antibiotics, NSAIDS, antihypertensives, beta-blockers, photosensitizing 
medications, or vitamin D supplements during study.  Antihypertensive and beta-blocker 
exclusion applies to Part 1 only. 
x Use of any intranasally administered over-the-counter product or nasal irrigation (e.g., 
neti pot) during study. 
x Inability to give informed consent. 
x Persistent asthma or any medical condition that in the opinion of the investigator may 
compromise the subject’s ability to safely participate in the study.  
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x Subjects with any significant clinical abnormalities which may interfere with study 
participation. 
x Prior use of AO+ Mist. 
x Subjects with immunodeficiencies, nasal lesions, nasal polyps, or sinus infections. 
x Use of an investigational drug within 30 days before screening Visit 1. 
* Exclusionary criterion due to 1) medication use as marker of persistent comorbid allergic or 
inflammatory condition that may increase subject risk with study participation, 2) potential of 
medication to interfere with study outcome measures or results. 
 
7 
PARTICIPANT ENROLLMENT 
7.1 
Consenting Participants 
Informed consent for participation in the study must be obtained before performing any study-
specific procedures.  
Informed Consent Forms for enrolled participants and for participants who are not subsequently 
enrolled will be maintained at the study sites in secure study files. Consent will be obtained by 
trained research study staff trained in taking informed consent. The study will be explained with 
the opportunity for the participant to ask questions. If a participant wishes to enter the study, a 
consent form will be completed and signed. 
7.2 
Screening for Eligibility 
For Part 1, after informed consent has been obtained, screening assessments (Visit 1) will be 
performed within 3 weeks (-21 days to 0) prior to the Baseline visit (Visit 2) to determine 
participant eligibility for enrollment in the study.  All screening evaluations must be completed 
and reviewed to confirm that participants meet all eligibility criteria before randomization on 
Day 0 (Visit 2). Inclusion and exclusion criteria will be reviewed at Screening (Visit 1) and 
Baseline visit (Visit 2) to make sure nothing changed.  The participant’s medical history and 
medication use will be reviewed. A brief physical examination including height, weight, vital 
signs, nasal inspection, and 12-lead ECG will be performed.  Laboratory testing, including blood 
sample for a comprehensive metabolic panel, complete blood count, chemistry, lipid panel, and 
liver function test, as well as urinalysis, will also be performed as part of screening.  Urine 
pregnancy test will be performed on WOCBP. 
For Part 2, after informed consent has been obtained, screening assessments (Visit 1) will be 
performed within 3 weeks (-21 days to 0) prior to the NAC titration (Visit 2) and NAC #1 (Visit 
3) visits to determine participant eligibility for enrollment in the study.  All screening evaluations 
must be completed and reviewed to confirm that participants meet all eligibility criteria before 
randomization on Day 14 (Visit 3). Inclusion and exclusion criteria will be reviewed at 
Screening (Visit 1).  The participant’s medical history and medication use will be reviewed. A 
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brief physical examination including height, weight, vital signs, and nasal inspection, will be 
performed.  Laboratory testing, including blood sample for a comprehensive metabolic panel, 
complete blood count, chemistry, lipid panel, and liver function test, as well as urinalysis, will 
also be performed as part of screening.  Urine pregnancy test will be performed on WOCBP.  
Additionally, ragweed pollen allergen skin test (skin prick test) and/or blood collection for 
ragweed pollen specific IgE will be performed as part of screening if documentation of a positive 
test within the past 12 months for either method cannot be provided. 
All screening assessments are listed in the Schedule of Events Table (Appendix A).  A 
participant must meet all inclusion criteria, and none of the exclusion criteria, to be enrolled and 
randomized in this study. The Investigator and team will maintain a screening log to record 
details of all persons screened and to confirm eligibility or record reasons for screening failure, 
as applicable. 
 
7.3 
Study Withdrawal and Withdrawal from Investigational Product and Stopping 
Criteria  
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).  
 
The reason for participant study withdrawal will be recorded in the electronic Case Report Form 
(eCRF). Data from participants withdrawing from the study will be considered evaluable up to 
the point at which they are withdrawn using the same criteria for evaluability as for participants 
who complete the study. 
For Part 1 safety evaluation and Part 2 preliminary efficacy , AOBiome will include study 
stopping rules to pause study for safety review in the event that t1 subject reports a SAE, t2 
grade 3 (severe) AEs, or t3 grade 2 (moderate) AEs considered possibly, probably, or definitely 
related to the investigational product. 
 
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7.4 
Screen Failures 
Data for screen and baseline failures will be collected in source documentation at the sites 
but will not be transmitted to AOBiome. 
 
7.5 
Early Termination 
Participants who have discontinued the study early will be evaluated by the Investigator at the 
Unanticipated Visit.  See the list of assessments to be performed at the Unanticipated Visit in the 
Schedule of 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.  
 
8 
STUDY TREATMENT 
8.1 
Investigational Product 
Under normal conditions of handling and administration, IP is not expected to pose 
significant safety risks to site staff. A Material Safety Data Sheet describing the 
occupational hazards and recommended handling precautions will be provided to site 
staff if required by local laws or will otherwise be available from the Sponsor upon request. 
 
Investigational product must be stored in a secure area under the appropriate physical 
conditions for the product. 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 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 
B244,  
30ml/bottle 
Placebo, 30ml/bottle 
Dosage form: 
B244 suspension 
B244 suspension 
Vehicle solution 
Unit dose strength: 
1x109 cells/ml 
4x109 cells/ml 
50nM Na2HPO4-
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2mM MgCl2 (pH 7.6) 
Route/administration/duration: Intranasal application:  
Part 1: 1 pump 
(140ul) per nostril 
BID for 14 days  
Part 2: 1 pump 
(140ul) per nostril 
BID for 14 days 
Intranasal application:  
Part 1: 1 pump (140ul) 
per nostril BID for 14 
days  
Part 2: 1 pump (140ul) 
per nostril BID for 14 
days 
Intranasal application:  
Part 1: 1 pump 
(140ul) per nostril 
BID for 14 days  
Part 2: 1 pump 
(140ul) per nostril 
BID for 14 days 
Dosing instruction: 
Prime the spray with 5 
actuations (pumps) 
into air prior to first 
use only.  Apply 1 
pump per nostril (2 
pumps total) in the 
morning and repeat (2 
additional pumps) at 
night for 14 days of 
treatment. 
Prime the spray with 5 
actuations (pumps) 
into air prior to first 
use only.  Apply 1 
pump per nostril (2 
pumps total) in the 
morning and repeat (2 
additional pumps) at 
night for 14 days of 
treatment. 
Prime the spray with 
5 actuations (pumps) 
into air prior to first 
use only.  Apply 1 
pump per nostril (2 
pumps total) in the 
morning and repeat (2 
additional pumps) at 
night for 14 days of 
treatment. 
Physical description: 
Odorless, cloudy, 
light pink suspension 
Odorless, cloudy, light 
pink suspension 
Odorless, clear, and 
colorless suspension 
Manufacturer/source of 
procurement: 
AOBiome, LLC 
AOBiome, LLC 
AOBiome, LLC 
 
The contents of the label will be in accordance with all applicable regulatory requirements. B244 
and matching placebo will be packaged in identical 30 ml white bottles.  
 
8.2 
Dose Changes 
No dose changes are anticipated. 
 
8.3 
Storage Conditions 
All investigational drug supplies in the study will be stored in a secure, refrigerated (2-8OC) safe 
place, under the responsibility of the Investigator or other authorized individual prior to 
randomization and distribution to participants.  Once subjects are sent home with investigational 
product at visit 2 and visit 3 for Part 1 and Part 2 phases of the study, respectively, subjects will 
store the investigational product (IP) in room temperature (20-25 ºC) during use at home on the 
counter, being mindful to avoid extreme temperatures and areas of excessive heat (e.g., inside the 
car or trunk during heat) or cold (i.e., do not freeze). 
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8.4 
Description of Blinding Method 
This study will be double-blind: neither Investigator(s), nor study participants, nor those 
involved in the conduct of the trial (including sponsor staff) will be aware of the treatment the 
participants are receiving. 
8.5 
Treatment Assignments: 
This is a double blind study. Randomization will be stratified by site. 
When a subject completes the screening tests and procedures, the investigator will notify the 
Site’s pharmacist and the subject will be randomly assigned to one of three treatment groups in a 
1:1:1 ratio. Randomization and treatment assignment will be verified and coordinated by the 
Site’s pharmacist.  
 
Each participant scheduled to receive investigational product (IP) will receive a randomization 
number at the time of randomization. The randomization number will be used to identify the 
study medication kit assigned to the participant and indicate the treatment to be administered to 
that participant.  
8.6 
Treatment Compliance 
Study treatment must be dispensed or administered according to procedures described 
herein. Only participants enrolled in the study may receive study treatment. Only authorized site 
personnel may supply study treatment. Participants will record use of the study medication 
utilizing the study diary at the time of use each day. Participants will review study medication 
compliance with the Investigator or designee. Any missed doses, timing, and reason for missed 
dose will be recorded in the eCRF. There should be no doubling of doses to make up for missed 
doses. If a dose is missed, the next dose of study medication should be taken as scheduled. 
Each site participating in the trial will be instructed to assess subject’s compliance by weighing 
the investigational product and obtaining the weight of the bottle in grams pre and post 
application during Part 1 (at Visits 2 and 3) as well as during Part 2 (at Visits 3 and 4).  Sites will 
be provided scales, which will be calibrated prior to each use. Study personnel will be instructed 
to record measurements into the eCRF. 
 
8.7 
Treatment Application 
Subjects will receive one 30 ml bottle at the Baseline visit (Visit 2) for a 14 day application in 
Part 1. Subjects will receive one 30 ml bottle at the NAC #1 visit (Visit 3) for a 14 day 
application in Part 2. Subjects will be instructed in the use of the spray bottle and asked to self-
administer the Investigational Product as follows: 
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x Spray 1 pump of medication into each nostril twice a day for 14 days 
Detailed Investigational Product nasal spray instructions are as follows: 
x Shake bottle gently 
x Prime nozzle if unused; Pump until mist appears (5 times) 
x Place nozzle straight up one nostril and pump once 
x Sniff gently after spraying 
x Repeat other nostril 
x Wipe spray nozzle with tissue when done 
x Do not wash/rinse the application site or blow their nose for 30 min after applying the 
mist 
 
In Part 1, subjects will be asked to report for a screening visit and if all inclusion/exclusion 
criteria are met, subjects will be asked to report for a baseline visit and randomization. Subjects 
will then be trained on the use of the nasal spray, and have their first dose of treatment on site. 
Subjects will remain onsite for observation for 1 hour post dosing. Subjects will then be 
dispensed study medication for 14 days of at home dosing. While at home, subjects will dose 
twice daily (am and pm), and record their dosing, any occurrence of an AE, and Total Nasal 
Symptom Score (TNSS) using a study diary. Subjects will be asked to visit the site at Day 14 
(Week 2) and at follow-up visits at Day 21 (Week 3) and Day 42 (Week 6) for safety 
assessments and will additionally record Peak Nasal Inspiratory Flow (PNIF), intranasal fluid, 
intranasal nitric oxide (nNO) and nasal inflammation score. 
In Part 2, subjects will be asked to report for a screening visit (Visit 1) and if all 
inclusion/exclusion criteria are met, subjects will be asked to report for a NAC titration (ragweed 
pollen nasal allergen titration) visit to administer a single provocative challenge dose for each 
subject (Visit 2).  Subjects will then return 14 days after the washout period for basic health 
checks and baseline assessments (intranasal fluid, TNSS, PNIF, intranasal nitric oxide, and have 
a nasal inflammation score) followed by confirmatory NAC (NAC#1) (Visit 3). During this time 
subjects will use a study diary to complete TNSS at -30 (30 minutes prior to NAC), 0 (baseline 
prior to NAC), 15, 30, 45, 60, 90, and 120 minutes. PNIF at -30, 0, 15, 30, 60, and 120 minutes 
will also be obtained.  TNSS is the sum of four individual symptom scores (running nose, 
congestion, itchy nose, and sneezing) rated on a four point scale (0-3).  Subjects are required to 
meet the minimum qualifying TNSS score of a change of 6/12 from their baseline score, on at 
least 2 diary cards. Subjects will also provide samples for intranasal fluid at 0, 20, and 60 
minutes, measure intranasal nitric oxide (nNO) and have a nasal inflammation score assessed by 
the investigator using a nasal otoscope at 0 and 120 minutes, in addition to blood collection at 
120 min for biomarkers.  Subjects meeting the requirement will be randomized to one of the 
three treatment arms.  Randomization will be 1:1:1 so that equal number of subjects will be 
treated in each arm of the study. Subjects will then be trained on the use of the nasal spray, and 
have their first dose of treatment on site (self-dosing). Subjects will remain onsite for observation 
for 1 hour post dosing. Subjects will then be dispensed study medication for 14 days of at home 
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dosing. While at home, subjects will dose twice daily (am and pm), and record their dosing, and 
any occurrence of an AE and TNSS using a study diary.  Subjects will return to the clinic for the 
last dose following the 14 days of at home dosing for their final visit (Visit 4).  During this time 
subjects will again have their baseline assessments and be exposed to ragweed pollen challenge 
(NAC#2), and will use a study diary to complete TNSS at -30, 0, 15, 30, 45, 60, 90, and 120 
minutes and PNIF at -30, 0, 15, 30, 60, and 120 minutes.  Prior to and 20 and 60 minutes after 
NAC #2, subjects will additionally provide samples for intranasal fluid, and have nNO measured 
and have a nasal inflammation score assessed by the investigator using a nasal otoscope at 0 and 
120 min, in addition to blood collection at 120 min for biomarkers.  
 
8.8 
Treatment of Investigational Product Overdose 
The sponsor does not recommend specific treatment for an overdose.  
 
8.9 
Product Accountability 
In accordance with federal and local regulatory requirements, the Investigators and designated 
site personnel must document the amount of investigational product dispensed to study 
participants, the amount returned by study participants, and amount received and returned to the 
sponsor, when applicable. Product accountability records must be maintained throughout the 
course of the trial. Any quality issue noticed with the receipt or use of an IP (deficient IP in 
condition, appearance, pertaining documentation, labeling, expiry date, etc.) should be promptly 
reported to the Sponsor, who will initiate a complaint procedure.  
All investigational product must be stored in a secure locked room with access limited to the 
Investigator and designated site personnel. Study product is to be stored in a refrigerator between 
2-8 degrees C. Maintenance of a temperature log is required.  
Under no circumstances will the Investigator allow IP to be used other than as directed by this 
Clinical Trial Protocol, or dispose of IP in any other manner.  
8.10 Unblinding Procedures 
The Investigator may unblind a participant’s treatment assignment only in the case of emergency 
or in the event of a serious medical condition when knowledge of the study treatment is essential 
for the appropriate clinical management or welfare of the participant, as determined by the 
Investigator. It is preferred (but not required) that the Investigator first contacts the medical 
monitor to discuss options before unblinding the participant’s treatment assignment. The 
Investigator must notify the Sponsor as soon as possible when a participant’s treatment 
assignment is unblinded without revealing the treatment assignment of the unblinded participant 
unless that information is deemed important for the safety of participants currently in the study. 
The date and reason for the unblinding must be documented in the participant’s study record.  
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The Medical Monitor may unblind the treatment assignment for any participant with an SAE. If 
the SAE requires that an expedited regulatory report be sent to one or more regulatory agencies, 
a copy of the report, identifying the participant’s treatment assignment, may be sent to clinical 
Investigators in accordance with local regulations and/or sponsor policy. 
8.11 Retrieval and Destruction of Investigational Product 
All partially used or unused treatments will be returned to the sites as brought by study 
participants. A detailed treatment log of the returned IP shall be established.  
The sites will not destroy unused IP unless the Sponsor provides written authorization to the 
contrary.  All used and unused bottles will be shipped to the authorized drug depot at the end of 
the study. 
8.12 Permitted Medications 
All participants will be screened for concomitant medications prior to inclusion into the study.  
Any concomitant medication to treat adverse events will be recorded in the Concomitant 
Medication section of the eCRF. 
 
8.13 Prohibited Medications 
Participants will be prohibited from taking antihypertensives, beta blockers, photosensitising 
medications, systemic immunosuppressants and vitamin D supplements. NSAIDs or 
corticosteroids are also prohibited as they may affect BP.  Additional prohibited medications 
include antihistamines, leukotriene modifying medications, cromolyn, environmental allergen 
immunotherapy, Omalizumab, antibiotics, and any intranasally administered over-the-counter 
product or nasal irrigation (e.g., neti pot).  Complete list of prohibited medications will be 
provided to the site PIs conducting the study. 
8.14 Rescue Medications 
Rescue medications will not be allowed during the study.  Subject may not start additional non-
randomized medication and should immediately alert the study site PI. Subjects that need to start 
new rescue medication regimen should be immediately discontinued from study participation 
and would be considered Early Termination. 
 
8.15 Handling of Investigational Product 
Subjects will receive one 30 ml bottle for each Part of the study. Each bottle will be used for 14 
days and brought to study appointment. The bottle may be placed on the counter to be used 
during the treatment period. 
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Subjects will be asked not to subject the treatment kit to conditions which are unnatural or 
harmful to the product, such as excessive heat (temperatures over 77°F (25°C) and freezing 
temperatures (at 0oC)). Subjects may travel with their study medication but should not leave it in 
the hot car, outside in the cold temperatures etc. Subjects will also be asked not to tamper or 
cause damage to IP. 
9 
CONTRACEPTION REQUIREMENTS 
Effective contraception is required for all women physiologically capable of becoming pregnant 
during study participation. Women of child-bearing potential must agree to use an acceptable 
form of contraception for up to 2 weeks after the study completion. Women of childbearing 
potential are defined as any female who has experienced menarche and who is NOT permanently 
sterile or postmenopausal. Postmenopausal is defined as 12 consecutive months with no menses 
without an alternative medical cause. 
 
Effective contraception methods include:  
x 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.  
x Use of oral, injected or implanted hormonal methods of contraception or placement of 
an IUD or IUS or other forms of hormonal contraception that have comparable 
efficacy, for example hormone vaginal ring or transdermal hormone contraception.  
x 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. 
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10 STUDY PROCEDURES 
10.1 Screening 
Prior to initiation of the recruitment phase, participating Investigator will identify a pool of 
potential study subjects. The Investigator will identify potentially eligible subjects in advance, by 
either reviewing past medical records and diagnoses, screening in clinics, referral from other 
physicians, or other sources of recruitment, to identify those aged 18 to 65 meeting the inclusion 
criteria.  The accuracy of self-reported data, including height, weight and medical history, will be 
assessed during the initial on-site screening visit.  Individuals who fail to meet study inclusion 
criteria will be dismissed at that time.  Individuals who meet inclusion criteria will move on to 
on-site screening visit. 
 
10.2 Informed Consent Procedures 
Eligible participants may only be included in the study after providing a 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 
eCRF. 
10.3 Study Assessments  
In Part 1, study assessments will occur at Baseline and Week 2 (Day 14) visit.  Follow-up visit 
will be at Week 3 (Day 21) and Week 6 (Day 42) after treatment to perform safety assessments 
and clinical examination of the nasal mucosa, sinuses, and upper airway.   
In Part 2, study assessments will occur at NAC titration visit (Day 0), NAC#1 visit (Day 14) and 
NAC#2 visit (Day 28).  Preliminary efficacy assessments will be after nasal allergen challenge in 
a prophylaxis treatment setting. 
 
In Part 1, subjects will be asked to report for a screening visit and if all inclusion/exclusion 
criteria are met, subjects will be asked to report for a baseline visit and randomization. Subjects 
will then be trained on the use of the nasal spray, and have their first dose of treatment on site. 
Subjects will remain onsite for observation for 1 hour post dosing. Subjects will then be 
dispensed study medication for 14 days of at home dosing. While at home, subjects will dose 
twice daily (am and pm), and record their dosing, any occurrence of an AE, and Total Nasal 
Symptom Score (TNSS) using a study diary. Subjects will be asked to visit the site at Day 14 
(Week 2) and at follow-up visits at Day 21 (Week 3) and Day 42 (Week 6) for safety 
assessments and will additionally record Peak Nasal Inspiratory Flow (PNIF), intranasal fluid, 
intranasal nitric oxide (nNO) and nasal inflammation score. 
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In Part 2, subjects will be asked to report for a screening visit (Visit 1) and if all 
inclusion/exclusion criteria are met, subjects will be asked to report for a NAC titration (ragweed 
pollen nasal allergen titration) visit to administer a single provocative challenge dose for each 
subject (Visit 2).  Subjects will then return 14 days after the washout period for basic health 
checks and baseline assessments (intranasal fluid, TNSS, PNIF, intranasal nitric oxide, and have 
a nasal inflammation score) followed by confirmatory NAC (NAC#1) (Visit 3). During this time 
subjects will use a study diary to complete TNSS at -30 (30 minutes prior to NAC), 0 (baseline 
prior to NAC), 15, 30, 45, 60, 90, and 120 minutes. PNIF at -30, 0, 15, 30, 60, and 120 minutes 
will also be obtained.  TNSS is the sum of four individual symptom scores (running nose, 
congestion, itchy nose, and sneezing) rated on a four point scale (0-3).  Subjects are required to 
meet the minimum qualifying TNSS score of a change of 6/12 from their baseline score, on at 
least 2 diary cards. Subjects will also provide samples for intranasal fluid at 0, 20, and 60 
minutes, measure intranasal nitric oxide (nNO) and have a nasal inflammation score assessed by 
the investigator using a nasal otoscope at 0 and 120 minutes, in addition to blood collection at 
120 min for biomarkers.  Subjects meeting the requirement will be randomized to one of the 
three treatment arms.  Randomization will be 1:1:1 so that equal number of subjects will be 
treated in each arm of the study. Subjects will then be trained on the use of the nasal spray, and 
have their first dose of treatment on site (self-dosing). Subjects will remain onsite for observation 
for 1 hour post dosing. Subjects will then be dispensed study medication for 14 days of at home 
dosing. While at home, subjects will dose twice daily (am and pm), and record their dosing, and 
any occurrence of an AE and TNSS using a study diary.  Subjects will return to the clinic for the 
last dose following the 14 days of at home dosing for their final visit (Visit 4).  During this time 
subjects will again have their baseline assessments and be exposed to ragweed pollen challenge 
(NAC#2), and will use a study diary to complete TNSS at -30, 0, 15, 30, 45, 60, 90, and 120 
minutes and PNIF at -30, 0, 15, 30, 60, and 120 minutes.  Prior to and 20 and 60 minutes after 
NAC #2, subjects will additionally provide samples for intranasal fluid, and have nNO measured 
and have a nasal inflammation score assessed by the investigator using a nasal otoscope at 0 and 
120 min, in addition to blood collection at 120 min for biomarkers. 
At the final visit for each phase of the study (Visit 5 for Part 1 and Visit 4 for Part 2), subjects 
may be requested to complete a subject satisfaction survey. 
 
10.4 Inclusion Procedures  
Once all inclusion/exclusion criteria are fulfilled, the patient becomes eligible for randomization 
and inclusion into the treatment period for each of Part 1 and Part 2 phases of the study.  
Treatment allocation will be performed as stated above in Section 8.5. Study medication will be 
delivered as stated in Section 8.7.  Patients will be counseled on product application and diary 
completion. 
 
10.5 Description by Type of Visit – Part 1 
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10.5.1 Screening Visit (Visit 1; Study Day -1) (-21 to 0 Days Visit Window)  
x 
informed consent completed and signed  
x 
inclusion and exclusion criteria  
x 
demographic data  
x 
medical/surgical history 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate 
x 
physical exam 
x 
body weight 
x 
height measurement  
x 
nasal inspection 
x 
12-lead ECG 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, lipid panel, 
liver function test, urinalysis) 
x 
urine pregnancy test (for women of childbearing potential) 
x 
safety AEs 
x 
study diary 
10.5.2 Baseline ABPM Placement (-3 to 0 Days Visit Window) 
This visit should occur 24 hours prior to baseline visit (Visit 2) between the hours of 8am +/- 2 
hrs. 
x 
obtain in clinic blood pressure and heart rate 
x 
provide 24 hr ABPM device to the subject 
x 
train the subject on the use of the machine and provide instructions 
10.5.3 Baseline Visit (Visit 2; Study Day 0) (-3 to 1 Days Visit Window) 
This visit should occur between the hours of 8am +/- 2 hrs. 
x 
inclusion and exclusion criteria  
x 
medical/surgical history 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate (pre- and post-first dose) 
x 
physical exam 
x 
ambulatory blood pressure monitoring (ABPM) removal (after 24 hour measurement) and 
evaluation of technical success/failure criteria 
x 
randomization 
x 
nasal inspection (pre- and post-first dose) 
x 
delivery of the corresponding pack of Investigational Product  
x 
Investigational Product application (under medical supervision) 
x 
obtain study medication weight for Investigational Product compliance 
x 
study counseling 
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x 
safety AEs 
x 
nasal fluid collection for biomarkers 
x 
blood collection for biomarkers 
x 
peak nasal inspiratory flow (PNIF) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary 
10.5.4 Baseline Repeat ABPM Placement (if Necessary) (-3 to 1 Days Visit Window) 
If the ABPM technical acceptance criteria are not met during the baseline visit (Visit 2) reading, 
the ABPM session may be repeated. In the event of technical failure, study coordinators will not 
perform any study related activities outlined in Visit 2 prior to repeating the ABPM session.  
Repeat monitoring may be scheduled up to 48 hours after the failed monitoring.  
This visit should occur between the hours of 8am +/- 2 hrs. 
x 
provide 24 hr ABPM device to the patient 
x 
train the patient on the use of the machine and provide instruction 
The patient will come back 24 hours later to remove ABPM machine and check on the readings 
to make sure the equipment performed as expected.  
If the machine performed as expected, then Visit 2 activities will be performed as outlined in the 
section above.  
10.5.5 Week 2 ABPM Placement (±3 Days Visit Window) 
This visit should occur 24 hours prior to week 2 visit (Visit 3) between the hours of 8am +/- 2 
hrs. 
x 
obtain in clinic blood pressure and heart rate 
x 
provide 24 hr ABPM device to the subject 
x 
train the subject on the use of the machine and provide instructions 
10.5.6 Week 2 Visit (Visit 3; Study Day 14) (±3 Days Visit Window) 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate  
x 
physical exam 
x 
body weight 
x 
ABPM removal (after 24 hour measurement) and evaluation of technical success/failure 
criteria 
x 
nasal inspection 
x 
12-lead ECG 
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x 
Investigational Product application (last dose, if needed) 
x 
obtain study medication weight for Investigational Product compliance 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, lipid panel, 
liver function test, urinalysis) 
x 
safety AEs 
x 
nasal fluid collection for biomarkers 
x 
blood collection for biomarkers 
x 
peak nasal inspiratory flow (PNIF) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary 
10.5.7 Week 2 Repeat ABPM Placement (if Necessary) (±3 Days Visit Window) 
If the ABPM technical acceptance criteria are not met during the week 2 visit (Visit 3) reading, 
the ABPM session may be repeated. In the event of technical failure, study coordinators will not 
perform any study related activities outlined in Visit 3 prior to repeating the ABPM session.  
Repeat monitoring may be scheduled up to 48 hours after the failed monitoring.  
This visit should occur between the hours of 8am +/- 2 hrs. 
x 
provide 24 hr ABPM device to the patient 
x 
train the patient on the use of the machine and provide instruction 
The patient will come back 24 hours later to remove ABPM machine and check on the readings 
to make sure the equipment performed as expected.  
If the machine performed as expected, then Visit 3 activities will be performed as outlined in the 
section above.  
10.5.8 Follow-up #1 Visit (Visit 4; Study Day 21) (±3 Days Visit Window) 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate  
x 
physical exam 
x 
body weight 
x 
nasal inspection 
x 
safety AEs 
x 
peak nasal inspiratory flow (PNIF) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary 
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10.5.9 Follow-up #2 Visit (Visit 5; Study Day 42) (±3 Days Visit Window) 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate  
x 
physical exam 
x 
body weight 
x 
nasal inspection 
x 
12-lead ECG 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, lipid panel, 
liver function test, urinalysis) 
x 
safety AEs 
x 
nasal fluid collection for biomarkers 
x 
blood collection for biomarkers 
x 
peak nasal inspiratory flow (PNIF) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary 
x 
subject satisfaction survey 
 
10.6 Description by Type of Visit – Part 2 
10.6.1 Screening Visit (Visit 1; Study Day -1) (-21 to 0 Days Visit Window)  
x 
informed consent completed and signed  
x 
inclusion and exclusion criteria  
x 
demographic data  
x 
medical/surgical history 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate 
x 
physical exam 
x 
body weight 
x 
height measurement  
x 
allergen skin test (skin prick test) (if documentation of a positive test within the past 12 
months is not available) 
x 
blood collection for IgE (if documentation of a positive test within the past 12 months is 
not available) 
x 
nasal inspection 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, lipid panel, 
liver function test, urinalysis) 
x 
urine pregnancy test (for women of childbearing potential) 
x 
safety AEs 
x 
study diary 
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10.6.2 NAC Titration Visit (Visit 2; Study Day 0) (±3 Days Visit Window)  
x 
physical exam 
x 
nasal inspection 
x 
NAC 
x 
safety AEs 
x 
peak nasal inspiratory flow (PNIF) (to be completed pre (-30 and 0 min) and post NAC 
(15, 30, 60, and 120 min)) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary (TNSS to be completed pre (-30 and 0 min) and post NAC (15, 30, 45, 60, 90, 
and 120 min)) 
10.6.3 NAC #1 Visit (Visit 3; Study Day 14) (±3 Days Visit Window) 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate  
x 
physical exam 
x 
body weight 
x 
randomization 
x 
nasal inspection 
x 
NAC 
x 
delivery of the corresponding pack of Investigational Product  
x 
Investigational Product application (under medical supervision) 
x 
obtain study medication weight for Investigational Product compliance 
x 
study counseling 
x 
safety AEs 
x 
nasal fluid collection for biomarkers (to be completed pre (0 min) and post NAC (20 and 
60 min)) 
x 
blood collection for biomarkers (to be completed post NAC) 
x 
peak nasal inspiratory flow (PNIF) (to be completed pre (-30 and 0 min) and post NAC 
(15, 30, 60, and 120 min)) 
x 
intranasal nitric oxide (to be completed pre (0 min) and post NAC (120 min)) 
x 
nasal inflammation score (to be completed pre (0 min) and post NAC (120 min)) 
x 
study diary (TNSS to be completed pre (-30 and 0 min) and post NAC (15, 30, 45, 60, 90, 
and 120 min)) 
10.6.4 NAC #2 Visit (Visit 4; Study Day 28) (±3 Days Visit Window) 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate  
x 
physical exam 
x 
body weight 
x 
nasal inspection 
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x 
NAC 
x 
Investigational Product application (last dose, if needed) 
x 
obtain study medication weight for Investigational Product compliance 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, lipid panel, 
liver function test, urinalysis) 
x 
safety AEs 
x 
nasal fluid collection for biomarkers (to be completed pre (0 min) and post NAC (20 and 
60 min)) 
x 
blood collection for biomarkers (to be completed post NAC) 
x 
peak nasal inspiratory flow (PNIF) (to be completed pre (-30 and 0 min) and post NAC 
(15, 30, 60, and 120 min)) 
x 
intranasal nitric oxide (to be completed pre (0 min) and post NAC (120 min)) 
x 
nasal inflammation score (to be completed pre (0 min) and post NAC (120 min)) 
x 
study diary (TNSS to be completed pre (-30 and 0 min) and post NAC (15, 30, 45, 60, 90, 
and 120 min)) 
x 
subject satisfaction survey 
10.6.5 Unscheduled/Unanticipated Study Visit 
If an event arises that requires patient to come in to the research center, subjects should be 
scheduled for the Unscheduled visit.   
During the visit, the following will be obtained:  
x 
medical/surgical history 
x 
record concomitant medications  
x 
obtain in clinic blood pressure, heart rate, respiratory rate 
x 
physical exam 
x 
body weight 
x 
nasal inspection 
x 
obtain study medication weight for Investigational Product compliance 
x 
study counseling 
x 
laboratory testing (blood for metabolic panel, hematology, clinical chemistry, liver 
function test, urinalysis) 
x 
study AEs 
x 
nasal fluid collection for biomarkers 
x 
blood collection for biomarkers 
x 
peak nasal inspiratory flow (PNIF) 
x 
intranasal nitric oxide 
x 
nasal inflammation score 
x 
study diary 
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11 METHODS OF ASSESSMENTS 
11.1 Blood Pressure Measurement 
Blood pressure readings will be obtained at every visit as described in Appendix A (schedule of 
events). Subject should be asked to remove all clothing that covers the location of cuff 
placement. The individual should be comfortably seated in the chair, with the legs uncrossed, 
and the back and arm supported, such that the middle of the cuff on the upper arm is at the level 
of the right atrium (the mid-point of the sternum). Neither the patient nor the observer should 
talk during the measurement. After 5 minutes sitting, serial clinic BP measurements and heart 
(x3) rate will be performed, using the non-dominant arm with a calibrated electronic 
sphygmomanometer. The average of the second and third readings will be documented. 
Respiratory rate (number of breaths per minute) will also be recorded. 
11.2 Physical Examinations  
The physical examination will be performed at Screening (Visit 1), Baseline (Visit 2), Week 2 
(Visit 3), Follow-up #1 (Visit 4), and Follow-up #2 (Visit 5) during Part 1, Screening (Visit 1), 
NAC titration (Visit 2), NAC #1 (Visit 3), and NAC #2 (Visit 4) during Part 2, and at 
Unanticipated Visit should one occur. The physical examination includes an assessment of 
general appearance and a review of systems (dermatologic, head, eyes, ears, nose, 
mouth/throat/neck, thyroid, lymph nodes, respiratory, cardiovascular, gastrointestinal, 
extremities, musculoskeletal, neurologic systems).  
11.3 Allergen Skin Test (Skin Prick Test)  
For Part 2, subjects with a well-documented history of seasonal allergic rhinitis (specifically 
ragweed pollen) with documentation of sensitivity by positive skin testing and/or IgE testing to 
the relevant allergens 12 months prior to enrollment that correlate with clinical history are 
screened and enrolled.  Subjects may be confirmed with a ragweed positive skin prick test with a 
wheal diameter at least 5 mm larger than the negative control and/or a ragweed specific IgE 
greater or equal to 0.7 kU/L. 
A diluted ragweed pollen allergen is applied with a prick or a puncture on the surface of the skin.  
After the prick, the area of the skin is observed for about 15 minutes to see if a reaction develops. 
The “wheal”—a raised, red, itchy bump and surrounding “flare”—indicates the presence of the 
allergy antibody when the person is exposed to specific allergens. The larger the wheal and flare, 
the greater the sensitivity. 
11.4 Blood Collection for IgE  
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Alternative to a ragweed pollen allergen skin test (skin prick test), blood collection for ragweed 
pollen specific IgE may be performed as part of screening.  Subjects with a ragweed specific IgE 
greater or equal to 0.7 kU/L will be enrolled. 
11.5 Ambulatory Blood Pressure Monitoring (ABPM)  
11.5.1 ABPM Monitoring Schedule  
ABPM will occur twice during the study.  
Baseline ABPM placement visit should occur 24 hours prior to baseline visit (Visit 2) between 
the hours of 8am +/- 2 hrs.  Subjects should report to the office at 8 am +/- 2 hrs such that they 
are hooked up and dosed as close to 8:00 am as possible (window 6:00 am – 10:00 am). At the 
baseline visit (Visit 2), subjects will have their ABPM machine removed and readings checked to 
make sure equipment performed as expected.   
Week 2 ABPM placement visit should occur 24 hours prior to week 2 visit (Visit 3) between the 
hours of 8am +/- 2 hrs.  Subjects should report to the office at 8 am +/- 2 hrs such that they are 
hooked up and dosed as close to 8:00 am as possible (window 6:00 am – 10:00 am). At the week 
2 visit (Visit 3), subjects will have their ABPM machine removed and readings checked to make 
sure equipment performed as expected. 
11.5.2 Office Visit  
Subjects must arrive at the office for ABPM hookup NOT HAVING TAKEN that day’s dose of 
study medication, and at an appropriate time so that ABPM hookup, dosing of study medication, 
and start of the ABPM test is at 8 am +/- 2 hrs (window 6:00 am – 10:00 am) following the 
guidelines below. Subjects who are non-compliant will be rescheduled for the following day or 
later within the visit window.  
x Subject dosing of study medication must occur no earlier than 6:00 AM and no later than 
10:00 AM.  
x There should be at least one (1) manually initiated ABPM reading after dosing of study 
medication. If no manual post-dose reading was recorded, START TEST time will equal 
the 1st valid reading after dosing, provided that the reading occurs between 6:00 am and 
10:00 am.  
x There should be a final manual reading on or after 24 hours of recording. If a manual 
reading was not recorded, the first recording after 24 hours will be considered the last 
valid recording of the session.  
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11.5.3 ABPM Test  
The ABPM device will be placed on subject’s arm during the clinic visit and recording device 
will obtain 24-hour blood pressure and heart rate measurements. The ABPM device and cuff 
may only be removed at the office, after a final manual reading is performed using the ABPM 
device. The ABPM device will be automatically programmed to inflate every 20 minutes 
throughout the recording period. The duration of the ABPM test is 24 hrs.  
ABPM data will then be transmitted to the Core Lab. Based on pre-established criteria, the 
ABPM will be reviewed by the Core Lab to determine if it was a technically successful 
monitoring. If technically unsuccessful, the monitoring may be repeated.  
11.5.4 ABPM Technical Success/Failure Criteria  
The following are the technical acceptance criteria against which subject ABPM data will be 
evaluated:  
x There must be at least one (1) valid reading per hour with the following possible 
exception: no more than three (3) non-consecutive hours with zero (0) valid readings 
during the recording period and two (2) consecutive hours with zero (0) valid readings 
during the recording period.  
Failure to achieve any one of the above criteria will result in the ABPM being declared a 
technical failure.  
If a subject has a technical failure on their repeat recording at Baseline, those subjects will be 
excluded from the study. However, if subjects pass their baseline and fails the final repeat 
recording , data will be included in the in-clinic BP analysis but be excluded from the ABPM 
analysis. We would estimate that this will occur in less than 5% of patients.  
11.5.5 Repeat ABPM  
If the ABPM technical acceptance criteria are not met the ABPM session may be repeated. 
Repeat monitoring may be scheduled up to 48 hours after the failed monitoring.  
11.6 Nasal Inspection  
Nasal inspection will include clinical examination of the nasal mucosa, sinuses, and upper 
airway using an otoscope.  Nasal inspection will be performed at the following visits: Screening 
(Visit 1), Baseline (Visit 2), Week 2 (Visit 3), Follow-up #1 (Visit 4), and Follow-up #2 (Visit 5) 
during Part 1 and Screening (Visit 1), NAC titration (Visit 2), NAC #1 (Visit 3), and NAC #2 
(Visit 4) during Part 2, and at Unanticipated Visit should one occur. 
11.7 Nasal Allergen Challenge (NAC)  
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The NAC model is a transient and direct allergen exposure allergic rhinitis (AR) model in which 
allergen is instilled directly to the nasal mucosa via a metered, intranasal spray (10).  Typically, 
the model involves testing of diluent to exclude those patients who may respond inappropriately, 
perhaps even through non-allergic mechanisms. Patients who remain eligible are then tested with 
increasing doses of intranasal allergen in a step-wise fashion to find the provocative dose level at 
which a pre-defined level of allergic signs and symptoms are met. Signs can include objective 
measures of intranasal changes such as nasal congestion utilizing peak nasal inspiratory flow to 
look at nasal flow resistance. Symptoms typically include subjective report by patients on the 
nasal symptoms of allergic rhinitis such as nasal congestion, rhinorrhea, itchy nose and sneezing.  
NAC studies have previously examined the potential shift in this dose-response or utilized one 
provocative dose level to test therapy efficacy. The advantages of this approach are primarily the 
high level of experimental control over allergen exposure and patient compliance to both 
treatment, as well as the ability to capture in a well-timed fashion both signs and symptoms. 
Typically, objective measures are not possible to record in a field type trial. A disadvantage of 
this model is the concentrated and supra-natural mode of allergen exposure via direct instillation 
of concentrated allergen directly to the nasal mucosa. The amount of allergen utilized in this 
model may constitute too large of an allergic stimulus for putative anti-allergic medications to 
abate; and as such, medications could be evaluated as too weakly effective to continue in 
development. Another disadvantage of the direct instillation NAC is the inability to test 
exposures to multiple allergens and pollutants that patients are typically exposed to in a real-
world setting, as well as the inability to test on-symptom effects of drugs and thereby limiting 
this approach to testing prophylactic drug effects.  
The NAC has the advantage of running pilot studies with a small number of participants as well 
as allowing biological sampling. The inflammatory response that ensues, including early and late 
phase reactions (in a subset of participants), is similar to that observed in people with symptoms 
of AR during natural exposure (11-13). Disease pathophysiology and the effect of medications 
can be studied through monitoring cytokines, mediators, and inflammatory cells, allowing for 
accurate measurements of drug efficacy and onset/duration of action for each group of 
participants experiencing early and/or late phase responses as applicable to the mechanism of 
action (14). The NAC model has proven reliable through many clinical trials of anti-histamines 
(15-18), intranasal corticosteroids (19-22), and subcutaneous and sublingual immunotherapies 
(23-26), albeit through variable methodologies. 
For Part 2 of the study, aqueous extract of ragweed pollen (short ragweed; Ambrosia 
artemisiifolia; Stallergenes Greer, Lenoir, NC) at an allergen concentration (or dilution) 
sufficient to generate a provocative response will be prepared for the NAC titration visit.  0.1 mL 
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of sterile short ragweed pollen amb a1 stock solution (338 ug/mL, 10 mL vial; diluent 
composition:  0.25% NaCl, 0.27% sodium bicarbonate, 0.2% phenol, 50% glycerin; stored at 2-
8oC; Lot 325732; expiration date 20Mar2019; Stallergenes Greer, Lenoir, NC) will be diluted in 
2.5 mL of 0.9% sterile saline to prepare 96.5 micrograms/mL (or 0.0965 mg/mL) ragweed 
solution.  Similarly, 0.1 mL of 50% glycerin saline (50% glycerin, 0.091% sodium bicarbonate, 
0.166% sodium chloride, and water for injection q.s; 100 mL vial; stored at room temperature; 
Lot 2022817; expiration date Feb2023; Stallergenes Greer, Lenoir, NC) will be diluted in 2.5 mL 
of 0.9% sterile saline to prepare the diluent.  Both of these solutions will be prepared fresh for 
each day’s use using aseptic procedure to fill the Aptar Bidose System Liquid (BDSL) nasal 
pumps.  Each sterile Aptar bidose pump is filled using 230 microliters of the ragweed solution or 
diluent to deliver 100 microliters per pump to each nostril (2 pumps total per NAC; 100 
microliters per nostril of 9.65 micrograms ragweed; 19.3 micrograms total ragweed per subject). 
Baseline PNIF and TNSS will be recorded 30 minutes and immediately prior to administration of 
the NAC titration dose, which is a a single provocative nasal allergen dose (0.1 mL per nostril at 
0.0965 mg/mL amb a1).  Nasal administration of diluent will be made 30, 20 and 10 minutes 
prior to commencing nasal allergen dosing.  100 /RIWKHGLOXWHGDOOHUJHQ
will be sprayed into 
each nostril using a nasal pump (Aptar), and afterwards PNIF (15, 30, 60, and 120 min) and 
TNSS (15, 30, 45, 60, 90, and 120 min) will be reFRUGHG,ID31,)UHGXFWLRQRIEDVHOLQH25D7166VFRUHRI
6/12 were achieved on at least 2 diary cards, the qualifying 
concentration to be used at the NAC visit is established. If the target values are not reached, they 
will be excluded.  
At the NAC #1 visit (Visit 3) the qualifying allergen concentration from the titration visit will be 
used as the single concentration for nasal challenge. Nasal administration of diluent will be made 
30, 20 and 10 minutes prior to commencing nasal allergen dosing.  TNSS will be recorded by the 
participants before the challenge at -30 (30 minutes prior to NAC), 0 (baseline prior to NAC), 
15, 30, 45, 60, 90, and 120 minutes following the nasal challenge. PNIF at -30, 0, 15, 30, 60, and 
120 minutes will also be obtained.  Subjects are required to meet the minimum qualifying TNSS 
score of a change of 6/12 from their baseline score (must be ), on at least 2 diary cards to 
be randomized and enrolled into the study. 
Subjects will return to the clinic for the last dose following the 14 days of at home dosing for 
their final visit (Visit 4).  Nasal administration of diluent will be made 30, 20 and 10 minutes 
prior to commencing nasal allergen dosing. During this time subjects will again have their 
baseline assessments and be exposed to ragweed pollen challenge (NAC#2), and will use a study 
diary to complete TNSS at -30, 0, 15, 30, 45, 60, 90, and 120 minutes and PNIF at -30, 0, 15, 30, 
60, and 120 minutes.  Differences in the pre-and post-treatment values for NAC-induced nasal 
symptoms and PNIF will be compared. 
11.8 12-Lead ECG  
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12-lead ECG will be taken at Screening (Visit 1), Week 2 (Visit 3), and Follow-up #2 (Visit 5) 
during Part 1.  Standard 12-lead ECGs will be performed according to the site SOPs. ECGs 
should be performed after the subject has been resting supine for t 5 minutes. The ECG will 
include all 12 standard leads and a Lead II rhythm strip on the bottom of the tracing. The ECG 
will be recorded at a paper speed of 25 mm/sec. The following ECG parameters will be 
FROOHFWHG35LQWHUYDO456LQWHUYDO47LQWHUYDODQs correction). 
All ECGs must be evaluated by a qualified physician for the presence of abnormalities. 
11.9 Laboratory Assessments 
Blood sample will be taken for hematology and clinical chemistry, including standard metabolic 
panel, complete blood count (CBC), clinical chemistry, lipid panel, and liver function test.  
Approximately 15 ml of whole blood will be drawn.  Urinalysis will be performed as part of 
safety labs.  Laboratory assessments will be made at Screening (Visit 1), Week 2 (Visit 3), and 
Follow-up #2 (Visit 5) during Part 1, Screening (Visit 1) and NAC #2 (Visit 4) during Part 2, and 
at Unanticipated Visit should one occur. 
Blood samples should be taken using standard venipuncture techniques. Blood sampling will be 
performed according to the site SOPs. 
The following laboratory variables will be determined:  Fasting glucose, Uric acid, BUN (blood 
urea nitrogen), Creatinine, BUN/creatinine ratio, eGFR (estimated glomerular filtration rate), 
Sodium, Potassium, Chloride, Calcium, Albumin, Bilirubin, Alkaline phosphatase, AST 
(aspartate aminotransferase), ALT (alanine transaminase), total cholesterol, HDL, LDL, 
triglycerides, CBC, ACE (angiotensin converting enzyme), and proteinurea.  
Any value outside the normal range (except those affected by not fasting) will be flagged for the 
attention of the Investigator or designee at the site. The Investigator or designee will indicate 
whether or not the value is of clinical significance. If the result of the clinical chemistry test from 
the samples taken during the screening phase is indicated as clinically significant, the study 
subject will NOT be allowed into the study.  
11.10 Urine Pregnancy Test  
Standard urine pregnancy testing will be performed on WOCBP as part of screening. 
11.11 Nasal Fluid Samples for Biomarkers 
Nasosorption with paper and then synthetic absorptive matrices (SAM) has been used to sample 
nasal mucosal lining fluid (27). This technique can be considered as “precision mucosal 
sampling”, since it samples directly from the respiratory mucosa, and is free from the salivary 
contamination that occurs in breath and sputum sampling. Nasosorption sampling involves 
manipulating the synthetic absorptive matrix (SAM) up the lumen of the nasal cavity, and then 
holding it in position against the mucosa by external firm finger pressure. This is more 
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comfortable and less invasive than using a conventional swab, where rotation against the 
mucosal surface is generally required.  
Nasosorption will be performed by placing strips of a hydrophilic polyester absorptive matrix 
(Mucosal Diagnostics, Hunt Developments Ltd., Midhurst, UK: available as a CE-marked 
device) measuring 7 × 35 mm into each nostril for 1 min. When the SAM strip is removed, it will 
be frozen at í80 °C for biostorage until elution, processing, and biomarker testing.  After 
shipment to a designated lab, SAM strips will be ZDVKHGLQ3%6EXIIHUS+Ocontaining BSA (1%) and Triton X 100 (1%) within the cup of a spin filter insert (Costar® Spin-
X®). Mucosal lining fluid will be then eluted from the SAM by spin filter centrifugation (5 min 
at 16,000G at 4 °C), left and right nostril samples combined, and aliquots tested for biomarkers 
to evaluate the immune response.  
Nasal fluid for biomarkers will be collected at Baseline visit (Visit 2), Week 2 visit (Visit 3), and 
Follow-up #2 (Visit 5) during Part 1 and NAC #1 (Visit 3; pre (0 min) and post NAC (20, 60 
min)) and NAC #2 (Visit 4; pre (0 min) and post NAC (20, 60 min)) during Part 2 according to 
Appendix A.  Samples will also be collected in the event of an Unanticipated Visit. 
Samples will be evaluated for select biomarkers that may include a few of the following (27): 
1. Nasal inflammatory cytokines/chemokines/immune cell markers: PGD2, beta-tryptase, 
histamine, C3a/4a/5a, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, 
IL-13, IL-15, IL-16, IL-17, IL-18, TNF-alpha, GM-CSF, MIP-1 alpha, MIP-1 beta, MCP-
1, Eotaxin-1, MDC 
Optionally, samples may be evaluated for nasal microbiome (e.g., PCR) to confirm presence of 
B244. 
11.12 Blood Collection for Biomarkers 
In addition to the blood drawn for the safety laboratory assessments, additional whole blood will 
be collected for potential biomarker analysis at Baseline visit (Visit 2), Week 2 visit (Visit 3), 
and Follow-up #2 (Visit 5) during Part 1 and NAC #1 (Visit 3; post NAC (120 min)) and NAC 
#2 (Visit 4; post NAC (120 min)) during Part 2 according to Appendix A.  Samples will also be 
collected in the event of an Unanticipated Visit. 
Approximately 15 ml of whole blood will be drawn for biomarkers at each visit. Patients will be 
asked to fast for at least 8 hrs before blood for biomarkers is drawn. 
Samples will be processed to serum or plasma on site and stored at -80 |C until shipment to a 
designated central lab for biostorage. Upon Sponsor approval, biomarkers will be evaluated for 
immune response.  
Samples will optionally be evaluated for biomarkers that may include a few of the following 
select nasal inflammatory cytokines and chemokines (27): 
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1. Nasal inflammatory cytokines/chemokines/immune cell markers: PGD2, beta-tryptase, 
histamine, C3a/4a/5a, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, 
IL-13, IL-15, IL-16, IL-17, IL-18, TNF-alpha, GM-CSF, MIP-1 alpha, MIP-1 beta, MCP-
1, Eotaxin-1, MDC 
11.13 Peak Nasal Inspiratory Flow (PNIF)  
Peak Nasal Inspiratory Flow (PNIF) is another commonly used method for assessing nasal 
patency. PNIF provides an objective measurement of nasal airflow obstruction. It has the 
advantage of being simple, non-invasive and easily taught so participants can perform it on their 
own. The outcome is a direct representation of nasal congestion and therefore can provide 
objective confirmation of the subjective TNSS.  
During Part 1, PNIF will be obtained during baseline visit (Visit 2), week 2 visit (Visit 3), 
follow-up #1 (Visit 4), and follow-up #2 (Visit 5).   
During Part 2, -30 min and baseline (0 min) PNIF will be recorded prior to NAC titration.  After 
15, 30, 60, and 120 minutes of the single provocative NAC titration dose, PNIF will be recorded.  
Subjects will return 14 days after the washout period for basic health checks and baseline PNIF 
assessments followed by confirmatory NAC (NAC#1) (Visit 3). During this time subjects will 
obtain PNIF at -30 (30 minutes prior to NAC), 0 (baseline prior to NAC), 15, 30, 60, and 120 
minutes.  Subjects will return to the clinic for the last dose following the 14 days of at home 
dosing for their final visit (Visit 4).  During this time subjects will be exposed to ragweed pollen 
challenge (NAC#2) and obtain PNIF at -30, 0, 15, 30, 60, and 120 minutes.   
11.14 Intranasal Nitric Oxide  
Exhaled nasal nitric oxide (NO) will be measured from the nasal cavity by a NO analyzer. 
During Part 1, nasal NO will be obtained during baseline visit (Visit 2), week 2 visit (Visit 3), 
follow-up #1 (Visit 4), and follow-up #2 (Visit 5).   
During Part 2, baseline nasal NO will be recorded prior to NAC titration.  After 15 minutes of 
each NAC titration concentration, NO will be recorded.  Subjects will return 14 days after the 
washout period for basic health checks and baseline NO assessments followed by confirmatory 
NAC (NAC#1) (Visit 3). During this time subjects will obtain NO at 0 (baseline prior to NAC), 
and 120 minutes.  Subjects will return to the clinic for the last dose (15 min prior to NAC #2) 
following the 14 days of at home dosing for their final visit (Visit 4).  During this time subjects 
will be exposed to ragweed pollen challenge (NAC#2) and obtain NO at 0 and 120 minutes. 
 
11.15 Nasal Inflammation Score  
Nasal inflammation will be graded by a clinician using a 0-3 nasal inflammation scale based on 
otoscope measurement where 0 = none, 1 = mild, 2 = moderate, and 3 = severe inflammation.  
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During Part 1, nasal inflammation score will be obtained during baseline visit (Visit 2), week 2 
visit (Visit 3), follow-up #1 (Visit 4), and follow-up #2 (Visit 5).   
During Part 2, baseline nasal inflammation score will be recorded prior to NAC titration.  After 
15 minutes of each NAC titration concentration, nasal inflammation score will be recorded.  
Subjects will return 14 days after the washout period for basic health checks and baseline nasal 
inflammation score assessments followed by confirmatory NAC (NAC#1) (Visit 3). During this 
time subjects will obtain nasal inflammation score at 0 (baseline prior to NAC), and 120 minutes.  
Subjects will return to the clinic for the last dose (15 min prior to NAC #2) following the 14 days 
of at home dosing for their final visit (Visit 4).  During this time subjects will be exposed to 
ragweed pollen challenge (NAC#2) and obtain nasal inflammation score at 0 and 120 minutes. 
 
11.16 Total Nasal Symptom Score (TNSS)  
The Total Nasal Symptom Score (TNSS) is the sum of sub-scores for each of nasal congestion, 
sneezing, nasal itching, and rhinorrhea at each time point, using a four point scale (0–3; 0 = 
none; 1 = mild; 2 = moderate; 3 = severe), where 0 indicates no symptoms, a score of 1 for mild 
symptoms that are easily tolerated, 2 for awareness of symptoms which are bothersome but 
tolerable and 3 is reserved for severe symptoms that are hard to tolerate and interfere with daily 
activity. TNSS is calculated by adding the sub-score for each of the symptoms to a total out of 
12.  
During Part 1, subjects will dose twice daily (am and pm) at home for 2 weeks and record their 
dosing, any occurrence of an AE, and TNSS during treatment and through 28 days of follow-up 
using a study diary. 
During Part 2, -30 min and baseline (0 min) TNSS will be recorded prior to NAC titration.  After 
15, 30, 45, 60, 90, and 120 minutes of the single NAC titration concentration, TNSS will be 
recorded. Subjects are required to meet the minimum qualifying TNSS score of a change of 6/12 
from their baseline score, on at least 2 diary cards.  Subjects will return 14 days after the washout 
period for basic health checks and baseline TNSS assessments followed by confirmatory NAC 
(NAC#1) (Visit 3). During this time subjects will use a study diary to complete TNSS at -30 (30 
minutes prior to NAC), 0 (baseline prior to NAC), 15, 30, 45, 60, 90, and 120 minutes.  TNSS is 
the sum of four individual symptom scores (running nose, congestion, itchy nose, and sneezing) 
rated on a four point scale (0-3).  Subjects are required to meet the minimum qualifying TNSS 
score of a change of 6/12 from their baseline score, on at least 2 diary cards. Subjects meeting 
the requirement will be randomized to one of the three treatment arms.  Randomization will be 
1:1:1 so that equal number of subjects will be treated in each arm of the study. Subjects will then 
be trained on the use of the nasal spray, and have their first dose of treatment on site (self-
dosing). Subjects will remain onsite for observation for 1 hour post dosing. Subjects will then be 
dispensed study medication for 14 days of at home dosing. While at home, subjects will dose 
twice daily (am and pm), and record their dosing, and any occurrence of an AE and TNSS using 
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a study diary.  Subjects will return to the clinic for the last dose following the 14 days of at home 
dosing for their final visit (Visit 4).  During this time subjects will again have their baseline 
assessments and be exposed to ragweed pollen challenge (NAC#2), and will use a study diary to 
complete TNSS at -30, 0, 15, 30, 45, 60, 90, and 120 minutes.   
11.17 Study Diary 
Subjects will be asked to fill out a daily treatment diary during screening, baseline, treatment, 
follow-up, and challenge period for dosing, nasal symptom scores (TNSS) and AEs.   
Participants will record their nasal symptoms on diary cards that include symptoms of runny 
nose, nasal congestion, sneezing and nasal itching. Each symptom will be scored from 0–3, 0 
indicating the absence of the symptom and 3 describing the symptom as severe and intolerable as 
described in Section 11.16. 
AEs will be distinguished between immediate (onset of reaction is during the first 30 minutes 
after administration) and delayed (onset of action is after the first 30 minutes of administration) 
effects for safety reporting of adverse events. 
11.18 Sample Shipment 
All laboratory testing samples are to be shipped overnight in designated temperature conditions 
to the central laboratory where samples will be analyzed.  
All frozen serum and nasal fluid for biomarkers are to be shipped monthly on dry ice to the 
central laboratory.  Shipments should be made only on Mondays and Tuesdays to ensure receipt 
of the specimens by Friday. 
12 SAFETY ASSESSMENTS 
12.1 Compliance 
Participants will be asked to bring study medication with them to selected scheduled visits 
(Visits 2 and 3 during Part 1; Visits 3 and 4 during Part 2). Study sites will be provided with a 
scale and weight of the study medication will be obtained before the first use (at Visit 2 during 
Part 1; at Visit 3 during Part 2) and at each visit after use (Visit 3 during Part 1; Visit 4 during 
Part 2).  
At Baseline, study personnel will be asked to take out study medication bottle from the carton, 
weigh the bottle and record the weight. At each subsequent visit, study personnel will need to 
weigh the bottle without the carton and record weight. This procedure should be followed every 
time study medication is dispensed and returned. Sites will be provided scales, which will be 
calibrated prior to each use. Weight will be recorded in grams. Study personnel will be instructed 
to record measurements into the eCRF. 
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12.2 Safety Monitoring 
Local and systemic adverse events (AEs) related to intranasal route of administration during 14 
days of treatment and 28 days of follow-up (7 and 28 days after the last dose of the study) will be 
recorded for the safety phase of the study in healthy volunteers (Part 1).  Additionally, local and 
systemic AEs will be recorded in subjects with a history of seasonal allergic rhinitis during the 
efficacy phase of the trial (Part 2). 
Safety monitoring will include infectious complications related to B244 (by symptoms and/or 
testing; pulmonary, nasal, neurological), local and systemic AEs related to intranasal route 
during treatment and at follow-up.  Local AEs include runny nose, nasal congestion, sneezing, 
nasal itching, palate itching, anosmia, nasal ulceration, nasal bleeding, sore throat, cough, Bell’s 
palsy, other neurologic complications, fevers, chills, headache, muscle aches, decreased appetite, 
nausea, vomiting, and rash.  
Sponsor will distinguish between immediate (onset of reaction is during the first 30 minutes after 
administration) and delayed (onset of action is after the first 30 minutes of administration) effects 
for safety reporting of adverse events. 
Due to the nature of the nasal allergen challenge (in Part 2 of the study) which consists of 
delivering allergen directly into the airways of an allergic individual, a medical doctor trained for 
treating anaphylaxis must be present within the building.  A crash cart and the crash team 
telephone number must be readily available.  The nasal allergen challenge involves spraying a 
solution with very small amounts of ragweed antigen (allergy causing substance) into the nose.  
The side effects of this can be nasal blockage, excessive nasal discharge (similar to seasonal 
allergy symptoms), which may be delayed by several hours.  There is also a risk of 
bronchoconstriction (wheezing), especially if one has a history of asthma. The most serious risk 
of having a nasal challenge test is anaphylactic reaction. This reaction is a medical emergency, 
causing difficulty breathing and a dangerously low blood pressure. However, anaphylactic 
reactions with nasal change test are rare and the physician will be monitoring subjects closely 
after the challenge during the time they are in clinic. If subjects notice symptoms noted above 
after they have left the clinic, they should call 911 immediately.  
 
A 180 day follow up contact for collecting serious adverse events and new onset medical 
conditions for Part 1 will be the medical monitor described in section 15.7 (Larry Weiss, MD, 
lweiss@aobiome.com; 617-475-1605; or Carmen Margaritescu, MD, 
carmen.margaritescu@integrium.com; 714-328-7083).  Alternatively, the site will contact 
subjects 180 days after the last visit and after study close out to collect SAEs or new onset 
medical conditions. 
12.3 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.  
12.4 Study Completion 
A completed participant is one who has completed all study visits. Day 42 study visit is defined 
as the participant’s last visit (Visit 5) for Part 1, while Day 28 study visit is defined as the 
participant’s last visit (Visit 4) for Part 2. 
12.5 Internal Safety Committee 
An internal safety committee meeting will review the 2 week treatment safety data during Part 1 
phase of the study and, if there are no safety concerns, will proceed to Part 2 of the study to 
evaluate preliminary efficacy in subjects with a history of seasonal allergic rhinitis (SAR). 
12.6 Subject Withdrawal Criteria 
A subject may withdraw from the study at any time at his/her own request, or may be withdrawn 
at any time at the discretion of the Investigator for safety, behavioral, or administrative reasons.  
Reasons for withdrawal (subjects who refuse to return for any remaining study visits) or 
discontinuation (subjects who prematurely stop the application) at any time during the study may 
include, but are not limited, to the following: 
 For safety reasons, either at the discretion of the Investigator or at the subject’s request 
 For protocol violations at the discretion of AOBiome 
 Due to concomitant therapy that could interfere with the results of the study (the 
Investigator will report all such information on the CRFs and decide, in accordance with 
AOBiome, whether the subject is to be withdrawn).  
All premature discontinuations and their causes must be carefully documented by the 
Investigator on the CRF or if needed on the AE form.   
If, for any reason, a subject is withdrawn before completing the final visit, the reason for 
termination will be entered on the CRF.  All data gathered on the subject prior to termination will 
be made available to AOBiome. Subjects not completing the entire study should be fully 
evaluated when possible.  The appropriate CRFs should be completed. 
If the subject chooses to withdraw before completing the study, the subject should notify the 
study coordinator who will instruct the subject on completion of assessments for Unanticipated 
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visit (Appendix A).  For subjects who refuse to complete the assessments for their early 
termination, every attempt must be made to check on their status, using any mode of 
communication such as telephone, email, fax, or text. 
13 EFFICACY ASSESSMENTS 
Efficacy endpoints will be descriptively summarized and will include the number of 
observations, mean, median, standard deviation, minimum, and maximum of scores/values at all 
applicable time points and for all treatments in the ITT Population. 
For Part 2 phase of the study, the following secondary efficacy assessments will be obtained: 
x 
Change in TNSS from baseline to 14 days of prophylaxis treatment. 
x 
Change in subjective nasal symptom scores of nasal congestion, rhinorrhea, nasal itching, 
and sneezing from baseline to 14 days of prophylaxis treatment. 
x 
Nasal symptom-free response rate within each individual symptom scores and percentage 
of subjects with a 25% or 50% reduction in TNSS after 14 days of prophylaxis treatment. 
The following exploratory efficacy assessments will be obtained: 
x 
Change in Peak Nasal Inspiratory Flow (PNIF), nasal inflammation score using otoscope, 
intranasal nitric oxide levels, and cytokine concentration in nasal cavity and blood from 
baseline to 14 days of B244 application and during prophylaxis treatment setting. 
Fisher’s exact test will be used for efficacy parameters that report frequencies or incidence of 
measured events to make treatment group comparisons.  For the continuous endpoints a one-
sided two-sample equal-variance z-test will be used to compare treatment group means.   
14 STATISTICAL CONSIDERATIONS 
14.1 Sample Size 
Part 1 (safety and tolerability evaluation in healthy volunteers) will enroll 24 subjects.  Up to 12 
subjects per dose cohort (n=8 active, n=4 vehicle) will be enrolled, with a target of 20 subjects 
completed (10 subjects per dose cohort; n=7 active, n=3 vehicle) assuming a 16.7% drop out 
rate.  The two dose cohorts are 1x109 cells/ml and 4x109 cells/ml. 
Part 2 (preliminary efficacy evaluation in subjects with a history of SAR to ragweed pollen) will 
enroll 42 subjects.  Up to 14 subjects per arm (1x109 cells/ml; 4x109 cells/ml or vehicle) will be 
enrolled, with a target of 12 subjects per arm for a total of 36 subjects completed, assuming a 10-
15% drop-out rate. 
14.2 Populations for Analysis 
ITT: includes all randomized participants.   
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Safety: includes all subjects who received at least 1 dose of study medication.  
Per Protocol: subjects who administered at least 50 % of IP, have at least one baseline and post 
baseline in clinic visit and did not have any major protocol violations.  
14.3 Data Analysis 
The analyses will be conducted on all participant data when the trial ends.  Data will be 
presented by strata treatment and overall.   
Categorical variables will be summarized by frequency distributions (number and percentages of 
subjects) and continuous variables will be summarized by descriptive statistics (mean, standard 
deviation etc). 
Adverse events will be summarized by treatment using the Medical Dictionary for Regulatory 
Activities (MedDRA) system organ class and preferred term. Separate tabulations will be 
produced for all treatment-emergent AEs (TEAEs), treatment-related AEs (those considered by 
the Investigator as at least possibly drug related), SAEs, discontinuations due to AEs, and AEs 
*UDGHVHYHULW\%\
-subject listings will be provided for any deaths, SAEs, and AEs leading to 
discontinuation. 
Descriptive statistics for each sequence will be provided for clinical laboratory values (e.g., 
hematology, serum chemistry, and urinalysis) and vital signs data, presented as both actual 
values and changes from baseline relative to each on-study evaluation. Abnormal clinical 
laboratory values will be listed.  
All data will be provided in by-subject listings. 
14.3.1 Disposition 
A tabulation of the disposition of subjects will be presented, including the number enrolled, 
the number randomized, the number treated, and the reasons for study discontinuation will be 
reported. Summaries of the number in each analysis set will be summarized. Entry criteria 
and protocol deviations will be listed. 
14.3.2 Demographic and Baseline 
Demographic and baseline characteristic data summarization will be performed in order to 
descriptively assess the comparability of strata and treatments. Data to be tabulated will 
include age, race, ethnicity, height, weight, and BMI, as well as baseline characteristics 
related to medical history. 
14.4 Safety Analyses 
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14.4.1 Definitions  
All adverse events recorded during the study will be coded according to Medical Dictionary 
for Regulatory Activities.  
The primary analysis of safety will be based on the safety population, which is defined as all 
subjects receiving at least 1 dose of B244 or placebo. Adverse events (AEs) will be coded 
using the Medical Dictionary for Regulatory Activities (MedDRA®) for purposes of 
summarization. All AEs occurring during the study will be included in by-subject data 
listings and tabulated. Events leading to death, SAEs, and events resulting in study 
discontinuation will be summarized using standard descriptive statistics, and presented by 
treatment arm. Exposure to study drug and reasons for discontinuation of study treatment will 
be tabulated. Shift tables relative to the normal range will be provided for laboratory 
parameters.  Additional analyses will be performed, if warranted, upon review of the data. 
14.4.2 Adverse Events  
All adverse events (AEs) recorded during the study through the date of randomization 
through 28 days after the last dose of study drug will analyzed.  
All AE’s will be coded according to Medical Dictionary for Regulatory Activities and 
summarized using System Organ Class (SOC) and Preferred Term (PT).  
AE’s will be summarized using incidence rates. Therefore, each subject will only contribute 
once for a given adverse event SOC or PT.  
A summary of the incidence of any adverse event, SAE, and adverse events leading to 
discontinuation will be presented.  Summaries will display, by strata and treatment sequence, 
the incidence of patients with events, the frequency of patients with events within each 
primary system organ class and by preferred terms. For each preferred term and each system 
organ class a patient will be counted only once. For summaries on severe or drug-related AE, 
for a given patient, the highest severity or relationship for a specific preferred term will be 
considered.  
14.4.3 Deaths and Serious Adverse Events  
Serious adverse events and events leading to death will be summarized overall and by 
primary system organ class and preferred term.  
14.4.4 Adverse Events Leading to Treatment Discontinuation  
Adverse events leading to treatment discontinuation will be summarized overall and by 
primary system organ class and preferred term.  
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14.5 Efficacy Analyses 
Additional exploratory analyses will be defined in the Statistical Analysis Plan. 
 
14.5.1 Handling of Dropouts or Missing Data  
Missing data will not be imputed for analysis.  
Subjects who dropout after enrollment but prior to randomization will be replaced.  
14.6 Clinical Trial Protocol Deviations  
All the following deviations will be summarized on the all randomized patient population:  
x 
Inclusion or exclusion criteria not satisfied.  
x 
Deviations related to the Investigational Product administration  
x 
Not permitted concomitant medications.  
15 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. 
15.1 Definition of an AE 
An AE is any untoward medical occurrence in a study participant which is temporally associated 
with the use of a medicinal product, regardless of its potential relationship to the medicinal 
product.  An AE, therefore, can be any unfavorable or unintended sign, including an abnormal 
symptom, or disease (new or exacerbated), whether or not related to the investigational product 
(IP).   
Examples of an AE include: 
 Exacerbation of a pre-existing condition including either an increase in frequency and/or 
intensity of the condition. 
 New conditions detected or diagnosed after investigational product administration even 
though it may have been present prior to the start of the study. 
 Signs, symptoms of a drug interaction. 
 Signs, symptoms of a suspected overdose of either investigational product or a concurrent 
medication (overdose per se should not be reported as an AE/SAE). 
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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). 
15.2 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 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. 
15.3 Time Period, Frequency, and Method of Detecting AEs and SAEs 
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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”). 
15.4 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. 
15.5 Evaluating AEs and SAEs 
15.5.1 Severity Rating 
The severity of an adverse event (AE and SAE) is to be scored according to the following scale: 
Mild 
Awareness of 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 
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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 15.2 “Definition 
of a SAE”. 
15.5.2 Relationship to Investigational Product (IP)  
SAEs will be classified as “definitely not related”, “unlikely related”, “possibly related”, 
“probably related”, or “definitely 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 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. 
15.6 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. 
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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. 
15.7 Prompt Reporting of SAEs to the Sponsor 
In the case of a Serious Adverse Event the Investigator must immediately: 
x 
SEND (within 1 working day) a scan of the signed and dated corresponding page(s) in 
the Case Report Form and SAE form to the representative of the Monitoring Team whose 
name, phone number and email appear on the Clinical Trial Protocol (Larry Weiss, MD, 
lweiss@aobiome.com; 617-475-1605), or to a designated Safety contact provided by the 
Monitoring Team (Carmen Margaritescu, MD, carmen.margaritescu@integrium.com; 
714-328-7083), as well as to the Central Database number;  
x 
ATTACH a photocopy of all examinations carried out and the dates on which these 
examinations were performed. Care should be taken to ensure that the patient's identity is 
protected and the patient's identifiers in the Clinical Trial are properly documented on all 
copies of source documents provided to the Sponsor. For laboratory results, include the 
laboratory normal ranges;  
x 
Follow-up of any Serious Adverse Event that is fatal or life threatening should be 
provided within one additional calendar week. The treatment code will be unblinded for 
reporting of Serious Adverse Events that are unexpected and reasonably associated with 
the use of the Investigational Product.  
Follow-up  
x 
The Investigator should take all appropriate measures to ensure the safety of the patients, 
including referral to a specialist if indicated. Notably he/she should follow up the 
outcome of any adverse events (clinical signs, laboratory values or other, etc) until the 
return to normal or stabilization of the patient's condition;  
x 
In the case of any serious adverse event, the patient must be followed up until clinical 
recovery is complete and laboratory results have returned to normal, or until progression 
has been stabilized. This implies that follow-up may continue after the patient has left the 
Clinical Trial and that additional investigations may be requested by the Monitoring 
Team;  
x 
In case of any serious adverse event brought to the attention of the Investigator at any 
time after cessation of Investigational Product and considered by him/her to be caused by 
the Investigational Product with a reasonable possibility, this should be reported to the 
Monitoring Team.  
 
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Primary Contact 
AOBiome Reportable Events Hotline 
24 Hour Phone: 617-475-1605 
Email: lweiss@aobiome.com  
Call medical monitor to email a scanned report 
 
16 ETHICAL AND REGULATORY STANDARDS 
16.1 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 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. 
16.2 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.  
16.3 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 
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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 manual paper consent to take part in the study.  Participants below the 
age of majority in the municipality must give written assent to participate in this study.  This 
consent must be dated and retained by the Principal Investigator as part of the study records.  A 
downloadable digital copy shall be given to the person signing the form.  The informed consent 
process must be documented in the participant’s source documents. 
The Investigator agrees that the Sponsor, its employees or agents will have the right from time to 
time during the course of this study to audit and review pertinent medical records relating to this 
clinical trial.  A statement will be obtained from each person participating in the study permitting 
the release of his/her medical records as necessary for inspection by authorized personnel of the 
Sponsor, FDA, and the staff managing the clinical study.  
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). 
16.4 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.   
16.5 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 
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sites 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. 
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. 
17  ADMINISTRATIVE RULES 
17.1 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.  
17.2 Archiving of Study Documentation 
The Investigator must maintain confidential all study documentation, and take measures to 
prevent accidental or premature destruction of these documents.  
It is recommended that the Investigator retain the study documents at least fifteen (15) years after 
the completion or discontinuation of the Clinical Trial. However, applicable regulatory 
requirements should be taken into account in the event that a longer period is required.  
The Investigator must notify the Sponsor prior to destroying any study essential documents 
within the fifteen (15) year period following the Clinical Trial completion or discontinuation.  
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If the Investigator's personal situation is such that archiving can no longer be ensured by him/her, 
the Investigator shall inform the Sponsor and the relevant records shall be transferred to a 
mutually agreed upon designee.  
17.3 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 
potential safety signals during its routine blinded safety review. The details for the analysis plan 
will be documented in the trial’s Statistical Analysis Plan. 
18 STUDY MONITORING 
18.1 Responsibilities of the Investigator(s)  
The Investigator(s) undertake(s) to perform the Clinical Trial in accordance with this Clinical 
Trial Protocol, ICH guidelines for Good Clinical Practice and the applicable regulatory 
requirements.  
The Investigator is required to ensure compliance with all procedures required by the Clinical 
Trial Protocol and by study procedures provided by the Sponsor (including security rules). The 
Investigator agrees to provide reliable data and all information requested by the Clinical Trial 
Protocol (with the help of the Case Report Form [CRF], Discrepancy Resolution Form [DRF] or 
other appropriate instrument) in an accurate and legible manner according to the instructions 
provided and to ensure direct access to source documents to Sponsor representatives. If any 
particular circuits have to be defined (e.g., e-CRF, Fax), particular attention should be paid to the 
confidentiality of the patient's data to be transferred. The Investigator may appoint such other 
individuals as he/she may deem appropriate as Sub-Investigators to assist in the conduct of the 
Clinical Trial in accordance with the Clinical Trial Protocol. All Sub-Investigators shall be 
timely appointed and listed. The Sub-Investigators will be supervised by and under the 
responsibility of the Investigator. The Investigator will provide them with a Clinical Trial 
Protocol and all necessary information.  
18.2 Responsibilities of the Sponsor  
The Sponsor of this Clinical Trial is responsible to Health Authorities for taking all reasonable 
steps to ensure the proper conduct of the Clinical Trial Protocol as regards ethics, Clinical Trial 
Protocol compliance, integrity and validity of the data recorded on the Case Report Forms. Thus, 
the main duty of the Monitoring Team is to help the Investigator and the Sponsor maintain a high 
level of ethical, scientific, technical and regulatory quality in all aspects of the Clinical Trial. At 
regular intervals during the Clinical Trial, the sites will be contacted, through monitoring visits, 
letters or telephone calls, by a representative of the Monitoring Team to review study progress, 
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Investigator and patient compliance with Clinical Trial Protocol requirements, and any emergent 
problems. During these monitoring visits, the following but not exhaustive list of points will be 
scrutinized with the Investigator: patient informed consent, patient recruitment and follow-up, 
Serious Adverse Event documentation and reporting, outcome events documentation and 
reporting, Investigational Product allocation, patient compliance with the Investigational Product 
regimen, Investigational Product accountability, concomitant therapy use and quality of data.  
18.3 Source Document Requirements  
According to the ICH guidelines for Good Clinical Practice, the Monitoring Team must check 
the Case Report Form entries against the source documents, except for the preidentified source 
data directly recorded in the Case Report Form. The Informed Consent Form will include a 
statement by which the patient allows the Sponsor’s duly authorized personnel, the Ethics 
Committee (IRB/EC), and the regulatory authorities to have direct access to source data which 
supports the data on the Case Report Forms (e.g., patient's medical file, appointment books, 
original laboratory records, etc.). These personnel, bound by professional secrecy, must keep 
confidential all personal identity or personal medical information (according to confidentiality 
rules).  
18.4 Use and Completion of Case Report Forms (CRFs) and Additional Requests  
The sponsor or CRO will be responsible for activities associated with the data management 
of this study. This will include setting up a relevant database and data transfer mechanisms, 
along with appropriate validation of data and resolution of queries. Data generated within this 
clinical study will be handled according to the relevant SOPs of the data management and 
biostatistics departments of sponsor or CRO. 
For Electronic Data Capture (EDC): 
Study sites will enter data directly into an EDC system by completing the eCRF via a secure 
internet connection.  Data entered into the eCRF must be verifiable against source documents 
at the study sites. Any changes to the data entered into the EDC system will be recorded in an 
automated, secure audit trail and is Food and Drug Administration (FDA) Code of Federal 
Regulations (CFR) Title 21 Part 11 compliant. 
Data entered into the eCRF will be validated as defined in the Data Validation Specifications 
(DVS). Validation includes, but is not limited to, validity checks (for example, missing data, 
range checks) and consistency checks (logical checks between variables) to ensure that study 
data are accurately reported. Additionally, CRO Data Management will perform aggregate data 
review as defined in the DVS to ensure that the data are complete, consistent and reasonable. The 
electronic edit checks will run continually throughout the course of the study and queries 
reviewed by CRO personnel to assure validity as compared to source records. Manual queries 
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may also be entered into EDC by Monitoring or Data Management personnel to address 
identified discrepancies. 
Medical conditions/procedures will be coded using MedDRA and prior and concomitant 
medications will be coded using WHODrug. 
At the conclusion of the study, each site will be provided with their subject CRFs in Portable 
Document Format (PDF) for archival.  The CRF PDFs will contain subject data, audit trail 
information, queries including responses, and comments. 
19 PUBLICATIONS 
All data generated from this study are the property of AOBiome LLC and shall be held in strict 
confidence along with all information furnished by AOBiome.  Independent analyses and/or 
publication of these data by the Investigator or any member of his/her staff is not permitted 
without prior written consent of AOBiome.   
Any formal presentation or publication of data from this trial will be considered as a joint 
publication by the Investigator(s) and appropriate Sponsor personnel.  Authorship will be 
determined by mutual agreement. Written permission to the Investigator will be contingent on 
the review by the Sponsor of the methodology and statistical analysis and any publication or 
presentation will provide for nondisclosure of AOBiome confidential or proprietary information.  
In all cases, the parties agree to submit all manuscripts or abstracts to all other parties at least 30 
days prior to submission.  This will enable all parties to protect proprietary information and to 
provide comments based on information that may not yet be available to other parties.   
20 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. 
21 CONFIDENTIALITY  
All information disclosed or provided by the Sponsor (or any company/institution acting on their 
behalf), or produced during the Clinical Trial, including, but not limited to, the Clinical Trial 
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Protocol, the CRFs, the Investigator's Brochure and the results obtained during the course of the 
Clinical Trial, is confidential. The Investigator or any person under his/her authority agrees to 
undertake to keep confidential and not to disclose the information to any third party without the 
prior written approval of the Sponsor. However, the submission of this Clinical Trial Protocol 
and other necessary documentation to the Ethics Committee (IRB/EC) is expressly permitted, the 
IRB/EC members having the same obligation of confidentiality. The Sub-Investigators shall be 
bound by the same obligation as the Investigator. The Investigator shall inform the Sub-
Investigators of the confidential nature of the Clinical Trial. The Investigator and the Sub-
Investigators shall use the information solely for the purposes of the Clinical Trial, to the 
exclusion of any use for their own or for a third party's account. 
22 PROPERTY RIGHTS  
All information, documents and Investigational Product provided by the Sponsor or its designee 
are and remain the sole property of the Sponsor. The Investigator shall not mention any 
information or the Product in any application for a patent or for any other intellectual property 
rights. All the results, data, documents and inventions, which arise directly or indirectly from the 
Clinical Trial in any form, shall be the immediate and exclusive property of the Sponsor. The 
complete verified database will be shared with the Operations Committee, which shall have full 
access to all data. The Sponsor may use or exploit all the results at its own discretion, without 
any limitation to its property right (territory, field, continuance). The Sponsor shall be under no 
obligation to patent, develop, market or otherwise use the results of the Clinical Trial. As the 
case may be, the Investigator and/or the Sub-Investigators shall provide all assistance required by 
the Sponsor, at the Sponsor's expense, for obtaining and defending any patent, including 
signature of legal documents.  
23 DATA PROTECTION  
The patient's personal data and Investigator's personal data which may be included in the 
Sponsor database shall be treated in compliance with all applicable laws and regulations;  
When archiving or processing personal data pertaining to the Investigator and/or to the patients, 
the Sponsor shall take all appropriate measures to safeguard and prevent access to this data by 
any unauthorized third party.  
24 SPONSOR AUDITS AND INSPECTIONS BY REGULATORY AGENCIES  
For the purpose of ensuring compliance with the Clinical Trial Protocol, Good Clinical Practice 
and applicable regulatory requirements, the Investigator should permit auditing by or on the 
behalf of the Sponsor and inspection by applicable regulatory authorities.  
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The Investigator agrees to allow the auditors/inspectors to have direct access to his/her study 
records for review, being understood that this personnel is bound by professional secrecy, and as 
such will not disclose any personal identity or personal medical information.  
The Investigator will make every effort to help with the performance of the audits and 
inspections, giving access to all necessary facilities, data, and documents. As soon as the 
Investigator is notified of a future inspection by the authorities, he will inform the Sponsor and 
authorize the Sponsor to participate in this inspection.  
The confidentiality of the data verified and the protection of the patients should be respected 
during these inspections.  
Any result and information arising from the inspections by the regulatory authorities will be 
immediately communicated by the Investigator to the Sponsor.  
The Investigator shall take appropriate measures required by the Sponsor to take corrective 
actions for all problems found during the audit or inspections.  
25 PREMATURE DISCONTINUATION OF THE STUDY OR PREMATURE CLOSE-
OUT OF A SITE  
25.1 Decided by the Sponsor in the Following Cases: 
1. In the event the results of the Clinical Trial do not appear to be scientifically convincing 
to the Sponsor;  
2. If the aim of the Clinical Trial has become outdated or is no longer of interest;  
3. If the information on the product leads to doubt as to the benefit/risk ratio;  
4. If the Investigator has received from the Sponsor all Investigational Product, means and 
information necessary to perform the Clinical Trial and has not included any patient after 
a reasonable period of time mutually agreed upon;  
5. In the event of breach by the Investigator of a fundamental obligation under this 
agreement, including but not limited to breach of the Clinical Trial Protocol, breach of 
the applicable laws and regulations or breach of the ICH guidelines for GCP;  
6. If the total number of patients are included earlier than expected; In any case the Sponsor 
will notify the Investigator of its decision by written notice.  
25.2 Decided by the Investigator  
The Investigator must notify (30 days' prior notice) the Sponsor of his/her decision and give the 
reason in writing. In all cases (decided by the sponsor or by the Investigator), the appropriate 
Ethics Committee(s) (IRB/EC) and Health Authorities should be informed.  
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26 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. 
All appendices attached hereto and referred to herein are made part of this Clinical Trial 
Protocol. The Investigator should not implement any deviation from, or changes of the Clinical 
Trial Protocol without agreement by the sponsor and prior review and documented 
approval/favorable opinion from the IRB/EC of an amendment, except where necessary to 
eliminate an immediate hazard(s) to clinical trial patients, or when the change(s) involves only 
logistical or administrative aspects of the trial. Any change agreed upon will be recorded in 
writing, the written amendment will be signed by the Investigator and by the Sponsor and the 
signed amendment will be filed with this Clinical Trial Protocol. Any amendment to the Clinical 
Trial Protocol requires written approval/favorable opinion by the IRB/EC prior to its 
implementation, unless there are overriding safety reasons. In some instances, an amendment 
may require a change to the Informed Consent Form. The Investigator must receive an IRB/EC 
approval/favorable opinion concerning the revised Informed Consent Form prior to 
implementation of the change. 
27 APPENDIX A-Schedule of Events 
 
 
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Visit name (Day) 
Part 1 
Part 2 
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 1 
Visit 2 
Visit 313 
Visit 414 
Screening 
 
(Day -1) 
Baseline 
 
(Day 0) 
Week 2 
 
(Day 14) 
Follow-
up #1 
(Day 21) 
Follow-
up #2 
(Day 42) 
Screening 
 
(Day -1) 
NAC 
titration 
(Day 0) 
NAC #1 
 
(Day 14) 
NAC #2  
 
(Day 28) 
Unanticipated 
Visit 
Visit Window, in Days 
-21 to 0 
-3 to 1 
+/-3  
+/-3 
+/-3 
-21 to 0 
+/-3 
+/-3 
+/-3 
 
Informed Consent1 
X 
 
 
 
 
X 
 
 
 
 
Inclusion/Exclusion 
Criteria2 
O 
O 
 
 
 
X 
 
 
 
 
Demographics 
O 
 
 
 
 
X 
 
 
 
 
Medical History 
O 
O 
 
 
 
X 
 
 
 
O 
Concomitant Medications 
O 
O 
O 
X 
X 
X 
 
X 
X 
O 
HR, BP, and respiratory 
rate 
X 
O15 
O 
X 
X 
X 
 
X 
X 
 
 
O 
Brief Physical Exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
O 
Body Weight 
X 
 
X 
X 
X 
X 
 
X 
X 
O 
Height 
X 
 
 
 
 
X 
 
 
 
 
Allergen Skin Test (Skin 
Prick Test) 
 
 
 
 
 
X21 
 
 
 
 
Blood Collection for IgE 
 
 
 
 
 
X21 
 
 
 
 
ABPM3 
 
X 
X 
 
 
 
 
 
 
 
Randomization4 
 
O 
 
 
 
 
 
X 
 
 
Nasal inspection5 
X 
X15 
X 
X 
X 
X 
X 
X 
X 
X 
NAC 
 
 
 
 
 
 
X 
X 
X 
 
12-lead ECG 
X 
 
X 
 
X 
 
 
 
 
 
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1. Informed consent can be obtained up to 21 days prior to the baseline visit. 
2. Inclusion and exclusion criteria will be reviewed at Screening and at Baseline visit to make sure nothing changed. 
3. Ambulatory Blood Pressure Monitoring for 24 hours to be performed for 24 hours prior to baseline and Week 2 visits.  Subjects to 
visit site 24 hours prior to baseline and Week 2 visits to initiate ABPM. ABPM removal (after 24 hour measurement) at Visits 2 and 3 
followed by evaluation of technical success/failure criteria. 
4. Subject’s demographic data and medical history are blinded to the statistician.   
5. Nasal inspection will be performed by clinical examination of the nasal mucosa, sinuses, and upper airway. 
6. Subjects will be counseled on the application of the nasal spray for the duration of the study. 
Delivery of Investigational 
Product 
 
X 
 
 
 
 
 
X 
 
 
Investigational Product 
application 
 
O 
O 
 
 
 
 
X 
X 
 
Investigational Product 
compliance 
 
X 
O 
 
 
 
 
X 
X 
X 
Counseling6 
 
O 
 
 
 
 
 
X 
 
X 
Laboratory Testing7 
X 
 
X 
 
X 
X 
 
 
X 
X 
Urinary pregnancy test8 
X 
 
 
 
 
X 
 
 
 
 
Safety AEs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Nasal fluid9 
 
O 
X 
 
X 
 
 
X16 
X16 
X 
Blood collection10 
 
X 
X 
 
X 
 
 
X17 
X17 
X 
Peak nasal inspiratory flow 
 
X 
X 
X 
X 
 
X22 
X18 
X18 
X 
Intranasal nitric oxide 
 
X 
X 
X 
X 
 
X 
X19 
X19 
X 
Nasal inflammation 
score11 
 
X 
X 
X 
X 
 
X 
X19 
X19 
X 
Study diary12 
X 
X 
X 
X 
X 
X 
X22 
X20 
X20 
X 
Subject satisfaction survey 
 
 
 
 
X 
 
 
 
X 
 
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7. Fasting blood sample for a comprehensive metabolic panel, complete blood count, chemistry, lipid panel, and liver function test-15 
ml; include urinalysis.  Patients should fast for at least 8 hours before the test. Blood will be shipped to the central lab for processing. 
8. Urine pregnancy test to be done on WOCBP. 
9. Nasal fluid will be collected at baseline, treatment, and follow-up visits for inflammatory cytokines during Part 1.  Additionally, 
they will be collected during challenge visits per schedule of events.  Optionally, samples may be evaluated for nasal microbiome 
(e.g., PCR) to confirm presence of B244. 
10. Blood samples for biomarkers will be collected and processed on site. Serum and plasma samples will be frozen onsite and 
shipped to the Central lab for storage. 
11. Nasal inflammation score assessment based on otoscope measurement. 
12. Subjects will be asked to fill out a daily treatment diary during screening, baseline, treatment, follow-up, and challenge period for 
dosing, nasal symptom scores (TNSS) and AEs. 
13. Subjects will have their basic health checks and baseline assessments (intranasal fluid, PNIF, intranasal nitric oxide, and nasal 
inflammation score), challenged with NAC #1, challenge baseline assessments made (e.g., TNSS, PNIF, nNO, intranasal fluid, nasal 
inflammation score), and randomized . 
14. Subjects will be challenged with NAC #2 followed by assessments. 
15. Pre and post first dosing. 
16. To be completed pre (0 min) and post NAC (20 and 60 min). 
17. To be completed post NAC (120 min). 
18. To be completed pre (-30 and 0 min) and post NAC (15, 30, 60, and 120 min). 
19. To be completed pre (0 min) and post NAC (120 min). 
20. TNSS to be completed pre (-30 and 0 min) and post NAC (15, 30, 45, 60, 90, and 120 min). 
21. Perform either skin prick test or IgE test at screening unless documentation of a positive test in the past 12 months can be provided 
for either method. 
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22. To be completed pre (-30 and 0 min) and post NAC (15, 30, 45, 60, 90, and 120 min for TNSS and 15, 30, 60, and 120 min for 
PNIF). 
 
 
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