Clinical trial NCT02342691
Conditions: Gingival Inflammation
Keywords: Safety and efficacy of BLXA4
Sponsor: The Forsyth Institute
Start date: 2015-04-01; Completion date: 2019-08-30
Enrollment: 127 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The primary objective is to evaluate the safety of an investigational compound, BLXA4-ME, topically applied as a daily oral rinse in adults with gingivitis. Safety will be assessed by the incidence of adverse events, including mucosal inflammation and irritancy and findings from safety labs. Subjects will be monitored for development of periodontitis, and oral flora will be analyzed to detect an increase in opportunistic organisms.

The secondary objective is to assess preliminary efficacy of the oral rinse, by monitoring changes in the plaque index (PI), modified gingival index (MGI), bleeding on probing (BOP) and levels of interleukin -1β (IL-1β) in gingival crevicular fluid (GCF).

The study comprises three groups in a randomized, placebo-controlled double-blind clinical trial design. The treatment group (1.0 μM BLXA4-ME oral rinse) and the placebo rinse group will each include 50 subjects. The no-rinse control group will consist of 25 subjects. Subjects in the treatment and placebo rinse groups will receive oral rinse (BLXA4-ME or placebo) to be applied once daily after morning teeth brushing. Safety parameters will be assessed before and after 3, 7, 14, 21, and 28 days of treatment. Efficacy parameters will be assessed before and after 14 and 28 days of treatment.

Detailed description:

Arms: arm 1: The topical oral rinse dosage form of BLXA4-ME (also known as ClinRinse-1) will consist of drug substance prepared at a concentration of 1.0 μM in an aqueous vehicle solution arm 2: The placebo preparation will consist of formulated oral rinse without BLXA4-ME and will be identical to the test rinse in color, appearance and taste arm 3: The no-rinse control group will use no oral rinse, in order to assess the effect of the rinsing action independent of the active ingredients
Interventions: intervention 1: BLXA4 intervention 2: Placebo oral rinse
Intervention descriptions: intervention 1: BLXA4-ME is a member of a new class of chemically and metabolically stable lipoxin analogs featuring a replacement of the tetraene unit of native lipoxin-A4 (LXA4) with a substituted benzo-fused ring system. The full chemical name of the BLXA4-ME drug substance is (5S, 6R, E)-methyl 5,6-dihydroxy-8-(2-((R,E)-3-hydroxyoct-1-enyl) phenyl) oct-7-enoate. intervention 2: The placebo preparation will consist of formulated oral rinse without BLXA4-ME and will be identical to the test rinse in color, appearance and taste

Protocol documents:
Prot_000.pdf: 
Thomas E. Van Dyke 
Protocol 14-020-E, V8.0 
BLXA4-ME  
18May2017 
i 
 
 
 
 
A Phase 1/2 Clinical Trial to Assess the Safety 
and Preliminary Efficacy of Lipoxin Analog 
BLXA4-ME Oral Rinse for the Treatment of 
Gingivitis 
 
NIDCR Protocol Number:  14-020-E 
 
NIDCR Grant Number: 
 
U01 DE026934 
 
Principal Investigator: 
Hatice Hasturk, DDS, PhD 
The Forsyth Institute 
 
NIDCR Program Official: 
 
Jane Atkinson, DDS 
 
Director, Center for Clinical Research (CCR) 
Division of Extramural Research (DER) 
National Institute of Dental and Craniofacial Research (NIDCR) 
National Institutes of Health (NIH) 
IND Sponsor: 
 
Thomas Van Dyke, DDS, PhD 
The Forsyth Institute 
 
IND Number: 
107061 
 
 
Funding Agency: 
National Institute of Dental and Craniofacial Research (NIDCR) 
 
Version Number: 8.0 
 
18 May 2017 
Forsyth IRB approved: 
June 8, 2017 


Thomas E. Van Dyke 
Protocol 14-020-E, V8.0 
BLXA4-ME  
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STATEMENT OF COMPLIANCE 
 
The study will be conducted in accordance with the International Conference on 
Harmonisation (ICH) guidelines for Good Clinical Practice (ICH E6), the Code of 
Federal Regulations (CFR) on the Protection of Human Subjects (45 CFR Part 46), and 
the National Institute of Dental and Craniofacial Research (NIDCR) Clinical Terms of 
Award.  All personnel involved in the conduct of this study have completed human 
subjects protection training. 


Thomas E. Van Dyke 
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Signed: 
 
 
SIGNATURE PAGE 
 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and 
according to local legal and regulatory requirements and applicable US federal 
regulations and ICH guidelines. 
 
Principal Investigator or Clinical Site Investigator: 
 
 
 
Date: 


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TABLE OF CONTENTS 
 
PAGE 
STATEMENT OF COMPLIANCE ................................................................................................II 
SIGNATURE PAGE ...................................................................................................................III 
TABLE OF CONTENTS ............................................................................................................ IV 
LIST OF ABBREVIATIONS...................................................................................................... VII 
PROTOCOL SUMMARY........................................................................................................... IX 
1 
KEY ROLES AND CONTACT INFORMATION.................................................................13 
2 
INTRODUCTION:  BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ......15 
2.1 
Background Information ........................................................................................15 
2.2 
Preclinical Studies on Treatment of Inflammatory Periodontal Disease.................16 
2.3 
Investigational Product for the Current Study ........................................................18 
2.4 
Rationale ..............................................................................................................18 
2.5 
Potential Risks and Benefits .................................................................................19 
2.5.1 Potential Risks ..........................................................................................19 
2.5.2 Potential Benefits ......................................................................................19 
3 
OBJECTIVES ...................................................................................................................20 
3.1 
Study Objectives ...................................................................................................20 
3.2 
Study Outcome Measures ....................................................................................20 
3.2.1 Primary......................................................................................................20 
3.2.2 Secondary .................................................................................................21 
3.2.3 Exploratory ................................................................................................21 
4 
STUDY DESIGN ..............................................................................................................21 
5 
STUDY ENROLLMENT AND WITHDRAWAL ..................................................................22 
5.1 
Subject Inclusion Criteria ......................................................................................22 
5.2 
Subject Exclusion Criteria .....................................................................................23 
5.3 
Strategies for Recruitment and Retention .............................................................25 
5.4 
Treatment Assignment Procedures.......................................................................26 
5.4.1 Randomization Procedures .......................................................................26 
5.4.2 Masking Procedures..................................................................................26 
5.5 
Subject Withdrawal ...............................................................................................27 
5.5.1 Reasons for Withdrawal ............................................................................27 
5.5.2 Handling of Subject Withdrawals or Subject Discontinuation of Study 
Intervention ...............................................................................................27 
5.6 
Premature Termination or Suspension of Study....................................................27 
6 
STUDY INTERVENTION..................................................................................................28 
6.1 
Study Product Description ....................................................................................28 
6.1.1 BLXA4-ME Oral Rinse...............................................................................28 
6.1.2 Placebo Oral Rinse ...................................................................................29 
6.1.3 Acquisition.................................................................................................29 
6.1.4 Formulation, Packaging, and Labeling.......................................................29 
6.1.5 Product Storage and Stability ....................................................................29 
6.2 
Dosage, Preparation, and Administration of Study Product...................................29 
6.3 
Modification of Study Product Administration for a Subject ...................................30 
6.4 
Accountability Procedures for the Study Product ..................................................30 
6.5 
Assessment of Subject Compliance with Study Product Administration ................30 
6.6 
Concomitant Medications/Treatments ...................................................................30 
7 
STUDY SCHEDULE.........................................................................................................30 


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7.1 
Prescreening ........................................................................................................30 
7.2 
Screening .............................................................................................................31 
7.3 
Enrollment/Baseline ..............................................................................................32 
7.4 
Intermediate Study Visits ......................................................................................33 
7.5 
Final Study Visit ....................................................................................................34 
7.6 
Final Safety Evaluation .........................................................................................35 
7.7 
Withdrawal Visit ....................................................................................................36 
7.8 
Unscheduled Visit .................................................................................................36 
8 
STUDY PROCEDURES/EVALUATIONS .........................................................................36 
8.1 
Clinical Evaluations...............................................................................................36 
8.1.1 Safety Observations and Other Measurements .........................................36 
8.1.2 Clinical Periodontal Measurements ...........................................................37 
8.2 
Laboratory Procedures/Evaluations ......................................................................39 
8.2.1 Clinical Laboratory Evaluations .................................................................39 
8.2.2 Special Assays or Procedures...................................................................40 
8.2.3 Specimen Preparation, Handling, and Storage ..........................................41 
9 
ASSESSMENT OF SAFETY ............................................................................................41 
9.1 
Specification of Safety Parameters .......................................................................41 
9.1.1 Adverse Events .........................................................................................41 
9.1.2 Solicited Adverse Events from the Memory Aid .........................................42 
9.1.3 Serious Adverse Events ............................................................................42 
9.1.4 Unanticipated Problems ............................................................................43 
9.2 
Time Period and Frequency for Event Assessment and Follow-Up .......................43 
9.3 
Characteristics of an Adverse Event .....................................................................44 
9.3.1 Relationship to Study Intervention .............................................................44 
9.3.2 Expectedness............................................................................................44 
9.3.3 Severity of Event .......................................................................................45 
9.4 
Reporting Procedures ...........................................................................................45 
9.4.1 Unanticipated Problem Reporting to IRB and NIDCR ................................45 
9.4.2 Serious Adverse Event Reporting to NIDCR..............................................46 
9.4.3 Reporting of SAEs and AEs to FDA ..........................................................47 
9.4.4 Reporting of Pregnancy.............................................................................47 
9.5 
Rules for Study Suspension or Discontinuation.....................................................48 
10 
STUDY OVERSIGHT .......................................................................................................48 
11 
CLINICAL SITE MONITORING ........................................................................................49 
12 
STATISTICAL CONSIDERATIONS..................................................................................49 
12.1 
Study Hypotheses.................................................................................................49 
12.2 
Analysis Populations.............................................................................................49 
12.2.1 Safety Population ......................................................................................49 
12.2.2 Efficacy Population....................................................................................49 
12.3 
Sample Size Considerations .................................................................................50 
12.4 
Planned Interim Analyses .....................................................................................52 
12.5 
Final Analysis Plan ...............................................................................................53 
12.5.1 Primary Safety Analysis ............................................................................53 
12.5.2 Secondary Efficacy Analysis .....................................................................53 
13 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ..................54 
14 
QUALITY CONTROL AND QUALITY ASSURANCE ........................................................54 
15 
ETHICS/PROTECTION OF HUMAN SUBJECTS.............................................................55 
15.1 
Ethical Standard ...................................................................................................55 


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15.2 
Institutional Review Board ....................................................................................55 
15.3 
Informed Consent Process ...................................................................................55 
15.4 
Exclusion of Women, Minorities, and Children (Special Populations) ....................56 
15.5 
Subject Confidentiality ..........................................................................................56 
15.6 
Future Use of Stored Specimens and Other Identifiable Data ...............................56 
16 
DAT A HANDLING AND RECORD KEEPING ...................................................................57 
16.1 
Data Management Responsibilities .......................................................................57 
16.2 
Data Capture Methods ..........................................................................................57 
16.3 
Types of Data .......................................................................................................57 
16.4 
Schedule and Content of Reports .........................................................................57 
16.5 
Study Records Retention ......................................................................................58 
16.6 
Protocol Deviations ...............................................................................................58 
17 
PUBLICATION POLICY ...................................................................................................59 
18 
LITERATURE REFERENCES ..........................................................................................60 
APPENDICES...........................................................................................................................64 


Thomas E. Van Dyke 
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LIST OF ABBREVIATIONS 
 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
ATLa 
15-epi-16-para-fluoro-phenoxy-LXA4, aspirin-triggered lipoxin analog 
BOP 
bleeding on probing 
CAL 
clinical attachment level 
CCTR 
Center for Clinical and Translational Research, Forsyth Institute 
CFR 
Code of Federal Regulations 
CTCAE 
Common Terminology Criteria for Adverse Events 
DNA 
deoxyribonucleic acid 
DSMB 
Data and Safety Monitoring Board 
ELISA 
enzyme-linked immunosorbent assay 
FDA 
Food and Drug Administration 
GCF 
gingival crevicular fluid 
GM-CEJ 
distance from free gingival margin to cementoenamel junction 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IL-1β 
interleukin-1β 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
LXA4 
lipoxin A4 
MGI 
modified gingival index 


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NIDCR 
National Institute of Dental and Craniofacial Research 
NIH 
National Institutes of Health 
NSAID 
nonsteroidal anti-inflammatory drug 
OHRP 
Office for Human Research Protections 
OMAS 
Oral Mucositis Assessment Scale 
PD 
probing depth 
PI 
plaque index 
RBC 
red blood cell 
SAE 
serious adverse event 
SPM 
specialized pro-resolution mediator 
UP 
unanticipated problem 
WBC 
white blood cell 


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PROTOCOL SUMMARY 
 
Title: 
A Phase 1/2 Clinical Trial to Assess the Safety and Preliminary 
Efficacy of Lipoxin Analog BLXA4-ME Oral Rinse for the 
Treatment of Gingivitis 
Précis: 
The study comprises 3 groups in a randomized, 
placebo-controlled, double-blind clinical trial design. The 
treatment group (1.0 μM BLXA4-ME oral rinse) and the placebo 
rinse group will each include 50 subjects. The no-rinse control 
group will consist of 25 subjects.  Subjects in the treatment and 
placebo rinse groups will receive oral rinse (BLXA4-ME or 
placebo) to be applied once daily after morning teeth brushing. 
Safety parameters will be assessed before treatment and 3, 7, 14, 
21, and 28 days after initial treatment. Efficacy parameters will be 
assessed before treatment and 14 and 28 days after initial 
treatment. 
Objectives: 
The primary objective is to evaluate the safety of an 
investigational compound, BLXA4-ME, topically applied as a daily 
oral rinse in adults with gingivitis.  Safety will be assessed by the 
incidence of adverse events, including mucosal inflammation and 
irritancy, and findings from safety laboratory tests. Subjects will 
be monitored for development or progression of periodontitis and 
oral flora will be analyzed to detect an increase in opportunistic 
organisms. 
The secondary objective is to assess preliminary efficacy of the 
oral rinse, by monitoring changes in the plaque index (PI), 
modified gingival index (MGI), bleeding on probing (BOP), 
andlevels of interleukin-1β (IL-1β) in gingival crevicular fluid 
(GCF). Changes in pocket depth (PD) and clinical attachment 
level (CAL) will be assessed as exploratory efficacy analysis. 
Population: 
125 healthy adults, aged 18 through 65 years, with existing 
gingivitis (MGI ≥ 2.0) 
Phase: 
1/2 
Number of Sites: 
1 
Description of 
Intervention: 
The active preparation (test rinse) will contain BLXA4-ME at 
1.0 μM in a formulated oral rinse containing 10% ethanol and 
0.25% sodium lauryl sulfate. The compound BLXA4-ME is an 
analog of lipoxin A4, a locally acting lipid mediator biosynthesized 
from the essential fatty acid, arachidonic acid. In BLXA4-ME, a 
fused o-substituted benzo-ring is incorporated between 


Thomas E. Van Dyke 
Protocol 14-020-E, V8.0 
BLXA4-ME  
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2 positions of the tetraene unit of native lipoxin A4. The placebo 
preparation will consist of formulated oral rinse without 
BLXA4-ME. 
Study Duration: 
34 months 
Subject 
Participation 
Duration: 
Approximately 3 months (visits through 28 days plus safety 
follow-up phone call 2 months after last product use) 
Estimated Time 
to Complete 
Enrollment: 
30 months 


Thomas E. Van Dyke 
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BLXA4-ME  
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Schematic of Study Design 
 
 
 
Screening Visit 
 
Day -10 to -1 
 
 
 
 
 
 
Study Visit 1 
Day 0 
Enrollment/ 
Baseline 
Obtain informed consent for screening. Perform pregnancy test.  Record 
medical/dental history, medication history.  Perform oral examination and 
record screening periodontal assessments. Screen individuals by 
eligibility criteria.  Collect blood and urine for screening labs. 
 
 
 
 
Total N = 125. Obtain informed consent.  Confirm eligibility based on 
laboratory test results. Perform pregnancy test.  Record vital signs. 
Perform oral examination and record MGI, PD, clinical attachment level 
(CAL), BOP, and PI. Collect GCF for IL-1β; collect oral flora samples. 
Collect blood for baseline BLXA4-ME measurement. 
 
 
 
 
 
 
Randomize subjects; provide oral 
rinse and instructions for use. 
 
 
 
 
 
 
 
50 subjects 
1.0 μM BLXA4-ME 
oral rinse 
 
50 subjects 
Placebo oral rinse 
25 subjects 
No 
treatment 
 
 
 
 
 
 
Study Visit 2 
Day 3 ± 1 
 
 
 
 
Study Visit 3 
Day 7 ± 1 
 
Perform oral exam to assess mucosal irritation/inflammation.  Record vital 
signs.  Assess compliance, adverse events. Provide additional oral rinse. 
 
 
 
Perform oral exam to assess mucosal irritation/inflammation.  Record vital 
signs.  Assess compliance, adverse events. Provide additional oral rinse. 


Thomas E. Van Dyke 
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Study Visit 4 
Day 14 ± 1 
 
 
 
 
 
 
Study Visit 5 
Day 21 ± 1 
 
 
 
 
 
 
Study Visit 6 
Day 28 ± 1 
 
 
 
 
 
 
Telephone 
Follow-up 
Day 90 ± 14 
 
Perform oral exam and repeat assessments of MGI, PD, CAL, BOP, and 
PI.  Record vital signs.  Collect blood and urine for safety labs; collect GCF 
for IL-1β; collect oral flora samples. Assess compliance, adverse events. 
Provide additional oral rinse. 
 
 
 
Perform oral exam to assess mucosal irritation/inflammation.  Record vital 
signs.  Assess compliance, adverse events. Provide additional oral rinse. 
 
 
 
 
 
Perform oral exam and repeat assessments of MGI, PD, CAL, BOP, and 
PI.  Record vital signs.  Collect blood and urine for safety labs; collect 
blood for BLXA4-ME measurement; collect GCF for IL-1β; collect oral flora 
samples. Assess compliance, adverse events. 
 
 
 
 
 
Record adverse events reported by subjects. 
 
 
 
Assessment of Final Study 


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1 KEY ROLES AND CONTACT INFORMATION 
 
 
Principal 
Investigator: 
Hatice Hasturk, DDS, PhD 
Director, Center for Clinical and Translational Research 
Associate Member of the Staff 
Department of Applied Oral Sciences 
The Forsyth Institute 
245 First Street 
Cambridge, MA 02142 
Telephone:  617-892-8499 
Fax:  617-892-8505 
Email: hhasturk@forsyth.org 
 
 
IND Sponsor: 
Thomas E. Van Dyke, DDS, PhD 
Vice President for Clinical and Translational Research 
Chair, Department of Applied Oral Sciences 
Center for Periodontology 
The Forsyth Institute 
245 First Street 
Cambridge, MA 02142 
Telephone:  617-892-8503 
Fax:  617-892-8504 
Email: tvandyke@forsyth.org 
 
 
On-site Medical 
Monitor: 
Katherine P. Lemon, PhD, MD 
Department of Microbiology 
The Forsyth Institute 
245 First Street 
Cambridge, MA 02142 
Telephone:  617-892-8501 
Email: klemon@forsyth.org 
 
 
NIDCR Medical 
Monitor: 
Kevin McBryde, MD 
Medical Officer, Division of Extramural Research 
National Institute of Dental and Craniofacial Research 
National Institutes of Health, DHHS 
6701 Democracy Blvd., Room 638 
Bethesda, MD 20892-4878 
Telephone:  301-594-0170 
Email: mailto:mcbrydekd@mail.nih.gov 


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NIDCR Program 
Official: 
Jane C. Atkinson, DDS 
Director, Center for Clinical Research 
NIH/NIDCR/DER/CCR 
6701 Democracy Blvd., Room 634 
Bethesda, MD 20892-4878 
Telephone:  301-435-7908 
Email: jatkinso@mail.nih.gov 
 
 
Institution: 
The Forsyth Institute 
245 First Street 
Cambridge, MA 02142 
Telephone:  617-262-5200 
Funding Agency: 
National Institute of Dental and Craniofacial Research 
National Institutes of Health, DHHS 
Bethesda, MD 20892-2190 
Telephone:  301-496-4261 


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2 INTRODUCTION:  BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
 
2.1 
Background Information 
 
Inflammation is the normal host tissue response to infection and injury.  However, 
uncontrolled and unresolved inflammation contributes to a range of acute and chronic 
human conditions such as arthritis and cardiovascular diseases. The chronic 
inflammation in these conditions is characterized by the production of inflammatory 
cytokines, arachidonic acid-derived eicosanoids (prostaglandins, thromboxanes, 
leukotrienes, and other oxidative derivatives), reactive oxygen species, and adhesion 
molecules (Gallin and Snyderman 1999, Tilley, Coffman et al. 2001, Nathan 2002). 
Periodontitis is a similar progressive inflammatory disease in which microbial etiologic 
factors induce an inflammatory cascade that leads to destruction of the organ 
supporting the teeth, the periodontium, including soft tissues and bone (Van Dyke, 
Lester et al. 1993, Giannobile, Lynch et al. 1995, Kornman, Page et al. 1997, Kinane 
and Lappin 2002).  As with all inflammatory diseases, the arachidonate-derived 
eicosanoids play a key role in the initiation and pathogenesis of the inflammatory lesion 
in periodontitis (Offenbacher, Odle et al. 1986, Birkedal-Hansen 1993, Birkedal-Hansen, 
Moore et al. 1993).  In the case of periodontitis, the associated gram negative bacteria, 
such as Porphyromonas gingivalis (P. gingivalis), initiate an influx of neutrophils and 
neutrophil cyclooxygenase-2 activation leading to increased prostaglandin E2 in situ 
(Pouliot, Clish et al. 2000).  Indeed, many of the early pathophysiological events in 
periodontal disease and its chronicity can be attributed to lipid mediators (Offenbacher 
1996).  Leukotriene B4, produced mainly by activated leukocytes, initiates accumulation 
and superoxide generation by neutrophils within inflamed sites, stimulating the release 
of granule-associated enzymes and bone resorption (Samuelsson, Dahlen et al. 1987, 
Varani and Ward 1994).  Prostaglandin E2 is a potent activator of osteoclast-mediated 
bone resorption, the hallmark of periodontal disease (Offenbacher, Odle et al. 1986, 
Tsai, Hong et al. 1998). Together with other eicosanoids, it mediates inflammation and 
periodontal tissue destruction (Smith, Braswell et al. 1993, Gronert, Kantarci et al. 
2004). 
 
Resolution of inflammation is an actively regulated program rather than the passive 
termination of inflammation, as was once widely believed (Levy, Clish et al. 2001, 
Serhan 2004, Bannenberg, Chiang et al. 2005, Serhan and Savill 2005). The crucial 
identification of the cellular events and molecular signals that determine the end of 
inflammation and beginning of resolution has led to a new appreciation of pathogenesis 
in inflammatory diseases (Serhan, Clish et al. 2000, Lawrence, Willoughby et al. 2002, 


Thomas E. Van Dyke 
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Heasman, Giles et al. 2003, Serhan, Brain et al. 2007). Neutrophils are present mainly 
in inflamed or injured tissues, and their effective elimination is a prerequisite for 
complete resolution of an inflammatory response (Ariel, Fredman et al. 2006).  Most 
current therapeutic approaches attempt to block activation of inflammation using 
anti-inflammatory drugs that are pathway inhibitors (nonsteroidal anti-inflammatory 
drugs [NSAIDs], or tumor necrosis factor inhibitors), or to promote healing with agents 
such as transforming growth factor β1, bridging molecules, and phagocyte receptors 
(Kantarci, Hasturk et al. 2006). Prostaglandins and leukotrienes play essential roles in 
orchestrating inflammation and are well-appreciated autacoids or locally acting 
mediators (Samuelsson, Dahlen et al. 1987). Cyclooxygenase inhibitors are examples 
of widely used anti-inflammatory drugs that act by blocking prostaglandin biosynthesis 
(Flower 2003); however, their use may actually attenuate the active resolution pathways 
mediated by lipoxins, resolvins, and protectins (Levy, Clish et al. 2001, Serhan 2004). 
 
A rapidly emerging body of evidence demonstrates that endogenous pro-resolving lipid 
mediators actively participate in regulating host responses and orchestrate resolution of 
inflammation (Serhan, Hong et al. 2002, Hong, Gronert et al. 2003). Lipoxins, the 
product of lipoxygenase and lipoxygenase interactions, are trihydroxy derivatives of 
arachidonic acid that actively drive resolution of inflammation; they are typified by lipoxin 
A4 (LXA4). In addition, the previously unappreciated role of aspirin-triggered 
transformation circuits in producing the endogenous anti-inflammatory 15-epi- form of 
LXA4 has led to a better understanding of pro-resolution signaling networks, including a 
series of complex cellular and chemical reactions and tissue trafficking events (Serhan 
2007).  For example, lipoxins not only reduce the influx of neutrophils, but also stimulate 
the nonphlogistic uptake of apoptotic neutrophils by tissue macrophages (Godson, 
Mitchell et al. 2000, Serhan 2004). 
 
2.2 
Preclinical Studies on Treatment of Inflammatory Periodontal Disease 
 
Current therapies for gingivitis and periodontitis remain inadequate. To examine the 
potential of pro-resolving molecules for the treatment of inflammatory oral diseases, a 
rabbit model of periodontitis has been used (Serhan, Jain et al. 2003, Hasturk, Kantarci 
et al. 2006, Hasturk, Kantarci et al. 2007). In this model system, P. gingivalis induced 
significant periodontal disease.  An initial study examined an LXA4 analog 
(aspirin-triggered lipoxin analog [15-epi-16-para-fluoro-phenoxy-LXA4], 15-epi-LXA4 
analog, denoted ATLa) to treat 6 rabbits with experimentally induced periodontitis. A 
topical dose of 6 μg 3 times per week for 6 weeks around the second premolar was 
found to reduce microbe-initiated, neutrophil-mediated tissue damage and bone 
destruction (Serhan, Jain et al. 2003). 


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Based on these observations, a series of chemically and metabolically stable 
benzo-LXA4 analogs were produced (Petasis, Keledjian et al. 2008). These analogs 
featured a modification of the tetraene portion via substitution of a benzo-fused ring 
system while retaining the biological activity of the lipid chain moieties.  Noted 
advantages of this series of compounds were longer half-life in vitro and ease of 
synthesis through iterative palladium-mediated coupling methods. In comparison with 
native LXA4, BLXA4-ME and related analogs were not readily converted to inactive 
forms in an in vitro stability assay system containing eicosanoid oxido-reductase, and 
this particular analog was most effective at inhibiting polymorphonuclear neutrophil 
infiltration in a murine peritonitis model (approximately 32% inhibition [n = 5-10, 
p < 0.005 compared with the vehicle control]). In the same model system, the 
benchmark compound, ATLa, gave 40% inhibition of polymorphonuclear neutrophil 
infiltration (p < 0.05) (Sun, Tjonahen et al. 2009).  Based on these preliminary efficacy 
assessments, BLXA4-ME was chosen as the lead compound for clinical development in 
this series. 
 
The observation of reduced tissue damage and bone loss in rabbits treated with a 
lipoxin analog suggests a paradigm shift in periodontitis treatment approachesa move 
away from a purely mechanistic and antimicrobial approach to one that considers the 
driving force of the disease, namely uncontrolled inflammation.  In this emerging 
paradigm, it is suggested that the infection can be controlled if the inflammation can be 
controlled.  In one study, pushing the inflammatory response of an experimental 
periodontitis lesion towards a resolution of inflammation resulted not only in tissue 
repair/regeneration, but also in significant reversion of the subgingival microflora to one 
consistent with health (Hasturk, Kantarci et al. 2007). This shift in microflora occurred in 
the absence of any antimicrobial intervention, and it highlights the potential of resolution 
agonists like BLXA4-ME to play an active role in antimicrobial activities in resolving 
gingival inflammation (Campbell, Serhan et al. 2011). The expected outcome of treating 
gingivitis with BLXA4-ME is resolution of gingival inflammation with a return of the 
gingival microflora profile to one associated with health. 
 
The remarkable potency of lipoxins and their analogs compared with NSAIDs supports 
a new mechanistic modality for the treatment of oral inflammatory conditions such as 
gingivitis and periodontitis through the resolution of inflammation rather than through 
interference with regulatory circuits controlling the initiation and maintenance of the 
inflammatory response.  Although less active than native LXA4, benzo-lipoxin analogs 
have proven activity in the nano- to microgram range in murine assays, and these 
doses are logarithmic orders of magnitude more potent than traditional NSAIDs, such as 
aspirin or indomethacin, and steroids (both classes requiring milligram/kilogram doses 
in vivo). The use of pro-resolving agonists could result in new therapies that reduce 


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inflammation without the unwanted side effects of traditional anti-inflammatory drugs 
(e.g., NSAIDs and steroids). 
 
2.3 
Investigational Product for the Current Study 
 
The investigational compound BLXA4-ME was designed as a new drug entity with 
increased chemical and metabolic stability for use as a pro-resolving anti-inflammatory 
agent. It is an analog of the naturally occurring autacoid LXA4. 
 
The investigational compound, (5S, 6R, E)-methyl 5,6-dihydroxy-8-(2-((R,E)-3- 
hydroxyoct-1-enyl) phenyl) oct-7-enoate, was originally designated as 9,12-LXA4 in 
certain preclinical studies. The name of the compound has been changed to 
BLXA4-ME to more accurately reflect the nomenclature used by the manufacturer and 
analytical laboratory.  The “-ME” designation reflects the methyl ester form of the drug 
substance. BLXA4-ME is converted by nonspecific esterase activity under physiological 
conditions in vivo to the more active “free-acid” form designated as BLXA4-FA.  Both 
the methyl ester and free-acid forms of BLXA4 are pharmacologically active. 
 
The active preparation (test rinse) for the study will contain BLXA4-ME at 1.0 μM as a 
formulated oral rinse containing 10% ethanol and 0.25% sodium lauryl sulfate in water. 
The placebo preparation will consist of formulated oral rinse without the BLXA4-ME. 
 
2.4 
Rationale 
 
The prevention of gingival inflammation or gingivitis has been an area of intense 
research and development for many years.  In spite of these efforts, only 2 types of oral 
rinse products have been marketed with an antigingivitis claim, those containing 
chlorhexidine and those containing essential oils.  The chlorhexidine products have 
significant side effects that limit their routine usefulness. The essential oils have limited 
effectiveness.  Both oral rinse products are antimicrobials; there are no products that 
directly address the inflammatory aspects of gingival inflammation or gingivitis.  The 
hypothesis to be tested in the current study is that the lipoxin analog BLXA4-ME, 
applied topically in an oral rinse formulation, will be safe in humans and will enhance the 
resolution of gingival inflammation. 
 
For reasons of subject compliance and routine oral hygiene habits, a once-a-day 
application was chosen. The dose was based on preclinical experiments in animals for 
topical application on a mg/kg-body weight basis (see Investigator’s Brochure for 
preclinical data). 


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2.5 
Potential Risks and Benefits 
 
2.5.1 
Potential Risks 
 
BLXA4-ME topical oral rinse is intended for administration as an oral rinse (mouthwash) 
and is not intended for ingestion.  For this study, BLXA4-ME will be applied topically as 
an oral rinse that will be held in the mouth for 45 seconds each day for 28 days. 
Potential risks include oral irritation from use of the rinse.  In a preclinical 28-day oral 
irritancy study in rats with abraded oral mucosa, transient erythema and edema of the 
oral mucosa were noted in animals treated with either BLXA4-ME or a saline solution 
used as a control.  Microscopic examination of oral tissues showed no differences 
between rats treated with BLXA4-ME and control rats treated with saline.  In this 
Phase 1/2 safety study, oral tissues will be closely monitored for signs of inflammation 
and irritancy, and product administration will be discontinued if toxicity develops. 
Because the investigational compound may be absorbed through the oral mucosa, 
blood and urine for safety laboratory tests will be collected from all subjects at screening 
and 14 and 28 days after initial product administration.  Treatment of gingivitis has the 
potential to conceal the presence of the more serious condition of periodontitis; 
therefore, subjects will be monitored for development or progression of periodontitis, 
using clinical periodontal measurements. In addition, oral flora will be analyzed to 
detect any increase in pathogenic organisms. 
 
Following the Screening Visit, individuals with gingivitis or periodontitis who are not 
eligible for participation in the trial will be informed of their disease status.  Those with 
gingivitis will be provided with oral hygiene instructions. Those with periodontitis will be 
advised to seek treatment and will be provided with information about dental facilities 
that could provide care.  Subjects who enroll in the trial will have inflammatory 
periodontal disease (gingivitis and/or periodontitis), and it is possible they will 
experience no improvement in their condition during the study.  Subjects will be 
informed of their disease status at enrollment and will be told that they may need to 
seek periodontal treatment after their participation in the trial is completed.  At the 
conclusion of their study participation, those with gingivitis will be provided with oral 
hygiene guidance, and those with periodontitis will be advised to seek treatment and will 
be provided with information about dental facilities that could provide care. 
 
2.5.2 
Potential Benefits 
 
As this is a Phase 1/2 safety study, the benefits to subjects from participation are not 
known.  This study has the potential to provide safety and preliminary efficacy 
information that could support further studies of the BLXA4-ME compound to assess its 
efficacy in the treatment of gingivitis or periodontal disease. 


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3 OBJECTIVES 
 
 
3.1 
Study Objectives 
 
The primary objective is to evaluate the safety of an investigational compound, 
BLXA4-ME, topically applied for 28 days as a daily oral rinse in adults with gingival 
inflammation as determined by a modified gingival index (MGI) score of 2.0 or more. 
Safety will be assessed based on the incidence of adverse events (AEs), including 
mucosal inflammation and irritancy, and findings from oral examinations and safety 
laboratory tests.  Subjects will be monitored for the development of periodontitis or 
progression of existing periodontitis. Oral flora will be analyzed to detect any increase 
in pathogenic organisms. 
 
A secondary objective is to assess preliminary efficacy of the oral rinse. Efficacy will be 
measured by changes in the plaque index (PI) (Silness and Loe 1964), MGI (Gordon, 
Lamster et al. 1985), bleeding on probing (BOP), and levels of interleukin-1β (IL-1β) in 
gingival crevicular fluid (GCF). 
 
3.2 
Study Outcome Measures 
 
3.2.1 
Primary 
 
The primary outcome measure of this study is safety, which will be assessed by the 
incidence of AEs, development or progression of periodontitis and changes in the oral 
flora. 
 
Adverse events will be recorded throughout the study, and may include events reported 
by subjects or changes observed in oral cavity examinations or vital signs (assessed at 
baseline, Days 3, 7, 14, 21, and 28).  Blood and urine will be collected for safety 
laboratory tests at Days 14 and 28 after product administration. Subjects will undergo 
close monitoring for mucosal inflammation and irritancy. 
 
Subjects will be monitored for the development of or the progression of existing 
periodontitis, using standard clinical periodontal measurements. Development or 
progression of periodontitis will be determined by an increase of 2 mm or more in 
pocket depth or clinical attachment level (CAL) from the Day 0 baseline measurement 
(Goodson et al, 1982). 
 
Each subject’s oral flora will be analyzed in a pooled plaque sample collected from the 
mesiobuccal surfaces of the Ramfjord teeth at the Baseline Visit and at Days 14 and 28 
of the experimental period.  Using the deoxyribonucleic acid (DNA)-DNA hybridization 
(checkerboard) method (Socransky, Smith et al. 1994, Socransky, Haffajee et al. 2004), 


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samples will be analyzed for the presence of the following species:  Fusobacterium 
nucleatum subspecies vincentii, Campylobacter concisus, Campylobacter rectus, 
Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Porphyromonas 
gingivalis, Capnocytophaga sputigena, Streptococcus oralis, Actinomyces naeslundii, 
Actinomyces israeli, Eubacterium brachy, Eikenella corrodens, and spirochetes. 
 
3.2.2 
Secondary 
 
The secondary outcome measures will consist of the change from baseline in the 
preliminary efficacy measures. These secondary efficiacy measures include MGI 
(Gordon, Lamster et al. 1985), BOP, PI (Silness and Loe 1964), and levels of IL-1β in 
GCF.  In addition, plaque and gingivitis severity scores will be calculated using methods 
previously described (Panagakos, Volpe et al. 2005).  Levels of IL-1β, a marker of 
inflammation, will be determined in the GCF collected at baseline and 14 and 28 days 
after treatment. Gingival crevicular fluid will be collected and quantified, and IL-1β 
levels in the GCF will be determined by enzyme-linked immunosorbent assay (ELISA). 
 
3.2.3 
Exploratory 
 
Differences among the treatment arms with respect to the change from baseline in 
pocket depth and clinical attachment level at Day 28 will be assessed as exploratory 
analyses. 
 
4 STUDY DESIGN 
 
This Phase 1/2 study comprises 3 groups in a randomized, placebo-controlled, 
double-blind clinical trial design. Subjects will be healthy adults, aged 18 through 
65 years, with gingivitis as defined by MGI ≥ 2.0. The treatment group (1.0 μM 
BLXA4-ME oral rinse) and the placebo rinse group will each consist of 50 subjects. The 
no-rinse control group will consist of 25 subjects. The group sizes allow for a 20% 
dropout rate. 
 
The treatment group will receive the active agent BLXA4-ME at a concentration of 
1.0 μM in an oral rinse applied once daily (after morning teeth brushing) for 28 days. 
The placebo rinse control subjects will rinse once daily after morning teeth brushing with 
the same rinse formulation, except that it will not contain BLXA4-ME. The no-rinse 
control group will use no oral rinse to assess the effect of the rinsing action independent 
of the active ingredients.  At the Enrollment/Baseline Visit, eligible individuals will be 
randomly assigned to 1 of the 3 treatment groups. The baseline clinical measurements, 
i.e., MGI, probing depth (PD), CAL, PI, and BOP, will be recorded. Plaque, GCF, and 
blood will be collected as described in the Manual of Procedures. The subjects 
randomized to rinse groups will be given the BLXA4-ME oral rinse formulation or 


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placebo rinse with instructions for use in addition to their regular oral hygiene.  Subjects 
in all groups will be instructed to maintain their regular oral hygiene procedures, except 
for using the study-provided rinse, if applicable. 
 
Subjects will return to the clinic on Days 3, 7, 14, 21, and 28 to undergo an oral 
examination to assess mucosal irritancy and inflammation, to participate in an interview 
to solicit AE information, and to renew their supply of oral rinse (excluding Day 28). 
Additional procedures on Days 14 and 28 will be recording of periodontal 
measurements to monitor for development of periodontitis, collection of blood and urine 
for safety laboratory tests, collection of plaque samples for analysis of oral flora, and 
collection of GCF for measurement of IL-1β. To assess absorption of BLXA4-ME 
through the oral mucosa, subjects will provide blood samples at Day 0 and Day 28, for 
measurement of plasma levels of BLXA4-ME, and for profiling of specialized lipid 
mediators in plasma and serum. Subjects will be contacted via telephone 
approximately 2 months after completion of treatment for safety follow-up. The total 
duration of an individual subject’s participation in the study is 3 months. 
 
 
5 STUDY ENROLLMENT AND WITHDRAWAL 
 
Approximately 125 healthy adult male and female subjects with gingival inflammation, 
determined by mean full mouth MGI of at least 2.0, will participate in the study. The 
inclusion and exclusion criteria are as follows: 
 
5.1 
Subject Inclusion Criteria 
 
1.  Signed consent form; 
 
2.  Good general health as evidenced by medical history; 
 
3.  Age 18 through 65 years; 
 
4.  Stable address; 
 
5.  Availability for the duration of the study (if a subject does not meet this 
criterion, he/she may be rescreened for study participation when he/she does 
meet this inclusion criterion); 
 
6.  Minimum of 20 natural teeth, excluding third molars; 
 
7.  Mean full mouth MGI of at least 2.0; 
 
8.  Willing to use prescribed oral hygiene procedures and products; 


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9.  Stability of medications for chronic conditions for at least 3 months prior to 
enrollment (if a subject does not meet this criterion, he/she may be 
rescreened for study participation when he/she does meet this inclusion 
criterion); 
 
10. For women of reproductive potential, use of licensed hormonal contraception 
or double-barrier methods; 
 
11. For men of reproductive potential, agreement to use condoms; 
 
12. Liver function test (aspartate aminotransferase [AST], alanine 
aminotransferase [ALT], alkaline phosphatase, and total bilirubin) levels equal 
to or less than 1.5 times the upper limit of normal; 
 
13. Serum creatinine levels equal to or less than the upper limit of normal; 
 
14. Subjects with complete blood count levels within 10% of the normal laboratory 
range and erythrocyte sedimentation rate equal to or less than 2 times the 
upper limit of normal. 
 
5.2 
Subject Exclusion Criteria 
 
1.  Presence of orthodontic appliances or removable partial dentures; 
 
2.  Presence of a soft tissue tumor of the oral cavity; 
 
3.  Presence of gross plaque or calculus as determined by the investigator; 
 
4.  Presence of extensive restorations that could affect the marginal gingiva (at 
the investigator’s discretion); 
 
5.  Preexisting oral pathology requiring immediate treatment or ulcerations of the 
mucosa; 
 
6.  Preexisting carious lesions requiring immediate treatment (if a subject meets 
this criterion, he/she may be rescreened for study participation when he/she 
no longer meets this exclusion criterion); 
 
7.  Current participation in another clinical trial or product test; 
 
8.  Pregnant or breastfeeding; 
 
9.  Residence in the same household as a subject currently enrolled in the study 
(due to potential blinding and compliance issues); 


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10. Periodontal therapy other than prophylaxis within the past 6 months or 
endodontic therapy within 1 month of enrollment (if a subject meets this 
criterion, he/she may be rescreened for study participation when he/she no 
longer meets this exclusion criterion); 
 
11. History of early onset periodontitis or acute necrotizing ulcerative gingivitis; 
 
12. Chronic disease with concomitant oral manifestations, such as autoimmune 
or immunosuppressive diseases (e.g., human immunodeficiency virus, severe 
combined immunodeficiency, neutropenia, juvenile arthritis, systemic lupus 
erythematosus, sickle cell anemia, Crohn's disease, rheumatoid arthritis, 
Sjögren’s syndrome) or immunocompromised status due to cancer 
chemotherapy, hematopoietic stem cell or solid organ transplant, head and 
neck radiotherapy, splenectomy, chronic corticosteroid usage; 
 
13. Recent history of chronic alcohol consumption of more than five 1.5-ounce 
servings of 80-proof distilled spirits, five 12-ounce servings of beer, or five 
5-ounce servings of wine per day; 
 
14. Tobacco use (former tobacco users may be enrolled, provided they have 
been tobacco-free for 1 year or more); 
 
15. Diabetes mellitus; 
 
16. Subjects with urinalysis results suggestive of infection (if a subject meets this 
criterion, he/she may be rescreened for study participation when he/she no 
longer meets this exclusion criterion); 
 
17. Medical conditions that the investigator considers significant and that may 
interfere with the examination or the safety of the subject; 
 
18. Chronic (2 weeks or more) use of medication known to affect periodontal 
status within 1 month of enrollment, such as ≥ 81 mg aspirin per day, 
phenytoin, calcium antagonists such as nifedipine, NSAIDs, warfarin, 
cyclosporine, ≥ 10 mg/day atorvastatin or equivalent dose of another statin 
(Subramanian, Emami et al. 2013) (if a subject meets this criterion, he/she 
may be rescreened for study participation when he/she no longer meets this 
exclusion criterion); 
 
19. Treatment with antibiotics within 1 month prior to enrollment (if a subject 
meets this criterion, he/she may be rescreened for study participation when 
he/she no longer meets this exclusion criterion); 


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20. Medical condition for which antibiotic treatment during the study period is 
likely or a condition for which antibiotic prophylaxis is recommended before 
dental procedures (American Heart Association 2007 guidelines [Geist, 
Fitzpatrick et al. 2007] will be followed); 
 
21. Known hypersensitivity to any component of the test or placebo products; 
 
22. Anything that, in the opinion of the investigator, would place the subject at 
increased risk or prevent the subject from fully complying with or completing 
the study. 
 
The on-site medical monitor will review all laboratory test results and ensure that 
subjects meet the inclusion criteria for the study and do not meet any exclusion criteria. 
Additionally, the on-site medical monitor will decide whether subjects with an abnormal 
laboratory test result should have the laboratory test repeated.  Subjects with a second 
abnormal laboratory test result will be referred to their primary care physician. 
 
5.3 
Strategies for Recruitment and Retention 
 
Enrollment of 125 subjects is expected to take place over approximately 30 months. 
Having an already identified subject pool at the Forsyth Institute Center for Clinical and 
Translational Research (CCTR) should allow recruitment of subjects required to conduct 
the study in a timely manner.  Subjects will be recruited from the CCTR subject pool, 
from area health centers, and through advertisements in local newspapers and 
electronic media outlets.  All recruitment information and advertising will be submitted to 
the Institutional Review Board (IRB) prior to release. 
 
Prospective subjects will receive a free periodontal examination and close dental and 
periodontal surveillance during the study.  In addition, each subject will be compensated 
for visits attended, and parking fees will be paid for individuals who attend screening or 
study visits. 
 
Subjects who miss a scheduled visit without notifying the study staff will be contacted by 
telephone, email, or text message to encourage their continued participation in the 
study and provide them with another appointment. If the subject declines further 
participation, he/she will be referred to a qualified practitioner for continued care. If a 
subject cannot be contacted by one of the methods listed above or he/she does not 
keep the next scheduled appointment, he/she will be sent a certified letter advising of 
the need for further follow-up and offering referral to a qualified practitioner for 
continued care. 


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Every effort will be made to encourage full participation until the end of the study.  If a 
participant appears to be considering ending participation in the study, specific retention 
activities will be conducted to encourage him or her to continue. Strategies to promote 
adherence to study requirements will include after-hours visits for those who have tight 
work or personal schedules. To encourage compliance with study procedures, 
participants will be provided written instructions for product use and important details 
about the study protocol, and will be reminded of these at each study visit and with 
phone calls. Based on the participant’s preferred communication method, reminder 
postcards, emails, telephone calls, and/or telephone texts will be provided prior to clinic 
visits.  Appointment cards with important information on the reverse side (for example, 
contact person and phone number in case of an AE) will be given to all subjects after 
each completed visit. 
 
5.4 
Treatment Assignment Procedures 
 
5.4.1 
Randomization Procedures 
 
Subjects will be assigned to treatment groups using a block randomization schedule. 
 
5.4.2 
Masking Procedures 
 
The examiner will be masked to the assigned treatment.  Subjects receiving oral rinse 
will not know whether they are in the active or placebo rinse group.  A qualified and 
trained individual not involved in the examination procedures will be responsible for 
making assignments to treatment groups, dispensing the products to the subjects with 
instructions, collecting information on subject compliance, and soliciting AE reports from 
subjects. 
 
Test materials will be coded and the identity of the products concealed, so the 
examiners and subjects will remain blinded.  Subjects will be reminded to avoid 
discussing product use with their examiners at any time during their participation. 
 
The product code may be broken for an individual subject if he or she experiences a 
serious adverse event (SAE) and cannot be adequately treated without knowing the 
identity of the study product.  Every effort will be made to inform the study investigators 
prior to breaking the blind, or in the event of an emergency, as soon as possible 
thereafter. 


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5.5 
Subject Withdrawal 
 
5.5.1 
Reasons for Withdrawal 
 
Subjects are free to withdraw from participation in the study at any time upon request. 
 
An investigator may terminate a study subject’s participation in the study if: 
 
 Any clinical AE, laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best 
interest of the subject. 
 
 The subject meets an exclusion criterion (either newly developed or not 
previously recognized) that precludes further study participation. 
 
5.5.2 
Handling of Subject Withdrawals or Subject Discontinuation of Study 
Intervention 
 
Should a subject elect to withdraw from the study and cease use of product, efforts will 
be made to complete as much follow-up of safety information as possible. Telephone 
contact with the subject is acceptable if it is not possible to arrange a follow-up 
appointment. Subjects who withdraw prior to the Day 14 Visit will be replaced. 
 
5.6 
Premature Termination or Suspension of Study 
 
This study may be suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to the investigator, 
the Investigational New Drug (IND) sponsor, the National Institute of Dental and 
Craniofacial Research (NIDCR), and the Food and Drug Administration (FDA).  If the 
study is prematurely terminated or suspended, the principal investigator will promptly 
inform the IRB and will provide the reason(s) for the termination or suspension. 
 
Circumstances that may warrant termination include, but are not limited to: 
 
 Determination of unexpected, significant, or unacceptable risk to subjects; 
 
 Insufficient adherence to protocol requirements; 
 
 Data that are not sufficiently complete and/or evaluable; 
 
 Plans to modify, suspend, or discontinue the development of the study drug; 
 
 Determination of futility.  No formal futility analysis is planned. However, if 
serious safety concerns require the unblinding of all study subjects, a lack of 


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efficacy may be considered along with the safety concerns as a reason for the 
premature termination of the study. 
 
6 STUDY INTERVENTION 
 
 
6.1 
Study Product Description 
 
6.1.1 
BLXA4-ME Oral Rinse 
 
BLXA4-ME is a member of a new class of chemically and metabolically stable lipoxin 
analogs featuring a replacement of the tetraene unit of native LXA4 with a substituted 
benzo-fused ring system. The full chemical name of the BLXA4-ME drug substance is 
(5S, 6R, E)-methyl 5,6-dihydroxy-8-(2-((R,E)-3-hydroxyoct-1-enyl) phenyl) oct-7-enoate. 
 
The topical oral rinse dosage form of BLXA4-ME (also known as ClinRinse-1) will 
consist of drug substance prepared at a concentration of 1.0 μM in an aqueous vehicle 
solution containing the inactive components shown in Table 6-1. 
 
Table 6-1 Inactive Components of BLXA4-ME 
 
COMPONENT 
AMOUNT 
(%w/w) 
Saccharin sodium 
0.03 
Ethanol (95%) 
10.00 
Propylene glycol 
7.00 
Sodium lauryl sulfate 
0.25 
Sorbitol 
10.00 
Flavoring oil 
0.145 
Water 
72.575 
 
 
 
The physical and chemical properties of BLXA4-ME, its anti-inflammatory and 
pro-resolving actions, and the procedures for preparing the oral rinses are described in 
the Investigator’s Brochure. 


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6.1.2 
Placebo Oral Rinse 
 
The placebo preparation will consist of formulated oral rinse without BLXA4-ME and will 
be identical to the test rinse in color, appearance, and taste. 
 
6.1.3 
Acquisition 
 
The BLXA4-ME compound and the oral rinse formulation containing the 1.0 μM dose of 
BLXA4-ME (ClinRinse-1) will be prepared by Avanti Polar Lipids, Alabaster, Alabama, a 
facility that adheres to the principles of Good Manufacturing Practice. The topical oral 
rinse will be packaged into amber high-density polyethylene bottles for the clinical 
studies and shipped to the Forsyth Institute. 
 
6.1.4 
Formulation, Packaging, and Labeling 
 
The formulation of ClinRinse-1 and placebo rinse is described in the Investigator’s 
Brochure. Each bottle of study product will contain a volume sufficient for 8 doses plus 
an excess volume to accommodate for an out-of-window study visit.  The residual 
volume will be measured to assess compliance with product use.  Labels on the rinse 
bottles will comply with the regulations for labeling of an investigational product (21 CFR 
Part 312.6), and the active and placebo rinses will be identified by codes, to maintain 
the blinding of subjects and investigators. 
 
6.1.5 
Product Storage and Stability 
 
Prior to its distribution to study participants, the BLXA4-ME oral rinse (ClinRinse-1) and 
placebo rinse will be stored at 2-8°C in high-density polyethylene bottles in a secure 
area reserved for study materials at the Forsyth Institute CCTR.  Subjects will be 
instructed to store the bottle at room temperature during the week of use. Storage with 
the subject’s toothbrush is intended to encourage compliance with the protocol-specified 
product use. Testing of the drug product has demonstrated that it is stable at 2-8°C for 
up to 6 months, and stable at 25°C/60% relative humidity for up to 4 weeks. 
 
6.2 
Dosage, Preparation, and Administration of Study Product 
 
ClinRinse-1 containing 1.0 μM BLXA4-ME and placebo rinse will be provided to study 
participants in the original bottles supplied by Avanti.  A 30 mL polypropylene dosing 
cup with volume markings will be provided with the rinse. Subjects will rinse under 
supervision on the day they pick up study drug.  They will be instructed to use the rinse 
(30 mL for 45 seconds) daily after brushing their teeth in the morning.  Subjects will be 
provided with a timer so that 45 seconds of use can be standardized.  Subjects will be 
resupplied with study drug on Days 3, 7, 14, and 21. 


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6.3 
Modification of Study Product Administration for a Subject 
 
See Sections 5.5 and 5.6 for information on product discontinuation. 
 
6.4 
Accountability Procedures for the Study Product 
 
All study products will be stored at 2-8°C in a secure area reserved for study materials 
at the Forsyth Institute CCTR.  ClinRinse-1 and placebo rinses will be segregated to 
maintain effective control of study products and ensure proper distribution to subjects. 
Documentation to maintain study product accountability will occur at all visits.  The 
product number and amount of medication provided will be recorded, and information 
will be collected to track subject compliance. To assist with product accountability, 
subjects will be instructed to bring the bottle containing unused product to each 
follow-up visit.  A trained and qualified individual not involved in the examination 
procedures will distribute product, provide rinse instructions, receive the returned 
bottles, and measure and record the residual volume of rinse. 
 
6.5 
Assessment of Subject Compliance with Study Product Administration 
 
Compliance will be recorded at all follow-up study visits by measurement of the volume 
of remaining product. This will be done to estimate under- and over-usage of study 
drug.  All subjects will be resupplied with rinse preparations at each study visit through 
the Day 21 Visit.  In addition, the subjects will be provided with memory aid cards, and 
will be queried as to their compliance with the required schedule of product use. 
 
6.6 
Concomitant Medications/Treatments 
 
Subjects will receive instructions to avoid, during the 28 days of product use, any 
medications and dental procedures that could affect periodontal status (see exclusion 
criteria). At each study visit, subjects will be asked to report all concomitant 
medications and any dental care received or procedures performed during the study 
period. 
 
7 STUDY SCHEDULE 
 
 
7.1 
Prescreening 
 
Prescreening of potential subjects may be completed using a telephone script with 
questions that assess preliminary eligibility. 


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7.2 
Screening 
 
Screening Visit (Day -10 to -1) 
 
 Obtain and document consent from potential subject on screening consent form. 
 
 Record demographics information. 
 
 Review and record medical/dental history, including alcohol/tobacco use, to 
determine eligibility based on inclusion/exclusion criteria. 
 
 Review and record medications history to determine eligibility based on 
inclusion/exclusion criteria. 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure, 
weight). 
 
 Perform intraoral and extraoral examinations needed to determine eligibility, 
including hard and soft tissue assessments (face, lymph nodes, lips, buccal 
mucosa, floor of the mouth, tongue, hard and soft palate, gingiva, edentulous 
ridges, and teeth). 
 
 Perform the MGI to ensure mean full mouth MGI of at least 2.0. Subjects with a 
mean MGI of less than 2.0 will not be enrolled in the study. 
 
 Assess for the presence of gross plaque and calculus.  Gross plaque and 
calculus are based on investigator discretion.  Subjects with the presence of 
gross plaque or calculus will not be enrolled in the study. 
 
 Obtain urine pregnancy test on women of childbearing potential. Study staff will 
record the test kit lot, expiration date, and result. Test must be documented as 
negative to meet eligibility criteria. 
 
 Obtain urine for urinalysis. 
 
 Obtain approximately 15 mL blood for complete blood count, erythrocyte 
sedimentation rate, creatinine, blood urea nitrogen, alkaline phosphatase, AST, 
ALT, total bilirubin, and electrolytes (sodium, potassium, chloride, and 
bicarbonate). 
 
 Schedule study visits for individuals who are potentially eligible (eligibility is 
confirmed after receipt of laboratory results). 
 
 For individuals with periodontal disease who are found to be ineligible, provide, 
as appropriate, oral hygiene instructions or information about dental facilities that 
could provide periodontal treatment. 


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7.3 
Enrollment/Baseline 
 
Study Visit 1 - Enrollment/Baseline Visit (Day 0) 
 
 Obtain and document consent from subject on study consent form. 
 
 Review and confirm medical and dental history, including alcohol and tobacco 
use history, for study inclusion. 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure). 
 
 Record current concomitant medications and dental procedures and update 
medication history for study inclusion. 
 
 Obtain urine pregnancy test on women of childbearing potential. Study staff will 
record the test kit lot, expiration date, and result. Test must be documented as 
negative before continuing to conduct baseline procedures that follow. 
 
 Perform intraoral and extraoral examinations, to include hard and soft tissue 
assessments (face, lymph nodes, lips, buccal mucosa, floor of the mouth, 
tongue, hard and soft palate, gingiva, edentulous ridges, and teeth). 
 
 Collect GCF for baseline measurements of IL-1. 
 
 Assess and record baseline MGI. 
 
 Assess and record baseline PI. 
 
 Obtain supragingival plaque samples for baseline analyses of oral bacteria. 
 
 Assess and record baseline PD and GM-CEJ distance (to calculate CAL). 
 
 Assess and record baseline BOP. 
 
 Reconfirm eligibility prior to subject randomization based on information collected 
during Screening Visit and study Visit 1. 
 
 Obtain approximately 15 mL blood for baseline BLXA4 level and specialized 
pro-resolution mediator (SPM) (lipoxins and resolvins) profiling. 
 
 Randomize subject to 1 of the study arms, using a block randomization schedule. 
 
 Provide study drug with instructions for use and a timer to be used while rinsing. 
A study team member other than the examiner will supervise the subject’s first 
use of the medication. 
 
 Provide subject with the memory aid and instructions for its use. 
 
 Provide subject with a study contact number to call for AEs or questions. 


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7.4 
Intermediate Study Visits 
 
Study Visit 2 (Day 3 ± 1) 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure). 
 
 Update medical and dental histories. 
 
 Record concomitant medications and dental procedures. 
 
 Perform intraoral and extraoral examinations; evaluate mucosal irritancy using 
edema/erythema score and record other findings. 
 
 Review memory aid and document AEs and product experience. 
 
 Review product use and compliance. 
 
 Record volume of unused product in bottle. 
 
 Provide additional product, if needed. 
 
Study Visit 3 (Day 7 ± 1) 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure). 
 
 Update medical and dental histories. 
 
 Record concomitant medications and dental procedures. 
 
 Perform intraoral and extraoral examinations; evaluate mucosal irritancy using 
edema/erythema score and record other findings. 
 
 Review memory aid and document AEs and product experience. 
 
 Review product use and compliance. 
 
 Record volume of unused product in bottle. 
 
 Provide additional product. 
 
Study Visit 4 (Day 14 ± 1) 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure). 
 
 Update medical and dental histories. 
 
 Record concomitant medications and dental procedures. 
 
 Obtain approximately 15 mL blood for complete blood count, erythrocyte 
sedimentation rate, creatinine, blood urea nitrogen, alkaline phosphatase, AST, 


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ALT, total bilirubin, and electrolytes (sodium, potassium, chloride, and 
bicarbonate). 
 
 Obtain urine for urinalysis. 
 
 Perform intraoral and extraoral examinations; evaluate mucosal irritancy using 
edema/erythema score and record other findings. 
 
 Collect GCF for measurements of IL-1. 
 
 Assess and record MGI. 
 
 Assess and record PI. 
 
 Obtain supragingival plaque samples for analyses of oral bacteria. 
 
 Assess and record PD and GM-CEJ distance (to calculate CAL). 
 
 Assess and record BOP. 
 
 Review memory aid and document AEs and product experience. 
 
 Review product use and compliance. 
 
 Record volume of unused product in bottle. 
 
 Provide additional product. 
 
Study Visit 5 (Day 21 ± 1) 
 
 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure). 
 
 Update medical and dental histories. 
 
 Record concomitant medications and dental procedures. 
 
 Perform intraoral and extraoral examinations; evaluate mucosal irritancy using 
edema/erythema score and record other findings. 
 
 Review memory aid and document AEs and product experience. 
 
 Review product use and compliance. 
 
 Record volume of unused product in bottle. 
 
 Provide additional product. 
 
7.5 
Final Study Visit 
 
Study Visit 6 (Day 28 ± 1) 


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 Record vital signs (temperature, pulse rate, respiratory rate, blood pressure, 
weight). 
 
 Update medical and dental histories. 
 
 Record concomitant medications and dental procedures. 
 
 Obtain approximately 15 mL blood for complete blood count, erythrocyte 
sedimentation rate, creatinine, blood urea nitrogen, alkaline phosphatase, AST, 
ALT, total bilirubin, and electrolytes (sodium, potassium, chloride, and 
bicarbonate). 
 
 Obtain approximately 15 mL blood for BLXA4 level and SPM (lipoxins and 
resolvins) profiling. 
 
 Obtain urine for urinalysis. 
 
 Perform intraoral and extraoral examinations; evaluate mucosal irritancy using 
edema/erythema score and record other findings. 
 
 Collect GCF for measurements of IL-1. 
 
 Assess and record MGI. 
 
 Assess and record PI. 
 
 Obtain supragingival plaque samples for analyses of oral bacteria. 
 
 Assess and record PD and GM-CEJ distance (to calculate CAL). 
 
 Assess and record BOP. 
 
 Review memory aid and document AEs and product experience. 
 
 Review product use and compliance. 
 
 Record volume of unused product in bottle. 
 
7.6 
Final Safety Evaluation 
 
Telephone Call for Safety Follow-up (3 months ± 2 weeks) 
 
The subject will be contacted via telephone to collect information on any AEs that may 
have occurred since the final study visit.  At the end of the telephone interview, subjects 
will be reminded that they may need periodontal treatment, and will be provided with 
information on dental facilities that could provide care. 


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7.7 
Withdrawal Visit 
 
If a subject withdraws early or is withdrawn by the investigator, efforts will be made to 
complete as much follow-up of safety information as possible. The subject will be 
invited to attend a withdrawal visit at which safety assessments will be completed. 
Telephone contact with the subject is acceptable if it is not possible to arrange a 
follow-up appointment. 
 
7.8 
Unscheduled Visit 
 
Unscheduled visits will be documented, recording the time and the reason for the visit 
and the disposition of the subject at the end of the visit. 
 
8 STUDY PROCEDURES/EVALUATIONS 
 
 
8.1 
Clinical Evaluations 
 
8.1.1 
Safety Observations and Other Measurements 
 
A complete medical history will be obtained at the Screening Visit. The medical history 
will also include demographic information, alcohol and tobacco history, medications, 
dental procedures, and dental history information. 
 
Vital signs, including oral temperature, pulse rate, respiratory rate, and blood pressure, 
will be measured at all study visits beginning with the Screening Visit and through the 
Day 28 Visit. Weight will be collected only at the Screening and Day 28 Visits. 
 
An intraoral and extraoral examination will be completed at each of the visits from 
Baseline through the Day 28 Visit.  During the intraoral examination, the oral cavity will 
be evaluated for ulceration and erythema using the Oral Mucositis Assessment Scale 
(OMAS) (Sonis, Eilers et al. 1999). Subjects will have the OMAS scored by 
2 independent examiners at each study visit, until 10 subjects have completed the 
Day 28 visit.  The OMAS will be used to evaluate ulceration and erythema at the 
following sites: 
 
 Upper and lower lips 
 
 Hard and soft palate/fauces 
 
 Right and left buccal mucosa 
 
 Ventral and lateral surfaces on the tongue 
 
 Floor of mouth 


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Ulceration and erythema will be scored using the system shown in Table 8-1. 
 
Table 8-1 Scoring System for Ulceration and Erythema 
 
Oral cavity for Ulceration 
Oral cavity for Erythema 
Grade 0 
No lesion 
Grade 0 
None 
Grade 1 
Lesion < 1 cm2 
Grade 1 
Not severe 
Grade 2 
Lesion 1 to 3 cm2 
Grade 2 
Severe 
Grade 3 
Lesion > 3 cm2 
 
 
Subject’s periodontal measurements (Section 8.1.2) will be monitored for the 
development of or the progression of periodontitis.  Safety laboratory tests will be 
obtained and reviewed by the on-site medical monitor as described in Section 8.2. At 
each visit except Screening and Baseline, the subject will provide the completed 
memory aid, and the clinician will interview the subject and collect information regarding 
solicited AEs. The clinician will also record concomitant medications and confirm that 
the subject has not had any dental procedures. 
 
8.1.2 
Clinical Periodontal Measurements 
 
The clinical periodontal measurements described below will be obtained from the full 
mouth at Baseline and on Days 14 and 28.  In addition, plaque and gingivitis severity 
scores will be calculated using the method described in (Panagakos, Volpe et al. 2005). 
These severity scores represent the ratio of surfaces with high PI (scores 2 and 3 
assessed with the Silness and Löe PI) and high gingival index (scores 3 and 4 
assessed with the MGI) to total scored surfaces. 
 
 Modified gingival index (Gordon, Lamster et al. 1985): The MGI will be assessed 
at 6 sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and 
mesiolingual aspects of the tooth), and will be recorded as follows: 
 
0 = absence of inflammation 
 
1 = mild inflammation; slight change in color, little change in texture of any 
portion of, but not the entire, marginal or papillary gingival unit 
 
2 = mild inflammation; criteria as above but involving the entire marginal and 
papillary gingival unit 


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3 = moderate inflammation; glazing, redness, edema, and/or hypertrophy of 
the marginal or papillary gingival unit 
 
4 = severe inflammation; marked redness, edema, and/or hypertrophy of the 
marginal or papillary gingival unit; spontaneous bleeding, congestion, or 
ulceration 
 
 Probing depth: Periodontal pocket depth will be determined at 6 sites per tooth 
using a UNC-15 periodontal probe, with measurements rounded to the next lower 
whole millimeter. 
 
 Distance from free gingival margin to cementoenamel junction: The GM-CEJ will 
be measured to calculate the CAL. The GM-CEJ is measured with a periodontal 
probe at 6 sites per tooth, and is recorded in millimeters rounded to the next 
lower whole number. Millimeters of recession will be recorded as a negative 
number, and when the free gingival margin is coronal to the cementoenamal 
junction, the GM-CEJ will be recorded as a positive number. 
 
 Clinical attachment level: The CAL will be calculated by subtracting the GM-CEJ 
from the PD. 
 
 Bleeding on probing:  BOP is a dichotomous measurement that will be recorded 
at the same 6 sites per tooth: 
 
0 = no bleeding in 15 seconds after probing 
 
1 = bleeding in 15 seconds after probing 
 
 Plaque index (Silness and Loe 1964): Plaque will be assessed at 6 sites per 
tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual 
aspects of the tooth). The PI will be recorded as follows: 
 
0 = no plaque 
 
1 = film at the gingival margin 
 
2 = moderate (easily visible) 
 
3 = abundance of material 
 
Periodontal measurements obtained on Days 14 and 28 will be compared with 
measurements obtained at Baseline to monitor subjects for development of or 
progression of periodontitis, defined for this study as an increase of 2 mm or more in 
pocket depth or CAL from the Day 0 baseline measurement. 


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8.2 
Laboratory Procedures/Evaluations 
 
Blood and urine for safety laboratory tests will be collected prior to treatment and on 
Days 14 and 28.  Safety laboratory test results will be reviewed by the on-site medical 
monitor. 
 
8.2.1 
Clinical Laboratory Evaluations 
 
8.2.1.1 
Pregnancy Test 
 
A urine pregnancy test will be performed for women of childbearing potential at the 
Screening and Baseline Visits.  The results must be available and negative before 
initiation of product use and continuation in the study. 
 
8.2.1.2 
Blood and Urine Analyses for Safety Assessments 
 
Blood will be sent to a clinical laboratory for the following tests: 
 
 Complete blood count: hemoglobin, hematocrit, red blood cell (RBC) count, 
white blood cell (WBC) count with differential and absolute counts by WBC type, 
platelet count, and RBC indices (mean corpuscular volume, mean corpuscular 
hemoglobin concentration, RBC distribution width) 
 
 Erythrocyte sedimentation rate 
 
 Serum chemistry tests: creatinine, blood urea nitrogen, alkaline phosphatase, 
AST, ALT, total bilirubin, and electrolytes (sodium, potassium, chloride, and 
bicarbonate). 
 
 Urinalysis:  color, appearance, specific gravity, pH, glucose, bilirubin, ketones, 
occult blood, protein, nitrite, leukocyte esterase, WBCs, RBCs, squamous and 
other epithelial cells, bacteria, casts, and crystals. 
 
The on-site medical monitor will review the results of safety laboratory tests to 
determine whether a laboratory test should be redrawn.  Subjects with serum creatinine 
values greater than 50% above their baseline level will be retested, even if within 
normal range. Subjects with persistent out-of-normal values at the retest will be 
referred to their primary care physician and will be followed until the values have 
returned to the normal range or are confirmed as stable with an adequate explanation. 
Additionally, the on-site medical monitor will review the laboratory results to assist the 
principal investigator with assessing and monitoring AEs. 


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8.2.2 
Special Assays or Procedures 
 
8.2.2.1 
Plaque Sample Collection, Processing and Analysis for Safety 
Assessments 
 
A plaque sample will be obtained from every subject at the Baseline Visit, Day 14, and 
Day 28, the conclusion of the experimental period. These plaque samples of oral flora 
will be analyzed for any increase in opportunistic or pathogenic bacteria.  Supragingival 
plaque will be obtained from 6 sites in the mouth (the mesiobuccal surfaces of the 
Ramfjord teeth). Samples will be placed in transport media for DNA checkerboard 
probe analysis (DNA-DNA hybridization assay) and analyzed according to the method 
of Socransky (Socransky, Smith et al. 1994, Socransky, Haffajee et al. 2004). Samples 
will be analyzed for the presence of Fusobacterium nucleatum subspecies vincentii, 
Campylobacter concisus, Campylobacter rectus, Tannerella forsythensis, Prevotella 
intermedia, Prevotella nigrescens, Porphyromonas gingivalis, Capnocytophaga 
sputigena, Streptococcus oralis, Actinomyces naeslundii, Actinomyces israeli, 
Eubacterium brachy, Eikenella corrodens, and spirochetes. The technique has been 
validated by several laboratories and found to be accurate, sensitive (detects 104 or 
more bacteria), and specific when carefully selected DNA probes are used. 
 
8.2.2.2 
Gingival Crevicular Fluid Collection and Analysis 
 
Crevicular fluid samples from the mesiobuccal sites of the 4 first molar teeth will be 
collected using paper strips (Periopaper) furnished by the manufacturers of the 
Periotron 8000, the instrument used for measurement of crevicular fluid volume. The 
sample strip will be placed in a prelabeled vial, snap frozen in liquid nitrogen, and stored 
at -80°C. Levels of IL-1β in GCF will be determined by ELISA. 
 
8.2.2.3 
Plasma Levels of BLXA4-ME and SPM Profiling in Plasma and 
Serum 
 
Blood will be collected at Baseline and on Day 28 to measure absorption of BLXA4-ME 
through the oral mucosa.  Labeled plasma specimens will be sent to Avanti Polar Lipids 
for assay using a liquid chromatography-tandem mass spectrometry technique. 
 
In addition, SPMs (lipoxins and resolvins) will be profiled in serum and plasma samples. 
Labeled plasma and serum specimens will be sent to laboratory of Dr. Charles Serhan 
at Brigham and Women’s Hospital, Boston for lipidomics assay using liquid 
chromatography-tandem mass spectrometry technique. 


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8.2.3 
Specimen Preparation, Handling, and Storage 
 
Specimen preparation for the various assays is described in the Manual of Procedures. 
Assays on plaque and GCF samples will be performed at the Forsyth Institute 
laboratories; these samples are stored at -80°C until time of assay.  Blood and urine for 
safety labs will be sent to a commercial clinical laboratory on the day the samples are 
collected.  Frozen plasma specimens for BLXA4 measurement will be sent to the 
laboratory at Avanti Polar Lipids.  Additional frozen plasma and serum specimens for 
SPM profiling will be sent to Dr. Serhan’s laboratory at Brigham and Women’s Hospital, 
Boston. 
 
9 ASSESSMENT OF SAFETY 
 
 
9.1 
Specification of Safety Parameters 
 
Parameters used to assess safety will include AEs spontaneously reported by the 
subject or observed by the investigator, solicited AEs captured by review of the memory 
aid while interviewing the subject, clinical laboratory values (reviewed by the on-site 
medical monitor), oral cavity examination, vital signs, and increases in pathogenic 
bacterial flora identified in plaque samples.  All AEs will be coded by system organ class 
and preferred term according to the terminology of the Medical Dictionary for Regulatory 
Activities. 
 
Monitoring of AEs will be conducted throughout the study.  New AEs, including SAEs, 
will be captured through the final study visit.  In addition, all AEs will be captured at the 
final telephone follow-up conducted 2 months after the last use of study product. 
Adverse events will be followed in accordance with Good Clinical Practice.  Serious 
adverse events will be immediately reported to the IND sponsor, to NIDCR, and to the 
IRB within 24 hours of becoming aware of the event, and will be monitored until they are 
resolved or are clearly determined to be due to a subject’s stable or chronic condition or 
intercurrent illness(es).  Unanticipated problems (UPs) will also be captured and 
reported to the IRB and FDA as required by regulation. 
 
9.1.1 
Adverse Events 
 
International Conference on Harmonisation (ICH) E6 defines an AE as any untoward 
medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product regardless of its causal relationship to the study treatment.  An 
AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of medicinal 
(investigational) product. The occurrence of an AE may come to the attention of study 


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personnel during study visits and interviews of a study subject presenting for medical 
care, or upon review by a study monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs should 
be captured. Information to be collected includes event description, time of onset, 
clinician’s assessment of severity, relationship to study product (assessed only by those 
with the training and authority to make a diagnosis, which would include MD, DO, 
physician assistant, nurse practitioner, DDS or DMD, dental hygienist), and time of 
resolution/stabilization of the event.  All AEs occurring while on study must be 
documented appropriately regardless of relationship.  All AEs will be followed to 
adequate resolution or stabilization. 
 
Any medical condition that is present at the time that the subject is screened should be 
considered as baseline and not reported as an AE. However, if it deteriorates at any 
time during the study, it should be recorded as an AE. 
 
9.1.2 
Solicited Adverse Events from the Memory Aid 
 
When the subject returns for follow-up visits, he or she will be instructed to bring the 
memory aid. Study staff will review the data recorded on the memory aid.  The 
information obtained from the interview will be recorded for the study. 
 
9.1.3 
Serious Adverse Events 
 
An SAE is an AE that meets 1 or more of the following criteria: 
 
 Results in death 
 
 Is life-threatening (places the subject at immediate risk of death from the event 
as it occurred) 
 
 Results in inpatient hospitalization or prolongation of existing hospitalization 
 
 Results in a persistent or significant disability or incapacity 
 
 Results in a congenital anomaly or birth defect 
 
An important medical event that may not result in death, be life-threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical 
judgment, the event may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. 


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All SAEs will be: 
 
 Reviewed and evaluated by a study clinician; 
 
 Recorded for the study; 
 
 Followed through resolution by a study clinician. 
 
9.1.4 
Unanticipated Problems 
 
The Office for Human Research Protections (OHRP) considers UPs involving risks to 
subjects or others to include, in general, any incident, experience, or outcome that 
meets all of the following criteria: 
 
 Unexpected in terms of nature, severity, or frequency given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 
 Related or possibly related to participation in the research (“possibly related” 
means there is a reasonable possibility that the incident, experience, or outcome 
may have been caused by the procedures involved in the research); and 
 
 Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
Because the occurrence of an incident, experience, or outcome that meets the above 
3 criteria will generally require changes in the research to protect the safety or welfare 
of subjects or others, any such occurrence will be promptly reported to the IRB, the 
medical monitor, and the Data and Safety Monitoring Board (DSMB) Chair. 
 
Unanticipated problems will be captured and reported throughout the study. 
 
9.2 
Time Period and Frequency for Event Assessment and Follow-Up 
 
Adverse events will be followed until resolved or considered stable. Phone calls or 
extra visits may be performed to follow up the AE. The subject will be questioned on 
the AE and whether it continues or is resolved at the time of the study visit, and whether 
the subject received any treatment specifically for the AE. The AE data will be updated 
and a follow-up report will be submitted to the IND Sponsor, NIDCR, and the IRB. 
 
All unresolved AEs will be followed by the investigator until the events are resolved, the 
subject is lost to follow-up, or the AE is otherwise explained.  At the last scheduled visit, 
the investigator will instruct each subject to report any subsequent event(s) that the 


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subject, or the subject’s personal physician, believes might reasonably be related to 
participation in this study.  The investigator will notify the IND sponsor, NIDCR, and the 
IRB of any death or AE occurring at any time after a subject has discontinued or 
terminated study participation, if the death or AE may reasonably be related to this 
study. 
 
9.3 
Characteristics of an Adverse Event 
 
All AEs must be graded for relationship to study product, expectedness, and severity. 
 
9.3.1 
Relationship to Study Intervention 
 
The clinician’s assessment of an AE's relationship to test article is part of the 
documentation process, but it is not a factor in determining what is or is not reported in 
the study.  If there is any doubt as to whether a clinical observation is an AE, the event 
should be reported. All AEs must have their relationship to study product assessed 
using the following terms: 
 
 Related (possible, probable, definite) events meet 1 or more of the following 
criteria: 
 
o The event is known to occur with the study intervention. 
o There is a temporal relationship between the intervention and event onset. 
o The event abates when the intervention is discontinued. 
o The event reappears upon a rechallenge with the intervention. 
 Not related (unlikely, not related) events meet 1 of the following criteria: 
 
o There is no temporal relationship between the intervention and event 
onset. 
 
o An alternate etiology has been established. 
 
9.3.2 
Expectedness 
 
This trial involves the first use of the BLXA4-ME oral rinse in humans; therefore, no 
human data exist to classify expectedness of events.  In a preclinical 28-day oral 
irritancy study in rats, transient erythema and edema of the oral mucosa were noted in 
animals treated with either BLXA4-ME or a saline solution used as a control.  For this 
study, the IND sponsor, the study investigator, and the NIDCR medical monitor will be 
responsible for assessing AEs that may be related to product use. An AE will be 
considered unexpected if the nature, severity, or frequency of the event is not consistent 
with the risk information described in the protocol or the Investigator’s Brochure or 
discussed with the subject during the informed consent process. 


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9.3.3 
Severity of Event 
 
Severity of AEs will be graded according to the National Cancer Institute’s Common 
Terminology Criteria for Adverse Events (CTCAE), Version 4.0: 
 
 Grade 1:  Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated 
 
 Grade 2:  Moderate; minimal, local, or noninvasive intervention indicated; limiting 
activities of daily living 
 
 Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting 
self-care activities of daily living 
 
 Grade 4: Life-threatening consequences; urgent intervention indicated 
 
 Grade 5:  Death related to AE 
 
Changes in the severity of an AE should be documented to allow an assessment of the 
duration of the event at each level of intensity.  Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
 
9.4 
Reporting Procedures 
 
9.4.1 
Unanticipated Problem Reporting to IRB and NIDCR 
 
Incidents or events that meet the OHRP criteria for UPs require the creation and 
completion of a UP report form.  The OHRP recommends that investigators include the 
following information when reporting an AE, or any other incident, experience, or 
outcome as a UP to the IRB: 
 
 Appropriate identifying information for the research protocol, such as the title, 
investigator’s name, and the IRB project number; 
 
 A detailed description of the AE, incident, experience, or outcome; 
 
 An explanation of the basis for determining that the AE, incident, experience, or 
outcome represents a UP; 
 
 A description of any changes to the protocol or other corrective actions that have 
been taken or are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following 
timeline: 
 
 Unanticipated problems that are SAEs will be reported to the IRB within 24 hours 
and to NIDCR within 1 week of the investigator becoming aware of the event. 


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 Any other UP will be reported to the IRB and to NIDCR within 2 weeks of the 
investigator becoming aware of the problem. 
 
 All UPs should be reported to appropriate institutional officials (as required by an 
institution’s written reporting procedures), the supporting agency head (or 
designee), and OHRP within 1 month of the IRB’s receipt of the report of the 
problem from the investigator. 
 
All UPs will be reported to NIDCR’s centralized reporting system via Rho Product 
Safety: 
 
 Product Safety Fax Line (US):  1-888-746-3293 
 
 Product Safety Fax Line (International): 919-287-3998 
 
 Product Safety Email:  rho_productsafety@rhoworld.com 
 
General questions about SAE reporting can be directed to the Rho Product Safety Help 
Line (available 8:00 AM – 5:00 PM Eastern Time): 
 
 US:  1-888-746-7231 
 
 International: 919-595-6486 
 
9.4.2 
Serious Adverse Event Reporting to NIDCR 
 
Any AE meeting the specified SAE criteria will be submitted on an SAE form to NIDCR’s 
centralized safety system via Rho Product Safety.  This report may be sent by fax or 
email. Once submitted, Rho Product Safety will send a confirmation email to the 
investigator within 1 business day.  The investigator should contact Rho Product Safety 
if this confirmation is not received.  This process applies to both initial and follow-up 
SAE reports. 
 
SAE reporting contact information: 
 
 Product Safety Fax Line (US):  1-888-746-3293 
 
 Product Safety Fax Line (International): 919-287-3998 
 
 Product Safety Email:  rho_productsafety@rhoworld.com 
 
General questions about SAE reporting can be directed to the Rho Product Safety Help 
Line (available 8:00 AM – 5:00 PM Eastern Time): 
 
 US:  1-888-746-7231 
 
 International: 919-595-6486 


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The study clinician will complete an SAE form and submit via fax or email within the 
following timelines: 
 
 All deaths and immediately life-threatening events, whether related or unrelated, 
will be recorded on the SAE form and submitted to Product Safety within 
24 hours of site awareness. 
 
 Serious adverse events other than death and immediately life-threatening events, 
regardless of relationship, will be reported by fax within 72 hours of site 
awareness. 
 
All SAEs will be followed until resolution or stabilization. 
 
9.4.3 
Reporting of SAEs and AEs to FDA 
 
Following notification from the investigator, the IND sponsor will report events that are 
both serious and unexpected and that are related to study product to the FDA within the 
required timelines as specified in 21 CFR Part 312.32:  fatal and life-threatening events 
within 7 calendar days (by phone or fax) and all other SAEs in writing within 15 calendar 
days.  All serious events designated as “not related” to study product will be reported to 
the FDA at least annually in a summary format. 
 
9.4.4 
Reporting of Pregnancy 
 
Subjects who are pregnant, nursing, or not using an acceptable birth control method at 
the time of the screening will not be enrolled in the study.  A urine pregnancy test will be 
performed for women of childbearing potential at the Screening and Baseline Visits to 
rule out pregnancy.  The results must be available and negative before study drug 
dispensation. The investigator will ensure that nonpregnant subjects are advised to 
avoid pregnancy and nursing during their participation in the research. 
 
Subjects will be advised to notify the principal investigator immediately should they 
become pregnant.  If a woman becomes pregnant or suspects she is pregnant while 
participating in this study, she must inform her treating dental provider immediately and 
permanently discontinue study drug.  A subject who becomes pregnant during study 
participation will be discontinued from study participation because of her additional 
health concerns during pregnancy and because of the need to avoid unnecessary risk 
to the fetus. If a female subject reports a pregnancy or a male subject reports a 
partner’s pregnancy, the investigator will report the pregnancy condition to the IND 
sponsor, safety monitoring team, and the IRB as soon as he/she becomes aware of the 
event. The investigator will attempt to obtain and report information about the 
pregnancy and the birth outcome. 


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9.5 
Rules for Study Suspension or Discontinuation 
 
The NIDCR medical monitor will review all SAEs and UPs in an expedited fashion. In 
the event of unacceptable toxicity, the trial will be stopped prematurely to prevent harm 
to subjects. In addition, any injuries, SAEs, or other unexpected AEs involving risk to 
subjects or others, and occurring at a frequency above that considered acceptable by 
the investigators, IND sponsor, NIDCR, or IRB, may result in a temporary or permanent 
halt to the study.  The study may be suspended for a safety review and consideration of 
study discontinuation if: 
 
 One or more subjects experiences a clinically significant event or laboratory 
abnormality determined to be Grade 3 or higher and related (possibly, probably, 
definitely) to use of the investigational product; 
 
 Two of more subjects experience a similar event determined to be Grade 2 or 
higher and related (possibly, probably, definitely) to use of the investigational 
product; 
 
 One or more subjects experiences a Grade 2 or higher event that does not 
resolve within 14 days. 
 
The NIDCR medical monitor or DSMB may request to be unblinded to the entire study 
when evaluating whether to suspend or discontinue the study.  In the event of study 
unblinding, a non-study statistician will be assigned as the “unblinded statistician” to 
assist in unblinding and any safety review. 
 
Any proposed changes in the consent form or research procedures resulting from any 
safety findings are to be submitted by the principal investigator to the IRB for approval. 
 
10 STUDY OVERSIGHT 
 
In addition to the principal investigator’s responsibility for oversight, study oversight will 
be under the direction of an independent DSMB constituted by NIDCR and composed of 
members with expertise in safety, statistics of early safety studies, and gingival and 
periodontal disease. The DSMB will operate under the rules of a charter approved at 
the organizational meeting of the DSMB.  Most data elements needed for the DSMB 
reviews will be collected in the clinical database, and the DSMB will meet at least 
annually.  The DSMB will assess safety and preliminary efficacy data for the study. The 
DSMB will be convened if a safety signal emerges prior to the completion of study 
enrollment. The DSMB will provide recommendations to the NIDCR. 


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11 CLINICAL SITE MONITORING 
 
Site monitoring is conducted to ensure that the human subjects protections, study 
procedures, laboratory procedures, study product administration, and data collection 
processes are of high quality and meet ICH E6 and regulatory guidelines.  The NIDCR 
is funding the study, and NIDCR or its designee will be responsible for conducting the 
on-site monitoring visits.  A study initiation visit will be performed by NIDCR prior to 
subject enrollment. The purpose of this visit is to ensure site staff and facilities are 
prepared to conduct the study as described in this protocol. Interim site monitoring 
visits will be performed as necessary to evaluate the conduct of the study, quality and 
integrity of data collection and regulatory documentation, and compliance with Good 
Clinical Practice. Scheduling of interim site monitoring visits will be dependent upon 
subject enrollment.  Following each site visit, the clinical site will be provided with 
documentation of actions for follow-up or resolution. A separate monitoring plan 
document will be developed to further describe the methods for monitoring, the 
frequency of monitoring, and the level of detail for monitoring. 
 
12 STATISTICAL CONSIDERATIONS 
 
 
12.1 
Study Hypotheses 
 
No formal statistical hypothesis testing will be performed in the primary safety analysis. 
The safety data will be summarized as described in Section 12.5.1. 
 
The efficacy hypothesis to be tested is that the efficacy outcomes differ among the 
study arms at the later post-baseline study visits. 
 
12.2 
Analysis Populations 
 
12.2.1  Safety Population 
 
The Safety Population will consist of all subjects who are randomized into a study arm 
and complete their Baseline Visit. 
 
12.2.2  Efficacy Population 
 
The Efficacy Population will consist of all subjects of the Safety Population who have a 
baseline and at least 1 post-baseline assessment of 1 or more of the secondary efficacy 
outcome measures. 


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12.3 
Sample Size Considerations 
 
Sample size determination is based on the power to detect AEs (the primary safety 
objective) with additional consideration given to detecting post-baseline study arm 
differences with respect to the MGI (a secondary efficacy outcome). 
 
The probability of observing a specific AE for the study’s sample size can be estimated. 
This probability depends on the sample size within a treatment group and on the true 
underlying probability of an individual experiencing the specific AE in that treatment 
group. Table 12-1 summarizes the probabilities of observing an AE given a sample size 
and true underlying event rate. For example, if the true probability of a subject 
experiencing a specific AE is 5%, the probability of observing 1 or more of that specific 
AE within a treatment group of 40 subjects is 87.1%. 
 
The study’s test oral rinse, placebo oral rinse, and no-rinse control arms are assigned 
treatment group sample sizes of 40, 40, and 20 subjects, respectively.  For the test oral 
rinse and the placebo oral rinse arms, a sample size of 40 subjects per treatment group 
should have probabilities of 80.5%, 87.1%, 91.6%, and 96.4% of observing an AE 
having underlying incidence probabilities of 4%, 5%, 6%, and 8%, respectively.  The 
no-rinse control arm is considered to be of lesser importance and, thus, to conserve the 
overall sample size, is assigned a sample size of 20 subjects, which should have 
probabilities of 55.8%, 64.2%, 71.0%, and 81.1% of observing an AE having underlying 
incidence probabilities of 4%, 5%, 6%, and 8%, respectively.  Thus, the above sample 
sizes of 40, 40, and 20 subjects should give adequate power (> 80%) to detect AEs that 
occur at true underlying frequencies of 4% or greater in the test and placebo oral rinse 
study arms, and adequate power (> 80%) to detect AEs that occur at true underlying 
frequencies of 8% or greater in the no-rinse control arm. 


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Table 12-1 
Probability of Observing One or More Adverse Events within a Study 
Arm 
 
"True" Underlying 
Probability of an Individual 
Experiencing an Adverse 
Event 
Probability of Observing One or More Adverse 
Events within a Study Arm 
No-Rinse Control Study 
Arm 
(n = 20) 
Test and Placebo Oral 
Rinse Study Arms 
(n = 40) 
0.01% 
0.20% 
0.40% 
0.10% 
2.0% 
3.9% 
0.25% 
4.9% 
9.5% 
0.50% 
9.5% 
18.2% 
1.00% 
18.2% 
33.1% 
2.00% 
33.2% 
55.4% 
3.00% 
45.6% 
70.4% 
4.00% 
55.8% 
80.5% 
5.00% 
64.2% 
87.1% 
6.00% 
71.0% 
91.6% 
8.00% 
81.1% 
96.4% 
10.0% 
87.8% 
98.5% 
Sample size (n) refers to the final sample size per treatment group of enrolled subjects who complete the 
study. 
 
Power estimates for the secondary efficacy endpoint (MGI) are found in Table 12-2. In 
this power analysis, MGI refers to the MGI scores averaged over all tooth sites within 
each subject and visit.  The power estimates in the table are based upon 
50,000 simulations of mixed models analyses. The mixed models simulations include 
the MGI as the dependent variable, and fixed effects for treatment group (test oral rinse, 
placebo oral rinse, and no-rinse control), visit (Baseline, 14 Days, and 28 Days), and 
their treatment group-by-visit interaction. The model includes random effects for subject 
using a compound symmetry variance structure (i.e., exchangeable covariance). These 
power analyses assume an overall within-study group standard deviation of 0.3 MGI 
units at each visit and a difference in the mean change from baseline MGI at 14 or 
28 days between the test oral rinse and the placebo rinse or between the test oral rinse 
and the no-rinse control of 0.2 MGI units.  Correlation of MGI within subject among visits 


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was assumed to be 0.3. The treatment group sample sizes of 40, 40, and 20 subjects 
for the test oral rinse, placebo oral rinse, and no-rinse control arms, respectively, are 
estimated to have powers of 71% and 54% to detect significant differences of 0.2 in the 
mean MGI scores between the test oral rinse and placebo oral rinse study arms, and 
between the test oral rinse and the no-rinse control study arms, respectively.  Even 
though these sample sizes would be considered under-powered for testing efficacy in a 
Phase 3 clinical trial, for this Phase 1/2 protocol, with efficacy as a secondary objective, 
these study sample sizes can provide evidence (significant or not) of efficacy and 
provide efficacy estimates with their associated standard deviations needed for planning 
future Phase 2 or Phase 3 trials. 
 
It is estimated that 50, 50, and 25 subjects will need to be enrolled to have final sample 
sizes of 40, 40, and 20 subjects for the test oral rinse, placebo oral rinse, and no-rinse 
control study arms, respectively, assuming a 20% subject attrition rate before 
completing the study. 
 
Table 12-2 
Mixed Models Power Analysis for MGI (Secondary Efficacy Outcome) 
Showing the Power for Several Sample Size Scenarios 
 
Enrolled and Eligible 
Subjects 
Subjects after 20% 
Drop-out 
Power (%) 
Test 
(n) 
Placebo 
(n) 
Control 
(n) 
Test 
(n) 
Placebo 
(n) 
Control 
(n) 
Trt – 
a 
Placebo 
Trt – 
Controlb 
50 
50 
25 
40 
40 
20 
70.9 
53.8 
Control = no-rinse control study arm; MGI = modified gingival index; placebo = placebo oral rinse study 
arm; test = test oral rinse study arm; trt = treatment (placebo or test oral rinse) study arm. 
a 
Trt-Placebo: Power (probability) for finding a statistically significant difference (alpha = 0.05) in the 
change from baseline MGI at 14 or 28 days between the test oral rinse and the placebo oral rinse study 
arms. 
b 
Trt-Control: Power (probability) for finding a statistically significant difference (alpha = 0.05) in the 
change from baseline MGI at 14 or 28 days between the test oral rinse and the no-rinse control study 
arms. 
 
12.4 
Planned Interim Analyses 
 
No interim analysis of efficacy endpoints is planned. Safety monitoring will occur 
throughout the study period. 


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12.5 
Final Analysis Plan 
 
12.5.1  Primary Safety Analysis 
 
Safety data will be summarized for all subjects in the Safety Population. Adverse event 
and SAE reporting requirements are found in Section 9. 
 
Safety for study treatment groups will be evaluated by the incidence, severity, and type 
of AEs, and by changes from baseline in subjects’ oral examination findings, vital signs, 
and clinical laboratory test results. Concomitant medications taken during the trial will 
be summarized and listed. 
 
The number and percentage of subjects who experience an AE, SAE, treatment-related 
AE, treatment-related SAE, and AE leading to discontinuation from the study will be 
summarized.  All AEs will be coded by system organ class and preferred term according 
to the terminology of the Medical Dictionary for Regulatory Activities, and severity will be 
graded according to the National Cancer Institute’s CTCAE, Version 4.0. 
 
All AEs will be summarized by system organ class and preferred term and by maximum 
CTCAE severity grade.  Summaries will also be presented by relationship to study 
treatment.  For AE summaries, emphasis will be placed on the summary of 
treatment-emergent AEs, defined as new AEs reported after initiation of treatment or 
pre-existing conditions that worsen during the treatment period. 
 
Subjects who withdraw from study treatment because of an AE will be listed. 
 
The incidence of development or progression of periodontal disease at Day 28 will be 
reported for each study arm as part of the primary safety analysis.  Development of 
disease or progression of existing periodontitis is defined as an increase from baseline 
of 2 mm or more in either the PD or CAL measures. 
 
12.5.2  Secondary Efficacy Analysis 
 
The study is not fully powered to find statistical differences among the study arms with 
respect to the secondary efficacy outcomes. As such, the main objectives of the 
secondary efficacy analyses are to:  1) obtain some evidence of efficacy (either a 
statistically significant effect or a nonsignificant trend); 2) obtain estimates of the size 
and variance of possible treatment effects that can then be used to plan later Phase 3 
clinical trials; and 3) explore which of several outcomes may best be used to measure 
efficacy.  As secondary analyses, no corrections will be made for multiple comparisons. 
 
The secondary efficacy measures of PI, MGI, IL-1β, and percentage of tooth sites with 
BOP will be analyzed using mixed models with a random effect for subject, and fixed 


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effects for baseline value, visit, study arm, and a study arm-by-visit interaction.  Contrast 
statements within the mixed models analyses will compare study arms with respect to 
the efficacy measures. These statistical models allow comparisons to be made 
between study arms, between visits, and between treatment groups at each visit. 
These mixed models analyses assume the efficacy outcomes are approximately normal 
in distribution. If residual analysis shows the normality assumption to be unreasonable 
for any efficacy outcomes, then generalized linear mixed models with the appropriate 
links and distributional assumptions will be used to analyze these efficacy outcomes. 
 
13 SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS 
 
The investigative team at the Forsyth Institute will be responsible for completion of all 
source documents, in compliance with ICH E6, Section 4.9 and regulatory and 
institutional requirements for the protection of confidentiality of subjects. The 
investigator will permit authorized representatives of NIDCR, FDA, and other regulatory 
authorities to inspect facilities and records relevant to this study if needed. 
 
Source data are all information, original records of clinical findings, observations, or 
other activities in a clinical trial necessary for the reconstruction and evaluation of the 
trial. Data collected for this trial will include demographic information; vital signs; 
medical/dental history; oral examination results; medication history; records of collection 
for blood, urine, GCF, and plaque samples; measurements of MGI, PD, GM-CEJ, BOP, 
and PI; study medication administration and accountability records; clinical laboratory 
results; and AEs. 
 
Source documents will include the original documents, data, and records (e.g., hospital 
records, clinical and office charts, safety laboratory test results and other laboratory test 
results, memoranda, subjects' diaries or evaluation checklists, subject files, and records 
kept at the Forsyth Institute and at the laboratories involved in the clinical trial).  Some 
data will be recorded directly into the study data system. The data captured in the study 
data system must be consistent with source documents; any discrepancies will be 
explained. 
 
14 QUALITY CONTROL AND QUALITY ASSURANCE 
 
Quality management is the overall process of establishing and ensuring the quality of 
processes, data, and documentation associated with clinical research activities.  It 
encompasses both quality control and quality assurance activities.  All sites conducting 
research that are funded by NIDCR are required to have a plan in place for assuring the 
quality of the research being conducted. 
 
This clinical trial will have a separate quality management plan to describe: 


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 How data will be evaluated for compliance with the protocol and for accuracy in 
relation to source documents; 
 
 Which documents will be reviewed (e.g., electronic case report forms, product 
accountability records, specimen tracking logs), who is responsible for reviewing, 
and the frequency for reviews; 
 
 Who will be responsible for addressing quality assurance issues (correcting 
procedures that are not in compliance with protocol) and quality control issues 
(correcting errors in data entry); 
 
 Staff training methods and how such training will be tracked; 
 
 
Calibration exercises conducted prior to and during the study to train examiners 
and maintain acceptable intra- and inter-examiner agreement. 
 
15 ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
 
15.1 
Ethical Standard 
 
The investigator will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report:  Ethical Principles and Guidelines for the 
Protection of Human Subjects of Research, as drafted by the United States National 
Commission for the Protection of Human Subjects of Biomedical and Behavioral 
Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the ICH E6. 
 
15.2 
Institutional Review Board 
 
The protocol, consent forms, recruitment materials, and all subject materials will be 
submitted to the Forsyth Institute IRB for review and approval.  Approval of both the 
protocol and the consent forms must be obtained before any subject is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the 
changes are implemented in the study. 
 
15.3 
Informed Consent Process 
 
Informed consent is a process that is initiated prior to the individual agreeing to 
participate in the study and continues throughout study participation. Extensive 
discussion of risks and possible benefits of study participation will be provided to 
subjects. A consent form describing in detail the study interventions/products, 
procedures, and risks will be given to the subject.  Consent forms will be IRB-approved, 
and the subject is required to read and review the document or have the document read 
to him or her. The investigator or designee will explain the research study to the subject 


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and answer any questions that may arise. The subject will sign the informed consent 
document prior to any study-related assessments or procedures. Subjects will be given 
the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate. They may withdraw consent at any time throughout the course 
of the study.  A copy of the signed informed consent document will be given to subjects 
for their records. The rights and welfare of the subjects will be protected by 
emphasizing to them that the quality of their clinical care will not be adversely affected if 
they decline to participate in this study. 
 
The consent process will be documented in the clinical or research record. 
 
15.4 
Exclusion of Women, Minorities, and Children (Special Populations) 
 
Adults of any gender or racial/ethnic group may participate. 
 
15.5 
Subject Confidentiality 
 
Subject confidentiality is strictly held in trust by the investigators, study staff, and the 
IND sponsor(s) and their agents. The study protocol, documentation, data, and all other 
information generated will be held in strict confidence. No information concerning the 
study or the data will be released to any unauthorized third party without prior written 
approval of the IND sponsor. 
 
Information from this study and from a subject’s medical record may be reviewed and 
photocopied by the FDA and/or state and federal regulatory agencies such as the 
OHRP as applicable, and the IRB of the Forsyth Institute.  Information from this study 
and from a subject’s medical record may be used for research purposes and may be 
published; however, a subject’s name will not be used in any publications. A subject’s 
data and samples will be protected by using coded designations only.  The list that 
matches a subject’s name to the code number will be kept in a password-protected 
computer file and destroyed permanently after study completion. A subject will not be 
personally identified by any of the research materials in this study. 
 
The study monitor or other authorized representatives of NIDCR may inspect all study 
documents and records required to be maintained by the investigator, including but not 
limited to medical records (office, clinic, or hospital) for the study subjects. The clinical 
study site will permit access to such records. 
 
15.6 
Future Use of Stored Specimens and Other Identifiable Data 
 
No specimens will be retained for future use. 


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16 DATA HANDLING AND RECORD KEEPING 
 
The investigators are responsible for ensuring the accuracy, completeness, legibility, 
and timeliness of the data reported. All source documents should be completed in a 
neat, legible manner to ensure accurate interpretation of data. The investigators will 
maintain adequate case histories of study subjects, including accurate case report 
forms and source documentation. 
 
Details of the study data system will be described in a separate Data Management Plan. 
 
16.1 
Data Management Responsibilities 
 
The investigative team at the Forsyth Institute will be responsible for data management 
including data entry, data security, and specimen tracking.  Unanticipated problems and 
AEs must be reviewed by the investigator or designee. 
 
16.2 
Data Capture Methods 
 
Data will be captured and stored electronically in a study-specific, password-protected 
database, with data quality checks built into the system. 
 
Data including medical and dental history, demographics, AEs, concomitant 
medications, oral examination results, and vital signs will be entered into a data system 
designed for this study.  Clinical measurements including MGI, PD, GM-CEJ, BOP, and 
PI will be entered chairside to an electronic data form, using special software installed 
on a study-designated laptop computer.  For chairside data collection, a data recorder 
will work with the study examiner to enter data during the oral examination.  Data from 
laboratory test results and special assays will be recorded by the laboratory and entered 
into the data system by study staff. 
 
16.3 
Types of Data 
 
Data for this study will include demographic and medical/dental history data, vital signs, 
information on concomitant medications, safety observations (records of AEs), oral 
examination findings, clinical outcome measures (i.e., PD, CAL, PI, MGI, BOP, plaque 
and gingivitis severity scores), safety laboratory and other laboratory values (i.e., IL-1β 
in GCF, BLXA4-ME levels in plasma, SPM profiles in plasma and serum), and microbial 
identification data. 
 
16.4 
Schedule and Content of Reports 
 
The primary outcome measures are the safety parameters, which will be assessed by 
recording all AEs and SAEs, as reported by subjects or observed by oral examination, 


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measurement of vital signs, and review of safety laboratory results. The clinical 
investigator and the IND sponsor will review these data and work with the data 
coordinating center to provide regular reports to NIDCR (the study funder), the DSMB, 
and the IRB. The NIDCR medical monitor will track safety data in real time.  If the trial 
is terminated prematurely for any reason, an abbreviated report will be prepared. To 
ensure confidentiality, coding will be used to identify subjects. 
 
16.5 
Study Records Retention 
 
Study documents should be retained for a minimum of 2 years after the last approval of 
a marketing application in an ICH region and until there are no pending or contemplated 
marketing applications in an ICH region, or until at least 2 years have elapsed since the 
formal discontinuation of clinical development of the investigational product. These 
documents should be retained for a longer period, however, if required by local 
regulations.  No records will be destroyed without the written consent of the IND 
sponsor. It is the responsibility of the IND sponsor to inform the investigator when these 
documents no longer need to be retained. 
 
Study records will be maintained for at least 3 years from the date that the grant final 
financial report is submitted to the National Institutes of Health (NIH). 
 
16.6 
Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical study protocol, Good Clinical 
Practice, or Manual of Procedures requirements. The noncompliance may be on the 
part of the subject, the investigator, or study staff. As a result of deviations, corrective 
actions are to be developed by the study staff and implemented promptly. 
 
These practices are consistent with investigator and sponsor obligations in ICH E6: 
 
 Compliance with Protocol, Sections 4.5.1, 4.5.2, 4.5.3, and 4.5.4 
 
 Quality Assurance and Quality Control, Section 5.1.1 
 
 Noncompliance, Sections 5.20.1 and 5.20.2 
 
All deviations from the protocol must be addressed in study subject source documents 
and promptly reported to NIDCR and the local IRB, according to their requirements.  A 
completed copy of the Protocol Deviation Form must be maintained in the regulatory 
file, as well as in the subject’s source document file. The principal investigator and 
study staff are responsible for knowing and adhering to the IRB requirements. 


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17 PUBLICATION POLICY 
 
Following completion of the study, the investigator is expected to publish the results of 
this research in a scientific journal. Any publications based on the results of the trial will 
conform to the NIH Grants Policy and the publication policy of the Forsyth Institute. 
This study will comply with the NIH Public Access Policy, which ensures that the public 
has access to the published results of NIH-funded research. It requires scientists to 
submit final peer-reviewed journal manuscripts that arise from NIH funds to the digital 
archive PubMed Central upon acceptance for publication. 
 
The International Committee of Medical Journal Editors (ICMJE) member journals have 
adopted a clinical trials registration policy as a condition for publication. The ICMJE 
defines a clinical trial as any research project that prospectively assigns human subjects 
to intervention or concurrent comparison or control groups to study the cause-and-effect 
relationship between a medical intervention and a health outcome. Medical 
interventions include drugs, surgical procedures, devices, behavioral treatments, 
process-of-care changes, and the like.  Health outcomes include any biomedical or 
health-related measures obtained in patients or participants, including pharmacokinetic 
measures and AEs. The ICMJE policy requires that all clinical trials be registered in a 
public trials registry such as ClinicalTrials.gov, which is sponsored by the National 
Library of Medicine. Other biomedical journals are considering adopting similar policies. 
For interventional clinical trials performed under NIDCR grants and cooperative 
agreements, it is the grantee’s responsibility to register the trial in an acceptable 
registry, so the research results may be considered for publication in ICMJE member 
journals. The ICMJE does not review specific studies to determine whether registration 
is necessary; instead, the committee recommends that researchers who have questions 
about the need to register err on the side of registration or consult the editorial office of 
the journal in which they wish to publish. 
 
United States Public Law 110-85 (Food and Drug Administration Amendments Act of 
2007), Title VIII, Section 801 mandates that a "responsible party" (i.e., the sponsor or 
designated principal investigator) register and report results of certain "applicable 
clinical trials," described as: 
 
 Trials of drugs and biologics: controlled clinical investigations, other than Phase I 
investigations, of a product subject to FDA regulation; 
 
 Trials of devices: controlled trials with health outcomes of devices subject to FDA 
regulation, other than small feasibility studies, and pediatric postmarket 
surveillance required by FDA. 
 
National Institutes of Health grantees must take specific steps to ensure compliance 
with NIH implementation of the Food and Drug Administration Amendments Act of 2007. 


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18 LITERATURE REFERENCES 
 
Ariel, A., G. Fredman, Y. P. Sun, A. Kantarci, T. E. Van Dyke, A. D. Luster and C. N. 
Serhan (2006). "Apoptotic neutrophils and T cells sequester chemokines during immune 
response resolution through modulation of CCR5 expression." Nat Immunol 7(11): 
1209-1216. 
 
Bannenberg, G. L., N. Chiang, A. Ariel, M. Arita, E. Tjonahen, K. H. Gotlinger, S. Hong 
and C. N. Serhan (2005). "Molecular circuits of resolution: formation and actions of 
resolvins and protectins." J Immunol 174(7): 4345-4355. 
 
Birkedal-Hansen, H. (1993). "Role of cytokines and inflammatory mediators in tissue 
destruction." J Periodontal Res 28(6 Pt 2): 500-510. 
 
Birkedal-Hansen, H., W. G. Moore, M. K. Bodden, L. J. Windsor, B. Birkedal-Hansen, A. 
DeCarlo and J. A. Engler (1993). "Matrix metalloproteinases: a review." Crit Rev Oral 
Biol Med 4(2): 197-250. 
 
Campbell, E. L., C. N. Serhan and S. P. Colgan (2011). "Antimicrobial aspects of 
inflammatory resolution in the mucosa: a role for proresolving mediators." J Immunol 
187(7): 3475-3481. 
 
Flower, R. J. (2003). "The development of COX2 inhibitors." Nat Rev Drug Discov 2(3): 
179-191. 
 
Gallin, J. I. and R. Snyderman (1999). Inflammation : basic principles and clinical 
correlates. Philadelphia, PA, Lippincott Williams & Wilkins. 
 
Geist, S. M., S. Fitzpatrick and J. R. Geist (2007). "American Heart Association 2007 
guidelines on prevention of infective endocarditis." J Mich Dent Assoc 89(9): 50-56. 
 
Giannobile, W. V., S. E. Lynch, R. G. Denmark, D. W. Paquette, J. P. Fiorellini and R. 
C. Williams (1995). "Crevicular fluid osteocalcin and pyridinoline cross-linked 
carboxyterminal telopeptide of type I collagen (ICTP) as markers of rapid bone turnover 
in periodontitis. A pilot study in beagle dogs." J Clin Periodontol 22(12): 903-910. 
 
Godson, C., S. Mitchell, K. Harvey, N. A. Petasis, N. Hogg and H. R. Brady (2000). 
"Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic 
neutrophils by monocyte-derived macrophages." J Immunol 164(4): 1663-1667. 
 
Gordon, J. M., I. B. Lamster and M. C. Seiger (1985). "Efficacy of Listerine antiseptic in 
inhibiting the development of plaque and gingivitis." J Clin Periodontol 12(8): 697-704. 
 
Gronert, K., A. Kantarci, B. D. Levy, C. B. Clish, S. Odparlik, H. Hasturk, J. A. Badwey, 
S. P. Colgan, T. E. Van Dyke and C. N. Serhan (2004). "A molecular defect in 
intracellular lipid signaling in human neutrophils in localized aggressive periodontal 
tissue damage." J Immunol 172(3): 1856-1861. 
 
Hasturk, H., A. Kantarci, E. Goguet-Surmenian, A. Blackwood, C. Andry, C. N. Serhan 
and T. E. Van Dyke (2007). "Resolvin E1 regulates inflammation at the cellular and 
tissue level and restores tissue homeostasis in vivo." J Immunol 179(10): 7021-7029. 


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Hasturk, H., A. Kantarci, T. Ohira, M. Arita, N. Ebrahimi, N. Chiang, N. A. Petasis, B. D. 
Levy, C. N. Serhan and T. E. Van Dyke (2006). "RvE1 protects from local inflammation 
and osteoclast- mediated bone destruction in periodontitis." FASEB J 20(2): 401-403. 
 
Heasman, S. J., K. M. Giles, C. Ward, A. G. Rossi, C. Haslett and I. Dransfield (2003). 
"Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage 
phagocytosis of apoptotic cells: implications for the resolution of inflammation." J 
Endocrinol 178(1): 29-36. 
 
Hong, S., K. Gronert, P. R. Devchand, R. L. Moussignac and C. N. Serhan (2003). 
"Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in 
murine brain, human blood, and glial cells. Autacoids in anti-inflammation." J Biol Chem 
278(17): 14677-14687. 
 
Kantarci, A., H. Hasturk and T. E. Van Dyke (2006). "Host-mediated resolution of 
inflammation in periodontal diseases." Periodontol 2000 40: 144-163. 
 
Kinane, D. F. and D. F. Lappin (2002). "Immune processes in periodontal disease: a 
review." Ann Periodontol 7(1): 62-71. 
 
Kornman, K. S., R. C. Page and M. S. Tonetti (1997). "The host response to the 
microbial challenge in periodontitis: assembling the players." Periodontol 2000 14: 33- 
53. 
 
Lawrence, T., D. A. Willoughby and D. W. Gilroy (2002). "Anti-inflammatory lipid 
mediators and insights into the resolution of inflammation." Nat Rev Immunol 2(10): 
787-795. 
 
Levy, B. D., C. B. Clish, B. Schmidt, K. Gronert and C. N. Serhan (2001). "Lipid 
mediator class switching during acute inflammation: signals in resolution." Nat Immunol 
2(7): 612-619. 
Nathan, C. (2002). "Points of control in inflammation." Nature 420(6917): 846-852. 
Offenbacher, S. (1996). "Periodontal diseases: pathogenesis." Ann Periodontol 1(1): 
821-878. 
 
Offenbacher, S., B. M. Odle and T. E. Van Dyke (1986). "The use of crevicular fluid 
prostaglandin E2 levels as a predictor of periodontal attachment loss." J Periodontal 
Res 21(2): 101-112. 
 
Panagakos, F. S., A. R. Volpe, M. E. Petrone, W. DeVizio, R. M. Davies and H. M. 
Proskin (2005). "Advanced oral antibacterial/anti-inflammatory technology: A 
comprehensive review of the clinical benefits of a triclosan/copolymer/fluoride 
dentifrice." J Clin Dent 16 Suppl: S1-19. 
 
Petasis, N. A., R. Keledjian, Y. P. Sun, K. C. Nagulapalli, E. Tjonahen, R. Yang and C. 
N. Serhan (2008). "Design and synthesis of benzo-lipoxin A4 analogs with enhanced 
stability and potent anti-inflammatory properties." Bioorg Med Chem Lett 18(4): 1382- 
1387. 


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Pouliot, M., C. B. Clish, N. A. Petasis, T. E. Van Dyke and C. N. Serhan (2000). "Lipoxin 
A(4) analogues inhibit leukocyte recruitment to Porphyromonas gingivalis: a role for 
cyclooxygenase-2 and lipoxins in periodontal disease." Biochemistry 39(16): 4761-4768. 
 
Samuelsson, B., S. E. Dahlen, J. A. Lindgren, C. A. Rouzer and C. N. Serhan (1987). 
"Leukotrienes and lipoxins: structures, biosynthesis, and biological effects." Science 
237(4819): 1171-1176. 
 
Serhan, C. N. (2004). "A search for endogenous mechanisms of anti-inflammation 
uncovers novel chemical mediators: missing links to resolution." Histochem Cell Biol 
122(4): 305-321. 
 
Serhan, C. N. (2007). "Resolution phase of inflammation: novel endogenous anti- 
inflammatory and proresolving lipid mediators and pathways." Annu Rev Immunol 25: 
101-137. 
 
Serhan, C. N., S. D. Brain, C. D. Buckley, D. W. Gilroy, C. Haslett, L. A. O'Neill, M. 
Perretti, A. G. Rossi and J. L. Wallace (2007). "Resolution of inflammation: state of the 
art, definitions and terms." FASEB J 21(2): 325-332. 
 
Serhan, C. N., C. B. Clish, J. Brannon, S. P. Colgan, N. Chiang and K. Gronert (2000). 
"Novel functional sets of lipid-derived mediators with antiinflammatory actions generated 
from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and 
transcellular processing." J Exp Med 192(8): 1197-1204. 
 
Serhan, C. N., S. Hong, K. Gronert, S. P. Colgan, P. R. Devchand, G. Mirick and R. L. 
Moussignac (2002). "Resolvins: a family of bioactive products of omega-3 fatty acid 
transformation circuits initiated by aspirin treatment that counter proinflammation 
signals." J Exp Med 196(8): 1025-1037. 
 
Serhan, C. N., A. Jain, S. Marleau, C. Clish, A. Kantarci, B. Behbehani, S. P. Colgan, G. 
L. Stahl, A. Merched, N. A. Petasis, L. Chan and T. E. Van Dyke (2003). "Reduced 
inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase 
and endogenous anti-inflammatory lipid mediators." J Immunol 171(12): 6856-6865. 
 
Serhan, C. N. and J. Savill (2005). "Resolution of inflammation: the beginning programs 
the end." Nat Immunol 6(12): 1191-1197. 
 
Silness, J. and H. Loe (1964). "Periodontal Disease in Pregnancy. Ii. Correlation 
between Oral Hygiene and Periodontal Condtion." Acta Odontol Scand 22: 121-135. 
 
Smith, M. A., L. D. Braswell, J. G. Collins, D. L. Boyd, M. K. Jeffcoat, M. Reddy, K. L. Li, 
S. Wilensky, R. Vogel, M. Alfano and et al. (1993). "Changes in inflammatory mediators 
in experimental periodontitis in the rhesus monkey." Infect Immun 61(4): 1453-1459. 
 
Socransky, S. S., A. D. Haffajee, C. Smith, L. Martin, J. A. Haffajee, N. G. Uzel and J. 
M. Goodson (2004). "Use of checkerboard DNA-DNA hybridization to study complex 
microbial ecosystems." Oral Microbiol Immunol 19(6): 352-362. 
 
Socransky, S. S., C. Smith, L. Martin, B. J. Paster, F. E. Dewhirst and A. E. Levin 
(1994). ""Checkerboard" DNA-DNA hybridization." Biotechniques 17(4): 788-792. 


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Sonis, S. T., J. P. Eilers, J. B. Epstein, F. G. LeVeque, W. H. Liggett, Jr., M. T. Mulagha, 
D. E. Peterson, A. H. Rose, M. M. Schubert, F. K. Spijkervet and J. P. Wittes (1999). 
"Validation of a new scoring system for the assessment of clinical trial research of oral 
mucositis induced by radiation or chemotherapy. Mucositis Study Group." Cancer 
85(10): 2103-2113. 
 
Subramanian, S., H. Emami, E. Vucic, P. Singh, J. Vijayakumar, K. M. Fifer, A. Alon, S. 
S. Shankar, M. Farkouh, J. H. Rudd, Z. A. Fayad, T. E. Van Dyke and A. Tawakol 
(2013). "High-dose atorvastatin reduces periodontal inflammation: a novel pleiotropic 
effect of statins." J Am Coll Cardiol 62(25): 2382-2391. 
 
Sun, Y. P., E. Tjonahen, R. Keledjian, M. Zhu, R. Yang, A. Recchiuti, P. S. Pillai, N. A. 
Petasis and C. N. Serhan (2009). "Anti-inflammatory and pro-resolving properties of 
benzo-lipoxin A(4) analogs." Prostaglandins Leukot Essent Fatty Acids 81(5-6): 357- 
366. 
 
Tilley, S. L., T. M. Coffman and B. H. Koller (2001). "Mixed messages: modulation of 
inflammation and immune responses by prostaglandins and thromboxanes." J Clin 
Invest 108(1): 15-23. 
 
Tsai, C. C., Y. C. Hong, C. C. Chen and Y. M. Wu (1998). "Measurement of 
prostaglandin E2 and leukotriene B4 in the gingival crevicular fluid." J Dent 26(2): 97- 
103. 
 
Van Dyke, T. E., M. A. Lester and L. Shapira (1993). "The role of the host response in 
periodontal disease progression: implications for future treatment strategies." J 
Periodontol 64(8 Suppl): 792-806. 
 
Varani, J. and P. A. Ward (1994). "Mechanisms of neutrophil-dependent and neutrophil- 
independent endothelial cell injury." Biol Signals 3(1): 1-14. 


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APPENDICES 
 
Appendix A:  Schedule of Events 


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APPENDIX A: 


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SCHEDULE OF EVENTS 
 
Procedures 
Screen 
Day 
-10 to -1 
Enrollment/ 
Baseline 
Visit 1 
Day 0 
Visit 2 
Day 3 
± 1 day 
Visit 3 
Day 7 
± 1 day 
Visit 4 
Day 14 
± 1 day 
Visit 5 
Day 21 
± 1 day 
Visit 6 
Day 28 
± 1 day 
F/U 
Day 90 
± 14 days 
Obtain informed consent 
X 
X 
Medical/dental history 
Xa,b 
Xa 
X 
X 
X 
X 
X 
Record concomitant medications and dental 
procedures 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy test 
X 
X 
Vital signsc 
X 
X 
X 
X 
X 
X 
X 
Intraoral and extraoral examinations 
X 
X 
X 
X 
X 
X 
X 
Assessment of plaque and calculus 
X 
Solicit adverse events 
X 
X 
X 
X 
X 
X 
Modified gingival index 
X 
X 
X 
X 
Gingival crevicular fluid sample for IL-1β 
X 
X 
X 
Probing depth; measure GM-CEJ to 
determine clinical attachment level 
X 
X 
X 
Bleeding on probing 
X 
X 
X 
Plaque index 
X 
X 
X 
Plaque samples to analyze oral flora 
X 
X 
X 
Blood for plasma BLXA4-ME level and SPM 
profiling in serum and plasma 
X 
X 
Blood for eligibility or safety laboratory testsd 
X 
X 
X 
Urine for eligibility or safety laboratory testsd 
X 
X 
X 


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Procedures 
Screen 
Day 
-10 to -1 
Enrollment/ 
Baseline 
Visit 1 
Day 0 
Visit 2 
Day 3 
± 1 day 
Visit 3 
Day 7 
± 1 day 
Visit 4 
Day 14 
± 1 day 
Visit 5 
Day 21 
± 1 day 
Visit 6 
Day 28 
± 1 day 
F/U 
Day 90 
± 14 days 
Assess compliance 
X 
X 
X 
X 
X 
Provide oral rinse 
X 
X 
X 
X 
X 
Hygiene instructions or referral for 
periodontal treatment 
X 
F/U = safety follow-up telephone call; GM-CEJ = distance from free gingival margin to cementoenamel junction; IL-1 = interleukin-1; SPM = specialized 
pro-resolution mediator 
a 
Includes history of alcohol and tobacco use. 
b 
Includes demographic information. 
c 
Pulse rate, respiratory rate, blood pressure, and oral body temperature are collected at all visits marked with “X”. Weight is collected at the Screening 
Visit and Visit 6 (Day 28). 
d 
The on-site medical monitor will review the results of safety laboratory tests to determine whether a laboratory test should be redrawn. 

SAP_001.pdf: 
 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
STATISTICAL ANALYSIS PLAN 
17 May 2018 
 
 
 
 
A Phase 1/2 Clinical Trial to Assess the Safety and Preliminary Efficacy of 
Lipoxin Analog BLXA4-ME Oral Rinse for the Treatment of Gingivitis 
 
 
NIDCR Protocol Number:  14-020-E  
Principal Investigator:  
Hatice Hasturk, DDS, PhD 
The Forsyth Institute  
245 First Street 
Cambridge, MA 02142 
Telephone: 617-892-8499 
Fax: 617-892-8505 
 
IND Sponsor: 
Thomas E. Van Dyke, DDS, PhD 
The Forsyth Institute 
245 First Street 
Cambridge, MA 02142 
Telephone: 617-892-8503 
Fax: 617-892-8504 
 
 
Funding Agency: 
National Institute of Dental and Craniofacial Research (NIDCR) 
Program Official: Jane Atkinson, DDS 
Grant Number: U01DE026934-01 
 
 
 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 2 of 35 
 
PREPARED BY 
 
Robert James, MS, MStat, Senior Biostatistician III 
 Rho, Inc.  
6330 Quadrangle Drive, Suite 500 
Chapel Hill, NC  27517 
Telephone:  (919) 408-8000 
Fax:  (919) 408-0999 
 
 
 
This document is confidential and proprietary to the IND sponsor, Thomas E. Van Dyke. Acceptance of this 
document constitutes agreement by the recipient that no unpublished information contained herein will be 
reproduced, published, or otherwise disclosed without the prior written approval of Thomas E. Van Dyke except 
that this document may be disclosed to appropriate Institutional Review Boards under the condition that they keep 
the information confidential. 
DOCUMENT VERSION CONTROL 
Version Number 
Date 
Comments/Changes 
            0.1 
22 August 2017 
First Draft 
            0.2 
27 October 2017 
Clarified the SAP as to the 
handling of missing values at 
DSMB request. 
            1.0 
02 February 2018 
Changed “worst observation 
carried forward” to last 
observation carried forward” 
Minor changes to the General 
Analysis and Reporting 
Conventions, and the 
Demographic and Other Baseline 
Characteristics. 
             1.1 
02 May 2018 
Updated sections 8.1 and 9.1.1 to 
describe analyses of subjects that 
receive the wrong drug treatment. 
2.0 
03 May 2018 
General formatting; Second 
approved version 
            3.0                                        17 May 2018 
Modified analysis populations in 
sections 4.1 and 4.2 to describe 
how to handle subjects that 
receive the wrong drug treatment.  
Removed the sentences 
describing handling of subjects 
that received the wrong treatment 
from sections 8.1 and 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 3 of 35 
9.1.1.Section 9.3.2 added 
clarification that all efficacy 
analyses were performed using 
the efficacy population. Added 
section 11 Changes to the 
Analyses Planned in the Protocol 
 
 
 
 
 
 
 
 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 4 of 35 
APPROVALS 
Approved: 
 
Date: 
 
 
Name: Thomas Van Dyke, DDS, PhD 
Title: IND Sponsor 
 
 
 
 
Date: 
 
 
Name: Hatice Hasturk, DDS, PhD 
Title: Principal Investigator 
 
 
 
 
Date: 
 
 
Name: Robert James, MS, MStat 
Title: Senior Biostatistician III 
 
 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 5 of 35 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ........................................................................................... 7 
1. 
PURPOSE OF THIS DOCUMENT ........................................................................... 8 
2. 
PROTOCOL SUMMARY ......................................................................................... 9 
2.1 Study Objectives .................................................................................................... 9 
2.2 Overall Study Design and Plan ................................................................................ 9 
2.3 Study Population.................................................................................................... 9 
2.4 Blinding ................................................................................................................ 9 
2.5 Sample Size Determination ................................................................................... 10 
3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS ................................ 13 
4. 
ANALYSIS POPULATIONS .................................................................................. 14 
4.1 Safety Population ................................................................................................. 14 
4.2 Efficacy Population .............................................................................................. 14 
5. 
STUDY SUBJECTS ................................................................................................ 15 
5.1 Disposition of Subjects ......................................................................................... 15 
5.2 Protocol Deviations .............................................................................................. 15 
6. 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ....................... 16 
7. 
MEASUREMENTS OF TREATMENT COMPLIANCE .......................................... 17 
8. 
SAFETY EVALUATION ........................................................................................ 18 
8.1 Overview of Safety Analysis Methods .................................................................... 18 
8.2 Progression of Periodontitis................................................................................... 18 
8.3 Extent of Exposure ............................................................................................... 19 
8.4 Adverse Events .................................................................................................... 19 
8.5 Deaths, Serious Adverse Events, and Other Significant Adverse Events ..................... 20 
8.6 Clinical Laboratory Evaluation .............................................................................. 20 
8.7 Microbial Profiles in Pooled Supragingival Plaque ................................................... 21 
8.8 Vital Signs, Physical Findings, and Other Observations Related to Safety ................... 21 
8.8.1 
Vital Signs .......................................................................................... 21 
8.8.2 
Oral Exam .......................................................................................... 22 
8.8.3 
Oral Mucositis Assessment Scale Ulceration and Erythema Scores ............ 22 
8.8.4 
Tooth Status ........................................................................................ 22 
9. 
EFFICACY EVALUATION.................................................................................... 23 
9.1 Overview of Efficacy Analysis Issues ..................................................................... 23 
9.1.1 
Handling of Dropouts and Missing Data ................................................. 23 
9.2 Efficacy Endpoints ............................................................................................... 23 
9.2.1 
Primary Efficacy Endpoints .................................................................. 23 
9.2.2 
Secondary Efficacy Endpoints ............................................................... 23 
9.2.2.1 
Plaque Index (PI) .................................................................... 24 
9.2.2.2 
Modified Gingival Index (MGI) ................................................ 24 
9.2.2.3 
Bleeding on Probing (BOP) ...................................................... 24 
9.2.2.4 
Plaque and Gingivitis Severity Indices ....................................... 24 
9.2.2.5 
Interleukin-1β (IL-1β) .............................................................. 25 
9.2.3 
Exploratory Efficacy Endpoints ............................................................. 25 
9.2.3.1 
Pocket Depth .......................................................................... 25 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
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9.2.3.2 
Clinical Attachment Loss ......................................................... 25 
9.3 Analysis Methods ................................................................................................ 25 
9.3.1 
Primary Efficacy Analyses .................................................................... 25 
9.3.2 
Secondary Efficacy Analyses ................................................................ 25 
9.3.2.1 
Secondary Efficacy Endpoints .................................................. 25 
9.3.2.2 
Sensitivity Analyses of Secondary Efficacy Outcomes ................. 27 
9.3.3 
Exploratory Efficacy Analyses .............................................................. 27 
10. INTERIM ANALYSES AND DATA MONITORING .............................................. 29 
11. CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL ....................... 30 
12. LIST OF PLANNED TABLES, FIGURES, AND LISTINGS .................................... 31 
13. APPENDICES ........................................................................................................ 32 
13.1 
Study Flow Chart .......................................................................................... 32 
13.2 
Schedule of Events ........................................................................................ 34 
 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 7 of 35 
LIST OF ABBREVIATIONS 
AE 
adverse event 
ALT 
Alanine aminotransferase 
AST  
Aspartate aminotransferase 
BLXA4-ME 
Investigational drug, a stable lipoxin analog 
BOP 
Bleeding on Probing 
CAL 
Clinical Attachment Level 
CI 
Confidence Interval 
CS 
Clinically Significant 
DBP 
Diastolic Blood Pressure 
GCF 
Gingival Crevicular Fluid 
GSI 
Gingivitis Severity Index 
IL-1β 
Interleukin-1β 
LXA4 
Lipoxin A4 
MedDRA 
Medical Dictionary for Regulatory Activities 
MGI 
Modified gingival index 
OMAS 
Oral Mucositis Assessment Scale 
PD 
Pocket depth 
PI 
Plaque Index 
PSI 
Plaque Severity Index 
PT 
Preferred Term 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SBP 
Systolic Blood Pressure 
SD 
Standard Deviation 
SE 
Standard Error 
SOC 
System Organ Class 
TEAE 
Treatment-Emergent Adverse Event 
  
 


 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 8 of 35 
1. PURPOSE OF THIS DOCUMENT 
This statistical analysis plan (SAP) for protocol NIDCR Protocol 14-020-E is being developed 
before database lock and the statistical analyses of the data.  The objective of this document is to 
(1) briefly summarize the purpose of the study and study design, (2) detail the planned statistical 
methodology, (3) define original and derived variables necessary to complete the analyses, and 
(4) delineate the most appropriate summary tables, listings, and graphical displays to be included 
in the study report. 
This SAP will be presented to the Principal Investigator for approval.  The statistical analyses 
and their corresponding tables, listings, and figures, as outlined in this document will be 
presented to the PI at the completion of the study analyses. 
  
 


 
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Statistical Analysis Plan 
Protocol No.14-020-E 
 
17 May 2018 
Version:  Hasturk Lipoxin SAP 3.0_2018_05_17_Clean.doc 
 
Page 9 of 35 
2. PROTOCOL SUMMARY 
2.1 Study Objectives 
The primary objective is to evaluate the safety of an investigational compound, BLXA4-ME, 
topically applied for 28 days as a daily oral rinse in adults with gingival inflammation.  Safety 
will be assessed by the incidence of adverse events, including mucosal inflammation and 
irritancy, and findings from oral examinations and safety laboratory tests.  Additionally, subjects 
will be monitored for development or progression of periodontitis and oral flora will be analyzed 
to detect an increase in opportunistic organisms.   
The secondary objective is to assess preliminary efficacy of the oral rinse, by monitoring 
changes in the plaque index (PI), modified gingival index (MGI), bleeding on probing (BOP), 
and levels of interleukin-1β (IL-1β) in gingival crevicular fluid (GCF). 
2.2 Overall Study Design and Plan 
This study has a randomized parallel placebo-controlled double-blind study design with one 
treatment arm and two control arms (placebo oral rinse, and no rinse controls).  Subjects in the 
treatment (N=50) and placebo rinse (N=50) arms will be double-blinded and receive oral rinse 
(BLXA4-ME or placebo rinse) to be applied once daily after morning teeth brushing.  The no-
rinse control arm (N=25) will be blinded from the investigators and apply no oral rinse.  Subjects 
will perform their randomized treatment for 28 days.  Safety parameters will be assessed before 
treatment and at Days 3, 7, 14, 21, and 28 after the initial treatment.  Efficacy parameters will be 
assessed before treatment and Day 14 and Day 28 after the initial treatment.  Total duration of 
subject participation will be 3 months (in clinic visits through 28 days, and a follow-up safety 
phone call at 2 months after last product use). 
The Study Flow Chart and the Schedule of Events can be found in Appendices 13.1 and 13.2, 
respectively. 
2.3 Study Population 
Approximately 125 healthy adult male and female subjects with gingival inflammation, 
determined by mean full mouth MGI of at least 2.0 at screening and baseline, will participate in 
the study. 
2.4 Blinding 
This is a double-blind study.  The PI, study coordinators, study examiners, data managers, and 
statisticians will be masked to the assigned treatment.  Subjects receiving oral rinse will not 
know whether they are in the active or placebo rinse group.  The no-rinse control will be blinded 
only from the PI, study coordinators, and study examiners since it is not possible to blind the 
subject from not having an oral rinse.  The product code may be broken for an individual subject 
if he or she experiences a serious adverse event (SAE) and cannot be adequately treated without 
knowing the identity of the study product.  The study blind will be broken after database lock. 
 


 
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2.5 Sample Size Determination 
Sample size determination is based on the power to detect AEs (a primary safety outcome) with 
additional consideration given to detecting post-baseline study arm differences with respect to 
the MGI (a secondary efficacy outcome). 
The probability of observing a specific AE for the study’s sample size can be estimated.  This 
probability depends on the sample size within a treatment group and on the true underlying 
probability of an individual experiencing the specific AE in that treatment group.  Table 2.5.1 
summarizes the probabilities of observing an AE given a sample size and true underlying event 
rate.  For example, if the true probability of a subject experiencing a specific AE is 5%, the 
probability of observing 1 or more of that specific AE within a treatment group of 40 subjects is 
87.1%. 
The study’s test oral rinse, placebo oral rinse, and no-rinse control arms are assigned treatment 
group sample sizes of 40, 40, and 20 subjects, respectively.  For the test oral rinse and the 
placebo oral rinse arms, a sample size of 40 subjects per treatment group should have 
probabilities of 80.5%, 87.1%, 91.6%, and 96.4% of observing an AE having underlying 
incidence probabilities of 4%, 5%, 6%, and 8%, respectively.  The no-rinse control arm is 
considered to be of lesser importance and, thus, to conserve the overall sample size, is assigned a 
sample size of 20 subjects, which should have probabilities of 55.8%, 64.2%, 71.0%, and 81.1% 
of observing an AE having underlying incidence probabilities of 4%, 5%, 6%, and 8%, 
respectively.  Thus, the above sample sizes of 40, 40, and 20 subjects should give adequate 
power (> 80%) to detect AEs that occur at true underlying frequencies of 4% or greater in the 
test and placebo oral rinse study arms, and adequate power (> 80%) to detect AEs that occur at 
true underlying frequencies of 8% or greater in the no-rinse control arm.  Conversely, if no AEs 
occur with a sample size of 20 and 40 subjects then the upper one-sided 95% CI of the 
underlying AE incidence rate would be 14% and 7.5%, respectively. 


 
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Table 2.5.1 
Probability of Observing One or More Adverse Events within a Study Arm 
"True" Underlying 
Probability of an Individual 
Experiencing an Adverse 
Event 
Probability of Observing One or More Adverse Events 
within a Study Arm 
No-Rinse Control Study 
Arm 
(n = 20) 
Test and Placebo Oral Rinse 
Study Arms 
(n = 40) 
0.01% 
0.20% 
0.40% 
0.10% 
2.0% 
3.9% 
0.25% 
4.9% 
9.5% 
0.50% 
9.5% 
18.2% 
1.00% 
18.2% 
33.1% 
2.00% 
33.2% 
55.4% 
3.00% 
45.6% 
70.4% 
4.00% 
55.8% 
80.5% 
5.00% 
64.2% 
87.1% 
6.00% 
71.0% 
91.6% 
8.00% 
81.1% 
96.4% 
10.0% 
87.8% 
98.5% 
Sample size (n) refers to the final sample size per treatment group of enrolled subjects who complete the study. 
 
Power estimates for the secondary efficacy endpoint (MGI) are found in Table 2.5.2.  In this 
power analysis, MGI refers to the MGI scores averaged over all tooth sites within each subject 
and visit.  The power estimates in the table are based upon 50,000 simulations of mixed models 
analyses.  The mixed models simulations include the MGI as the dependent variable, and fixed 
effects for treatment group (test oral rinse, placebo oral rinse, and no-rinse control), visit 
(Baseline, Day 14, and Day 28), and their treatment group-by-visit interaction.  The model 
includes random effects for subject using a compound symmetry variance structure (i.e., 
exchangeable covariance).  These power analyses assume an overall within-study group standard 
deviation of 0.3 MGI units at each visit and a difference in the mean change from baseline MGI 
at 28 days between the test oral rinse and the placebo rinse or between the test oral rinse and the 
no-rinse control of 0.2 MGI units.  Correlation of MGI within subject among visits was assumed 
to be 0.3.  The treatment group sample sizes of 40, 40, and 20 subjects for the test oral rinse, 
placebo oral rinse, and no-rinse control arms, respectively, are estimated to have powers of 71% 
and 54% to detect significant differences of 0.2 in the mean MGI scores between the test oral 
rinse and placebo oral rinse study arms, and between the test oral rinse and the no-rinse control 
study arms, respectively.  Even though these sample sizes would be considered under-powered 
for testing efficacy in a Phase 3 clinical trial, for this Phase 1/2 protocol, with efficacy as a 
secondary objective, these study sample sizes can provide evidence (significant or not) of 
efficacy and provide efficacy estimates with their associated standard deviations needed for 
planning future Phase 2 or Phase 3 trials.  


 
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It is estimated that 50, 50, and 25 subjects will need to be enrolled to have final sample sizes of 
40, 40, and 20 subjects for the test oral rinse, placebo oral rinse, and no-rinse control study arms, 
respectively, assuming a 20% subject attrition rate before completing the study. 
 
Table 2.5.2. 
Mixed Models Power Analysis for MGI (Secondary Efficacy Outcome) 
Showing the Power for Several Sample Size Scenarios 
Enrolled and Eligible 
Subjects 
Subjects after 20%  
Drop-out 
Power (%)  
Test  
(n) 
Placebo 
  (n) 
Control 
  (n) 
Test 
(n) 
Placebo 
  (n) 
Control 
  (n) 
Trt – 
Placeboa 
Trt – Controlb 
50 
50 
25 
40 
40 
20 
70.9 
53.8 
Control = no-rinse control study arm; MGI = modified gingival index; placebo = placebo oral rinse study arm; test = 
test oral rinse study arm; trt = treatment (placebo or test oral rinse) study arm. 
a 
Trt-Placebo:  Power (probability) for finding a statistically significant difference (alpha = 0.05) in the 
change from baseline MGI at 28 days between the test oral rinse and the placebo oral rinse study arms. 
b 
Trt-Control:  Power (probability) for finding a statistically significant difference (alpha = 0.05) in the 
change from baseline MGI at 28 days between the test oral rinse and the no-rinse control study arms. 
 
 


 
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3. GENERAL ANALYSIS AND REPORTING CONVENTIONS 
The following is a list of general analysis and reporting conventions to be applied for this study. 
If departures from these general conventions are present in the specific evaluations section of this 
SAP, then those conventions will take precedence over these general conventions. 
 All analysis will be performed using the SAS System version 9.4 or later. 
 All subject data listings and summary listings will be sorted for presentation in order of 
treatment group and subject number in the following order: BLXA4-ME rinse, placebo rinse, 
and non-rinse control.   
 Missing values for both numeric and character variables will be presented as blanks in a table 
or listing. 
 Mixed case will be used for all titles, footnotes, column headers, and programmer-supplied 
formats.  
 All date values will be presented as DDMMMYYYY (e.g. 21AUG2008) format.  A 4-digit 
year is required for all dates.  Partial dates will dashes to represent missing days or months 
(e.g. - -JUL2009). 
 All observed time values will be presented using a 24-hour clock HH:MM format (e.g., 
01:35, or 17:26).  
 Population sizes will be presented for each treatment group in the column heading as 
(N=XX).  
 The sample size (n) shown as a summary statistic will be the number of subjects with non-
missing values. 
 Categorical variables will be summarized using counts (n) and percentages (%) and will be 
presented in the form n (%).  All summaries for categorical variables will include all 
categories, even if none of the subjects had a response in a particular category, to ensure 
completeness.  Continuous variables will be summarized using descriptive statistics: n, mean, 
median, standard deviation (SD) or standard error (SE), minimum, and maximum.  The mean 
and median will be reported at 1 more significant digit than the precision of the data.  The SD 
and/or SE will be reported at 2 more significant digits than the precision of the data.  The 
minimum and maximum will be reported to the same level of precision as the original 
observations.   
 Calculated variables will not be rounded prior to analysis.  Rounding will only occur after 
analysis.  If any values are calculated to have more significant digits than the original data, 
then the value will be rounded so that it is the same level of precision as the original data. 
 P-values will be reported to 3 decimal places if greater than 0.001.  If less than 0.001 then p-
values will be reported as ‘<0.001’.   


 
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4. ANALYSIS POPULATIONS 
The exclusion criterion of periodontitis was removed in October 2015 for Protocol Version 4.0.  
This will possibly change the study population.  To explore the possible effects of including 
study participants with periodontitis, sensitivity analyses will be performed as described in 
section 9.3.2.2. 
4.1 Safety Population 
The Safety Population will consist of all subjects who are randomized into a study arm and 
complete their Baseline Visit. For the safety evaluations, subjects will be analyzed according to 
the treatment actually received.  Any subjects that in error were administered both the BLXA4-
ME and the placebo rinse at different times during the study will be analyzed in the BLXA4-ME 
treatment arm.  Subjects that in error were administered both the non-rinse control treatment and 
either the BLXA4-ME or placebo rinse will be analyzed according to the rinse administered. 
 
4.2 Efficacy Population 
The Efficacy Population will consist of all subjects of the Safety Population who have a baseline 
and at least 1 post-baseline assessment of 1 or more of the secondary efficacy outcome measures.  
Subjects that dropout or withdraw prior to Day 14 will be replaced in the efficacy population. 
For the efficacy evaluations, subjects will be analyzed according to the treatment actually 
received.  Any subjects that began the study administering the treatment for one of the study 
arms and then later in error switched treatments to another study arm will not be included in the 
efficacy population.   
 
 
 


 
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5. STUDY SUBJECTS 
5.1 Disposition of Subjects 
The disposition of all subjects and the study populations to which they belong will be presented 
in the subject listings and summarized in a table as counts by treatment allocation.  The subject 
dispositions will include subjects randomized, completing the study, discontinuing/ withdrawing, 
and the number of subjects in both the safety and efficacy analysis populations.  The reasons for 
discontinuing/ withdrawing from the study will be included in the disposition listing and table. 
5.2 Protocol Deviations 
Protocol deviations will be presented in a listing and summarized in a table by treatment 
allocation and type of deviation.  Types of deviations that will be summarized as counts and 
percentages in the table include: 
 Consent deviation  
 Eligibility deviation  
 Missed visit  
 Procedure performed outside of visit window 
 Study procedure not completed 
 Study procedure deviation 
 Other  
 
Table 5.2.1 
Assessment Time Windows 
 
Study Visit 
Time window 
(study day ± assessment window) 
Visit 1 – Enrollment, Baseline 
Day 0 
Visit 2 
Day 3 ± 1 
Visit 3 
Day 7 ± 1 
Visit 4 
Day 14 ± 1 
Visit 5 
Day 21 ± 1 
Visit 6 – Final Study Visit 
Day 28 ± 1 
Visit 7 – Telephone Safety 
Follow-up 
Day 90 ± 14 
 
 
 


 
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6. DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS 
Baseline data (e.g., demography, medical history, dental history, and concurrent medications) 
will be summarized. 
Demographic baseline characteristics will be summarized descriptively for both the Safety and 
Efficacy Populations.  The demographic variables of sex, age, ethnicity, and race will be listed 
and summarized in a table.  The categorical variables of sex, ethnicity, and race will be 
summarized by frequencies.  Age will be summarized as mean, SD, median, minimum, and 
maximum.   
To assess overall health of the study participants prior to study treatment, any significant medical 
history will be included in the subject listings.  The number and percentage of subjects with each 
medical history event will be presented by body system or oral structure.   
 
Results of the baseline oral exam will be listed by area/oral structure examined and will denote 
an abnormal or normal result; and, if abnormal, the associated description of the abnormality will 
be listed.   
Tooth status at baseline will be presented in a listing by subject and tooth, and will denote 
whether the tooth is missing, sound, decayed, filled, fractured, was crowned, and/or is a dental 
implant. 
All prior and concomitant medications will be coded to preferred drug names and therapeutic 
drug class using the WHO Drug Dictionary.  Use of concomitant medications will be 
summarized for each therapeutic drug class and each preferred drug name. Concomitant 
medications will include medications reported on the Concomitant Medications page of the CRF. 


 
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7. MEASUREMENTS OF TREATMENT COMPLIANCE 
Compliance will be recorded at all study visits by measurement of the volume of remaining 
product.  Expected volume returned is calculated as the volume dispensed - (number of days 
since last study visit x 30ml).  A subject will have “over used” the rinse if the percent volume 
used is > 120%.  A subject will have “under used” the rinse if the percent volume used is < 80%.  
A subject listing will capture under- and over-usage of study drug and non-compliance as 
determined by the volume of returned study drug and as reported by the subject queries.  If more 
than 12 subjects are out of study drug compliance, a table will summarize the frequencies of 
under- and over-usage of study drug by treatment arm.  
 
 


 
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8. SAFETY EVALUATION 
8.1 Overview of Safety Analysis Methods 
All safety analyses will be carried out using the Safety Population.  
 Adverse events (AEs) 
 Unanticipated problems 
 Vital signs  
 Laboratory determinations (hematology, serum chemistry, and urinalysis) 
 Physical examinations 
 Dental examinations 
 
With the exception of the progression of periodontitis based on the dental examinations, no 
statistical comparisons between the treatment groups are planned for the safety variables.   
 
8.2 Progression of Periodontitis 
Progression of periodontitis will be defined as an increase of 2 mm or more in pocket depth (PD) 
or clinical attachment level (CAL) from the Day 0 baseline measurement.  The frequency 
(percent) of subjects with progression of periodontitis will be summarized by study arm, visit, 
and overall.  Generalized linear mixed models regression based on a binomial distribution and 
logit link will compare the odds ratios of the overall incidence of the progression of periodontitis 
among the study arms at the Day 14 and Day 28 time points.  The results of the generalized 
linear mixed model will be presented in a table and include p-values and odds ratios (with 95% 
CI) for the treatment arm comparisons at the two study visits.  The p-values will not be adjusted 
for multiplicity. 
The generalized linear mixed models analyses will automatically adjust for any missing values 
provided the missing values are missing at random; and thus, no additional imputation is 
necessary for missing values that are missing at random.   
Missing observations are “not missing at random” if their missing-ness is possibly correlated 
with disease progression or efficacy.  Missing PD or CAL observations due to missed visits or 
early study withdrawal will be deemed “not missing at random” if there were any treatment-
emergent AE (TEAE) prior to or concurrent with a subject’s missed visit and the relationship 
between the TEAE and study drug was rated as “Possible”, ”Probable”, or ”Definite”. 
If more than 5% of the PD or CAL measures are missing, or any missing PD or CAL measures 
are deemed “not missing are random”, then a sensitivity analysis will be performed by repeating 
the above generalized linear mixed model with all missing values imputed as having progression 
of periodontitis.  The sensitivity analysis will also be triggered if for any other reason the 
principal investigator suspects that missing values may bias the analysis.  The reasons a 
sensitivity analysis is triggered will be footnoted in the sensitivity analysis table. 


 
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8.3 Extent of Exposure 
Summary statistics will be presented for the treatment exposure.  Treatment exposure will be 
defined as the number of days that the subject performed their allocated oral rinse treatment and 
the total volume of rinse used.  
8.4 Adverse Events 
All AEs will be coded to a system organ class (SOC) and preferred term (PT) using the most 
current version of the Medical Dictionary for Regulatory Activities (MedDRA).  All tabular AE 
summaries will be for TEAEs.  TEAEs will be defined as those AEs with an onset on or after the 
date of first dose of study drug (study visit 1: Day 0).  If an AE was recorded prior to the first 
dose of study drug and there was an increase in its severity, the AE will be considered a TEAE.  
All other AEs will be classified as non-treatment-emergent.  Treatment-emergent AEs will be 
flagged in all AE data listings. 
An overall summary table will be developed to report the number of events and the incidence of 
participants having at least one event in the following categories:  
 TEAEs 
 TEAEs indicated as serious (SAEs) 
 TEAEs that lead to study drug discontinuation 
 TEAEs with an outcome of death 
 TEAEs that were reported as having a definite/certain or possible relation to  study drug 
 TEAEs reported as having a severity rating of severe 
In addition, a summary table of TEAEs classified by system organ class (SOC) and preferred 
term (PT) will be provided for each of the following: 
 TEAEs 
 TEAEs by maximum severity  
 TEAEs by relationship to study drug 
The summary of all TEAEs will present both the number of TEAEs and the incidence of TEAEs.  
The summary of TEAEs by severity, relationship to study drug, and week of treatment will only 
report the incidence of TEAEs.  When reporting the number of TEAEs, if the same TEAE occurs 
for a subject on multiple occasions the event will be counted once for each occurrence.  When 
reporting the incidence, a subject will only be counted once if they ever experience an event 
within the SOC and ever experience the individual PT.  
For the summary of TEAEs by severity, if the severity of the TEAE is not reported, then the 
severity of the TEAE will be counted as severe.  If the same TEAE occurs for a subject on 
multiple occasions, the TEAE will be categorized according to the highest severity rating for that 
TEAE in that subject.  For the summary of TEAEs by relationship to study drug, if the 
relationship is missing, it will be counted as definite/certain.  If the same TEAE occurs for a 
subject on multiple occasions, the TEAE will be categorized according to the closest relationship 
to study drug reported for that TEAE in that subject. 


 
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8.5 Deaths, Serious Adverse Events, and Other Significant Adverse Events 
In addition to these summary tables, the incidence of adverse events leading to death, the 
incidence of SAEs, and incidence of TEAEs that led to study drug being discontinued will be 
presented in summary tables and listings.  The summary listings will provide all of the 
information reported for that AE and will include the number of days from date of first dose to 
date of the occurrence of the AE. 
8.6 Clinical Laboratory Evaluation 
Subject clinical chemistry, hematology, and urinalysis samples will be obtained at the baseline, 
Day 14 and Day 28 visits.  All lab results will be listed by subject, laboratory parameter, and 
time.  Out-of-range flags (H: high; or L: low) when available will be used to denote abnormal 
values.   
All treatment-emergent abnormal values, as determined on the basis of the normal values 
provided by the central laboratory, will be listed with relevant subject information.  In addition, 
all treatment-emergent values that were deemed clinically significant (CS) by the on-site medical 
monitor will be flagged in the listing.  An additional listing will only list records whose values 
were deemed clinically significant. 
The quantitative hematology, clinical chemistry, and urinalysis parameters and their change from 
baseline will be summarized in tables by study arm and study visit.  Summary statistics will 
include n, mean, SD, median, minimum, maximum values, and the number and percent of 
subjects with missing values for the laboratory test.  Repeat or ad hoc laboratory tests will not be 
summarized, but they will be included in the listings.  The order of presentation will cluster the 
laboratories by test and time of measurement in three tables for clinical chemistry, hematology, 
and urinalysis data, respectively. 
For hematology, clinical chemistry, and urinalysis laboratory parameters, shift tables will be 
presented for change from baseline at study Day 14 and Day 28.  For the shift tables, the 
Baseline laboratory parameter values will be cross-referenced with their associated study visit 
Day 14 and Day 28 laboratory parameter values with respect to the n and percent of parameter 
values flagged as low, normal, high, or abnormal based on the laboratory provided normal ranges 
for each of the lab parameters.   The shift tables will include an extra column for baseline values 
that shift to missing values due to early withdrawal or otherwise. 


 
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8.7  Microbial Profiles in Pooled Supragingival Plaque 
The pooled supragingival plaque samples collected from the mesiobuccal surfaces of the 
Ramfjord teeth at the baseline, Day 14 and Day 28 visits will be analyzed for the presence of 
pathogenic organisms.  The frequency and percent of the presence of the following organisms 
will be summarized (tables, listings and checkerboard plots) by study arm and visit:  
Fusobacterium nucleatum subspecies vincentii, Campylobacter concisus, Campylobacter rectus, 
Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Porphyromonas 
gingivalis, Capnocytophaga sputigena, Streptococcus oralis, Actinomyces naeslundii, 
Actinomyces israeli, Eubacterium brachy, Eikenella corrodens, and spirochetes.  The table and 
figure will also capture the frequency and percent of subjects with microbial counts missing. 
8.8 Vital Signs, Physical Findings, and Other Observations Related to Safety 
8.8.1 
Vital Signs 
The vital sign parameters will include sitting pulse rate, respiration rate, blood pressure (systolic 
(SBP) and diastolic (DBP)), and body temperature (oral).  All vital signs and weight values will 
be listed, by subject.  Out-of-range flags (H or L) will be used to denote abnormal values.  The 
abnormal values will be identified based upon the clinically significant criteria CS as listed in 
Table 9.8.1.1.  In addition, all treatment-emergent CS vital sign values or weights will be listed 
separately, by subject, with relevant subject information.   
The actual vital sign values and the change from baseline values will be summarized at all 
assessment times.  Summary statistics will include n, mean, SD, median, minimum, maximum 
values, and the number and percent of subjects with missing values for the vital sign measure. 
Table 8.8.1.1  CS Criteria for Vital Signs 
Parameter 
PCS Criteria 
Vital Signs 
 
Blood pressure 
(sitting) 
 
    Diastolic 
< 50 mmHg, %, with decrease from BL ≥15 mmHg; or 
> 105 mmHg, with increase from BL ≥15 mmHg 
    Systolic 
< 90 mmHg, %, with decrease from BL ≥20 mmHg; or 
> 180 mmHg, with increase from BL ≥20 mmHg 
 
 
Heart rate (sitting) 
< 50 BPM, with decrease from BL ≥10 BPM; or 
> 120 BPM; or 
Change from BL ≥ 30 BPM 
Temperature (oral) 
> 101F or 38.4C, with increase from BL ≥ 2F or 0.75C 
Weight 
Change from BL ≥ 7% body weight 
BL=Baseline 


 
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8.8.2 
Oral Exam 
All oral exam findings will be listed by subject.  Any treatment-emergent abnormal findings will 
be listed.  Subjects will be considered to have a treatment-emergent abnormal finding if the 
subject has a normal assessment for a specific oral structure at the baseline visit and is assessed 
as having an abnormal finding at a post-baseline study visit.  A table will summarize by the 
number and percent of subjects that have treatment-emergent abnormal findings for each oral 
structure.  This table will also summarize for each oral structure the number and percent of 
subjects that improve or have no change in normal/abnormal status from the Baseline visit.  The 
number and percent of subjects with missing values due to early withdrawal or otherwise will be 
summarized in the table for each oral structure. 
8.8.3 
Oral Mucositis Assessment Scale Ulceration and Erythema Scores 
The Oral Mucositis Assessment Scale (OMAS) score for ulceration and erythema will be listed 
for each oral structure by subject and visit.  A table will summarize by the number and percent of 
subjects for each oral structure with ulceration and erythema scores that worsen, improve, or stay 
the same from baseline.  The table will also summarize the number and percent of subjects with 
the OMAS scores missing due to early withdrawal or otherwise. 
8.8.4 
Tooth Status 
The tooth status will be listed for each tooth by subject and visit.  A table will summarize by the 
number and percent of subjects and by the number and percent of teeth for each tooth attribute at 
each study visit that worsen, improve, stay the same from baseline, or have missing values due to 
early study withdrawal or otherwise .  Tooth status attributes recorded include: missing, sound, 
decayed, filled, fractured, crown, and dental implant. 


 
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9. EFFICACY EVALUATION 
9.1 
Overview of Efficacy Analysis Issues 
9.1.1 
Handling of Dropouts and Missing Data 
.   
Subjects that dropout or are withdrawn prior to Day 14 will be replaced in the Efficacy 
Population.  The mixed models analyses (described in the sections below) will automatically 
adjust for any missing values provided the missing values are missing at random; and thus, no 
additional imputation is necessary for missing values that are missing at random.   
Missing observations are “not missing at random” if their missing-ness is possibly correlated 
with disease progression or efficacy.  Missing efficacy observations due to missed visits or early 
study withdrawal will be deemed “not missing at random” if there were any treatment-emergent 
AE (TEAE) prior to or concurrent with a subject’s missed visit and the relationship between the 
TEAE and study drug was rated as “Possible”, ”Probable”, or ”Definite”. 
If more than 5% of a given efficacy endpoint is missing, or any missing observations are deemed 
“missing not at random” then a sensitivity analysis will be performed by repeating the given 
efficacy analysis with all missing values imputed using “last observation carried forward”.  The 
sensitivity analysis will also be triggered if for any other reason the principle investigator 
suspects that missing values may bias the analysis.  The reasons any sensitivity analyses are 
triggered will be footnoted in the sensitivity analysis tables. 
9.2 
Efficacy Endpoints 
9.2.1 
Primary Efficacy Endpoints 
This is a Phase 1/2 study and there are no primary efficacy endpoints as listed in the protocol; 
however, the secondary endpoints of modified gingival index (MGI) followed by bleeding on 
probing (BOP), both at Day 28, are considered the efficacy endpoints of chief importance. 
9.2.2 
Secondary Efficacy Endpoints 
At baseline, study Day 14, and study Day 28, the following secondary efficacy variables: 
modified gingival index (MGI), bleeding on probing (BOP), IL-1β, plaque index (PI), plaque 
severity index (PSI) and gingivitis severity index (GSI) will be recorded.  PI, MGI, and BOP will 
be scored for each of the 6 tooth-sites for each tooth.  IL-1β will be measured in the GCF 
collected from one tooth (assigned by algorithm) in each quadrant.  All efficacy analyses will be 
performed on the Efficacy Population. 
The time point of greatest interest would be Day 28, but analysis will also be performed for Day 
14. 
The exploratory analysis pocket depth (PD) and clinical attachment level (CAL) will be 
descriptive. 


 
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9.2.2.1 Plaque Index (PI) 
Plaque Index will be assessed at 6 sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, 
lingual, and mesiolingual surfaces of the tooth).  Each tooth site will have its PI scored as 
follows: 
0 = No plaque; 
1 = Film at gingival margin; 
2 = Moderate (easily visible); 
3 = Abundance of material. 
The PI will be summarized as the whole-mouth average for each participant and study visit as 
follows:  First the PI tooth-site measures will be averaged for each tooth; and then, these tooth 
averages will be averaged over the entire mouth for a given participant. 
9.2.2.2 Modified Gingival Index (MGI) 
Modified Gingival Index (MGI) will be assessed at 6 sites per tooth (mesiobuccal, buccal, 
distobuccal, distolingual, lingual, and mesiolingual surfaces of the tooth).  Each tooth-site will 
have its MGI scored as follows: 
0 = Absence of Inflammation 
1 = Mild inflammation; slight change in color, little change in texture of any portion of, 
but not the entire marginal or papillary gingival unit 
2 = Mild inflammation; criteria as above but involving the entire marginal and papillary 
gingival unit 
3 = Moderate inflammation; glazing, redness, edema, and/or hypertrophy of the 
marginal or papillary gingival unit 
4 = Severe inflammation; marked redness, edema, and/or hypertrophy of the marginal 
or papillary gingival unit, spontaneous bleeding, congestion, or ulceration 
The MGI will be summarized as the whole-mouth average for each participant and study visit as 
follows:  First the MGI tooth-site measures will be averaged for each tooth; and then, these tooth 
averages will be averaged over the entire mouth for a given participant. 
9.2.2.3 Bleeding on Probing (BOP) 
Bleeding on probing is a dichotomous measurement that will be recorded at the same six sites 
per tooth as PI and MGI.  The BOP will be reduced to one record per subject as percent of sites 
with BOP. 
9.2.2.4 Plaque and Gingivitis Severity Indices 
Plaque Severity Index (PSI) : This index allows for a comparison of the tooth surface sites from 
each group that received the most severe Silness and Loe Plaque Index scores; that is, a Silness 
and Loe Plaque Index score of 2 and 3. The mean Plaque Severity Index will be calculated for 
each subject by dividing the total number of tooth surface sites scored either 2 or 3 by the total 
number of tooth surface sites scored in the mouth for plaque formation (number of teeth scored 
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Gingivitis Severity Index (GSI):  This index allows for a comparison of the gingival sites from 
each group that received the most severe Modified Gingival Index scores; that is, a Modified 
Gingival Index score of 3 or 4, by the total number of sites scored in the entire mouth for 
gingivitis (number of teeth scored multiplied by six). 
9.2.2.5 Interleukin-1β (IL-1β) 
The IL-1 will be summarized as the arithmetic and geometric means of the IL-1 
concentrations over the 4 GCF samples taken at each visit for a study participant.  These means 
will be referred to as the 4-sample IL-1 means in the remainder of this document. 
9.2.3 
Exploratory Efficacy Endpoints 
9.2.3.1 Pocket Depth 
The pocket depth (PD) will be measured at each of the 6 tooth-sites for each tooth of each 
participant during the baseline and study Day 28 visit. 
9.2.3.2 Clinical Attachment Loss 
The clinical attachment level (CAL) will be calculated at each of the 6 tooth-sites for each tooth 
of each participant during the baseline and study Day 28 visit.  CAL will be calculated as PD 
minus the cementoenamel junction. 
9.3 Analysis Methods 
9.3.1 
Primary Efficacy Analyses 
All efficacy analyses are considered secondary analyses. 
9.3.2 
Secondary Efficacy Analyses 
The main objectives of the secondary efficacy analyses are to:  1) obtain some evidence of 
efficacy (either a statistically significant effect or a trend); 2) obtain estimates of the size and 
variance of possible treatment effects that can then be used to plan later  clinical trials; and 3) 
explore which of several outcomes may best be used to measure efficacy.  All efficacy analyses 
will be based on the efficacy population.  
 
9.3.2.1 Secondary Efficacy Endpoints 
Secondary analyses will test for treatment arm differences in the change from baseline of the 
efficacy outcomes: MGI, percent of tooth sites with BOP, PI, GSI, PSI, and IL-1β, at the study 
Day 14 and 28.  These secondary endpoints will be analyzed using mixed models with change 
from baseline of the whole mouth average of the efficacy endpoint as the dependent variable, 
subject as a random effect, a covariate adjustment for baseline, and fixed effects for visit, study 
arm, and a study arm-by-visit interaction.   
 


 
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The following SAS code using mixed models will be used to analyze the secondary endpoints:  
MGI, PI, GSI, PSI , IL-1 (4-sample mean), and percent of sites with BOP. 
 
Proc Mixed data=dataset; 
    class subject visit study_arm; 
    model  change  = baseline study_arm visit study_arm *visit ; 
    repeated visitn / sub=subject  type=cs; 
    lsmeans   study_arm*visit / pdiff; 
run; 
 
where:  change = change in the efficacy endpoints from baseline. 
        
These mixed models analyses assume the efficacy outcomes are approximately normal in 
distribution.  A compound symmetry variance structure is assumed for the mixed models; 
however, if the Schwarz Bayesian criterion indicates that an unstructured variance structure 
better fits the model then the unstructured variance structure for the repeated effects will be used.  
Residual analysis will test the underlying assumptions of the mixed models analyses.  The choice 
of using the arithmetic 4-sample IL-1 means or the geometric 4-sample IL-1 means will be 
based upon the models with the better residual fit to model assumptions.  If for any efficacy 
endpoint, the normality assumption is found to be unreasonable, then log-transformation will be 
used to normalize the data.  Furthermore, if log-transformation fails to normalize the data then 
generalized linear mixed models with the appropriate links and distributional assumptions or will 
be used to analyze these efficacy outcomes. 
 
Within the each of the mixed models analysis of the efficacy endpoints, pairwise contrasts will 
be made to test for statistical differences among the 3 study arms with respect to the change from 
baseline at the Day 14 and Day 28 visits.  Further pairwise contrasts will test each endpoint for 
statistical differences between the Day 14 and Day 28 visits within each study arm.   
 
Since this is a safety phase 1 / 2 with safety measures as the primary endpoints, the secondary 
efficacy endpoints will not be adjusted for multiplicity.   However, the contrast between the 
treatment and placebo rinse arms with respect to the MGI change from baseline at Day 28 will be 
considered the secondary endpoint of chief importance.  The same statistical contrast but with 
BOP change from baseline as the dependent variable will be considered the statistical contrast 
next in order of importance. 
 
Tables will summarize the p-values, contrasts, and least squares means (with standard errors) of 
the change from baseline and their differences among the treatment groups and visits for each of 
the mixed models analyses described above. 
 
Summary statistics of the secondary endpoints and their change from baseline, including n, 
means, minimums, maximums, standard deviations, and standard errors, will be summarized in a 
table by study arm and study visit.  Raw values of all the secondary efficacy endpoints, both on 
the tooth-site level and the subject level will be listed. 


 
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9.3.2.2 Sensitivity Analyses of Secondary Efficacy Outcomes 
The exclusion criterion for periodontitis was removed in October 2015 for Protocol Version 4.0.  
This may change the study population by adding study participants with more advanced disease 
and possibly increase the variability in the effect sizes of the secondary efficacy outcomes.  To 
explore the possible effects of changing the study population, sensitivity analyses will be 
performed for each of the secondary efficacy analyses.  These sensitivity analyses will be 
performed similar to the secondary efficacy analyses but with the exclusion of subjects with 
periodontitis that would have been excluded in the original protocol.  The results of the 
sensitivity analyses will then be compared to the secondary efficacy analyses to understand how 
removing the exclusion for periodontitis may affect the efficacy results. 
9.3.3 
Exploratory Efficacy Analyses 
Exploratory analyses will test for treatment arm differences in the change from baseline of the 
exploratory efficacy outcomes: PD and CAL at study Day 28.  The PD and CAL change from 
baseline at Day 28 will be analyzed using ANCOVA with the PD or CAL change from baseline 
as the dependent variable, a covariate adjustment for baseline, and fixed effects study arm.   
 
The following SAS code using mixed models will be used to analyze the secondary endpoints: 
PGSS, whole-mouth averages of PI, and MGI, IL-1 4-sample mean, and percent of sites with 
BOP. 
 
Proc GLM data=dataset; 
    class subject study_arm; 
    model  change  = baseline study_arm  ; 
    lsmeans   study_arm / pdiff; 
run; 
 
where:  change = change in the efficacy endpoints at study Day 28 from baseline. 
        
The ANCOVA analyses assume the outcomes are approximately normal in distribution with 
homogeneity of variance.  Residual analysis will test these underlying assumptions. If these 
underlying assumptions are found to be unreasonable for the ANCOVA analyses then the 
nonparametric Wilcoxon rank sum will be used to test for pairwise differences between the 3 
treatment arms with respect to the PD or CAL change from baseline at study Day 28. 
 
Tables will summarize the p-values, and least squares means (with standard errors) of the change 
from baseline and their differences among the treatment groups and visits for each of the mixed 
models analyses.  If the nonparametric Wilcoxon rank sum test is employed then p-values with 
their associated Hodges-Lehmann estimators will be reported along with the median, minimum, 
maximum, 10th, and 90th percentiles for the changes from baseline of the PD and CAL measures 
for each treatment arm. 
Raw values of all the PD and CAL measures, both on the tooth-site level will be listed. 
A sensitivity analysis will be performed to compare generalized estimating equations (GEE) 
methods to the mixed models results in the secondary efficacy analyses.  These generalized 


 
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estimating equations (GEE) method with a linear link and exchangeable covariance matrix (using 
a within subject correlation structure) will be used to estimate the treatment group effects on the 
change from baseline in MGI with a covariate adjustment for the baseline MGI.   These 
exploratory GEE models will be performed at both the whole-mouth and tooth-site levels. 
 
 
 
 


 
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10. INTERIM ANALYSES AND DATA MONITORING 
No formal interim analysis is planned. 
 


 
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11. CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
The safety and efficacy populations were expanded on to describe the handling of subjects that 
received the wrong treatment. 


 
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12. LIST OF PLANNED TABLES, FIGURES, AND LISTINGS 
Lists of planned tables, figures, and listings will be provided in a separate document. 


 
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13. APPENDICES 
13.1 Study Flow Chart 


 
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13.2 Schedule of Events 
Procedures  
Screen 
 
Day 
-10 to -1 
Enrollment/
Baseline 
Visit 1 
 
Day 0 
Visit 2 
 
Day 3 
± 1 day 
Visit 3 
 
Day 7 
± 1 day 
Visit 4 
 
Day 14 
± 1 day 
Visit 5 
 
Day 21 
± 1 day 
Visit 6 
 
Day 28 
± 1 day 
F/U 
 
Day 90 
± 14 days 
Obtain informed consent 
X 
X 
 
 
 
 
 
 
Medical/dental history  
Xa,b 
Xa 
X 
X 
X 
X 
X 
 
Record concomitant medications and dental 
procedures  
X 
X 
X 
X 
X 
X 
X 
 
Urine pregnancy test 
X 
X 
 
 
 
 
 
 
Vital signsc 
X 
X 
X 
X 
X 
X 
X 
 
Intraoral and extraoral examinations 
X 
X 
X 
X 
X 
X 
X 
 
Assessment of plaque and calculus 
X 
 
 
 
 
 
 
 
Solicit adverse events 
 
 
X 
X 
X 
X 
X 
X 
Modified gingival index 
X 
X 
 
 
X 
 
X 
 
Gingival crevicular fluid sample for IL-1β  
 
X 
 
 
X 
 
X 
 
Probing depth; measure GM-CEJ to 
determine clinical attachment level 
 
X 
 
 
X 
 
X 
 
Bleeding on probing 
 
X 
 
 
X 
 
X 
 
Plaque index  
 
X 
 
 
X 
 
X 
 
Plaque samples to analyze oral flora 
 
X 
 
 
X 
 
X 
 
Blood for plasma BLXA4-ME level and SPM 
profiling in serum and plasma 
 
X 
 
 
 
 
X 
 
Blood for eligibility or safety laboratory testsd 
X 
 
 
 
X 
 
X 
 


 
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Procedures  
Screen 
 
Day 
-10 to -1 
Enrollment/
Baseline 
Visit 1 
 
Day 0 
Visit 2 
 
Day 3 
± 1 day 
Visit 3 
 
Day 7 
± 1 day 
Visit 4 
 
Day 14 
± 1 day 
Visit 5 
 
Day 21 
± 1 day 
Visit 6 
 
Day 28 
± 1 day 
F/U 
 
Day 90 
± 14 days 
Urine for eligibility or safety laboratory testsd 
X 
 
 
 
X 
 
X 
 
Assess compliance 
 
 
X 
X 
X 
X 
X 
 
Provide oral rinse  
 
X 
X 
X 
X 
X 
 
 
Hygiene instructions or referral for 
periodontal treatment 
 
 
 
 
 
 
 
X 
F/U = safety follow-up telephone call; GM-CEJ = distance from free gingival margin to cementoenamel junction; IL-1 = interleukin-1; SPM = specialized 
pro-resolution mediator 
a 
Includes history of alcohol and tobacco use. 
b 
Includes demographic information. 
c 
Pulse rate, respiratory rate, blood pressure, and oral body temperature are collected at all visits marked with “X”.  Weight is collected at the Screening 
Visit and Visit 6 (Day 28). 
d 
The on-site medical monitor will review the results of safety laboratory tests to determine whether a laboratory test should be redrawn.