Clinical trial NCT05086276
Conditions: Hearing Loss, Sensorineural Noise Induced Hearing Loss Sudden Hearing Loss
Keywords: Intratympanic administration Restoration of hearing loss
Sponsor: Frequency Therapeutics
Start date: 2021-10-12; Completion date: 2022-12-30
Enrollment: 142 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a Phase 2, prospective, randomized, double-blind, placebo-controlled, single-dose, multicenter study to evaluate the efficacy of FX-322, administered by intratympanic injection, in adults with acquired sensorineural hearing loss (SNHL).

Detailed description:
This is a Phase 2, prospective, randomized, double-blind, placebo-controlled, single-dose, multicenter study to evaluate the efficacy of FX-322, administered by intratympanic injection, in adults with acquired sensorineural hearing loss (SNHL).

Previous human studies in subjects 18 to 65 years inclusive (FX-322-103, FX-322-201, FX-322-111) and subjects 66-85 inclusive (FX-322-112) demonstrated that a single dose FX-322 was well tolerated in patients with acquired SNHL with no treatment-related serious adverse events. Adverse events in these studies were generally common to and associated with the intratympanic injection procedure with mild and transient discomfort in patients both with the drug and the placebo.

Frequency Therapeutics' Investigators intend to evaluate efficacy with a single dose of FX-322 in subjects 18 to 65 years inclusive with acquired SNHL.

Arms: arm 1: FX-322, 1 dose arm 2: Placebo, 1 dose
Interventions: intervention 1: FX-322 intervention 2: Placebo
Intervention descriptions: intervention 1: Active Comparator intervention 2: Placebo

Protocol documents:
Prot_000.pdf: 
 
 
 
 
Clinical Study Protocol 
 
 
 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, Single-Dose, 
Multicenter Study to Evaluate the Efficacy of FX-322 Administered by Intratympanic Injection in 
Adults with Acquired Sensorineural Hearing Loss 
 
 
 
NCT Number: 05086276 
 
 
Protocol Date: 05 Jan 2022 
 
 
 
 
 
*Per clinicaltrials.gov results posting guidelines, this cover page is attached to the Protocol FX-
322-208 v3.0 for investigational product FX-322  


 
 
FX-322-208 V3.0 05Jan2022                                                                                Page 1 of 64 
 
CLINICAL STUDY PROTOCOL 
 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-
Controlled, Single-Dose, Multicenter Study to Evaluate the Efficacy of 
FX-322 Administered by Intratympanic Injection in Adults with 
Acquired Sensorineural Hearing Loss 
 
Protocol Number: 
FX-322-208 
Investigational Product: 
FX-322 
Phase: 
2 
Sponsor: 
Frequency Therapeutics 
75 Hayden Ave – Suite 300 
Lexington, MA 02421 
USA 
 
Protocol Date: 
05 Jan 2022 
Protocol Version: 
3.0 
CONFIDENTIAL 
 
 
This protocol may not be reproduced or communicated to a third party without the 
written permission of Frequency Therapeutics


 
FX-322-208 V3.0 05Jan2022                                                                            Page 2 of 64 
 
1 
PROTOCOL APPROVAL SIGNATURES 
Protocol Title: 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, 
Single-Dose, Multicenter Study to Evaluate the Efficacy of FX-322 
Administered by Intratympanic Injection in Adults with Acquired 
Sensorineural Hearing Loss 
Protocol Number: 
FX-322-208 
Protocol Version:  
3.0, 05 Jan 2022 
 
The trial will be conducted in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and applicable United States (US) Code of Federal Regulations (CFR). 
The Principal Investigator will assure that no deviation from, or changes to the protocol will take 
place without prior agreement from the sponsor and documented approval from the Institutional 
Review Board (IRB), except where necessary to eliminate an immediate hazard(s) to the trial 
participants. All personnel involved in the conduct of this study have completed Human Subjects 
Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval. Approval of both the protocol and the consent form 
must be obtained before any participant is enrolled. Any amendment to the protocol will require 
review and approval by the IRB before the changes are implemented to the study.  All changes to the 
consent form will be IRB approved; a determination will be made regarding whether a new consent 
needs to be obtained from participants who provided consent, using a previously approved consent 
form. 
 
 
Sponsor Signature 
 
 
 
 
 
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 3 of 64 
 
2 
SYNOPSIS 
Protocol Number: 
FX-322-208 
 
Title: 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, Single-Dose, 
Multicenter Study to Evaluate the Efficacy of FX-322 Administered by Intratympanic 
Injection in Adults with Acquired Sensorineural Hearing Loss  
 
Investigational Product: 
FX-322 
 
Study Centers: 
Approximately 25 centers in the US 
 
 
Phase: 
Phase 2 
 
Objectives: 
Primary Objective 
• To assess efficacy following a single dose of FX-322 versus Placebo, in subjects with 
acquired sensorineural hearing loss. 
 
Secondary Objective(s) 
• To assess safety following a single dose of FX-322 or Placebo in subjects with 
acquired sensorineural hearing loss. 
 
Endpoints: 
 
Primary Efficacy Endpoint 
• Word Recognition in quiet (WR) improvements through Day 90 
 
Secondary Endpoint(s) 
• Words-in-Noise improvements through Day 90 
• Audiometry 
o Standard Pure Tone (air 0.25-8 kHz; bone 0.5-4 kHz) 
o Extended High Frequency (air 9-16 kHz) 
• 
 Qualitative Questionnaire 
• Tinnitus Functional Index (TFI) Questionnaire 
• Research Assessment on 
 Quality of Life 
Questionnaire 
• Patient Global Impression of Change (PGI-C) Hearing Loss Scale 
• Patient Global Impression of Change (PGI-C) Daily Impacts Scale 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 4 of 64 
 
 
Exploratory Endpoint(s) 
• Audiometry, speech perception measure improvements, and safety assessments 
beyond Day 90 
 
Safety Endpoint(s) 
• Adverse events 
• Changes in: 
o Otoscopy 
o Tympanometry 
o C-SSRS 
 
Study Design: 
This is a Phase 2, prospective, randomized, double-blind, placebo-controlled, single-dose, 
multicenter study to evaluate the efficacy of FX-322, administered by intratympanic 
injection, in adults with acquired sensorineural hearing loss (SNHL).  
 
Previous human studies in subjects 18 to 65 years inclusive (FX-322-103, FX-322-201, FX-
322-111) and subjects 66-85 inclusive (FX-322-112) demonstrated that a single dose FX-322 
was well tolerated in patients with acquired SNHL with no treatment-related serious adverse 
events. Adverse events in these studies were generally common to and associated with the 
intratympanic injection procedure with mild and transient discomfort in patients both with the 
drug and the placebo (McLean et al., 2021). 
 
In previous single-dose human studies in subjects 18-65 years inclusive (FX-322-201, FX-
322-111) it was demonstrated that treatment with FX-322 was associated with statistically 
significant and clinically meaningful improvements in auditory measures 90 days after a 
single administration of FX-322, compared to either placebo or untreated ears. 
 
We intend to further establish efficacy with a single dose of FX-322 in subjects 18 to 65 
years inclusive with acquired SNHL. 
 
The study will have two Parts as described below: 
Part A: Screening/Start of Lead-in/Visit 1Lead-in/Visit 2 
Part B: Randomization, Treatment, Follow-up, and Observation 
Part A 
Screening/Start of Lead-In:  Visits can occur 30-40 days prior to study drug administration 
[Visit 3 (Day 1)]. All subjects will be evaluated to determine study eligibility. For the 
purpose of ensuring the integrity of the trial and minimizing bias, certain study design details 
are further described within a separate, restricted-access unmasked protocol addendum. As 
part of recording the medical history of subjects, concurrent use of hearing aids during the 
study will be documented, as well as hearing loss etiology and laterality (unilateral, bilateral).  
Eligible subjects will be required to return for Lead-in/Visit 2 (14-18 days after Visit 1) to 
complete assessments.  Subjects will be instructed to contact the Investigator or relevant staff 
if a medical change has occurred during the lead-in period (Visit 1 to pre-dose Visit 3).  
Eligible subjects will return for Visit 3 and will be evaluated to determine whether they meet 
appropriate Inclusion/Exclusion criteria required for randomization.   
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 5 of 64 
 
Part B 
Randomization, Treatment, Follow-up, and Observation: Approximately 124 subjects are 
planned to be randomized in this study.  The subjects will be randomized to receive FX-322 or 
placebo according to the different treatment group assignments listed below: 
 
Treatment 
Group 
# of 
Subjects 
Day 1 
1 
62 
FX-322 
2 
62 
Placebo 
 
Once a subject has been identified as meeting all Inclusion/Exclusion criteria, including the 
criteria described within the unmasked addendum which will be determined via the electronic 
data capture (EDC) system, the subject will be randomized. FX-322 or placebo will be 
administered at Visit 3 (Day 1). The syringes containing either placebo or FX-322 will be 
obscured to maintain the blind for the injecting Otolaryngologist.  
Each subject will be placed in the supine position. Topical anesthesia (with the exception of 
phenol) will be administered directly to the tympanic membrane. Under a microscope, a 25-
gauge needle will be used to inject FX-322 or placebo into the middle ear at the junction of the 
posterior inferior and posterior superior quadrant with the needle tip directed towards the round 
window. The posterior superior quadrant should be avoided to prevent injury to the ossicles.  
After injection, the subject will continue to lie with the injected ear facing up for 20-30 minutes. 
The injections will be performed at an appropriate location designated by a Board-certified 
Otolaryngologist trained and experienced in performing intratympanic injections. Safety 
monitoring will include recording of adverse events (AEs), monitoring of audiology, 
tympanometry, otoscopic exams, and C-SSRS. 
Follow up: Subjects will be required to return to clinic for safety, otologic, and audiologic 
assessments at Days 30 (Visit 4), 60 (Visit 5), and 90 (Visit 6) following the study injection. 
Unscheduled visits may occur as needed. 
Observation: Subjects will return to clinic for safety, otologic, and audiologic assessments at 
Days 180 (Visit 7) and 270 (Visit 8).   
 
Number of Subjects to be Randomized:  
Approximately 124 randomized subjects in order to obtain a target of 112 evaluable subjects. 
 
Study Duration: 
Part A: 30-40 days 
Part B: 90 days 
 
Study Population: 
Male and female adults (18-65 years inclusive), otherwise healthy with acquired 
sensorineural hearing loss, with screening PTA values between 35-85 dB. Subject must have 
a medical history that is consistent with adult-onset SNHL that is acquired based on medical 
histories consistent with exposure to noise (Johnson et al., 2017) or as a result of sudden 
sensorineural loss. 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 6 of 64 
 
Statistical Analysis: 
Sample Size:  The sample size of 112 evaluable subjects, in a 1:1 allocation ratio of FX-322 
and Placebo provides 80% to show improvements in WR. 
 
Statistical Methods: Descriptive summaries will be provided for patient disposition, 
demographic and baseline disease data by treatment group.   
Efficacy analysis of continuous endpoints will be examined via a Mixed Model for Repeated 
Measures (MMRM).  Inferential analyses for categorical endpoints will be analysed using 
univariate methods and/or Generalized Estimating Equations (GEEs) (or appropriate 
longitudinal methods as detailed in the SAP).  When appropriate, models will include 
adjustments for baseline as well as the stratification factors used at randomization and 
clinically relevant confounders as identified in the SAP.  Departures of these models from the 
statistical assumptions will be assessed and alternative approaches maybe employed.   
Summary statistics for efficacy endpoints will be provided by treatment group over the study 
and at specific timepoints. Comparisons between FX-322 and placebo and 95% confidence 
interval of the difference will also be calculated.  
The incidence of treatment-emergent adverse events will be presented by preferred term and 
body system using the Medical Dictionary for Regulatory Activities (MedDRA®).  
Tabulations will be provided by seriousness, severity, and relationship to study drug.  
Audiology, tympanometry and otologic data will be presented as summary statistics at each 
timepoint as well as changes from baseline and/or shift tables. All subjects exposed to study 
drug will be included in the Safety Analysis Set and included in the safety analyses according 
to the actual treatment group regardless of the randomized assignment.   
Details of all planned statistical analyses and methods will be provided in the SAP, and will 
be finalized prior to study blind break for the primary analysis. 
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 7 of 64 
 
3 
PROTOCOL AMENDMENT 
3.1 
Protocol Version 3.0 Amendments 
Item 
No. 
Change 
Section and Page Number(s) 
1. 
Updated date and version of 
protocol. 
Title page, pg. 1 
Section 1, Signature page, pg. 2 
Section 20, Investigator Signature page, pg. 
54 
Footer, all pages 
2. 
Clarified primary and secondary 
endpoints to show improvement 
through Day 90. Added 
Exploratory Endpoints. 
Section 2, Synopsis, pgs. 3-4 
Section 9, Study Objectives and Endpoints, 
pgs. 22-23 
3. 
Removed ‘Day -40 to Day -30’ 
from Screening/Start of Lead-
in/Visit 1 and removed ‘Day -15’ 
from Lead-in/Visit 2. Added 
Observational visits (Visit 7 and 
Visit 8). Updated the visit 
windows for Visits 2 and 3. 
Section 2, Synopsis, pgs. 4-5 
Section 10.2, Description, pgs. 23-24 
Section 12, Timing of Study Procedures, 
pgs. 36-38 
Section 12.9, Duration of Treatment, pg. 39 
Section 13, Study Assessments, pgs. 39-41 
Section 14, Adverse Events, pg. 42 
 
4. 
Clarified that summary statistics 
for efficacy endpoints will be 
provided by the treatment group 
over the study and at specific 
timepoints. Further clarified 
primary efficacy analysis. 
Section 2, Synopsis, pg. 6 
Section 15.5, Efficacy Analyses, pg. 47 
5.  
Updated protocol to confirm that 
interim analysis is no longer 
planned. 
Section 2, Synopsis, pg. 6 
Section 10.8.1, Blinding, pg. 33 
Section 15.4, Interim Analyses, pg. 47 
6.  
Updated Introduction to align with 
Investigator’s Brochure, v7.0. 
Section 7, Introduction, pgs. 19-21 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 8 of 64 
 
Item 
No. 
Change 
Section and Page Number(s) 
Section 8.1, Known Potential Benefits, pg. 
21 
7. 
Added reference for McLean et al., 
2021. 
Section 8.2, Known Potential Risks, pg. 22 
Section 19, References, pg. 52 
8. 
Updated Study Design schematic. 
Section 10.1.2 Study Design, pg. 25 
9. 
Updated Schedule of Assessments 
to clarify visit windows and add 
Observational Visits 7 and 8. 
Section 10.2.2, Schedule of Assessments, 
pgs. 26-27 
10. 
Clarified end of study definition to 
be considered a subject who has 
completed all follow-up visits, 
including Visit 6 (Day 90). 
Section 10.4, End of Study Definition, pg. 
28 
11. 
Updated protocol to allow for 
rescreening with sponsor approval. 
Section 10.5.5, Screen Failures, pg. 30 
12. 
Updated protocol to confirm that 
all unused and used study drug 
‘should’ be retained at the site 
until inventoried by the site 
monitor, ‘in compliance with the 
site’s SOPs’. 
Section 10.7.4, Disposal, Return or 
Retention of Study Drug, pg. 33 
13. 
Changed bilateral ‘NIHL’ to 
bilateral ‘hearing loss’. 
Section 10.8.4, Selection and Timing of 
Dose for Each Subject, pg. 34 
14. 
Updated Prohibited 
Medication/Therapy section to 
clarify that oral and intratympanic 
steroids are prohibited through 
Visit 6 (Day 90). 
Section 10.9.1, Prohibited 
Medication/Therapy, pg. 35 
15. 
Updated Discontinuation details to 
include observational visits and 
confirm early termination 
procedures should be Visit 6 (Day 
90) assessments. 
Section 11.1, Discontinuation, pg. 35 
16. 
Further defined who will be 
considered Lost to Follow-up. 
Section 11.2, Lost to Follow-up, pg. 35 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 9 of 64 
 
Item 
No. 
Change 
Section and Page Number(s) 
17. 
Clarified use of center frequencies 
for audiometry. 
Section 13.1.4, Extended High Frequency 
Audiometry, pg. 40 
18. 
Clarified that subjects should rest 
for at least 5 minutes prior to vital 
signs. 
Section 13.2.3, Vital Signs, pg. 41 
19. 
Clarified that AESIs will be 
captured through Visit 6 (Day 90) 
only. 
Section 14, Adverse Events, pg. 44 
20. 
Removed ‘clinical laboratory 
measurements’ and added 
‘pregnancy tests.’ Removed details 
of partial date imputations. This 
will be captured in the Statistical 
Analysis Plan.  
Section 15.6, Safety Analyses, pg. 48 
 
3.2 
Protocol Version 2.0 Amendments 
Item 
No. 
Change 
Section and Page Number(s) 
1. 
Updated date and version of 
protocol. 
Title page, pg. 1 
Section 1, Signature page, pg. 2 
Section 22, Investigator Signature page, pg. 
49 
Footer, all pages 
2. 
Added Patient Global Impression 
of Change Hearing Loss and Daily 
Impacts.  
Section 2, Synopsis – Secondary 
Endpoints, pg. 3 
Section 9, Study Objectives and Endpoints, 
pg. 18 
Section 10.2.2, Schedule of Assessments, 
pg. 22 
Section 12.6, Follow-up Visit, pg. 33 
Section 13.1, Efficacy Measurements 
Assessed, pgs. 35-36 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 10 of 64 
 
Item 
No. 
Change 
Section and Page Number(s) 
Section 23.5 and 23.6, Appendix, pgs. 58-
59 
3.  
Added safety and efficacy 
information about studies FX-322-
111 and FX_322-112 from 
Investigator’s Brochure v7.0. 
Section 2, Synopsis – Study Design, pg. 4 
Section 7, Introduction, pg. 16 
Section 10, Investigational Plan, pg. 19 
Section 10.3, Scientific Rationale for Study 
Design, pg. 24 
4.  
Added PGI-S, PGI-C, SAP, and 
SRT to List of Abbreviations and 
Definitions of Terms. 
Section 6, List of Abbreviations and 
Definition of Terms, pgs. 14-15 
5. 
Study Design schematic updated 
Section 10.1.2 Study Design, pg. 21 
6.  
Added Speech Reception 
Threshold (SRT) as assessment. 
Section 10.2.2, Schedule of Assessments, 
pg. 22 
Section 12, Timing of Study Procedures, 
pgs. 32-34 
Section 13.3, Other Study Assessments, pg. 
37 
7. 
Removed ‘SL’ from ‘W-22 SL’. 
Updated throughout protocol 
8.  
Added Patient Global Impression 
of Severity Hearing Loss and 
Daily Impacts. 
Section 10.2.2, Schedule of Assessments, 
pg. 22 
Section 12.3, End of Lead-In/Treatment 
Visit 3 (Day 1), pg. 32 
Section 13.3, Other Study Assessments, pg. 
37 
Section 23.3 and 23.4, Appendix, pgs. 56-
57 
9. 
Updated Schedule of Assessments 
to add Inclusion/Exclusion 
assessment at Visit 2 (Day -15), 
clarify that Visit 6 procedures 
should be conducted for subjects 
who early terminate from the 
Section 10.2.2, Schedule of Assessments, 
pgs. 22-23 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 11 of 64 
 
Item 
No. 
Change 
Section and Page Number(s) 
study, added SRT, PGI-S, and 
PGI-C. 
10. 
Updated Inclusion #6 to note 
examples of two forms of 
contraception that would include 
the use of a condom. 
Section 10.5.2, Inclusion Criteria, pg. 24 
11. 
Clarified procedures to be 
conducted for Early Termination 
visits. 
Section 11.1, Discontinuation, pg. 31 
12. 
Updated Visit 2 (Day -15) 
procedures to include reassessment 
of Inclusion/Exclusion eligibility. 
Section 12.2, Lead-in Visit 2 (Day -15), pg. 
32 
13. 
Added SRT and PGI-S scales to 
Section 13.3 ‘Other Study 
Assessments’ and updated Section 
13 from ‘Efficacy and Safety 
Assessments’ to ‘Study 
Assessments’. 
Section 13, Study Assessments, pgs. 34-37 
14. 
Clarified that 
 
 Qualitative 
Questionnaire is completed by the 
subject. 
Section 13.1.6, 
 
Qualitative Questionnaire, pg. 35 
15. 
Clarified that adverse events 
should be collected starting from 
the time of study treatment and 
new adverse events should be 
documented for worsening events. 
Section 14, Adverse Events, pgs. 37-38 
16.  
Added references for: Johnson, et 
al 2017, Meikle et al 2012, Posner 
et al 2011, US FDA Oct 2018 and 
alphabetized all references. 
Section 2, Synopsis – Study Population, pg. 
5 
Section 13.1.5, Tinnitus Functional Index, 
pg. 35 
Section 13.2.6, Columbia Suicide Severity 
Rating Scale, pg. 36 
Section 21, References, pgs. 47-48 
17. 
Removed ‘Baseline’ from Visit 3 
(Day 1). Updated references to 
Updated throughout protocol 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 12 of 64 
 
Item 
No. 
Change 
Section and Page Number(s) 
Visits by visit number instead of 
days.  
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 13 of 64 
 
4 
TABLE OF CONTENTS 
1 
PROTOCOL APPROVAL SIGNATURES ............................................................ 2 
2 
SYNOPSIS .................................................................................................................. 3 
3 
PROTOCOL AMENDMENT .................................................................................. 7 
3.1 
Protocol Version 3.0 Amendments ........................................................................... 7 
3.2 
Protocol Version 2.0 Amendments ........................................................................... 9 
4 
TABLE OF CONTENTS ........................................................................................ 13 
5 
LIST OF TABLES ................................................................................................... 16 
6 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ........................ 17 
7 
INTRODUCTION ................................................................................................... 19 
8 
RISK/BENEFIT ASSESSMENT ........................................................................... 21 
8.1 
Known Potential Benefits ........................................................................................ 21 
8.2 
Known Potential Risks ............................................................................................ 22 
8.3 
Assessment of Potential Risks and Benefits ........................................................... 22 
9 
STUDY OBJECTIVES AND ENDPOINTS ......................................................... 22 
10 
INVESTIGATIONAL PLAN ................................................................................. 23 
10.1 Overall Study Design and Plan: ............................................................................. 23 
10.2 Description ................................................................................................................ 23 
10.2.1 
Study Design ............................................................................................. 25 
10.2.2 
Schedule of Assessments .......................................................................... 26 
10.3 Scientific Rationale for Study Design .................................................................... 28 
10.4 End of Study Definition ........................................................................................... 28 
10.5 Selection of Study Population ................................................................................. 28 
10.5.1 
Number of Planned Subjects .................................................................... 28 
10.5.2 
Inclusion Criteria ...................................................................................... 28 
10.5.3 
Exclusion Criteria ..................................................................................... 29 
10.5.4 
Withdrawal of Subjects From Study Assessments ................................... 29 
10.5.5 
Screen Failures .......................................................................................... 30 
10.6 Investigational Products .......................................................................................... 31 
10.6.1 
Investigational Products Administered ..................................................... 31 
10.6.2 
Identity of Investigational Products .......................................................... 31 
10.7 Preparation and Dispensing .................................................................................... 31 
10.7.1 
Packaging and Labeling ............................................................................ 33 
10.7.2 
Supply, Storage and Handling .................................................................. 33 


 
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10.7.3 
Compliance and Drug Accountability ...................................................... 33 
10.7.4 
Disposal, Return or Retention of Study Drug ........................................... 33 
10.8 Randomization and Blinding .................................................................................. 33 
10.8.1 
Blinding .................................................................................................... 33 
10.8.2 
Un-blinding ............................................................................................... 34 
10.8.3 
Method of Assigning Subjects to Treatment Groups ............................... 34 
10.8.4 
Selection and Timing of Dose for Each Subject ....................................... 34 
10.9 Prior and Concomitant Therapy ............................................................................ 34 
10.9.1 
Prohibited Medication/Therapy ................................................................ 35 
10.9.2 
Rescue Medication .................................................................................... 35 
10.9.3 
Treatment Compliance .............................................................................. 35 
11 
DISCONTINUATION AND LOST TO FOLLOW-UP ....................................... 35 
11.1 Discontinuation ........................................................................................................ 35 
11.2 Lost to Follow-up ..................................................................................................... 35 
12 
TIMING OF STUDY PROCEDURES .................................................................. 36 
12.1 Screening/Start of Lead-In Visit (Visit 1; [Day -40 to -30]) ................................. 36 
12.2 Lead-In Visit (Visit 2; [14-18 days after Visit 1]).................................................. 36 
12.3 End of Lead-In/Treatment (Visit 3, 30-40 days after Visit 1 [Day 1]) ................ 36 
12.4 Follow-up (Visit 4, [Day 30 +/- 5 days]) ................................................................. 37 
12.5 Follow-up (Visit 5, [Day 60 +/- 5 days]) ................................................................. 37 
12.6 Follow-up (Visit 6, [Day 90 +/- 5 days]) ................................................................. 38 
12.7 Observation (Visit 7, [Day 180 +/- 7 days]) ........................................................... 38 
12.8 Observation (Visit 8, [Day 270 +/- 7 days]) ........................................................... 38 
12.9 Duration of Treatment ............................................................................................ 39 
13 
STUDY ASSESSMENTS ........................................................................................ 39 
13.1 Efficacy Measurements Assessed ........................................................................... 39 
13.1.1 
Word Recognition in Quiet (WR) ............................................................. 39 
13.1.2 
Words-In-Noise Testing (WIN) ................................................................ 39 
13.1.3 
Standard Pure Tone Audiometry .............................................................. 39 
13.1.4 
Extended High Frequency Audiometry .................................................... 40 
13.1.5 
Tinnitus Functional Index (TFI) ............................................................... 40 
13.1.6 
Qualitative Questionnaire .................................. 40 
13.1.7 
Research Assessment on 
 
 Quality of Life 
 ................................................................. 40 
13.1.8 
Patient Global Impression of Change Hearing Loss Scale ....................... 40 
13.1.9 
Patient Global Impression of Change Daily Impacts Scale ...................... 40 
13.2 Safety Measurements Assessed ............................................................................... 41 
13.2.1 
Tympanometry .......................................................................................... 41 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 15 of 64 
 
13.2.2 
Concomitant Medication .......................................................................... 41 
13.2.3 
Vital Signs ................................................................................................ 41 
13.2.4 
Physical Exam (PE) .................................................................................. 41 
13.2.5 
Otoscopy ................................................................................................... 41 
13.2.6 
Columbia Suicide Severity Rating Scale (C-SSRS) ................................. 41 
13.3 Other Study Assessments ........................................................................................ 41 
13.3.1 
Speech Reception Threshold (SRT) ......................................................... 41 
13.3.2 
Patient Global Impression of Severity (PGI-S) Hearing Loss .................. 42 
13.3.3 
Patient Global Impression of Severity (PGI-S) Daily Impacts ................. 42 
14 
ADVERSE EVENTS ............................................................................................... 42 
14.1 Serious Adverse Events ........................................................................................... 44 
14.2 Expedited Safety Reporting .................................................................................... 46 
15 
STATISTICAL METHODS ................................................................................... 46 
15.1 Sample Size ............................................................................................................... 46 
15.2 Analysis Sets ............................................................................................................. 46 
15.3 Subject Disposition, Demographics and Baseline Disease Status ........................ 47 
15.4 Interim Analyses ...................................................................................................... 47 
15.5 Efficacy Analyses ..................................................................................................... 47 
15.6 Safety Analyses ......................................................................................................... 48 
15.6.1 
Adverse Events ......................................................................................... 48 
15.6.2 
Prior and Concomitant Medications ......................................................... 48 
15.7 Handling of Missing Data and Subject Withdrawals ........................................... 48 
16 
QUALITY ASSURANCE AND QUALITY CONTROL ..................................... 48 
16.1 Audit and Inspection ............................................................................................... 48 
16.2 Monitoring ................................................................................................................ 49 
16.3 Data Management and Coding ............................................................................... 49 
17 
RECORDS AND SUPPLIES .................................................................................. 49 
17.1 Drug Accountability ................................................................................................ 49 
17.2 Financing and Insurance ......................................................................................... 50 
18 
ETHICS .................................................................................................................... 50 
18.1 Independent Ethics Committee or Institutional Review Board .......................... 50 
18.2 Regulatory Authorities ............................................................................................ 50 
18.3 Ethical Conduct of the Study .................................................................................. 50 
18.4 Informed Consent .................................................................................................... 50 
18.5 Subject Confidentiality ............................................................................................ 51 


 
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18.6 Reporting and Publication, Including Archiving ................................................. 51 
19 
REFERENCES ........................................................................................................ 52 
20 
INVESTIGATOR SIGNATURE PAGE ............................................................... 54 
21 
APPENDICES .......................................................................................................... 55 
21.1 Appendix A: 
 Qualitative Questionnaire 
 
 ................................................................................................................ 55 
21.2 Appendix B: Research Assessment on 
 
 Quality of Life 
.................................. 57 
21.3 Appendix C: Patient Global Impression of Severity (PGI-S) Hearing 
Loss Scale .................................................................................................................. 61 
21.4 Appendix D: Patient Global Impression of Severity (PGI-S) Daily 
Impacts Scale ............................................................................................................ 62 
21.5 Appendix E: Patient Global Impression of Change (PGI-C) Hearing 
Loss Scale .................................................................................................................. 63 
21.6 Appendix F: Patient Global Impression of Change (PGI-C) Daily 
Impacts Scale ............................................................................................................ 64 
 
 
 
5 
LIST OF TABLES 
Table 1: FX-322 Group Dosing Schedule ............................................................................... 24 
Table 2: Schedule of Assessments ........................................................................................... 26 
Table 3:  FX-322 Study Drug .................................................................................................. 32 
Table 4: Dosages of Active Agents to be Injected in Humans ................................................ 32 
 
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 17 of 64 
 
6 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Abbreviation 
or Term 
Explanation 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
ASHA 
American Speech-Language-Hearing Association 
BMI 
Body mass index 
CNC 
Consonant-nucleus-consonant  
CONSORT 
Consolidation Standards of Reporting Trials 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
C-SSRS 
Columbia Suicide Severity Rating Scale 
dB 
Decibel 
DMSO 
Dimethyl sulfoxide 
EDC 
Electronic Data Capture System 
FAS 
Full Analysis Set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GSK 
Glycogen Synthase Kinase 
HDAC 
Histone deacetylase 
HCG 
Human chorionic gonadotrophin 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
LGR5+ 
Leucine-rich repeat-containing G-protein coupled receptor 5 positive 
MedDRA  
Medical Dictionary for Regulatory Activities 
MMRM 
Mixed Model for Repeated Measures 
NIHL 
Noise Induced Hearing Loss 
NU-6 
Northwestern University Auditory Test No. 6 
PE 
Physical exam 
PGI-S 
Patient Global Impression of Severity 
PGI-C 
Patient Global Impression of Change 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
PPAS 
Per Protocol Analysis Set 
PTA 
Pure Tone Average 
 
Research Assessment on 
 
Quality of Life 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SfAS 
Safety Analysis Set 
SL 
Sensation Level 
SNHL 
Sensorineural hearing loss  
SNR 
Signal-to-noise ratio 
SRT 
Speech Reception Threshold  
SSNHL 
Sudden Sensorineural hearing loss 


 
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Abbreviation 
or Term 
Explanation 
SOP 
Standard Operating Procedure 
SPL 
Sound pressure level 
SUSAR 
Suspected Unexcepted Serious Adverse Reaction 
TFI 
Tinnitus Functional Index 
TM 
Tympanic membrane 
WHO 
World Health Organization 
WIN 
Words-in-Noise  
WR 
Word Recognition in quiet 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 19 of 64 
 
7 
INTRODUCTION 
Worldwide, an estimated 1.5 billion people currently experience hearing loss due to exposure 
to damaging levels of sound, with an anticipated 2.5 billion afflicted by 2050, including 1.1 
billion young people at risk for hearing loss due to loud sounds over long periods (WHO 
2021). According to the US National Institutes of Health, approximately 90 percent of those 
with hearing loss are affected by SNHL. Excessive sound levels or loud sounds for extended 
periods of time hyperstimulate cochlear hair cells, which can lead to increased production of 
reactive oxygen species and oxidative cell death. Structural damage to hair cells results in 
SNHL, which can be characterized by an attenuation and distortion of response to incoming 
auditory stimuli. Presently, no curative treatments exist, but rather assistive devices such as 
hearing aids and cochlear implants are used to address the symptoms of SNHL. These device 
options, however, do not address the cause of SNHL, whereas the proposed drug product FX-
322 is designed to activate cochlear progenitor cells leading to the development of new 
sensory hair cells and new progenitor cells, and these regenerative mechanistic steps translate 
to a restoration of hearing function in patients with SNHL.  
SNHL accounts for about 90% of all cases of hearing loss (Li 2017). Leading causes include 
noise exposure, ototoxic medications, advanced age, inherited, and autoimmune disorders. 
SNHL is usually irreversible and managed with hearing aids or cochlear implants. Noise is a 
major occupational and environmental hazard, causing hearing loss, sleep disturbance, 
fatigue, and hypertension (Hong 2013). SNHL has long been recognized as the primary and 
direct health effect of excessive noise exposure (Basner 2015). In the United States, an 
estimated 48 million people or 20.3% of the population 12 years or older has hearing loss in 
one or both ears (Lin 2011). The World Health Organization reported that 16% of the 
disabling hearing loss in adults is attributable to occupational noise exposure (Nelson 2005). 
Hearing loss is associated with increased disability, dementia, clinically relevant depression, 
anxiety, stress and other mental health disorders, especially in the elderly (Choi 2016) 
(McGilton 2016) (Deal 2016) (Brody 2018) (Curhan 2019) (Hsu 2016) (Cosh 2019) (Amieva 
2018) (Jayakody 2018).  
FX-322 intratympanic injection is indicated for improvement in speech perception in patients 
with acquired sensorineural hearing loss (SNHL). FX-322 was developed to improve how a 
patient with SNHL feels, functions or survives, assessed using a reliable measure of speech 
perception. FX-322 is a combination of the GSK3 inhibitor, laduviglusib and sodium 
valproate. The drug product contains
 
 
 FX-322 has been developed 
as the maximal feasible dose (MFD) that allows the highest amount of the actives which can 
be contained in the formulation without affecting its gelation properties. 
 is 
contained in several pharmaceutical products approved for human use including at 15.1% 
(w/w) for intratympanic use. 
FX-322 has been shown to target cellular mechanisms, including GSK and HDAC inhibition, 
which act synergistically to induce a regenerative response in cochlear tissue by causing 
asymmetric division of the Lgr5+ progenitor cells to become inner ear hair cells while 
retaining progenitor cells (which also function as a defined subset of supporting cells) 
(McLean et al., 2017) and thus may restore hearing function in patients with acquired SNHL. 
In preclinical experiments, a fixed ratio dose combination of FX03 and Valproate Sodium 
demonstrates: 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 20 of 64 
 
• Expansion of the Lgr5+ progenitor cells and subsequent conversion into hair cells 
using newborn and adult cells. Substantial Lgr5+ cell expansion occurs with the 
combination of agents but not with the individual agents.  
• Expansion of progenitor cells from adult primate inner ear, and expansion and 
subsequent conversion of progenitor cells into hair cells from the adult human inner 
ear. 
• Formation of new hair cells in ototoxin damaged mouse cochlear explants. 
• Significant improvement in hearing and hair cell numbers in an adult mouse model of 
noise-induced hearing loss. 
 FX-322 is a combination of 
laduviglusib and sodium valproate 
 
 FX-322 is administered by intratympanic injection into the 
middle ear and deposited onto the surface of the oval and round window membranes where 
the active ingredients diffuse into the cochlea of the inner ear and are absorbed.  
Minimal systemic exposure occurred after a single intratympanic injection of FX-322 in three 
previous clinical trials. In the first of these trials, FX-322-103, nine patients undergoing 
cochlear implantation surgery received a single unilateral intratympanic injection of FX-322 
(6 patients) or placebo (3 patients) to study tolerability, systemic drug exposure, and cochlear 
drug exposure. The results demonstrated acceptable local and systemic acute tolerability and 
very limited systemic exposure that was unmeasurable within 24 hours post injection. In 
addition, the cochlear FX-322 drug concentration data demonstrated the drug was present at 4 
hours after intratympanic injection in one patient but unmeasurable after 24 hours in the 
perilymph in two subjects. 
The second clinical trial, FX-322-110, was a safety and tolerability study conducted in 
patients with hearing loss with a planned cochlear implant surgery to measure concentrations 
of FX-322 in cochlear fluid and plasma post intratympanic injection. FX-322 was well 
tolerated, as all AEs recovered without sequelae and no serious or severe adverse events were 
observed. FX-322 showed acceptable local and systemic acute tolerability and very limited 
systemic exposure after intratympanic injection. 
The third clinical trial was a Phase ½ (FX-322-201) randomized, double-blind, placebo-
controlled single-dose study in adults with mild to moderately severe stable sensorineural 
hearing loss. The objectives of this study were to assess: 1) the systemic safety of two dose 
levels of FX-322; 2) the plasma pharmacokinetic profile of FX-322; and 3) the effect of FX-
322 on otologic and audiologic measures. After intratympanic injection of FX-322, there 
were no serious adverse events (SAEs) or AEs that led to a subject withdrawal or death. The 
most frequently reported TEAEs that were reported, such as ear discomfort and pain, were all 
transient, generally mild and typical of the intratympanic injection procedure and not related 
to the material injected. One subject treated with FX-322H was observed with a pinhole 
tympanic membrane perforation on Day 15 that resolved by Day 30. There were no other 
TEAEs observed in the otoscopic examinations. No clinically significant safety issues were 
observed in ECGs, physical examinations, vital sign measurements, or on otologic or 
audiologic measures. Additionally, the systemic PK profile of FX-322 was minimal and dose-
proportionate with both drugs cleared from the circulation within approximately 24 hours. 
Over the course of the study, a post-hoc analysis showed a statistically significant 
improvement in measures of hearing function for patients treated with FX-322 versus patients 
given placebo. FX-322 patients showed an improvement in hearing function from baseline to 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 21 of 64 
 
Day 90. The notable improvements in these measures of hearing function were generally 
observed in FX-322 patients within 15-30 days of treatment, they were sustained for 90 days, 
and when responders were retested 1-2 years later the improvements in auditory function 
were maintained. 
 
Preliminary results are available from a recently completed open-label, single-dose study of 
FX-322 (FX-322-111) designed to evaluate the impact of injection conditions on tolerability. 
In the multi-center, randomized study, subjects with mild to severe SNHL (n=33) were injected 
in one ear with FX-322, with the untreated ear as the control. Hearing function was tested over 
the course of 90 days following dosing. At this timepoint, thirty-four percent (34%) of subjects 
achieved a ten percent (10%) or greater absolute improvement in word recognition scores in 
the treated ear, which was clinically meaningful and statistically significant compared to the 
untreated ear (p <0.05).  
 
A Phase 1b (FX-322-112) randomized, double blind, placebo controlled, single dose, 
multicenter, safety study in adults with age related sensorineural hearing loss did not show 
significant treatment effect with FX-322 administration compared to placebo. No subjects 
enrolled in this study had either noise induced or sudden sensorineural hearing loss, conditions 
where FX-322 associated hearing benefits were observed in prior studies. Results did show a 
favorable safety and tolerability profile. 
 
Frequency has completed a pre-defined interim analysis of Study FX-322-202. The interim 
analysis does not replicate the results we have seen from our other single dose trials, 
including FX-322-201, FX-322-201-2, and FX-322-111. The FX-322-202 trial may be 
flawed due to bias that may have been introduced to the trial from a subject who posted the 
informed consent on social media, causing potential subjects to consciously or unconsciously 
adjust their baseline evaluation scores with intentional incorrect responses to qualify for the 
study. Unfortunately, these factors may have compromised the ability of study FX-322-202 to 
precisely and accurately assess any treatment effects of FX-322 with multiple injections.  
 
As a result, efficacy results were confounded by bias issues affecting patient 
inclusion/exclusion and a potential placebo effect. 
 
8 
RISK/BENEFIT ASSESSMENT 
8.1 
Known Potential Benefits 
The active ingredients in FX-322 target two cellular mechanisms, histone deacetylase 
(HDAC) inhibition and GSK-3 inhibition, which act synergistically to induce a regenerative 
response in cochlear tissue. FX-322 could serve as a treatment for patients with SNHL 
because the active ingredients have demonstrated 1) the ability to induce progenitor cell 
expansion and subsequent conversion into new hair cells across species, including human, 2) 
restoration of hair cells in ototoxin-damaged mouse cochlear implants, and 3) a significant 
improvement in hearing and hair cell numbers in an adult mouse model of noise-induced 
hearing loss. 
Most recently it has been shown in a number of studies that in humans with stable mild to 
moderately severe SNHL, a single intratympanic dose of FX-322 may improve hearing 
function to some degree in humans.  


 
FX-322-208 V3.0 05Jan2022                                                                            Page 22 of 64 
 
8.2 
Known Potential Risks 
The risks of intratympanic injection of FX-322 have been carefully studied in nonclinical 
toxicology studies, as well as in a number of clinical trials. Based on these studies, the risks 
appear to be similar to those experienced with intratympanic injections of other materials, 
such as steroids, that are not FDA-approved. The risks of single intratympanic injections are 
characterized as persistent eardrum perforation, pain or bleeding with injection, temporary 
dizziness, middle ear infection, and conductive or sensorineural hearing loss. A published 
clinical trial using multiple intratympanic injections of a steroid in SSNHL patients has 
shown the majority of adverse events to be associated with the injection procedure, with 
eardrum perforations reported at less than 4% (Rauch et al., 2011; McLean et al., 2021). 
8.3 
Assessment of Potential Risks and Benefits 
Based on the very low potential for risk, which is primarily associated with the intratympanic 
injection procedure, compared to the potential benefit which is the possibility of restoration 
of hearing, the risk/benefit profile is considered acceptable to conduct the study described 
herein. 
9 
STUDY OBJECTIVES AND ENDPOINTS 
Primary Objective 
• To assess efficacy following a single dose of FX-322 versus Placebo, in subjects with 
acquired sensorineural hearing loss. 
 
Secondary Objective(s) 
• To assess safety following a single dose of FX-322 or Placebo in subjects with 
acquired sensorineural hearing loss. 
 
Endpoints: 
 
Primary Efficacy Endpoint 
• Word Recognition in quiet (WR) improvements through Day 90 
 
Secondary Endpoints 
• Words-in-Noise improvements through Day 90 
• Audiometry 
o Standard Pure Tone (air 0.25-8 kHz; bone 0.5-4 kHz) 
 Extended High Frequency (air 9-16 kHz) 
• 
Qualitative Questionnaire 
• Tinnitus Functional Index (TFI) Questionnaire 
• Research Assessment on 
 Quality of Life 
Questionnaire 
• Patient Global Impression of Change (PGI-C) Hearing Loss Scale 
• Patient Global Impression of Change (PGI-C) Daily Impacts Scale 
 
Safety Endpoint(s) 
• Adverse events 
• Changes in: 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 23 of 64 
 
o Otoscopy 
o Tympanometry 
o C-SSRS 
 
Exploratory Endpoint(s) 
• Audiometry, speech perception measure improvements, and safety assessments 
beyond Day 90 
 
 
10 INVESTIGATIONAL PLAN 
10.1 Overall Study Design and Plan:  
10.2 Description 
This is a Phase 2, prospective, randomized, double-blind, placebo-controlled, single-dose, 
multicenter study to evaluate the efficacy of FX-322, administered by intratympanic 
injection, in adults with acquired sensorineural hearing loss (SNHL).  
 
Previous human studies in subjects 18 to 65 years inclusive (FX-322-103, FX-322-201, FX-
322-111) and subjects 66-85 inclusive (FX-322-112) demonstrated that a single dose of FX-
322 was well tolerated in patients with acquired SNHL with no treatment-related serious 
adverse events. Adverse events in these studies were common to and associated with the 
intratympanic injection procedure with mild and transient discomfort in patients both with the 
drug and the placebo. 
 
In previous single-dose human studies in subjects 18-65 years inclusive (FX-322-201, FX-
322-111) it was demonstrated that treatment with FX-322 was associated with statistically 
significant and clinically meaningful improvements in auditory measures 90 days after a 
single administration of FX-322, compared to either placebo or untreated ears. 
 
We intend to further establish efficacy with a single dose of FX-322 in subjects 18 to 65 
years inclusive with acquired SNHL. 
 
The study will have two Parts as described below: 
Part A: Screening/Start of Lead-in/Visit 1, Lead-in/Visit 2  
Part B: End of Lead-in/Treatment/Visit 3 (Day 1), Randomization (Double-blind), Follow-up 
(Days 30, 60 and 90), and Observation (Days 180 and 270) 
 
Part A 
Screening/Start of Lead-In:  Visit can occur 30-40 days prior to study drug administration 
[Visit 3 (Day 1)]. All subjects will be evaluated to determine study eligibility. For the 
purpose of ensuring the integrity of the trial and minimizing bias, certain study design details 
are further described within a separate, restricted-access unmasked protocol addendum. As 
part of recording the medical history of subjects, concurrent use of hearing aids during the 
study will be documented, as well as hearing loss etiology and laterality (unilateral, bilateral).  
Eligible subjects will be required to return for Lead-in Visit 2 (14-18 days after Visit 1) to 
complete assessments. Subjects will be instructed to contact the Investigator or relevant staff 
if a medical change has occurred during the lead-in period (Visit 1 to pre-dose Visit 3).  


 
FX-322-208 V3.0 05Jan2022                                                                            Page 24 of 64 
 
Eligible subjects will return for Visit 3 and will be evaluated to determine whether they meet 
appropriate Inclusion/Exclusion criteria required for randomization.   
 
Part B 
Randomization, Treatment, and Follow-up: Approximately 124 subjects are planned to be 
randomized in this study.  The subjects will be randomized to receive FX-322 or placebo 
according to the different treatment group assignments listed below: 
Table 1: FX-322 Group Dosing Schedule 
 
Treatment 
Group 
# of 
Subjects 
Day 1 
1 
62 
FX-322 
2 
62 
Placebo 
 
Once a subject has been identified as meeting all Inclusion/Exclusion criteria, including the 
criteria described within the unmasked addendum which will be determined via the electronic 
data capture (EDC) system, the subject will be randomized. FX-322 or placebo will be 
administered on Visit 3 (Day 1). The syringes containing either placebo or FX-322 will be 
obscured to maintain the blind for the injecting Otolaryngologist.  
Each subject will be placed in the supine position. Topical anesthesia (with the exception of 
phenol) will be administered directly to the tympanic membrane. Under a microscope, a 25-
gauge needle will be used to inject FX-322 or placebo into the middle ear at the junction of the 
posterior inferior and posterior superior quadrant with the needle tip directed towards the round 
window. The posterior superior quadrant should be avoided to prevent injury to the ossicles.  
After injection, the subject will continue to lie with the injected ear facing up for 20-30 minutes. 
The injections will be performed at an appropriate location designated by a Board-certified 
Otolaryngologist trained and experienced in performing intratympanic injections. Safety 
monitoring will include recording of adverse events (Aes), monitoring of audiology, 
tympanometry, otoscopic exams, and C-SSRS. 
 
Follow up: Subjects will be required to return to clinic for safety, otologic, and audiologic 
assessments at Days 30, 60, and 90 following the study injection. Unscheduled visits may 
occur as needed.  See the Schedule of Assessments for more information. 
 
Observation: Subjects will return to clinic for safety, otologic, and audiologic assessments at 
Days 180 and 270.  
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 25 of 64 
 
10.2.1 Study Design 
Figure 1: Study Design 
  
 
 
 
 
 
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 26 of 64 
 
10.2.2 Schedule of Assessments 
Table 2: Schedule of Assessments 
Visit  
Screening/ 
Start of 
Lead-In 
Lead-In 
Visit 
End of 
Lead-In/ 
Treatment 
In-Clinic 
Follow-up  
In-Clinic 
Follow up   
In-Clinic 
Follow-up / 
ETe 
In-Clinic 
Observation 
In-Clinic 
Observation 
Un-scheduled 
Visit 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
UNS 
Assessment/Day 
Day -40 to 
-30 
Visit 1 + 
16 days 
Visit 1 + 35 
days 
Day 30 
Day 60 
Day 90 
Day 180 
Day 270 
 
Visit Window 
N/A 
+/- 2 
days 
+/- 5 days +/- 5 days +/- 5 days +/- 5 days 
+/- 7 days 
+/- 7 days 
N/A 
Informed Consent 
X 
 
 
 
 
 
 
 
 
Inclusion/ Exclusion Criteria 
Xa 
Xa 
Xb 
 
 
 
 
 
 
Eligibility Assessment by EDC 
X 
X 
Xb 
 
 
 
 
 
 
Demographics 
Xa 
 
 
 
 
 
 
 
 
Medical History  
Xa 
Xa 
Xb 
 
 
 
 
 
 
Concomitant Medication  
Xa 
Xa 
Xb, c 
X 
X 
X 
X 
X 
X 
Physical Examination including weight and 
height 
X 
 
 
 
 
 
 
 
Xd 
Vital Signs (body temperature, pulse rate, bp) 
X 
X 
Xb 
X 
X 
X 
 
 
Xd 
Tympanometry  
X 
 
Xb 
X 
X 
X 
X 
X 
Xd 
Standard Pure Tone Audiometry 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 
Speech Reception Threshold (SRT) 
X 
X 
X 
X 
X 
X 
X 
X 
Xd 
Extended High Frequency Audiometry 
X 
X 
 
Xb  
X 
X 
X 
X 
X 
Xd 
Word recognition, quiet (Maryland CNC) 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 
Word recognition (W-22) 
X 
X 
Xb 
X 
X 
X 
X 
X 
Xd 
Words in noise (WIN) 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 27 of 64 
 
Visit  
Screening/ 
Start of 
Lead-In 
Lead-In 
Visit 
End of 
Lead-In/ 
Treatment 
In-Clinic 
Follow-up  
In-Clinic 
Follow up   
In-Clinic 
Follow-up / 
ETe 
In-Clinic 
Observation 
In-Clinic 
Observation 
Un-scheduled 
Visit 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
UNS 
Assessment/Day 
Day -40 to 
-30 
Visit 1 + 
16 days 
Visit 1 + 35 
days 
Day 30 
Day 60 
Day 90 
Day 180 
Day 270 
 
Visit Window 
N/A 
+/- 2 
days 
+/- 5 days +/- 5 days +/- 5 days +/- 5 days 
+/- 7 days 
+/- 7 days 
N/A 
Tinnitus Functional Index 
 
 
Xb 
 
 
X 
X 
X 
Xd 
Questionnaire 
Qualitative 
Questionnaire 
Columbia Suicide Severity Rating Scale 
 
 
 Xb 
X 
X 
X 
X 
X 
Xd 
PGI-S Hearing Loss Scale 
 
 
Xb 
 
 
 
 
 
 
PGI-S Daily Impacts Scale 
 
 
Xb 
 
 
 
 
 
 
PGI-C Hearing Loss Scale 
 
 
 
 
 
X 
 
 
 
PGI-C Daily Impacts Scale 
 
 
 
 
 
X 
 
 
 
Otoscopy 
X 
 
Xb 
X 
X 
X 
X 
X 
Xd 
Urine Pregnancy Test (women of childbearing 
potential only) 
X 
 
Xb 
 
 
X 
 
 
Xd 
Randomization by EDC 
 
 
Xb 
 
 
 
 
 
 
Study Medication (FX-322 or placebo) 
 
 
X 
 
 
 
 
 
 
Adverse Events 
 
 
Xc 
X 
X 
X 
X 
X 
X 
  
a – Performed during the Screening Visit and again if a subject notes a change in their medical status during the lead in period 
b – Assessment performed prior to injection  
c – Assessments performed after injection 
d – Perform at Investigator Discretion 
e – If a subject discontinues prior to Visit 6 (Day 90), all efforts should be made to complete the Visit 6 (Day 90) In-Clinic Follow-up/Early Termination (ET) Visit as soon as possible and, 
whenever possible, prior to starting any new medication or treatment.  
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 28 of 64 
 
10.3 Scientific Rationale for Study Design 
The active ingredients in FX-322 target two cellular mechanisms, histone deacetylase 
(HDAC) and GSK-3 inhibition, which act synergistically to induce progenitor cell expansion 
and new hair cell formation in several ex vivo test systems. In vivo, significant improvements 
in hearing and increases in hair cell counts were observed in an adult mouse model of noise-
induced hearing loss after a single treatment. Most recently, it has been shown that in humans 
with stable SNHL, a single intratympanic dose of FX-322 was well tolerated and was 
associated with improvements in speech intelligibility as measured by WR and WIN, data 
that suggest FX-322 may improve hearing function to some degree in humans. 
Previous human studies in subjects 18 to 65 years inclusive (FX-322-103, FX-322-201, FX-
322-111) and subjects 66-85 inclusive (FX-322-112) demonstrated that a single dose FX-322 
was well tolerated in patients with acquired SNHL with no treatment-related serious adverse 
events. Adverse events in these studies were common to and associated with the 
intratympanic injection procedure with mild and transient discomfort in patients both with the 
drug and the placebo. 
 
In previous single-dose human studies in subjects 18-65 years inclusive (FX-322-201, FX-
322-111) it was demonstrated that treatment with FX-322 was associated with statistically 
significant and clinically meaningful increases in auditory measures 90 days after a single 
administration of FX-322, compared to either placebo or untreated ears. We intend to further 
establish efficacy with a single dose of FX-322 in subjects 18 to 65 years inclusive with 
acquired SNHL. 
 
10.4 End of Study Definition 
A participant is considered to have completed the study if he or she has completed all follow-
up visits, including Visit 6 (Day 90). 
10.5 Selection of Study Population 
10.5.1 Number of Planned Subjects  
Approximately 124 subjects are planned to be randomized 1:1 to FX-322 or Placebo. 
10.5.2 Inclusion Criteria 
To be eligible for study entry subjects must satisfy all the following criteria: 
1. Subject has read and voluntarily signed the Informed Consent Form (ICF) after all 
questions have been answered and prior to any study-mandated procedure. 
2. Adult aged 18-65 years inclusive at Screening. 
3. Documented medical history consistent with acquired, adult onset, sensorineural hearing 
loss associated with noise-induced SNHL (NIHL) or idiopathic sudden SNHL (SSNHL) 
(documented audiogram at least 6 months prior to screening required).  
4. A pure tone average at the Screening Visit of 35-85 dB at 500Hz, 1000Hz, 2000Hz, and 
4000Hz in the ear to be injected. 
5. Ability to communicate well with the Investigator and is willing to comply with and 
complete all the study procedures. 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 29 of 64 
 
6. Female subjects must be of non-childbearing potential or will need to utilize two methods 
of highly effective contraception during the study participation (e.g. hormonal 
contraception and condom or an intrauterine device and condom) or remain abstinent.  
Male subjects should use condoms with spermicide during the course of the study or 
remain abstinent.  Subjects should not donate sperm or ova during the study period. 
7. Have met additional masked criteria as determined by EDC. 
10.5.3 Exclusion Criteria 
Subjects will be excluded from the study if one or more of the following criteria are 
applicable: 
1. Subject has previously been randomized in a FX-322 clinical trial. 
2. Perforation of tympanic membrane or other tympanic membrane disorders that would 
interfere with the delivery and safety assessment of an intratympanic medication or 
reasonably be suspected to affect tympanic membrane healing after injection in study ear.  
This includes a current tympanostomy tube. 
3. Any conductive hearing loss of greater than 15 dB at a single frequency or greater than 
10dB at two or more contiguous octave frequencies in the study ear at the Screening visit. 
4. Active chronic middle ear disease or a history of major middle ear surgery, as an adult, in 
the ear to be injected. 
5. Subject has had an intratympanic injection in either ear within 3 months of the screening 
visit. 
6. Evidence of or previous diagnosis of auditory neuropathy, traumatic brain injury, 
“central” hearing loss, or genetic hearing loss.  
7. History of chronic, recurrent clinically significant vestibular symptoms. 
8. History of Bilateral Sudden Sensorineural Hearing Loss or Recurrent Sudden 
Sensorineural Hearing Loss. 
9. History of clinically significant systemic autoimmune disease (e.g. rheumatoid arthritis, 
Sjogren’s syndrome, multiple sclerosis, psoriasis). 
10. History of head or neck radiation, treatment, or exposure to platinum based chemotherapy 
drugs or aminoglycosides. 
11. Exposure to another investigational drug within 28 days prior to screening visit. 
12. Evidence of any active or chronic disease or condition that could interfere with, or for 
which the treatment of might interfere with, the conduct of the study, or that would pose 
an unacceptable risk to the subject in the opinion of the investigator following a detailed 
medical history, physical examination, and vital signs (systolic and diastolic blood 
pressure, pulse rate, body temperature). 
13. Positive urine pregnancy test or breast-feeding. 
14. Any known factor, condition, or disease that, in the view of the Investigator, might 
interfere with treatment compliance, study conduct or interpretation of the results. 
 
 
10.5.4 Withdrawal of Subjects From Study Assessments 
Subjects may withdraw from the study for any of the following reasons: 
• Adverse Event 
• Death 
• Lack of Efficacy  


 
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• Lost to Follow-up 
• Other 
• Physician Decision  
• Pregnancy 
• Progressive Disease 
• Protocol Violation 
• Recovery 
• Study Terminated by Sponsor 
• Technical Problem 
• Withdrawal by Subject 
 
Subjects are free to withdraw from the study at any time without providing reason(s) for 
withdrawal and without prejudice to further treatment. Should a subject withdraw early from 
the study the reason(s) for study withdrawal will be documented on the case report form 
(CRF).  All reasonable efforts will be made to have the subject return for one final 
assessment. Subjects withdrawing from the study will be encouraged to complete the same 
final evaluations as subjects completing the study according to this protocol, particularly 
safety evaluations. The aim is to record data in the same way as for subjects who completed 
the study. 
Pregnancy 
Subjects will be instructed that known or suspected pregnancy occurring during the study, in 
subjects or female partners of male subjects, should be confirmed and reported to the 
investigator.  All subjects will be followed until the end of the study, completing study 
assessments as appropriate during pregnancy. The Investigator should also be notified of 
pregnancy occurring during the study but confirmed after completion of the study. In the 
event that a subject is subsequently found to be pregnant after inclusion in the study, any 
pregnancy will be followed to term, and the status of mother and child will be reported to the 
sponsor after delivery. 
Full details will be recorded on the pregnancy page of the CRF at the conclusion of the 
pregnancy.  
Non-childbearing potential: 
Subjects that are deemed non-childbearing potential should have documented hysterectomy, 
bilateral oophorectomy, or bilateral tubal ligation, or be in menopause (no menstrual cycle for 
at least a year). 
10.5.5 Screen Failures 
Screen failures are defined as participants who consent to participate in the clinical trial but 
are not subsequently randomly assigned to the study intervention or entered in the study. A 
minimal set of screen failure information is required to ensure transparent reporting of screen 
failure participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) 
publishing requirements and to respond to queries from regulatory authorities. Minimal 
information includes informed consent, demography, and screen failure details. 
 
Rescreening may be permitted in the FX-322-208 study with sponsor approval. 


 
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10.6 Investigational Products 
10.6.1 Investigational Products Administered 
Approximately 124 subjects are planned to be randomized in this study.  The subjects will be 
randomized 1:1 to receive FX-322 or placebo. 
 
Table 1: FX-322 Group Dosing Schedule 
Treatment 
Group 
# of 
Subjects 
Day 1 
1 
62 
FX-322 
2 
62 
Placebo 
 
Topical anesthesia (with the exception of phenol) will be administered directly to the 
tympanic membrane.  After approximately 10 minutes, under a microscope, a 25-gauge 
needle will be used to inject of FX-322 into the middle ear at the junction of the posterior 
inferior and posterior superior quadrant with the needle tip directed towards the round 
window. The posterior superior quadrant should be avoided to prevent injury to the ossicles.  
 
After injection, the subject will continue to lie with the injected ear facing up for 20-30 
minutes. The injections will be performed at an appropriate location designated by a Board 
Certified Otolaryngologist trained and experienced in performing intratympanic injections.   
 
10.6.2 Identity of Investigational Products 
The investigational drug is FX-322 which is a fixed ratio dose combination of 2 small 
molecules: a glycogen synthase kinase (GSK) inhibitor (FX03) and valproate sodium (FX00), 
a histone deacetylase (HDAC) inhibitor. FX-322 is in the form of a poloxamer thermo-
sensitive gel matrix. 
10.7 Preparation and Dispensing 
The FX-322 investigational product is a 
 FX- 
322 is prepared by
 
  
 
An osmolarity matched placebo for FX-322 is a sterile liquid for intratympanic injection 
only. The placebo is prepared by 
 
 
 
The components in the study drug and placebo are shown in the following tables: 


 
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Table 3:  FX-322 Study Drug 
 
Active Ingredients 
Total in 
 
FX03 
 
Valproate Sodium 
 
Inactive Ingredients 
 
 
 
 
 
 
 
 
The matched placebo has been developed with similar pH, osmolality and gelation as FX-
322.  In a 
 injection, placebo contains 
 
 
   
 
Table 4: Dosages of Active Agents to be Injected in Humans  
 
 
FX-322 
Total 
(in a 
 injection) 
 
FX03 
0.628 mg 
 
FX00 
17.72 mg 
 
 
Frequency Therapeutics will supply sterile FX-322 and placebo, as a sealed sterile vial 
containing 
 and another sterile vial containing a 
. The placebo 
or FX-322 is prepared by mixing a 
 with a 
 
 
 
Each box will be labelled Study drug in compliance with applicable local regulations. FX-
322/Placebo and 
vials will be packaged in separate boxes.  
 
Detailed instructions on compounding the study drug will be provided in the pharmacy 
manual.  
 
All study drug will be transported, received, stored and handled strictly in accordance with 
the product label, the instructions provided to the investigational sites, the sites’ standard 
operation procedures and applicable regulations.   
 


 
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10.7.1 Packaging and Labeling 
For the injection, the study drug will be provided in a kit labelled for the FX-322-208 study.  
The 
will be taken from general stock at the site.  Topical anesthetic will be provided 
to the site.  More information can be found in the Pharmacy Manual.  The study drug will be 
prepared and drawn into a sterile 1 mL tuberculin syringe. The syringe will be labelled 
clearly with the details for each randomized patient.  The dose 
will be filled to the 
 mark.  Refer to the Pharmacy Manual for further instructions. 
 
The label(s) for the investigational product will include sponsor name and address, the 
protocol number, investigational product name, dosage form, amount of investigational 
product per container, batch number, unique kit number, storage conditions, and required 
caution statements and/or regulatory statements, as applicable. 
 
10.7.2 Supply, Storage and Handling 
Empty vials and containers should be retained until the conclusion of the study, after the site 
monitor has performed accountability and in compliance with the site’s SOP.  
10.7.3 Compliance and Drug Accountability 
Accountability for the study drug at the study site is the responsibility of the Investigator, or 
their designee. The Investigator will ensure that the study drug is used only in accordance 
with this protocol. Drug accountability records indicating the drug’s delivery date to the site, 
inventory at the site, use by each subject, and return (of unused vials) to Frequency 
Therapeutics (or destruction, if approved by Frequency Therapeutics) will be maintained by 
the clinical site. These records will adequately document that the subjects were provided the 
doses as specified in the protocol and should reconcile all study drug received from 
Frequency Therapeutics or its designee. Accountability records will include dates, quantities, 
batch/serial numbers, and subject identification numbers. The site monitor will review drug 
accountability at the site on an ongoing basis during monitoring visits. 
10.7.4 Disposal, Return or Retention of Study Drug 
All unused and used study drug should be retained at the site until inventoried by the site 
monitor, in compliance with the site’s SOPs. All used, unused or expired study drug will be 
returned to Frequency Therapeutics or if authorized, disposed of at the study site in 
accordance with governing regulations and documented.  
10.8 Randomization and Blinding 
10.8.1 Blinding 
The subjects, all Frequency Therapeutics staff and representatives, Investigators and site 
personnel involved in administering study assessments will be blinded to the study drug 
assignment. The otolaryngologist will be blinded to treatment. The pharmacy staff who 
prepare the study drug will be unblinded and will not perform any other roles on the study 
other than drug preparation and accountability.  The independent statistician and/or 
independent statistical programmer who is not otherwise involved with the study will 
generate the randomization schedule and will be un-blinded. 


 
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In addition to being blinded to the study drug assignment, Investigators and site personnel 
will be masked to certain study design elements, selection criteria, procedures, and statistical 
methods. The pharmacy staff will also be masked to certain study design elements, selection 
criteria, procedures, and statistical methods. These criteria will be specified in an unmasked 
addendum to this protocol. The EDC system will be programmed to determine eligibility of 
subjects and randomize eligible subjects as per these design elements.  
10.8.2 Un-blinding 
Only in the case of emergency, when knowledge of the study drug administered is essential 
for the clinical management or welfare of the subject, may the Investigator un-blind a 
subject’s treatment group assignment. Under such conditions, the identity of the study drug 
will be obtained either via the unblinding functionality within the RTSM system or by 
contacting the CRO. 
 
If possible, the Medical Monitor and Frequency Therapeutics should be consulted prior to 
breaking the blind. If the blind is broken for any reason, the Investigator must notify 
Frequency Therapeutics and the Medical Monitor immediately of the un-blinding incident 
without revealing the subject’s study treatment group assignment to Frequency Therapeutics 
and the Medical Monitor. In the event that the treatment group assignment is broken, the date, 
the signature of the person who broke the code and the reason for breaking the code must be 
recorded on the source documents.  Any code-breaks that occur must be immediately 
reported to the Medical Monitor.  Any subject whose treatment group assignment has been 
un-blinded will be followed up for safety purposes.  
 
Upon completion of the study and unblinding, subjects who received placebo and completed 
study visits through Day 90, may have the opportunity to receive FX-322 as part of an open-
label extension. Further details will be provided in a separate protocol.  
 
10.8.3 Method of Assigning Subjects to Treatment Groups 
Subjects will be randomized 1:1 to one of two groups (FX-322 or placebo).  A single 
injection of FX-322 or placebo will be administered on Day 1. 
 
10.8.4 Selection and Timing of Dose for Each Subject 
For subjects with bilateral hearing loss, if both ears meet the inclusion and exclusion criteria 
as determined by the Investigator, EDC will select the study ear at Visit 3 (Day 1). Subjects 
will be administered a total of 1 dose of FX-322 or placebo.  Study injection will take place 
on Day 1. 
  
10.9 Prior and Concomitant Therapy 
At each study visit the study personnel will question the subject about any medication taken, 
including vitamin supplements and herbal remedies. Any concurrent medications will be 
recorded in the subject’s records and the CRF. Any changes in doses or introduction of a new 
medication during the course of the study will be recorded. Any medications taken in the 14 
days prior to the Screening visit will be recorded on the Concomitant Medication log. 


 
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10.9.1 Prohibited Medication/Therapy 
Oral and intratympanic steroids are prohibited through the follow up period [through Visit 6 
(Day 90)]. The subject’s best medical care should guide the investigator in the management 
of conditions that develop during the observational period after 90 days. Aminoglycosides 
and platinum-based chemotherapies are excluded during the study. 
 
10.9.2 Rescue Medication 
No rescue medication will be provided as no FDA approved treatment for sensorineural 
hearing loss exists. The Investigator should treat any adverse events as medically appropriate 
and per their standard of care.   
10.9.3 Treatment Compliance 
Treatment will be administered by the Investigator to each subject on Day 1 only, therefore 
compliance will not be recorded if the subject has their study treatment. 
 
11 DISCONTINUATION AND LOST TO FOLLOW-UP 
11.1 Discontinuation  
If a clinically significant finding is identified (including, but not limited to changes from 
baseline) after randomization, the investigator or qualified designee will determine if any 
change in participant management is needed. Any new clinically relevant finding will be 
reported as an adverse event (AE). 
 
The data to be collected at the time of study discontinuation will include the following: 
• All efforts will be made for the subject to complete all of the follow up and 
observational visits. 
• If the subject does not wish to continue the follow up period, all efforts will be made 
for the subject to complete all designated assessments collected at the Visit 6 (Day 
90)/Follow-up ET Visit. 
 
11.2 Lost to Follow-up 
A subject will be considered lost to follow-up if he or she fails to return for 2 or more 
scheduled visits (or the final visit in the follow-up) and is unable to be contacted by the study 
site staff.  
 
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit: 
• The site will attempt to contact the participant and reschedule the missed visit within 
5 days and counsel the participant on the importance of maintaining the assigned visit 
schedule and ascertain if the participant wishes to and/or should continue in the study. 
• Before a participant is deemed lost to follow-up, the investigator or designee will 
make every effort to regain contact with the participant (3 telephone calls and, if 
necessary, a certified letter to the participant’s last known mailing address or local 
equivalent methods). These contact attempts should be documented in the 
participant’s medical record or study file.  
 


 
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Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
12 TIMING OF STUDY PROCEDURES 
Subjects will provide written informed consent before any study related procedures are 
performed. 
12.1 Screening/Start of Lead-In Visit (Visit 1; [Day -40 to -30]) 
The following procedures will be performed at the Screening Visit: 
 
• Obtain signed Informed Consent 
• Assess for eligibility (against the Inclusion and Exclusion criteria) 
• Collect full medical history, including targeted hearing loss history, concomitant 
illnesses/diseases, and concomitant medications 
• Record demographic data, including ethnic origin, date of birth, and sex 
• Perform a physical examination, including body weight and height 
• Record vital signs (blood pressure, body temperature, and heart rate) 
• Collect urine sample for pregnancy test, if applicable 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
• Enter audiologic assessment results into EDC and confirm eligibility 
 
After the Screening Period (signing the Informed Consent Form and completing all Screening 
Visit assessments), the subject will enter a Lead-In period of at least 30 days (period can be 
extended to 39 days but cannot exceed).   
 
12.2 Lead-In Visit (Visit 2; [14-18 days after Visit 1]) 
The following procedures will be performed at the Lead-in Visit: 
 
• Collect concomitant medications 
• Reassess for eligibility against the Inclusion and Exclusion criteria 
• Record any changes in medical history that have occurred since the previous visit 
• Record vital signs (blood pressure, body temperature, and heart rate) 
• Perform standard pure tone audiometry, speech reception threshold, extended high 
frequency audiometry, word recognition testing (Maryland CNC and W-22), and WIN 
testing (NU-6) 
• Enter audiologic assessment results into EDC and confirm eligibility 
 
 
12.3 End of Lead-In/Treatment (Visit 3, 30-40 days after Visit 1 [Day 1]) 
The following procedures will be performed at End of Lead-In/Treatment/Visit 3 (Day 1): 
• Before injection of study drug:  
o Reassess for eligibility against the Inclusion and Exclusion criteria 


 
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o Record any changes in medical history that have occurred since the previous 
visit 
o Collect concomitant medications and document topical anesthetic used on 
study ear 
o Perform a urine pregnancy test, if applicable 
o Perform standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-
22), WIN testing (NU-6), and tympanometry 
o Perform otoscopy 
o Enter audiologic assessment results into EDC and confirm eligibility  
o Record vital signs 
o Perform Tinnitus Functional Index and Columbia Suicide and Severity Scale 
o Perform 
 
o Perform PGI-S Hearing Loss Scale 
o Perform PGI-S Daily Impacts Scale 
o When all the above procedures have been performed and the Investigator and 
EDC has confirmed the subject’s eligibility for the study, the subject will be 
randomized. Each subject will receive a unique randomization number 
• Inject FX-322 or placebo via intratympanic injection to the study ear 
• After study drug injection: 
o Record any adverse events that have occurred since study treatment 
o Perform 
 Qualitative Questionnaire 
 
12.4 Follow-up (Visit 4, [Day 30 +/- 5 days]) 
The Follow-up Visit (Visit 4) will take place at Day 30 (± 5 days). The following procedures 
will be performed at the Follow-up Visit: 
• Record any AEs that have occurred since the last visit and any changes in concomitant 
medication 
• Perform Columbia Suicide and Severity Scale 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
• Record vital signs 
 
12.5 Follow-up (Visit 5, [Day 60 +/- 5 days]) 
The Follow-up Visit (Visit 5) will take place at Day 60 (± 5 days). The following procedures 
will be performed at the Follow-up Visit: 
• Record any AEs that have occurred since the last visit and any changes in concomitant 
medication 
• Perform Columbia Suicide and Severity Scale 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
• Record vital signs 
 


 
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12.6 Follow-up (Visit 6, [Day 90 +/- 5 days]) 
The Follow-up Visit (Visit 6) will take place at Day 90 (± 5 days). The following procedures 
will be performed at the Follow-up Visit: 
• Record any AEs that have occurred since the last visit and any changes in concomitant 
medication 
• Perform Tinnitus Functional Index and Columbia Suicide and Severity Scale 
• Perform 
 
• Perform PGI-C Hearing Loss Scale 
• Perform PGI-C Daily Impacts Scale 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
• Record vital signs 
• Collect urine sample for pregnancy test, if applicable 
 
12.7 Observation (Visit 7, [Day 180 +/- 7 days]) 
The Observational Visit (Visit 7) will take place at Day 180 (± 7 days). The following 
procedures will be performed at the Follow-up Visit: 
• Record any AEs that have occurred since the last visit and any changes in concomitant 
medication 
• Perform Tinnitus Functional Index and Columbia Suicide and Severity Scale 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
 
12.8 Observation (Visit 8, [Day 270 +/- 7 days]) 
The Observational Visit (Visit 8) will take place at Day 270 (± 7 days). The following 
procedures will be performed at the Follow-up Visit: 
• Record any AEs that have occurred since the last visit and any changes in concomitant 
medication 
• Perform Tinnitus Functional Index and Columbia Suicide and Severity Scale 
• Perform otoscopy, standard pure tone audiometry, speech reception threshold, extended 
high frequency audiometry, word recognition testing (Maryland CNC and W-22), 
WIN testing (NU-6), and tympanometry 
 


 
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12.9 Duration of Treatment 
The duration of treatment will be 1 day at Visit 3 (Day 1) with up to 9 months of follow-up 
and observation after the study treatment.  
 
13 STUDY ASSESSMENTS 
13.1 Efficacy Measurements Assessed 
13.1.1 Word Recognition in Quiet (WR) 
Word recognition in quiet will be measured with recorded Maryland CNC and W-22 word 
lists. The Maryland CNC Word lists were devised by Lehiste and Peterson (1959) and revised 
to give more uniform distribution of word familiarity by eliminating rare words and proper 
nouns (Peterson & Lehiste, 1962). Normative studies were performed by Causey et al., 
(1984). The W-22 word list was devised by Hirsh et al. (1952). W-22 words are comprised of 
speech sounds with the same relative frequency as English speech. The tests will consist of 
50 words presented to the subject and the percentage of words correctly identified will be 
recorded.  Word recognition will be performed at Visit 1, Visit 2, Visit 3, Visit 4, Visit 5, 
Visit 6, Visit 7, and Visit 8.  For Visit 1, testing will be performed at 30dB SL regarding the 
pure tone average of 0.5, 1, and 2 kHz at Screening/Visit 1. For Visit 2 and all subsequent 
visits (Visits 3, 4, 5, 6, 7, and 8) testing will be performed at 30dB SL regarding the pure tone 
average of 0.5, 1, and 2 kHz at Visit 2. If 30dB SL is at an uncomfortable listening level for 
the participant, testing will be performed at the participant’s most comfortable listening level. 
Word lists will be rotated at each visit. Audio recordings of subject responses during 
audiologic testing will be reviewed by a surveillance team for quality assurance. Additional 
details can be found in the Audiology Manual of Procedures.   
13.1.2 Words-In-Noise Testing (WIN) 
The Words-in-Noise Test (WIN), using the NU-6 list, was developed as an instrument to 
quantify the ability of listeners to understand monosyllabic words in background noise using 
multitalker babble (Wilson, 2003). Materials are recorded at 7 signal-to-noise ratios (0, 4, 8, 
12, 16, 20, 24 dB) that are presented in a descending manner. This test is conducted by 
presenting 2 lists of 35 recorded words to each ear. Overall accuracy will be recorded as the 
number of words identified correctly. This test will occur at Visit 1, Visit 2, Visit 3, Visit 4, 
Visit 5, Visit 6, Visit 7, and Visit 8. Testing will be performed at 80dB SPL. Word lists will 
be rotated at each visit. Audio recordings of subject responses during audiologic testing will 
be reviewed by a surveillance team for quality assurance. Additional details can be found in 
the Audiology Manual of Procedures. 
 
13.1.3 Standard Pure Tone Audiometry 
Standard Pure Tone Audiometry will be measured to determine a subject’s threshold for 
hearing at various frequencies and will be performed at Visit 1, Visit 2, Visit 3, Visit 4, Visit 
5, Visit 6, Visit 7, and Visit 8. This will be performed on a calibrated audiometer by a 
licensed audiologist.  The following frequencies will be obtained:  Air: 250 Hz, 500 Hz, 1000 
Hz, 2000 Hz, 3000 Hz, 4000 Hz, 6000 Hz, and 8000 Hz; Bone: 500 Hz, 1000 Hz, 2000 Hz, 
3000 Hz, and 4000 Hz. Audio recordings of subject responses during audiologic testing will 
be reviewed by a surveillance team for quality assurance. Additional details can be found in 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 40 of 64 
 
the Audiology Manual of Procedures. 
 
13.1.4 Extended High Frequency Audiometry 
Extended high frequency audiometry will be performed to determine a subject’s threshold for 
hearing at frequencies beyond those in standard pure tone audiometry and will be performed 
at Visit 1, Visit 2, Visit 3, Visit 4, Visit 5, Visit 6, Visit 7, and Visit 8. This will be performed 
on a calibrated audiometer by a licensed audiologist. The following frequencies will be 
obtained: Air: 9000 Hz, 10000 Hz, 11200 Hz, 12400 Hz, 14000 Hz, and 16000 Hz. Because 
some audiometers use center frequencies that vary by small amounts, sites should use the 
threshold at the closest listed center frequency (e.g., 11000 Hz may be used for 11200 Hz; 
12500 Hz may be used for 12400 Hz). Audio recordings of subject responses during 
audiologic testing will be reviewed by a surveillance team for quality assurance. Additional 
details can be found in the Audiology Manual of Procedures. 
 
13.1.5 Tinnitus Functional Index (TFI) 
The Tinnitus Functional Index (Meikle et al 2012) has eight subscales that address the 
intrusiveness of tinnitus, the sense of control the patient has, cognitive interference, sleep 
disturbance, auditory issues, relaxation issues, quality of life, and emotional distress.  The 
subject will report answers to each of the 25 questions using a scale of 0-10.  The TFI will be 
given to the subject to complete at Visit 3, Visit 6, Visit 7, and Visit 8. 
13.1.6 
Qualitative Questionnaire 
This questionnaire records subject experience 
 
 
13.1.7 Research Assessment on 
 Quality of 
Life 
 
This questionnaire is a patient reported outcome measure. 
 
 
 
 
13.1.8 Patient Global Impression of Change Hearing Loss Scale 
This questionnaire is a patient reported outcome measure. The subject will select their 
response to best describe the overall change in their hearing loss since they were administered 
the study medication. The scale will be given to all subjects to complete at Visit 6.  
13.1.9 Patient Global Impression of Change Daily Impacts Scale 
This questionnaire is a patient reported outcome measure. The subject will select their 
response to best describe the overall change in the impact of their hearing loss on daily 
activities since they were administered the study medication. The scale will be given to all 
subjects to complete at Visit 6.  


 
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13.2 Safety Measurements Assessed 
13.2.1 Tympanometry 
Tympanometry (an objective test of tympanic membrane mobility) tests the integrity of the 
tympanic membrane by varying air pressure in the ear canal. Middle ear compliance, peak 
pressure, and tympanogram type will be recorded.  Tympanometry will be performed Visit 1, 
Visit 3, Visit 4, Visit 5, Visit 6, Visit 7, and Visit 8.  The assessment will be performed by a 
licensed audiologist on a calibrated tympanometer.  A print-out containing the tympanometry 
from the machine will be collected and stored in the source document.   
13.2.2 Concomitant Medication 
Subjects will be asked about concomitant medications at time points outlined in the Schedule 
of Assessments.  All concomitant medication information will be recorded on the CRF. 
13.2.3  Vital Signs 
Vital signs (temperature, blood pressure and heart rate) will be recorded at time points 
outlined in the Schedule of Assessments and will be performed in a standardized manner, i.e., 
after the subject has rested for at least 5 minutes. 
 
13.2.4  Physical Exam (PE) 
A complete physical examination will be performed by a licensed provider at Visit 1. 
Complete physical examinations include: general appearance, head, ears, eyes, nose, throat, 
dentition, thyroid, chest (heart, lungs), abdomen, skin, neurological, extremities, back, neck, 
musculoskeletal, and lymph nodes and any pertinent system based on any prior findings. 
Physical examinations may be performed at various unscheduled time points, if deemed 
necessary by the Investigator. 
13.2.5  Otoscopy 
Microscopic otoscopy will be included to specifically record any abnormalities of the 
external ear canal, tympanic membrane and middle ear and will be performed at Visit 1, Visit 
3, Visit 4, Visit 5, Visit 6, Visit 7, and Visit 8. 
13.2.6   Columbia Suicide Severity Rating Scale (C-SSRS) 
This scale is used to determine if any suicide ideation or intention is present (Posner et al 
2011).  The questionnaire is read aloud to the subject and responses are recorded by the 
trained staff member. If any ideation or intention is present, the Investigator will interview 
the subject and have them follow up with their primary healthcare provider.  The C-SSRS 
will be completed at Visit 3 (Baseline/Screening version), Visit 4, Visit 5, Visit 6, Visit 7, 
and Visit 8 (Since Last Visit version). 
13.3 Other Study Assessments 
13.3.1 Speech Reception Threshold (SRT) 
Speech reception threshold is being used as a cross-check measure for the results of pure-tone 
audiometry. The pure tone average (PTA) shall be compared to the SRT to alert the tester to 
possible concerns about the accuracy of the pure tone results.  


 
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13.3.2 Patient Global Impression of Severity (PGI-S) Hearing Loss 
This questionnaire is a patient reported outcome measure. The subject will select their 
response to best describe the severity of their hearing loss over the past week. The scale will 
be given to all subjects to complete at Visit 3. 
13.3.3 Patient Global Impression of Severity (PGI-S) Daily Impacts 
This questionnaire is a patient reported outcome measure. The subject will select their 
response to best describe the impact of their hearing loss on their daily activities over the past 
week. The scale will be given to all subjects to complete at Visit 3. 
14 ADVERSE EVENTS 
Adverse Event Definition 
An AE is defined as any untoward medical occurrence in a clinical study subject 
administered a medicinal product which does not necessarily have a causal relationship with 
this 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 a 
medicinal (investigational) product, whether or not it is related to the medicinal 
(investigational) product. This includes an exacerbation of pre-existing conditions or events, 
intercurrent illnesses, drug interaction or the significant worsening of the indication under 
investigation that is not recorded elsewhere on the CRF under specific assessments. 
Anticipated fluctuations of pre-existing conditions, including the disease under study that do 
not represent a clinically significant exacerbation or worsening need not be considered AEs. 
It is the responsibility of the investigator to document all AEs that occur during the study. 
AEs will be elicited by asking the subject a nonleading question, for example, “Have you 
experienced any new or changed symptoms since we last asked/since your last visit?”.  AEs 
will be recorded from the time of study treatment (Visit 3) through the end of the 
observational period, or final study visit.  AEs should be reported on the appropriate page of 
the CRF. 
 Unexpected Adverse Event or Unexpected Suspected Adverse Reaction Definition 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed 
in the investigator brochure or is not listed at the specificity or severity that has been 
observed; or, if an investigator brochure is not required or available, is not consistent with the 
risk information described in the general investigational plan or elsewhere in the current 
application, as amended. 
“Unexpected,” as used in this definition, also refers to adverse events or suspected adverse 
reactions that are mentioned in the investigator brochure as occurring with a class of drugs or 
as anticipated from the pharmacological properties of the drug, but are not specifically 
mentioned as occurring with the particular drug under investigation. 
Assessment of Severity 
Each AE will be assigned a category by the investigator as follows: 


 
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Mild: 
An AE that is easily tolerated by the subject, causes minimal discomfort 
and does not interfere with everyday activities. 
Moderate: 
An AE that is sufficiently discomforting to interfere with normal 
everyday activities; intervention may be needed. 
Severe: 
An AE that prevents normal everyday activities; treatment or other 
intervention usually needed. 
If there is a worsening in severity of an AE, it must be recorded as a separate event. 
Assessment of Causality 
Every effort will be made by the investigator to assess the relationship of the AE, if any, to 
the study drug. Causality should be assessed using the categories presented in the following 
table: 
Not Related: 
Clinical event with an incompatible time relationship to study 
drug administration, and that could be explained by underlying 
disease or other drugs or chemicals or is incontrovertibly not 
related to the study drug. 
Unlikely: 
Clinical event whose time relationship to study drug 
administration makes a causal connection improbable, but that 
could plausibly be explained by underlying disease or other drugs 
or chemicals. 
Possible: 
Clinical event with a reasonable time relationship to study drug 
administration, but that could also be explained by concurrent 
disease or other drugs or chemicals. 
Related: 
Clinical event with a reasonable time relationship to study drug 
administration and is unlikely to be attributed to concurrent 
disease or other drugs or chemicals. 
Action Taken Regarding Study Drug 
Not Applicable: 
Adverse event began after study drug injection was complete.  
Dose Not Changed: 
An adverse event was experienced at the time of study drug 
injection, but no action was taken with the study drug in relation 
to the specific adverse event. 
Drug Withdrawn: 
Study drug injection was stopped permanently in relation to the 
specific adverse event. 


 
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Adverse Events of Special Interest (Audiometric and Otoscopic) 
If any of the following criteria are met through Visit 6 (Day 90), the event will be recorded as 
an adverse event of special interest (AESI) per ASHA guidelines (ASHA 1994): 
• Asymmetric loss of hearing greater than or equal to 20 dB at any one frequency in the 
treated ear compared to Visit 3. 
• Asymmetric loss of hearing greater than or equal to 10 dB at two adjacent frequencies 
in the treated ear compared to Visit 3. 
• Asymmetric loss of response at three consecutive test frequencies where responses 
were previously obtained in the treated ear compared to Visit 3. 
For word recognition testing, a follow up visit score through Visit 6 (Day 90) in the treated 
ear that falls below the lower limit of the 95% confidence interval of the Visit 3 word 
recognition score will be considered an AESI (Carney and Schlauch, 2007). 
Additionally, if the subject experiences a perforation greater than 25% of the tympanic 
membrane in the treated ear through Visit 6 (Day 90) this will be recorded as an AESI.  
If any subjects in the study experience the same AESI through Visit 6 (Day 90), the Medical 
Monitor will discuss these with the Principal Investigators and each subject’s condition will 
be discussed.   
 Follow-up of Adverse Events 
All AEs will be followed until the end of study participation and SAEs until the event is 
resolved or until, in the opinion of the investigator, the event is stabilized or determined to be 
chronic. Details of AE resolution must be documented in the CRF. 
Documentation and Reporting of Adverse Events 
AEs should be reported and documented in accordance with the procedures outlined below. 
All AEs occurring during the study (after study treatment has begun) must be documented on 
the relevant CRF pages. The following data should be documented for each AE: 
• Description of the symptom event, or underlying diagnosis 
• Classification of ‘serious’ or ‘not serious’ 
• Severity (see definitions above) 
• Date of first occurrence and date of resolution (if applicable) 
• Action taken (see definitions above) 
• Causal relationship (see definitions above) 
• Outcome of event (unknown, recovered, not yet recovered, recovering/resolving, 
recovered with sequelae, deaths [with date and cause reported]) 
14.1 Serious Adverse Events 
Serious Adverse Event Definition 
An SAE is any untoward medical occurrence or effect that, at any dose, 


 
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• Results in death. 
• Is life-threatening (an AE is life-threatening if the subject was at immediate risk of 
death from the event as it occurred, i.e., it does not include a reaction that might have 
caused death if it had occurred in a more serious form). 
• Requires or prolongs inpatient hospitalization. (Complications occurring during 
hospitalization are AEs and are SAEs if they cause prolongation of the current 
hospitalization. Hospitalization for elective treatment of a pre-existing 
non--worsening condition is not, however, considered an AE. The details of such 
hospitalizations must be recorded on the medical history or physical examination page 
of the CRF). 
• Results in persistent or significant disability/incapacity. (An AE is incapacitating or 
disabling if it results in a substantial and/or permanent disruption of the subject’s 
ability to carry out normal life functions). 
• Results in a congenital anomaly/birth defect. 
• Important Medical Event 
In addition, medical and scientific judgement is required to decide if prompt notification is 
required in situations other than those defined for SAEs above. This may include any event 
that the Investigator regards as serious that did not strictly meet the criteria above but may 
have jeopardized the subject or required intervention to prevent one of the outcomes listed 
above, or that would suggest any significant hazard, contraindication, side effect, or 
precaution that may be associated with the use of the investigational product. 
Reporting of Serious Adverse Events 
Any SAE must be reported by the Investigator if it occurs during the clinical study, whether 
or not the SAE is considered to be related to the investigational product. The Serious Adverse 
Event CRF must be completed, with as much information as is available, within 24 hours of 
when the investigative site becomes aware of the event. Once the CRF is completed, an EDC 
email notification is sent to 
    Additional details regarding the SAE 
reporting process can be found in the Investigator Site File. 
The Investigator should not wait to receive additional information to document fully the 
event before notification of a SAE, though additional information may be requested. 
Information from relevant laboratory results, hospital case records, and autopsy reports 
should be obtained. 
Instances of death, congenital abnormality, or an event that is of such clinical concern as to 
influence the overall assessment of safety, if brought to the attention of the Investigator at any 
time after cessation of study drug administration and linked by the investigator to this study, 
should be reported to the study monitor. 
The sponsor will promptly notify all relevant Investigators and the regulatory authorities of 
findings that could adversely affect the safety of subjects, impact on the conduct of the study 
or alter the independent ethics committee (IEC)/institutional review board (IRB) 
approval/favorable opinion of the study. In addition the sponsor will expedite the reporting to 
all concerned Investigators, to the IRBs, where required, and to the regulatory authorities of 
all adverse reactions that are both serious and unexpected. 
Details of the procedures to be followed if a pregnancy occurs are provided in Section 10.5.4. 


 
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14.2 Expedited Safety Reporting  
All serious and unexpected suspected adverse reactions (SUSARs) will be the subject of 
expedited reporting. The sponsor shall ensure that all relevant information about a SUSAR 
that is fatal or life-threatening is reported to the regulatory authorities and IRB within 7 days 
after knowledge by the sponsor of such a case and that relevant follow up information is 
communicated within an additional 8 days. All other SUSARs will be reported to the 
regulatory authorities and IRB within 15 days after knowledge by the sponsor of such a case. 
All investigators should follow up SUSARs until the event is resolved or until, in the opinion 
of the investigator, the event is stabilized or determined to be chronic. Post study SUSARs 
that occur after the subject has completed the clinical study must be reported by the 
investigator to the sponsor. 
15 STATISTICAL METHODS 
All analyses, data listings, and reports will be detailed in the Statistical Analysis Plan (SAP).  
This will be finalized prior to unblinding of the study.  Additional reports and/or analyses will 
be considered ad-hoc or post-hoc in nature and detailed in the CSR. 
In general, descriptive statistics are defined for continuous variables as the number of 
subjects (n), mean, standard deviation, minimum, median, and maximum; and as count and 
percentage for categorical variables. 
 
 
15.1 Sample Size 
The sample size of 112 evaluable subjects, in a 1:1 allocation ratio of FX-322 and Placebo, 
was selected to provide an overall statistical power of at least 80% to detect a difference in 
the primary endpoint for word recognition. The target enrolment of 124 subjects is an 
adjustment for an assumed 10% study dropout rate. 
 
15.2 Analysis Sets 
• Full Analysis Set (FAS) 
The Full Analysis Set is defined as all randomized subjects who receive one dose of study 
drug (regardless of whether a full dose was administered) in the qualified ear.  Subjects will 
be analysed according to their randomized treatment group.  The FAS will be the primary 
analysis set for all efficacy endpoints.   
 
• Per Protocol Analysis Set (PPAS) 
The Per Protocol Analysis Set is defined as all randomized subjects receiving one full dose of 
study drug in the qualified ear, treated per the randomization treatment schedule, and without 
major protocol deviations that could interfere with interpretation of the results.  If different 
from the FAS, the PPAS may be used in additional sensitivity analyses.  Details will be 
provided in the SAP. 
 
• Safety Analysis Set (SfAS) 


 
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The Safety Analysis Set (SfAS) will include all subjects who receive one dose of study drug 
(regardless of whether a full dose was administered) in any ear and will be analyzed 
according to the actual treatment group regardless of their randomized treatment assignment.  
For safety assessments completed by ear for each subject, actual treated and actual untreated 
ears will be summarized separately.  The SfAS will be used for the analysis of safety.             
 
15.3 Subject Disposition, Demographics and Baseline Disease Status 
Descriptive statistics for subject disposition, demographics, and baseline disease status will 
be provided.  Tabulations will be summarized by randomized group. 
15.4 Interim Analyses 
No unblinded interim analysis is planned.  
The blinded data will be continuously monitored for safety.  Blinded efficacy data monitoring 
will be conducted on an on-going basis in order to understand patterns of missingness in real-
time and to monitor data integrity. 
The sponsor reserves the option to conduct an interim analysis for the potential purposes of 
sample re-estimation and/or futility. Should the sponsor choose to utilize this option, the 
protocol and SAP would each be prospectively amended. If an interim analysis is to be 
conducted, the sponsor intends to keep it blinded to an independent Data Monitoring 
Committee, as well as to Frequency. If an interim analyses is conducted, there would be 
prespecified criteria around sample size re-estimation based on pooled variance estimates, as 
well as futility analysis.  
15.5 Efficacy Analyses 
Primary efficacy analysis will be conducted after all subjects have either completed follow-up 
visits or early discontinued from study. Primary analysis will be performed in an unblinded 
fashion for analysis purpose while subjects not completing observation visits will continue to 
be followed as being blinded to treatment assignment.  
Efficacy endpoints will be summarized descriptively at each study timepoint.  Calculations 
will include the observed data at each timepoint, and if relevant, the change from baseline 
(baseline defined as the average across the pre-treatment timepoints).   
For continuous endpoints means, medians, standard deviations, minimums and maximum 
will be provided.  For categorical endpoints the number and percent of subjects within each 
category will be calculated.  For descriptive analyses, subjects with missing data will be 
excluded for a particular calculation. 
Inferential statistical methods examining continuous endpoints will be examined via a Mixed 
Model for Repeated Measures (MMRM).  Inferential analyses for categorical endpoints will 
be analysed using univariate methods and/or Generalized Estimating Equations (GEEs) (or 
appropriate longitudinal methods as detailed in the SAP).  When appropriate, models will 
include adjustments for baseline as well as the stratification factors used at randomization and 
clinically relevant confounders as identified in the SAP.  Departures of these models from the 
statistical assumptions will be assessed and alternative approaches may be employed.   


 
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Efficacy endpoints will be analysed using the FAS, and complete details of these analyses 
will be included in the SAP.  
15.6 Safety Analyses 
Safety will be evaluated using the safety analysis set and will include the incidence of 
adverse events (AEs) and serious adverse events (SAEs).  Additionally, descriptive statistics 
will be provided by actual treatment group for vital signs, pregnancy tests,  concomitant 
medications, otology, questionnaire responses, and tympanometry and otoscopic assessments.   
15.6.1  Adverse Events 
The number and percentage of subjects reporting adverse events (AEs) will be summarized 
via the MedDRA system by organ class and preferred term.  Data will be tabulated by 
severity, physician assessment of relationship to study drug, serious AEs, and AEs leading to 
death or study withdrawal.  Adverse events of the ear and AESIs will include summaries by 
treated vs. non-treated ear. 
 
15.6.2  Prior and Concomitant Medications 
Prior and concomitant medications will be coded using the World Health Organization’s 
(WHO) Drug Dictionary.  The incidence of subjects using concomitant medications will be 
tabulated by Anatomical Therapeutic Chemical (ATC), preferred term, and dosing group.  
Prior medications will exclusively be displayed in line listings. 
 
Concomitant medications are defined as all medications that started on or after the 
administration of study drug, or were ongoing at the time of study drug administration.  Prior 
medications are all recorded medications that started and stopped prior to administration of 
study drug.  In the event that a subject begins a previously ended medication (prior 
medication) following dose of study drug, the post-dose use will be considered concomitant 
while the prior use will still be reported.   
 
15.7 Handling of Missing Data and Subject Withdrawals 
With the exception of partial dates to assess AEs and concomitant medications, safety data 
will not be imputed.  In general, safety endpoints will not be imputed and missing FAS 
analysis data will be excluded (outside of model-specific, required assumptions).  Details and 
any exceptions will be provided in the SAP. 
 
 
16 QUALITY ASSURANCE AND QUALITY CONTROL 
16.1 Audit and Inspection 
Study center(s) and study documentation may be subject to Quality Assurance audit during 
the course of the study by the sponsor or its nominated representative. In addition, inspections 
may be conducted by regulatory authorities at their discretion. 


 
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16.2 Monitoring 
Data for each subject will be recorded on a CRF. Data collection must be completed for each 
subject who signs an informed consent form (ICF) and is administered study drug. 
In accordance with GCP and ICH guidelines, the study monitor will carry out source 
document verification and request clarification at regular intervals to ensure that the data 
collected in the CRF are accurate, complete and reliable. Study monitor has the responsibility 
of assessing the progress of the study, of checking that the informed consent forms have been 
signed by the patient, ensuring adherence to and compliance with the study protocol and 
other study-related documents 
The investigator must permit the monitor, the IEC/IRB, the sponsor’s internal auditors, and 
representatives from regulatory authorities direct access to all study-related documents and 
pertinent hospital or medical records for confirmation of data contained within the CRFs.   
16.3 Data Management and Coding 
The sponsor will be responsible for activities associated with the data management of this 
study. This will include setting up a relevant database and data transfer mechanisms, along 
with appropriate validation of data and resolution of queries. Data generated within this 
clinical study will be handled according to the relevant standard operating procedures (SOPs) 
of the data management and biostatistics departments of the sponsor/CRO. 
Study centers will enter data directly into the CRF.  All data must be verifiable against source 
documents at the study center. Any changes to the data entered into the CRF will be recorded 
in the audit trail and will be FDA CFR 21 Part 11 compliant. 
Medical coding will use Medical Dictionary for Regulatory Activities (MedDRA) for 
concomitant diseases and AEs and WHO Drug for medications. 
Missing or inconsistent data will be queried for clarification. Subsequent modifications to the 
CRFs will be documented. 
17 RECORDS AND SUPPLIES 
17.1 Drug Accountability 
On receipt of the study drug, the Investigator (or designee) will conduct an inventory of the 
supplies and verify that study drug supplies are received intact and in the correct amounts 
before completing a supplies receipt. The Investigator will retain the original of this receipt at 
the study center and return a copy to the study monitor. The monitor may check the study 
supplies at each study center at any time during the study. 
It is the responsibility of the study monitor to ensure that the Investigator (or designee) has 
correctly documented the amount of the study drug received, dispensed, and returned on the 
dispensing log that will be provided. A full drug accountability log will be maintained at the 
study center at all times. The study monitor will arrange collection of unused study drug. The 
study monitor will also perform an inventory of study drug at the close-out visit to the study 
center. All discrepancies must be accounted for and documented. 


 
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17.2 Financing and Insurance 
Financing and insurance of this study will be handled by the Sponsor. 
18 ETHICS 
18.1 Independent Ethics Committee or Institutional Review Board 
Before initiation of the study at each study center, the protocol, the ICF, other written 
material given to the subjects, and any other relevant study documentation will be submitted 
to the appropriate IEC/IRB. Written approval of the study and all relevant study information 
must be obtained before the study center can be initiated or the study drug is released to the 
Investigator. Any necessary extensions or renewals of IEC/IRB approval must be obtained 
for changes to the study such as amendments to the protocol, the ICF or other study 
documentation. The written approval of the IEC/IRB together with the approved ICF must be 
filed in the study files. 
The Investigator will report promptly to the IEC/IRB any new information that may 
adversely affect the safety of the subjects or the conduct of the study. The Investigator will 
submit written summaries of the study status to the IEC/IRB as required. On completion of 
the study, the IEC/IRB will be notified that the study has ended. 
18.2 Regulatory Authorities 
Relevant study documentation will be submitted to the regulatory authorities of the 
participating country (USA only), according to local/national requirements, for review and 
approval before the beginning of the study. On completion of the study, the United States 
regulatory authorities will be notified that the study has ended. 
18.3 Ethical Conduct of the Study 
The Investigator(s) and all parties involved in this study should conduct the study in 
adherence to the ethical principles based on the Declaration of Helsinki, GCP, ICH 
guidelines, and the applicable national and local laws and regulatory requirements. 
18.4 Informed Consent 
The process of obtaining informed consent must be in accordance with applicable regulatory 
requirement(s) and must adhere to GCP. 
The Investigator is responsible for ensuring that no subject undergoes any study related 
examination or activity before that subject has given written informed consent to participate 
in the study. 
The Investigator or designated personnel will inform the subject of the objectives, methods, 
audio recordings, anticipated benefits, potential risks, and inconveniences of the study. The 
subject should be given every opportunity to ask for clarification of any points s/he does not 
understand and, if necessary, ask for more information. At the end of the interview, the 
subject will be given ample time to consider the study. Subjects will be required to sign and 
date the ICF. After signatures are obtained, the ICF will be kept and archived by the 
investigator in the investigator’s study file. A signed and dated copy of the subject ICF will 
be provided to the subject or their authorized representative. 


 
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It should be emphasized that the subject may refuse to enter the study or to withdraw from 
the study at any time, without consequences for their further care or penalty or loss of 
benefits to which the subject is otherwise entitled. Subjects who refuse to give or who 
withdraw written informed consent should not be included or continue in the study. 
If new information becomes available that may be relevant to the subject’s willingness to 
continue participation in the study, a new ICF will be approved by the IEC(s)/IRB(s) (and 
regulatory authorities, if required). The study subjects will be informed about this new 
information and reconsent will be obtained. 
18.5 Subject Confidentiality 
Monitors, auditors, and other authorized agents of the sponsor and/or its designee, the 
IEC(s)/IRB(s) approving this research, and the United States FDA, as well as that of any 
other applicable agency(ies), will be granted direct access to the study subjects’ original 
medical records for verification of clinical study procedures and/or data, without violating the 
confidentiality of the subjects to the extent permitted by the law and regulations. In any 
presentations of the results of this study or in publications, the subjects’ identity will remain 
confidential. 
All personal data collected and processed for the purposes of this study should be managed 
by the investigator and his/her staff with adequate precautions to ensure confidentiality of 
those data, and in accordance with the Health Insurance Portability and Accountability Act 
(1), applicable to national and/or local laws and regulations on personal data protection. 
18.6 Reporting and Publication, Including Archiving 
Essential documents are those documents that individually and collectively permit evaluation 
of the study and quality of the data produced. After completion of the study (end of study 
defined as the date of the last visit of the last subject), all documents and data relating to the 
study will be kept in an orderly manner by the Investigator in a secure study file. This file 
will be available for inspection by the sponsor or its representatives. Essential documents 
should be retained for 2 years after the final marketing approval in an ICH region or for at 
least 2 years since the discontinuation of clinical development of the investigational product. 
It is the responsibility of the sponsor to inform the study center when these documents no 
longer need to be retained. The Investigator must contact the sponsor before destroying any 
study related documentation. In addition, all subject medical records and other source 
documentation will be kept for the maximum time permitted by the hospital, institution, or 
medical practice. 
The sponsor must review and approve any results of the study or abstracts for professional 
meetings prepared by the investigator(s). Published data must not compromise the objectives 
of the study.  


 
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19 REFERENCES 
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American Speech-Language-Hearing Association. (1994). Guidelines for the audiologic 
management of individuals receiving cochleotoxic drug therapy. ASHA 36 (Suppl 12):11-19. 
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Basner, M., Brink, M., Bristow, A., de Kluezinaar, Y., Finegold , L., & Hong, J. (2015). ICBEN 
review of research on the biological effects of noise 2011-2014. Noise & Health, 57-82. 
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Carney, E., & Schlauch, R. S. (2007). Critical Difference Table for Word Recognition Testing 
Derived Using Computer Simulation. Journal of Speech, Language, and Hearing Research, 50(5), 
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Causey, G. D., Hood, L. J., Hermanson, C. L., & Bowling, L. S. (1984). The Maryland CNC 
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Choi, J., Betz, J., Li, L., Blake, C., Sung, Y., Contrera, K., & Lin, F. (July 2016). Association of 
Using Hearing Aids or Cochlear Implants With Changes in Depressive Symptoms in Older 
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Deal, J., Betz, J., Yaffe, K., Harris, T., Purchase-Helzner, E., Satterfield, S., & Pratt, S. (2017). 
Hearing impairment and incident dementia and cognitive decline in older adults: The health ABC 
study. Journals of Gerontology - Series A Biological Sciences and Medical Sciences, 703-709. 
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HIRSH, I.J., DAVIS, H., SILVERMAN, S. R., REYNOLDS, E. G., ELDERT, E., & BENSON, 
R. W. (1952). Development of materials for speech audiometry. Journal of Speech and Hearing 
Disorders, 17, 321-337. 
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Hong , O., Kerr, M., & Poling , G. (April, 2013). Understanding and preventing noise-induced 
hearing loss. ELSEVIER, 110-118. 
9. 
Johnson TA, Cooper S, Stamper GC, Chertoff M. Noise Exposure Questionnaire (NEQ): A Tool 
for Quantifying Annual Noise Exposure. Journal of the American Academy of Audiology 2017 
Jan; 28(1): 14-35. 
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Kuder, G. Frederic, and Marion W. Richardson. "The theory of the estimation of test 
reliability." Psychometrika 2.3 (1937): 151-160. 
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Lehiste, I. & Peterson, G. (1959). Linguistic considerations in the study of speech intelligibility. 
The Journal of the Acoustical Society of America, 31(3), 280-286. 
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Lehiste, I. & Peterson, G. (1962). Revised CNC lists for auditory tests. Journal of Speech, 
Language, and Hearing Research, 27(1), 62-70. 
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Li, L., Chao, T., Brant, J., O'Malley Jr, B., Tsourkas, A., & Li, D. (2017). Advances in Nano-
based inner ear delivery systems for the treatment of sensorineural hearing loss. Adv Drug 
Delivery. Advanced Drug Delivery Reviews. 
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Lin, F., Niparko, J., & Ferrucci, L. (November, 2011). Hearing loss prevalence in the United 
States. Arch Intern Med., 1851-1853. 
15. 
McGilton, K., Höbler, F., Campos, J., Dupuis, K., Labreche, T., & Guthrie, D. (2016). Hearing 
and vision screening tools for long-term care residents with dementia: protocol for a scoping 
review. BMJ Open. 
16. 
McLean WJ, et al. Improved speech intelligibility in subjects with stable sensorineural hearing 
loss following intratympanic dosing of FX-322 in a Phase 1b study. Otology & Neurotology 
(August 2021), 42 (7), 849-857. 
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McLean WJ, Yin X, Lu L, et al. Clonal expansion of Lgr5-positive cells from mammalian cochlea 
and high-purity generation of sensory hair cells. Cell Rep 2017;18:1917-29. 
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Meikle M.B., Henry J.A., Griest S.E., Stewart B.J., Abrams, H.B., McArdle R., Myers P.J., 
Newman C.W., Vernon J.A., et al. The tinnitus functional index: development of a new clinical 
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19. 
Nelson DI, Nelson RY, Concha-Barrientos M, Fingerhut M, (2005). The global burden of 
occupational noise-induced hearing loss. Am J Ind Med, 48(6): 446-458. 
20. 
Posner K., Brown GK, Stanley B, Brent DA, Yershova KV, Oquendo MA, Currier GW, Melvin 
GA, Greenhill L, Shen S, Mann JJ (2011) The Columbia-Suicide Severity Rating Scale: initial 
validity and internal consistency findings from three multisite studies with adolescents and adults, 
Am J Psychiatry 168: 1266-1277. 
21. 
Rauch SD, Halpin CF, Antonelli PJ, et al. Oral vs Intratympanic Corticosteroid Therapy for 
Idiopathic 
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Thornton AR, Raffin MJM: Speech discrimination scores modified as a binomial variable. J Sp 
Hear Res 1978;21:507–518. 
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US Department of Health and Human Services.  The Health Insurance Portability and 
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24. 
US Food and Drug Administration. Methods to Identify What is Important to Patients and Select, 
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25. 
Wilson RH, Abrams, HB, Pillion AL. (2003). A word-recognition task in multitalker babble 
using a descending presentation mode from 24 dB to 0 dB signal to babble. J Rehabil Res Dev. 
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26. 
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20 INVESTIGATOR SIGNATURE PAGE 
Protocol Title: 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, 
Single-Dose, Multicenter Study to Evaluate the Efficacy of FX-322 
Administered by Intratympanic Injection in Adults with Acquired 
Sensorineural Hearing Loss  
Protocol Number: 
FX-322-208 
Protocol Version:  
3.0, 05 Jan 2022 
 
Confidentiality and GCP Compliance Statement 
I, the undersigned, have reviewed this protocol (and amendments), and I will conduct the 
study as described in compliance with this protocol (and amendments), GCP, and relevant 
ICH guidelines. 
Once the protocol has been approved by the IEC/IRB, I will not modify this protocol without 
obtaining prior approval of Frequency Therapeutics and of the IRB. I will submit the protocol 
amendments and/or any ICF modifications to Frequency Therapeutics and IRB, and approval 
will be obtained before any amendments are implemented. 
I understand that all information obtained during the conduct of the study with regard to the 
subjects’ state of health will be regarded as confidential. No subjects’ names will be 
disclosed. All subjects will be identified by assigned numbers on all CRFs and source 
documents forwarded to the sponsor. Clinical information may be reviewed by the sponsor or 
its agents or regulatory agencies. Agreement must be obtained from the subject before 
disclosure of subject information to a third party. 
Information developed in this clinical study may be disclosed by Frequency Therapeutics, to 
other clinical investigators, regulatory agencies, or other health authority or government 
agencies as required. 
 
 


 
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21 APPENDICES 
21.1 Appendix A: 
Qualitative Questionnaire 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 56 of 64 
 
 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 57 of 64 
 
21.2  Appendix B: Research Assessment on 
 
Quality of Life 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 58 of 64 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 59 of 64 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 60 of 64 
 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 61 of 64 
 
 
21.3 Appendix C: Patient Global Impression of Severity (PGI-S) Hearing Loss Scale 
Instructions:  Please choose the response below that best describes the severity 
of your hearing loss over the past week. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
☐ None 
☐ Mild 
☐ Moderate 
☐ Severe 
☐ Very Severe 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 62 of 64 
 
 
21.4 Appendix D: Patient Global Impression of Severity (PGI-S) Daily Impacts Scale 
Instructions:  Please choose the response below that best describes the impact 
of your hearing loss on your daily activities over the past week. 
 
 
 
 
 
 
 
 
 
 
 
 
 
☐ None 
☐ Mild 
☐ Moderate 
☐ Severe 
☐ Very Severe 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 63 of 64 
 
21.5 Appendix E: Patient Global Impression of Change (PGI-C) Hearing Loss Scale 
Instructions:  Please choose the response below that best describes overall 
change in your hearing loss since you were administered the study medication.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
☐ Much better 
☐ A little better 
☐ No change 
☐ A little worse 
☐ Much worse 


 
FX-322-208 V3.0 05Jan2022                                                                            Page 64 of 64 
 
 
21.6 Appendix F: Patient Global Impression of Change (PGI-C) Daily Impacts Scale 
Instructions:  Please choose the response below that best describes overall 
change in the impact of your hearing loss on your daily activities since you 
were administered the study medication. 
 
 
 
 
☐ Much better 
☐ A little better 
☐ No change 
☐ A little worse 
☐ Much worse 

SAP_001.pdf: 
 
 
 
 
Statistical Analysis Plan 
 
 
 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, Single-Dose, 
Multicenter Study to Evaluate the Efficacy of FX-322 Administered by Intratympanic Injection in 
Adults with Acquired Sensorineural Hearing Loss 
 
 
 
NCT Number: 05086276 
 
 
Document Date: 19 Jan 2023 
 
 
 
 
 
*Per clinicaltrials.gov results posting guidelines, this cover page is attached to the Statistical 
Analysis Plan v2.0 for Protocol FX-322-208 for investigational product FX-322  


Frequency Therapeutics 
CONFIDENTIAL 
Statistical Analysis Plan
Protocol No. FX-322-208 
19 January 2023
 
 
Page 1 of 64 
Version Date: 2023-01-19 
Version: 2.0  
 
 
Frequency Therapeutics/
  
STATISTICAL ANALYSIS PLAN 
 
 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, Single-
Dose, Multicenter Study to Evaluate the Efficacy of FX-322 Administered by 
Intratympanic Injection in Adults with Acquired Sensorineural Hearing Loss 
 
FX-322-208 Protocol (Version 3.0, 05 January 2022) 
 
VERSION: 
2.0 
DATE: 
19 January 2023 
NCT#: 
 
COMPOUND #: 
FX-322 
SPONSOR: 
Frequency Therapeutics  
75 Hayden Avenue  
Lexington, MA 02421 
USA 
REGULATORY 
AGENCY 
IDENTIFIER 
NUMBER(S): 
 
PREPARED BY: 
 
 
 
 
 
 
 
 
 


Frequency Therapeutics 
CONFIDENTIAL 
Statistical Analysis Plan
Protocol No. FX-322-208 
19 January 2023
 
 
Page 2 of 64 
Version Date: 2023-01-19 
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Frequency Therapeutics/
  
STATISTICAL ANALYSIS PLAN 
ACKNOWLEDGMENT AND SIGNATURE SHEET 
 
A Phase 2, Prospective, Randomized, Double-Blind, Placebo-Controlled, Single-
Dose, Multicenter Study to Evaluate the Efficacy of FX-322 Administered by 
Intratympanic Injection in Adults with Acquired Sensorineural Hearing Loss 
 
 
 
 
 
 
Approved: 
Date: 
_______________ 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
Approved: 
 
Date: 
_______________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Approved: 
Date: 
_______________ 
 
 
 
 
 
 
 
 
 
 
 


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Protocol No. FX-322-208 
19 January 2023
 
 
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Version History 
 
SAP 
Version 
 
Approval Date 
 
Change(s) 
 
Rationale 
1.0 
26JUL2022   
Not Applicable 
Original Version 
2.0 
19JAN2023 
Modified primary analysis as well as the 
fixed-sequence testing procedure. 
Clarified Words-in-Noise key secondary 
efficacy endpoint. 
Added Patient Global Impression of Change 
as key secondary efficacy endpoint. 
Specified estimands for primary and key 
secondary endpoints. 
Clarified methods for missing data for 
primary and key secondary endpoints. 
Updated the denominators for the 
disposition summary. 
Clarified speech reception threshold 
summary will be generated for treated and 
untreated ears.  
Stated randomization stratification factors 
since randomization complete at the time 
of SAP Version 2.0.  
Version 2.0, finalized 
prior to database lock 
and treatment 
unblinding 
 


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Protocol No. FX-322-208 
19 January 2023
 
 
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Table of Contents 
List of Abbreviations ..................................................................................................................... 8 
1. 
Purpose of the Analyses ........................................................................................................10 
2. 
Protocol Summary ................................................................................................................10 
 
Study Objectives ........................................................................................................10 
 
Study Design .............................................................................................................10 
2.2.1. 
Part A: Screening/Start of Lead-In/Visit 1, Lead-in/Visit 2 ............................................11 
2.2.2. 
Part B: End of Lead-in/Treatment/Visit 3 (Day 1), Randomization (Double-blind), Follow-
up (Days 30, 60 and 90), and Observation (Days 180 and 270) ...................................................11 
 
Randomization ..........................................................................................................12 
 
Study Population .......................................................................................................12 
 
Sample Size Determination .........................................................................................12 
3. 
General Analysis and Reporting Conventions ...........................................................................12 
4. 
Analysis Sets ........................................................................................................................14 
5. 
Study Subjects......................................................................................................................14 
 
Disposition of Subjects ...............................................................................................14 
 
Demographic and Other Baseline Characteristics ...........................................................16 
6. 
Study Operations ..................................................................................................................16 
 
Study Conduct ...........................................................................................................16 
 
Protocol Deviations ....................................................................................................16 
7. 
Endpoint Evaluation ..............................................................................................................17 
 
Overview of Analysis Methods ....................................................................................17 
7.1.1. 
Pooling Algorithm ...................................................................................................17 
7.1.2. 
Assessment Time Windows .....................................................................................17 
7.1.3. 
Timing of Analyses ..................................................................................................18 
7.1.4. 
Multiplicity Adjustment...........................................................................................18 
7.1.5. 
Model Fitting .........................................................................................................19 
7.1.6. 
Imputation Rules for Partial/Missing Data .................................................................19 
7.1.7. 
Summary of Endpoints and Analysis Methods ............................................................19 
 
Primary and Key Secondary Efficacy Endpoint Estimands ................................................23 
7.2.1. 
Primary Efficacy Endpoint ........................................................................................23 
7.2.2. 
Key Secondary Efficacy Endpoint ..............................................................................24 
7.2.3. 
Other Key Secondary Efficacy Endpoints ...................................................................24 


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Word Recognition in Quiet: Maryland CNC and W-22 .....................................................24 
7.3.1. 
Improvement from baseline above Carney-Schlauch upper confidence limits ................25 
7.3.2. 
Percentage of recognized words ..............................................................................26 
7.3.3. 
Change from baseline in percentage of recognized words ...........................................27 
7.3.4. 
Improvement ≥10% (5 words) from baseline in percentage of recognized words ...........27 
7.3.5. 
Change from Baseline in Arcsine-transformed WR .....................................................28 
7.3.6. 
Improvement from baseline above Thornton-Raffin upper confidence limits ................28 
 
Words-In-Noise .........................................................................................................29 
7.4.1. 
Percentage of recognized words ..............................................................................29 
7.4.2. 
Change from baseline in percentage of recognized words ...........................................30 
7.4.3. 
Improvement ≥10% (7 words) from baseline in percentage of recognized words overall 30 
7.4.4. 
Spearman-Kärber estimates of dB thresholds for word recognition (25th, 50th (median) 
and 75th percentiles) .............................................................................................................31 
7.4.5. 
Change from baseline in median Spearman-Kärber estimates .....................................32 
7.4.6. 
Improvement ≥3 dB shifts in median Spearman-Kärber estimates from baseline ...........32 
 
Patient Global Impression of Severity/Patient Global Impression of Change .....................33 
7.5.1. 
PGI-S hearing loss scale responses ............................................................................33 
7.5.2. 
PGI-C hearing loss scale responses ...........................................................................33 
7.5.3. 
PGI-S daily impacts scale responses ..........................................................................34 
7.5.4. 
PGI-C daily impacts scale responses ..........................................................................34 
 
Pure Tone Audiometry ...............................................................................................34 
7.6.1. 
Air and Bone Conduction audiometry dB HL thresholds by frequency ...........................35 
7.6.2. 
Mean overall PTA (AC over 0.5, 1, 2, and 4 kHz) and BCA (BC over 0.5, 1, 2 and 4 kHz) ...35 
7.6.3. 
AC and BC improvement ≥10 and ≥15 dB HL from baseline, separately, for two contiguous 
frequencies ..........................................................................................................................36 
7.6.4. 
AC and BC improvement ≥15 and ≥20 dB HL from baseline, separately, for any frequency
 
36 
7.6.5. 
AC and BC composite improvement from baseline .....................................................36 
7.6.6. 
Change from baseline in Mean overall PTA and BCA ...................................................37 
7.6.7. 
Shifts from baseline in Mean overall PTA Categories ..................................................37 
7.6.8. 
Mean low AC dB HL threshold (AC over 0.25, 0.5, 1, and 2 kHz) ...................................38 
7.6.9. 
Mean high AC dB HL threshold (AC over 3, 4, 6 and 8 kHz) ..........................................38 
7.6.10. 
Air-Bone Imbalance at 0.5, 1, 2, 3, and 4 kHz .............................................................38 
7.6.11. 
Shift from baseline in Air-Bone Imbalance at 0.5, 1, 2, 3, and 4 kHz ..............................38 
 
Extended High Frequency Audiometry .........................................................................39 
7.7.1. 
Air audiometry dB HL thresholds by frequency ..........................................................39 


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7.7.2. 
Mean dB HL threshold (AC over 9-16 kHz) .................................................................40 
7.7.3. 
AC improvement ≥10 dB HL and ≥15 dB HL from baseline, separately, for two contiguous 
frequencies ..........................................................................................................................40 
7.7.4. 
AC improvement ≥15 and ≥20 dB HL from baseline, separately, for any frequency ........40 
7.7.5. 
AC composite improvement from baseline ................................................................41 
7.7.6. 
Shift from baseline of no signal to any signal by frequency ..........................................41 
7.7.7. 
Shift from baseline of no signal to any signal in two contiguous frequencies .................42 
7.7.8. 
Shift from baseline of no signal to any signal in three contiguous frequencies ...............42 
 
Examination of Subgroups ..........................................................................................42 
8. 
Safety Evaluation ..................................................................................................................43 
 
Overview of Safety Analysis Methods ...........................................................................43 
 
Extent of Exposure .....................................................................................................43 
 
Adverse Events ..........................................................................................................44 
8.3.1. 
Imputation Rules for Missing AE Date/Time of Onset/Resolution .................................44 
 
Deaths, Serious Adverse Events, and Other Significant Adverse Events ............................45 
 
Urine Pregnancy Tests ................................................................................................45 
 
Vital Signs, Physical Findings, and Other Observations Related to Safety ..........................46 
8.6.1. 
Vital Signs ..............................................................................................................46 
8.6.2. 
Physical Examinations .............................................................................................46 
8.6.3. 
Otoscopic Examinations ..........................................................................................46 
8.6.4. 
Tympanometry ......................................................................................................46 
8.6.5. 
Columbia-Suicide Severity Rating Scale Assessments ..................................................47 
8.6.6. 
Concomitant Medications .......................................................................................47 
9. 
Other Analyses .....................................................................................................................48 
 
 Qualitative Questionnaire .........................................................48 
9.1.1. 
Qualitative questionnaire responses .........................................................................48 
 
Tinnitus Functional Index ............................................................................................48 
9.2.1. 
Mean total score, individual subscale scores, and loudness score ................................48 
9.2.2. 
Change from baseline in mean total score, individual subscale scores, and loudness score
 
49 
 
Research Assessment 
 
49 
9.3.1. 
Mean total score, mean domain scores, and individual scores .....................................49 
9.3.2. 
Change from baseline in mean total score, mean domain scores, and individual scores ..49 
9.3.3. 
Domain level compliance ........................................................................................50 
 
Speech Reception Threshold (SRT) ...............................................................................50 


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10. Interim Analysis and Data Monitoring .....................................................................................50 
11. Changes to the Analyses Planned in the Protocol .....................................................................50 
12. References ..........................................................................................................................50 
13. Appendices ..........................................................................................................................52 
 
Study Flow Chart .......................................................................................................52 
 
Schedule of Assessments ............................................................................................53 
14. Attachments ........................................................................................................................55 
 
Thornton and Raffin (1978) Word Recognition Scoring Chart ..........................................55 
 
Carney-Schlauch (2007) Word Recognition Scoring Chart ...............................................56 
 
TFI Calculations..........................................................................................................57 
 
Calculations ...................................................................................................60 
 
 


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LIST OF ABBREVIATIONS 
AC 
Air Conduction 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
ANCOVA 
Analysis of Covariance 
ATC 
Anatomical Therapeutic Chemical 
BC 
Bone Conduction 
BCA 
Bone Conduction Average 
BMI 
Body Mass Index 
BP 
Blood Pressure 
C-SSRS 
Columbia-Suicide Severity Rating Scale 
CI 
Confidence Interval 
CMH 
Cochran-Mantel-Haenszel 
CNC 
Consonant-Nucleus-Consonant 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
dB 
Decibel 
EDC 
Electronic Data Capture 
EHFA 
Extended High Frequency Audiometry 
ET 
Early Termination 
FAS 
Full Analysis Set 
GEE 
Generalized Estimating Equation 
HL 
Hearing Level 
ICH 
International Conference on Harmonisation 
LOCF 
Last observation carried forward 
LS 
Least Squares 
MCAR 
Missing Completely At Random 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMRM 
Mixed-effects Model for Repeated Measures 
NIHL 
Noise-induced Sensorineural Hearing Loss 
PGI-C 
Patient Global Impression of Change 
PGI-S 
Patient Global Impression of Severity 
PPAS 
Per Protocol Analysis Set 
PRO 
Patient Reported Outcome 
PT 
Preferred Term 
PTA 
Pure Tone Average 
 
 
 


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SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SfAS 
Safety Analysis Set 
SL 
Sensation Level 
SOC 
System Organ Class 
SPL 
Sound Pressure Level 
SNHL 
Sensorineural Hearing Loss  
SSNHL 
Sudden Sensorineural Hearing Loss 
SRT 
Speech Reception Threshold 
TEAE 
Treatment-Emergent Adverse Event 
TFI 
Tinnitus Functional Index 
WR 
Word Recognition in Quiet 
WIN 
Words-in-Noise 
 
 


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19 January 2023
 
 
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1. 
PURPOSE OF THE ANALYSES 
The purpose of this statistical analysis plan (SAP) is to provide detailed information to aid in 
the implementation of the statistical analysis and reporting of the study data for use in the 
clinical study report (CSR).  It briefly summarizes the protocol, describes the analysis sets, 
and the planned analyses.  The details of the specific statistical methods that will be used 
for the pre-specified analyses of the primary and secondary endpoints are provided and 
were defined prior to unblinding of the data.  This SAP does not limit the analyses in the 
CSR, and additional analyses to supplement the CSR writing may be conducted and will be 
identified in the CSR as post hoc.  Table, figure, and listing specifications are in separate 
documents. 
This SAP was written with due consideration of the recommendations outlined in the most 
recent International Conference on Harmonisation (ICH) E9 Guideline entitled Guidance for 
Industry:  Statistical Principles for Clinical Trials and the most recent ICH E3 Guideline, 
entitled Guidance for Industry:  Structure and Content of Clinical Study Reports and was 
finalized prior to database lock. 
2. 
PROTOCOL SUMMARY 
 
Study Objectives 
Primary Objective 
• To assess efficacy following a single dose of FX-322 versus Placebo, in subjects with 
acquired sensorineural hearing loss (SNHL). 
Secondary Objective 
• To assess safety following a single dose of FX-322 or Placebo in subjects with acquired 
sensorineural hearing loss. 
 
Study Design 
This is a Phase 2, prospective, randomized, double-blind, placebo-controlled, single-dose, 
multicenter study to evaluate the efficacy of FX-322, administered by intratympanic 
injection, in adults with acquired sensorineural hearing loss.  
The study will have two Parts as described below: 
Part A: Screening/Start of Lead-in/Visit 1, Lead-in/Visit 2  
Part B: End of Lead-in/Treatment/Visit 3 (Day 1), Randomization (Double-blind), Follow-up 
(Days 30, 60 and 90), and Observation (Days 180 and 270) 


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2.2.1. Part A: Screening/Start of Lead-In/Visit 1, Lead-in/Visit 2 
Visits can occur 30-40 days prior to study drug administration [Visit 3 (Day 1)].  All subjects 
will be evaluated to determine study eligibility.  For the purpose of ensuring the integrity of 
the trial and minimizing bias, certain study design details are described within a restricted-
access unmasked protocol addendum.  As part of recording the medical history of subjects, 
concurrent use of hearing aids during the study will be documented, as well as hearing loss 
etiology and laterality (unilateral, bilateral).  Eligible subjects will be required to return for 
Lead-in/ Visit 2 (14-18 days after Visit 1) to complete assessments.  Subjects will be 
instructed to contact the Investigator or relevant staff if a medical change has occurred 
during the lead-in period (Visit 1 to pre-dose Visit 3).  Eligible subjects will return for Visit 3 
(Day 1) and will be evaluated to determine whether they meet appropriate 
Inclusion/Exclusion criteria required for randomization.   
2.2.2. Part B: End of Lead-in/Treatment/Visit 3 (Day 1), Randomization (Double-
blind), Follow-up (Days 30, 60 and 90), and Observation (Days 180 and 270) 
Approximately 124 subjects are planned to be randomized in this study in order to obtain a 
target of 112 evaluable subjects.  The subjects will be randomized to receive FX-322 or 
placebo according to the different treatment group assignments listed below: 
Treatment 
Group 
# of 
Subjects 
Day 1 
1 
62 
FX-322 
2 
62 
Placebo 
 
Once a subject has been identified as meeting all Inclusion/Exclusion criteria, including the 
criteria described within the unmasked protocol addendum which will be determined via 
the electronic data capture (EDC) system, the subject will be randomized.  FX-322 or 
placebo will be administered at Visit 3 (Day 1).  The syringes containing either placebo or 
FX-322 will be obscured to maintain the blind for the injecting Otolaryngologist.  
Each subject will be placed in the supine position.  Topical anesthesia (with the exception of 
phenol) will be administered directly to the tympanic membrane in the study ear.  Under a 
microscope, a 25-gauge needle will be used to inject FX-322 or placebo into the middle ear 
at the junction of the posterior inferior and posterior superior quadrant with the needle tip 
directed towards the round window.  The posterior superior quadrant should be avoided to 
prevent injury to the ossicles.  After injection, the subject will continue to lie with the 
injected ear facing up for 20-30 minutes.  The injections will be performed at an appropriate 
location designated by a Board-certified Otolaryngologist trained and experienced in 
performing intratympanic injections.  Safety monitoring will include recording of adverse 
events (AEs), monitoring of audiology, tympanometry, otoscopic exams, and Columbia-
Suicide Severity Rating Scale (C-SSRS) assessments. 


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Follow-up: Subjects will be required to return to clinic for safety, otologic, and audiologic 
assessments at Days 30 (Visit 4), 60 (Visit 5), and 90 (Visit 6) following the study injection.  
Unscheduled visits may occur as needed. 
Observation: Subjects will return to clinic for safety, otologic, and audiologic assessments at 
Days 180 (Visit 7) and 270 (Visit 8).   
 
Randomization 
Subjects will be randomized in a 1:1 ratio to one of two groups (FX-322 or placebo, 
respectively).  The randomization will be stratified for Visit 2 (Day -15) Maryland CNC Word 
Recognition Score (above or equal to 28% words in quiet correct and below 28% words in 
quiet correct) and unilateral versus bilateral hearing loss.  A single injection of FX-322 or 
placebo will be administered on Day 1 by intratympanic injection in the study ear. 
 
Study Population 
Male and female adults (18-65 years inclusive), otherwise healthy with acquired 
sensorineural hearing loss, associated with noise-induced SNHL (NIHL) or idiopathic sudden 
SNHL (SSNHL). 
 
Sample Size Determination 
The sample size of 112 evaluable subjects, in a 1:1 allocation ratio of FX-322 and Placebo, 
was selected to provide an overall statistical power of at least 80% to detect a difference in 
the primary endpoint for word recognition, as defined by the proportion of subjects that 
exceed the 95% upper confidence limit as described by Carney and Schlauch (2007) in the 
study ear.  The target enrollment of 124 subjects is an adjustment for an assumed 10% 
study dropout rate. Detailed sample size determination is provided in the unmasked 
protocol addendum. Each subject contributes one study ear and one non-study ear. 
3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS 
The following is a list of general analysis and reporting conventions to be applied for this 
study.  
• Categorical variables will be summarized using counts (n) and percentages (%) and 
will be presented in the form n (%).  If a count is 0, no percentage will be shown.  To 
ensure completeness, summaries for categorical and discrete variables will include 
all categories, even if no subjects had a response in a particular category.  Missing 
data for categorical variables will have a ‘Missing’ category added at the end and the 
count will be presented without a percentage.  Percentages for categorical variables 
will exclude the ‘Missing’ category. 
• Continuous variables will be summarized using number of evaluable subjects, mean, 
standard deviation (SD), minimum, maximum, and median.  The mean, median, and 


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confidence intervals (CI) will be rounded and reported to 1 more level of precision 
than the original observations, and the SD will be rounded and reported to 2 more 
levels of precision than the original observations.  The minimum and maximum will 
be the same precision as the original data.  
• Following SAS default rules, the median will be reported as the rounded average of 
the two middle numbers if the dataset contains even numbers. 
• P-values will be rounded and reported to 3 decimal places if greater than 0.001.  If 
the rounded p-value is less than 0.001, ‘<0.001’ will be reported.  If the rounded p-
value is >0.999, ‘>0.999’ will be reported.  P-values and significant levels will be 
reported as 0.05 rather than .05.  
• No preliminary rounding will be performed; rounding will only occur after analysis.  
To round, consider digit to right of last significant digit:  if < 5 then round down, if ≥5 
then round up. 
• All listings will be sorted in order of subject and time of assessment (e.g., visit, time, 
and/or event). 
• Dates in listings will be displayed as yyyy-mm-dd (e.g., 2022-01-22). 
• Age (in years) will be calculated using the date of birth and the Screening date in the 
following SAS algorithm:  floor((intck(‘month’, date of birth, Screening date) – 
(day(Screening date) < day(date of birth))))/12.  In the analysis datasets, tables, and 
listings, age will be reported as the integer part of the derived age, with no rounding. 
• For all efficacy endpoints, including word recognition in quiet (Maryland Consonant-
Nucleus-Consonant [CNC] and W-22) and words in noise (WIN), baseline values for 
analysis will be defined by ear as the average across the pre-treatment timepoints 
(Lead-in and Day 1) for left and right ears separately.  For the analyses of 
improvement from baseline above upper confidence limits, either per Carney-
Schlauch (2007) or Thornton-Raffin (1978), the rounded up average will be used for 
conservative approach.  Left and right ears will be mapped to treated and untreated 
for analysis.     
• For all safety endpoints and other analyses, baseline will be the last non-missing 
value before study drug administration.  
• For efficacy analyses, Day 90 values will be based on the scheduled Day 90 nominal 
case report form (CRF) recorded visit.  If no scheduled visit data is available for the 
nominal day 90 CRF recorded visits, the non-missing data in either ear from an early 
termination or unscheduled assessment performed closest to the target day will be 
used.  


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• All endpoints which reference a change in percentage of recognized words are 
calculated as and should be interpreted as a linear change (i.e., observed – baseline) 
and not a relative change (i.e. (observed – baseline)/baseline x 100). 
• For all shift from baseline analyses, percentages will be based on the number of 
subjects in each category at baseline and the post-baseline visit.  
• All analysis will be performed using the SAS System version 9.4 or higher. 
4. 
ANALYSIS SETS 
Analysis sets in this study are defined in the following: 
• Full Analysis Set  
The Full Analysis Set (FAS) includes all randomized subjects who receive one dose of study 
drug (regardless of whether a full dose was administered) in the study ear.  Subjects will be 
analyzed according to their randomized treatment group.     
• Per Protocol Analysis Set  
The Per Protocol Analysis Set (PPAS) includes subjects in the FAS receiving one full dose of 
study drug in the study ear to be treated per the randomization treatment schedule and 
without major protocol deviations as defined in Section 6.1.  PPAS subjects will be analyzed 
according to their randomized treatment group.   
• Safety Analysis Set  
The Safety Analysis Set (SfAS) includes all subjects who receive one dose of study drug 
(regardless of whether a full dose was administered) in any ear and will be analyzed 
according to the actual treatment received.  For safety assessments completed by ear for 
each subject, results from actual treated and actual untreated ears will be summarized 
separately.  The SfAS will be used for the analysis of safety.   
All main efficacy analyses will be based on FAS, with sensitivity analyses for primary and key 
secondary endpoint analyses in the PPAS. If more than 10% of subjects in the FAS are 
excluded from the PPAS, additional sensitivity analyses, other than for the primary and key 
secondary endpoints based on PPAS, may be performed. 
5. 
STUDY SUBJECTS 
 
Disposition of Subjects  
The disposition of all consented subjects will be tabulated by treatment group and overall.  
Subjects who failed screening or were not randomized will only be included in the overall 
column.  The following disposition information will be summarized: 


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• The number of subjects consented (signed Informed Consent) 
• The number and percentage of subjects who failed screening and the reasons for 
failure 
• The number of subjects not randomized and the number of subjects randomized 
• The number of subjects in the FAS 
• The number and percentage of subjects in the PPAS and SfAS analysis sets  
• The number and percentage of subjects who completed the study through Day 90 
(Visit 6) 
• The number and percentage of subjects who discontinued the study early and the 
reasons for withdrawal 
• The number and percentage of subjects who discontinued the study early for 
reasons related to COVID-19 
• The number and percentage of subjects who continued to the Observational period 
• The number and percentage of subjects who completed Day 180 (Visit 7) and Day 
270 (Visit 8) 
• The number and percentage of subjects who did not complete Day 180 (Visit 7) and 
Day 270 (Visit 8) and the reasons not completed 
• The number and percentage of subjects who did not complete Day 180 (Visit 7) and 
Day 270 (Visit 8) for reasons related to COVID-19 
Percentages for the number of subjects who failed screening and the reasons for failure will 
be based on the number of consented subjects.  All other percentages will be based on 
number of subjects in the SfAS.    
Duration of study follow up (from randomization date to study completion date or early 
discontinuation visit date) will be summarized in a Kaplan-Meier curve for all randomized 
subjects by treatment group. Subjects who completed the study will be censored at the 
study completion date. 
Subject disposition data for all randomized subjects will also be listed.  A separate by-
subject listing for screen failures will be generated along with reason for screen failure. 
A listing of all subjects affected by the COVID-19 pandemic (discontinued the study early, 
had remote visits performed, or missed visits for reasons related due to COVID-19) will be 
provided to list the impacts of the pandemic on the subject’s participation. 


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Demographic and Other Baseline Characteristics  
Descriptive statistics for demographic and other baseline characteristics will be summarized 
by treatment group and overall based on FAS.  Characteristics to be summarized include: 
• Demographic: age, race, ethnicity, sex, height, weight, and body mass index (BMI) 
• Hearing loss history: laterality, etiology of hearing loss for treated and untreated 
ears, duration of hearing loss for treated ears 
• History of noise exposure 
• Hearing aid use 
• Pure Tone Average (0.5, 1, 2, and 4 kHz) for the treated ear 
• Bone Conduction Average (0.5, 1, 2, and 4 kHz) for the treated ear 
• Word Recognition in Quiet (Maryland CNC) score for the treated ear 
• Word Recognition in Quiet (W-22) score for the treated ear 
• WIN score for the treated ear 
• Patient Global Impression of Severity (PGI-S) Hearing Loss response 
• Patient Global Impression of Severity (PGI-S) Daily Impacts response 
• Randomization stratum 
Demographic and baseline characteristics data will be listed for subjects in the FAS. 
6. 
STUDY OPERATIONS 
 
Study Conduct 
Enrollment will be summarized by site for all enrolled, randomized, and treated subjects, 
separately. A by-subject listing of batch numbers for all treated subjects will be provided. 
The number and percentage of study visits attended will be summarized for subjects in the 
FAS. 
 
Protocol Deviations 
Protocol deviations will be identified, documented, reviewed, and assessed according to the 
study protocol deviation guidance.  Important or major protocol deviations are those that 


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might significantly affect the completeness, accuracy, and/or reliability of key study data or 
that might significantly affect a subject’s rights, safety, or well-being.  
Major protocol deviations will be listed and tabulated by deviation category for subjects in 
the FAS by treatment group and overall. 
7. 
ENDPOINT EVALUATION 
 
Overview of Analysis Methods 
7.1.1. Pooling Algorithm 
This is a multi-center (US only) study. Unless stated otherwise, data from all participating 
centers in the study will be pooled for analyses. A site poolability assessment of the primary 
efficacy analysis will be conducted to assess heterogeneity of the treatment effect between 
sites. This will be conducted as a descriptive analysis for the primary efficacy endpoint. 
7.1.2. Assessment Time Windows 
For by-visit summaries, the nominal visit will be used for analysis. Unscheduled visits will be 
listed but will not be displayed separately in the summaries.  
Analysis windows listed in Table 7-1 will be used in the efficacy analyses for slotting actual 
efficacy assessment date into planned assessment schedule.  Early termination visit and 
unscheduled visit efficacy data will be included in the analyses if no scheduled visit data is 
available for the nominal CRF visit.  If more than one unscheduled assessment (early 
termination or unscheduled) is performed within an analysis window, the assessment 
performed closest to the target day will be used.   
Table 7-1  
Study Visit Windows  
 
Visit 
Visit 
Number 
Assessment
/Day 
 
Target Day 
 
Target Window 
Analysis 
Windowa  
Screening/ 
Start of Lead-In 
1 
-40 to -30  
N/A 
N/A 
N/A - 
Nominal 
CRF visit 
Lead-In Visit 
2 
-26 to -12 
Visit 1 + 16 days Target day (+/- 
2 days) 
N/A - 
Nominal 
CRF visit 
End of Lead-In/ 
Treatment 
3 
1  
Visit 1 + 35 days 1/Target day 
(+/- 5 days) 
1 
In-Clinic Follow-up 
4 
30 
30 
25-35  
(+/- 5 days) 
2-45 
In-Clinic Follow-up 
5 
60 
60 
55-65  
(+/- 5 days) 
46-75 


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In-Clinic Follow-up 
/Early Termination 
(ET) 
6 
90 
90 
85-95  
(+/- 5 days) 
76-135 
In-Clinic 
Observation 
7 
180 
180 
173-187  
(+/- 7 days) 
136-225 
In-Clinic 
Observation 
8 
270 
270 
263-277  
(+/- 7 days) 
≥226 
aAnalysis windows for the Screening and Lead-In Visit are not defined and visit assignments 
are based on the nominal CRF visits.  Analysis windows for visits on or after randomization 
are defined using the mid-points between each scheduled visit and will be used to map early 
termination or unscheduled visit data to scheduled visits.   
 
7.1.3. Timing of Analyses 
Primary efficacy analysis will be conducted after all subjects have either completed the 
follow-up visits (Day 30, 60, and 90 visits) or early discontinued from study.  Primary 
analysis will be performed in an unblinded fashion for analysis purposes while subjects not 
completing the observation visits will continue to be followed as being blinded to treatment 
assignment.  Final CSR analyses will be performed after the study is completed, and the 
database is locked. 
7.1.4. Multiplicity Adjustment 
A fixed-sequence testing procedure will be implemented to control the family-wise type I 
error rate at 5% for the primary and key secondary endpoints as presented graphically in 
Figure 1. The hypothesis of superiority on the primary and key secondary efficacy endpoints 
of FX-322 versus placebo will be tested in the hierarchical order.  The primary efficacy 
endpoint, incidence of subjects who have a Word Recognition in Quiet (Maryland CNC) 
improvement at Day 90, will be tested first, using the primary analysis approach described 
in Section 7.3.1. If superiority of FX-322 over placebo in improving speech perception for 
Word recognition in Quiet (WR) is established, the treatment effect in the key secondary 
endpoint, WIN improvement based on mean change from baseline in percentage of 
recognized words overall at Day 90, will be tested subsequently, using the analysis approach 
described in Section 7.4.2. If superiority of FX-322 over placebo is established for the key 
secondary endpoint, the treatment effect the next key secondary efficacy endpoint, Patient 
Global Impression of Change (PGI-C) Hearing Loss Scale responses at Day 90, will be tested 
subsequently, using the analysis approach described in Section 7.5.2.  Lastly, if superiority of 
FX-322 over placebo is established for the previous three endpoints, the treatment effect 
for PGI-C Daily Impacts Scale responses at Day 90 will be tested subsequently, using the 
analysis approach described in Section 7.5.4. 


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Figure 1 - Multiple Comparisons Procedure for Primary and Key Secondary Endpoints 
 
7.1.5. Model Fitting 
Inferential statistical methods examining continuous endpoints will be examined via a 
Mixed-effects Model for Repeated Measures (MMRM). Inferential analyses for categorical 
endpoints will be analyzed using univariate methods and/or Generalized Estimating 
Equations (GEE). The MMRM and GEE analyses will employ an unstructured working 
correlation matrix. 
Further details for each of the planned models and analyses are provided in subsequent 
sections. 
7.1.6. Imputation Rules for Partial/Missing Data 
Based on previous studies of FX-322, there is expected to be a very minimal amount of 
missing data in this study.  Missing data for endpoints for word recognition in quiet using 
Carney-Schlauch confidence intervals will be assumed missing at random and will be 
imputed using the last observation carried forward (LOCF).  Missing data for the key 
secondary endpoint for WIN improvement will be assumed missing at random and will be 
taken into account using the model-based MMRM approach.  Missing data for PGI-C 
analyses will not be imputed.  Missing data for other endpoints will remain missing in the 
planned statistical summaries and analyses. 
7.1.7. Summary of Endpoints and Analysis Methods 
To assess treatment effect if no rescue medication is available, for efficacy analyses, 
assessments performed on or after date of rescue medication or therapy taken during the 
follow up period will not be included in the analyses. Specifically, rescue medication 
includes intratympanic steroid administered in the treated ear after study drug 
administration through Day 90 visit.  A summary of endpoints and analysis methods is 
presented in Table 7-2. 
Table 7-2 
Endpoints and Analysis Methods 
Endpoint 
Analysis Method(s)  
Section 
Word Recognition in Quiet (Maryland CNC) and Word Recognition (W-22)* 
7.3 


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Endpoint 
Analysis Method(s)  
Section 
Improvement from baseline 
above Carney-Schlauch 
upper confidence limits 
(Primary Endpoint) 
• 
Descriptive summary for treated and untreated ears 
• 
Primary Analysis: Chi-Square test for treated ears 
• 
Sensitivity Analysis: Chi-Square test for treated ears 
in PPAS 
• 
Sensitivity Analysis: Chi-Square test for treated ears 
based on improvement from baseline above 80%, 
85%, and 90% Carney-Schlauch upper confidence 
limits 
• 
Sensitivity Analysis: CMH test for treated ears 
• 
Sensitivity Analysis: GEE in treated ears 
• 
Sensitivity Analysis: DerSimonian and Laird (1986) 
estimation for treated ears using both randomization 
factors (4 strata) as defined in Section 2.3.  If the 
analysis cannot be computed using both 
randomization factors, the analysis will be 
performed using the single randomization factor (2 
strata) that produces more similarly sized strata.  
• 
Scatterplots for treated and untreated ears 
• 
Descriptive summary for treated ears by site 
7.3.1 
Percentage of recognized 
words  
• 
Descriptive summary for treated and untreated ears 
7.3.2 
Change from baseline in 
percentage of recognized 
words  
• 
Descriptive summary for treated and untreated ears 
• 
Line graphs (with standard error bars) for observed 
mean change from baseline for treated ears 
7.3.3 
Improvement ≥10% (5 
words) from baseline in 
percentage of recognized 
words  
• 
Descriptive summary for treated ears 
• 
GEE in treated ears 
7.3.4 
Change from baseline in 
arcsine-transformed WR  
• 
Line graphs (with standard error bars) for adjusted 
least squared means in change from baseline for 
treated ears 
• 
MMRM in treated ears 
7.3.5 
Improvement from baseline 
above Thornton-Raffin 
upper confidence limits  
• 
Descriptive summary for treated and untreated ears 
• 
Scatterplots for treated and untreated ears 
7.3.6 
Words-In-Noise*  
7.4 
Percentage of recognized 
words 
• 
Descriptive summary for treated and untreated ears 
7.4.1 
Change from baseline in 
percentage of recognized 
• 
Descriptive summary for treated and untreated ears 
7.4.2 


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Endpoint 
Analysis Method(s)  
Section 
words (Key Secondary 
Efficacy Endpoint) 
• 
Line graphs (with standard error bars) for adjusted 
least squared means in change from baseline for 
treated ears 
• 
Primary Analysis: MMRM in treated ears 
• 
Sensitivity Analysis: MMRM in treated ears for PPAS 
Improvement ≥10% (7 
words) from baseline in 
percentage of recognized 
words  
• 
Descriptive summary for treated ears 
• 
GEE in treated ears 
7.4.3 
Spearman-Kärber estimates 
of decibel (dB) thresholds 
for word recognition (25th, 
50th (median) and 75th 
percentiles)  
• 
Descriptive summary for treated ears 
• 
Scatter plots for treated ears 
7.4.4 
Change from baseline in 
median Spearman-Kärber 
estimates 
• 
Descriptive summary for treated ears 
• 
Line graphs (with standard error bars) for adjusted 
least squared means in change from baseline for 
treated ears 
• 
MMRM in treated ears 
7.4.5 
Improvement ≥3 dB shifts in 
median Spearman-Kärber 
estimates from baseline 
• 
Descriptive summary for treated ears 
• 
GEE in treated ears 
7.4.6 
Patient Global Impression of Severity and Patient Global Impression of Change*  
7.5 
PGI-S hearing loss scale 
responses 
• 
Descriptive summary for treated ears 
7.5.1 
PGI-C hearing loss scale 
responses 
(Key Secondary Efficacy 
Endpoint) 
• 
Analysis of Covariance (ANCOVA) in treated ears 
7.5.2 
PGI-S daily impacts scale 
responses 
• 
Descriptive summary for treated ears 
7.5.3 
PGI-C daily impacts scale 
responses (Key Secondary 
Efficacy Endpoint) 
• 
ANCOVA in treated ears 
7.5.4 
Pure Tone Audiometry 
7.6 
Air Conduction (AC) and 
Bone Conduction (BC) 
audiometry dB hearing level 
(HL) thresholds by 
frequency  
• 
Descriptive summary for treated and untreated ears 
7.6.1 


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Endpoint 
Analysis Method(s)  
Section 
Mean overall pure tone 
average (PTA) (AC over 0.5, 
1, 2, and 4 kHz) and Bone 
Conduction Average (BCA) 
(BC over 0.5, 1, 2 and 4 kHz) 
• 
Descriptive summary for treated ears 
7.6.2 
AC and BC improvement 
≥10 and ≥15 dB HL from 
baseline, separately, for two 
contiguous frequencies  
• 
Descriptive summary for treated ears 
7.6.3 
AC and BC improvement 
≥15 and ≥20 dB HL from 
baseline, separately, for any 
frequency  
• 
Descriptive summary for treated ears 
7.6.4 
AC and BC composite 
improvement from baseline 
• 
Descriptive summary for treated ears 
• 
GEE in treated ears 
7.6.5 
Change from baseline in 
mean overall PTA and BCA 
• 
Descriptive summary for treated ears 
7.6.6 
Shift from baseline in mean 
overall PTA categories 
• 
Descriptive summary for treated ears 
7.6.7 
Mean low AC dB HL 
threshold (AC only over 
0.25, 0.5, 1, and 2 kHz 
frequencies)  
• 
Descriptive summary for treated ears 
7.6.8 
Mean high AC dB HL 
threshold (AC only over 3, 4, 
6, and 8 kHz frequencies)  
• 
Descriptive summary for treated ears 
7.6.9 
Air-Bone Imbalance at 0.5, 
1, 2, 3, and 4 kHz 
• 
Descriptive summaries for treated ears 
7.6.10 
Shift from baseline in Air-
Bone Imbalance at 0.5, 1, 2, 
3, and 4 kHz 
• 
Descriptive summary for treated ears 
7.6.11 
Extended High Frequency Audiometry (EHFA) 
7.7 
AC audiometry dB HL 
thresholds by frequency  
• 
Descriptive summary for treated and untreated ears 
7.7.1 
Mean dB HL threshold (AC 
over 9-16 kHz)  
• 
Descriptive summary for treated ears 
7.7.2 
AC improvement ≥10 and 
≥15 dB HL from baseline, 
• 
Descriptive summary for treated ears 
7.7.3 


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Endpoint 
Analysis Method(s)  
Section 
separately, for two 
contiguous frequencies  
AC improvement ≥15 and 
≥20 dB HL from baseline, 
separately, for any 
frequency  
• 
Descriptive summary for treated ears 
7.7.4 
AC composite improvement 
from baseline 
• 
Descriptive summary for treated ears 
• 
GEE in treated ears 
7.7.5 
Shift from baseline of no 
signal to any signal by 
frequency  
• 
Descriptive summary for treated ears 
7.7.6 
Shift from baseline of no 
signal to any signal in two 
contiguous frequencies 
• 
Descriptive summary for treated ears 
7.7.7 
Shift from baseline of no 
signal to any signal in three 
contiguous frequencies 
• 
Descriptive summary for treated ears 
7.7.8 
Examination of Subgroups* 
7.8 
Incidence of WR 
Improvement from baseline 
above Carney-Schlauch 
upper confidence limits 
• 
Forest plot  
 
*Efficacy endpoint 
 
 
Primary and Key Secondary Efficacy Endpoint Estimands 
7.2.1. Primary Efficacy Endpoint 
Variable: Response, defined as improvement from baseline at Day 90 above Carney-
Schlauch 95% upper confidence limits in Word Recognition in Quiet (Maryland CNC) in 
treated ears. 
Population: The analysis population will be the FAS. 
Intercurrent events: Subjects who discontinue the study early will be considered missing at 
random, and their last non-missing observation will be carried forward (i.e., if their Day 60 
improvement is above Carney-Schlauch 95% upper confidence limit, then their Day 90 
results will be assumed to also be above the limit). 


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Population-level summary: Comparison of responder proportions between treatment 
groups at the Day 90 visit. 
Additional details on the computation, analysis, and sensitivity analyses for the primary 
endpoint can be found in Section 7.3.1. 
7.2.2. Key Secondary Efficacy Endpoint 
Variable: Change from baseline in percentage of recognized words overall at Day 90 in 
Words-in-Noise in treated ears. 
Population: The analysis population will be the FAS. 
Intercurrent events: Subjects who discontinue the study early will be assumed missing at 
random and missing responses will be taken into account using the model-based approach 
of MMRM. 
Population-level summary: Comparison of mean difference between treatment groups at 
the Day 90 visit in change from baseline in percentage of recognized words. 
Additional details on the computation, analysis, and sensitivity analyses for the key 
secondary efficacy endpoint can be found in Section 7.4.2. 
7.2.3. Other Key Secondary Efficacy Endpoints 
Variables: PGI-C hearing loss scale response and PGI-C daily impacts scale response at Day 
90. 
Population: The analysis population will be the FAS. 
Intercurrent events: Subjects who do not have these assessments completed at Day 90 will 
be assumed missing at random, and no responses will be imputed. 
Population-level summary: Comparison of mean difference between treatment groups at 
the Day 90 visit. 
Additional details on the computation, analysis, and sensitivity analyses for these secondary 
efficacy endpoints can be found in Section 7.5.2 and 7.5.4. 
 
Word Recognition in Quiet: Maryland CNC and W-22 
Word recognition will be measured separately with recorded Maryland CNC and W-22 word 
lists.  Both tests will consist of 50 words presented to the subject and the percentage of 
words correctly identified will be recorded separately for each test.  If all 50 words are not 
assessed in an ear, the WR test score will not be calculated for that ear. 


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Word recognition will be performed at the Screening/Start of Lead-In, Lead-In, End of Lead-
In/Treatment, Day 30, Day 60, Day 90, Day 180, and Day 270 visits.  For Screening/Start of 
Lead-In, testing will be performed at 30 dB sensation level (SL) regarding the pure tone 
average of 0.5, 1, and 2 kHz at Screening/Start of Lead-In.  For Lead-In and all subsequent 
visits (End of Lead-In/Treatment, Day 30, Day 60, Day 90, Day 180, and Day 270) testing will 
be performed at 30 dB SL regarding the pure tone average of 0.5, 1, and 2 kHz at Lead-In.  If 
30 dB SL is at an uncomfortable listening level for the participant, testing will be performed 
at the participant’s most comfortable listening level.  Word lists will be rotated at each visit.   
For speech perception, WR in Quiet (Maryland CNC) will be used for the primary analyses. 
All analyses will be repeated using WR in Quiet (W-22) for supplementary analyses. 
Word Recognition in Quiet results will be presented in a data listing for subjects in the FAS.   
7.3.1. Improvement from baseline above Carney-Schlauch upper confidence limits   
• 
Computation of the Endpoint 
Improvement is defined as an increase in post-baseline WR score above the Carney-
Schlauch (2007) 95% upper limit based on baseline (average of Lead-In and End of Lead-
In/Treatment scores) assessment.  Subjects with improvement will be computed as 1, and 
as 0, otherwise for each visit and by ear (left, right).  Missing values will be imputed using 
LOCF.  Left and right ears will be mapped to treated and untreated for analysis.  The 
incidence of WR improvement will be computed as the proportion of subjects who have a 
WR in Quiet improvement. 
The Carney-Schlauch 95% confidence intervals (2007) reported in Section 14.2 will be used 
directly and not computed. 
• 
Analysis of the Endpoint 
The incidence of WR improvement will be summarized descriptively for treated and 
untreated ears separately by visit and treatment group for subjects in the FAS. 
The primary efficacy analysis will be conducted for the incidence of WR improvement at Day 
90 in treated ears for subjects in the FAS using a 1 degree of freedom Chi-Square test for a 
single 2x2 table without stratification.  Although the randomization was stratified by Visit 2 
WR Maryland CNC score (>= 28% vs < 28%) and laterality of hearing loss (bilateral vs 
unilateral), the randomization was done to maintain balance among the treatment groups 
across the randomization factors to help interpretation of the results rather than any 
prognostic status.  As such, it is believed the unstratified primary analysis will be more 
straightforward in interpretation, especially if some of the sample sizes in individual strata 
are small. 
• 
Sensitivity Analyses of the Endpoint 


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The Chi-Square test will be repeated using treated ears for subjects in the PPAS.  
Additionally, the Chi-Square test will be performed with improvement being defined as an 
increase in post-baseline WR score above the 90%, 85%, and 80% upper limit based on 
baseline assessment using Carney-Schlauch method for treated ears. The 90%, 85%, and 
80% upper limits will be determined based on simulation using the same approach used to 
compute the 95% limits described by Carney-Schlauch (2007). 
A two-sided 95% CI for the odds ratio for the incidence of improvement between the 
treatment groups for treated ears will also be computed using the Cochran-Mantel-
Haenszel (CMH) chi-square test, adjusted for the randomization stratification factors.  
A GEE model will be utilized to estimate an odds ratio and its associated 95% CI between 
the treatment groups at each visit for treated ears.  With the binomial distribution 
assumption and logit link, the model will include fixed effects of treatment group, visit, 
interaction between visit and treatment group, baseline WR score, interaction between 
baseline WR score and visit, and randomization stratification factors. The model will utilize 
an unstructured working correlation matrix.  If the model fails to converge, the 
randomization stratification factors will be excluded from the model.  The odds ratios along 
with 95% CIs and p-values between treatment groups will be reported by visit.   
An analysis will be conducted for the incidence of WR improvement at Day 90 using the 
DerSimonian and Laird method.  In treated ears only for subjects in the FAS, a 2-sided 95% 
CI for the difference between treatment groups in incidence of WR improvement at Day 90 
will be computed by the method of DerSimonian and Laird, using a fixed-effects model 
(setting the variance in treatment effects, ∆2, equal to zero), adjusting for the 
randomization stratification factors.  The weighted difference in the incidence of 
improvement, 95% CI, and p-value can be determined by the following formula: 
𝜃̂ =
∑
𝑤𝑖𝜃̂𝑖
4
𝑖=1
∑
𝑤𝑖
4
𝑖=1
~𝑁(𝜃, 1/ ∑𝑤𝑖
4
𝑖=1
) 
Where 𝜃̂𝑖 is the difference in the incidence of improvement at Day 90 of the ith stratum and 
𝑤𝑖= 1/var(𝜃̂𝑖).  The DerSimonian and Laird methodology is consistent with inverse 
variance weighting methodology.  If the analysis cannot be performed using both 
randomization factors (4 strata) due to no variation within 1 or more randomization strata, 
the analysis will be performed using the single randomization factor (2 strata) that produces 
more similarly sized strata.  Scatter plots displaying the percentage of recognized words 
(Section 7.3.2) at baseline versus post-baseline, along with the Carney-Schlauch confidence 
intervals, will be generated for each visit separately for treated and untreated ears. 
7.3.2. Percentage of recognized words 
• 
Computation of the Endpoint 


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The percentage of recognized words will be computed by visit as the number of recognized 
words divided by 50 and multiplied by 100, separately for each ear.  Missing data will not be 
imputed. 
• 
Analysis of the Endpoint 
The percentage of recognized words will be summarized descriptively for treated and 
untreated ears separately by visit and treatment group for subjects in the FAS. 
7.3.3. Change from baseline in percentage of recognized words 
• 
Computation of the Endpoint 
The change from baseline to each post-baseline visit in percentage of recognized words will 
be computed as 100*[(w/50) – (b/50)] where “w” is the frequency of words recognized at 
the post-baseline visit and “b” is the frequency of words recognized at baseline. 
• 
Analysis of the Endpoint 
The change from baseline in percentage of recognized words will be summarized 
descriptively for treated and untreated ears separately by visit and treatment group for 
subjects in the FAS. 
A Line graph (with standard error bars) will be created for the observed mean change from 
baseline in percentage of recognized words for treated ears by post-baseline visit and 
treatment group for subjects in the FAS.  Post-baseline visit will be plotted on the x-axis and 
the observed mean change from baseline in percentage of recognized words for treated 
ears will be plotted on the y-axis.  Each treatment group will be presented as a separate line 
in the plot.   
7.3.4. Improvement ≥10% (5 words) from baseline in percentage of recognized words  
• 
Computation of the Endpoint 
Improvement ≥10% in percentage of recognized words at each post-baseline visit for 
treated ears will be computed as 1 if the change from baseline in percentage of recognized 
words ≥10% and as 0, otherwise where change from baseline is non-missing.  Missing data 
will not be imputed. 
• 
Analysis of the Endpoint 
The incidence of improvement ≥10% in percentage of recognized words at each post-
baseline visit for treated ears will be summarized using frequencies and percentages by visit 
and treatment group for subjects in the FAS. 
In treated ears only for subjects in the FAS, the GEE model described in Section 7.3.1 will be 
repeated for improvement ≥10% in percentage of recognized words.  


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7.3.5. Change from Baseline in Arcsine-transformed WR  
• 
Computation of the Endpoint 
The arcsine-transformed WR (Studebaker, 1985) will be computed by visit for treated ears 
as  
𝑅= 𝑎𝑟𝑐𝑠𝑖𝑛√𝑤(𝑛+ 1)
⁄
+ 𝑎𝑟𝑐𝑠𝑖𝑛√(𝑤+ 1) (𝑛+ 1)
⁄
 
where “R” is the transformed radians, “w” is the frequency of words recognized, and “n” is 
the number of words in the word test (n = 50 for WR assessments).  Missing data will not be 
imputed. 
Change from baseline to each post-baseline visit in arcsine-transformed WR will be 
computed as R post-baseline – R at baseline. 
• 
Analysis of the Endpoint 
In treated ears only for subjects in the FAS, post-baseline treatment group comparisons 
through day 90 will be performed using a MMRM model for change from baseline in 
arcsine-transformed WR with fixed covariates for treatment group, visit, interaction 
between visit and treatment group, baseline WR score, the interaction between baseline 
WR score and visit, and randomization stratification factors. If the model fails to converge, 
the randomization stratification factors will be removed from the model.  An unstructured 
covariance will be used to model the within-subject errors. Linear contrasts will be 
constructed to estimate the treatment effect for the mean change from baseline in arcsine-
transformed WR by visit (e.g., estimated Least squares (LS) Mean differences by visit). 
A Line graph (with standard error bars) will be created for the adjusted (least squares) mean 
change from baseline in arcsine-transformed WR for treated ears by post-baseline visits 
through day 90 and treatment group for subjects in the FAS.  Post-baseline visit will be 
plotted on the x-axis and the adjusted (least squares) mean change from baseline in arcsine-
transformed WR for treated ears will be plotted on the y-axis.  Each treatment group will be 
presented as a separate line in the plot.   
7.3.6. Improvement from baseline above Thornton-Raffin upper confidence limits 
• 
Computation of the Endpoint 
Improvement is defined as an increase in post-baseline WR score above the Thornton-Raffin 
(1978) 95% upper limit (Section 14.1) based on baseline (average of Lead-In and End of 
Lead-In/Treatment scores) assessment.  Subjects with improvement will be computed as 1, 
and as 0, otherwise for each visit and by ear (left, right).  Missing data will not be imputed.  
Left and right ears will be mapped to treated and untreated for analysis.  The incidence of 
WR improvement will be computed as proportion of subjects who have a WR in Quiet 
improvement based on the Thornton-Raffin confidence limits. 


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The Thornton-Raffin 95% confidence intervals reported in Section 14.1 will be used directly 
and not computed. 
• 
Analysis of the Endpoint 
The incidence of WR improvement will be summarized descriptively for treated and 
untreated ears separately by visit and treatment group for subjects in the FAS.  
Scatter plots displaying the percentage of recognized words (Section 7.3.2) at baseline 
versus those at post-baseline, along with the Thornton-Raffin confidence intervals, will be 
generated for each visit separately for treated and untreated ears. 
 
Words-In-Noise 
The WIN test, using the NU-6 list, was developed as an instrument to quantify the ability of 
listeners to understand monosyllabic words in background noise using multitalker babble 
(Wilson, 2003).  Materials are recorded at 7 signal-to-noise ratios (0, 4, 8, 12, 16, 20, 24 dB) 
that are presented in a descending manner.  This test is conducted by presenting 2 lists of 
35 recorded words (70 total) to each ear.  The number of and percentage of correctly 
recognized words at each signal-to-noise ratio will be recorded separately by ear.  The 
overall WIN score will be recorded as the percentage of correctly recognized words across 
all signal-to-noise ratios by visit and ear.  If all 70 words are not assessed in each ear, the 
WIN test score will not be calculated.  This test will occur at the Screening/Start of Lead-In, 
Lead-In, End of Lead-In/Treatment, Day 30, Day 60, Day 90, Day 180, and Day 270 visits.  
Testing will be performed at 80 dB sound pressure level (SPL).  Word lists will be rotated at 
each visit.   
WIN results will be presented in a data listing for the FAS.   
7.4.1. Percentage of recognized words 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the number of words classified as correctly recognized 
at each signal-to-noise ratio will be recorded.  The overall WIN score will also be computed 
as the number of words correctly recognized across all signal-to-noise ratios.  Left and right 
ears will be mapped to treated and untreated for analysis.  The percentage of recognized 
words will be computed for each signal-to-noise ratio by visit as the number of recognized 
words divided by 10 and multiplied by 100, separately for each ear.  The percentage of 
recognized words overall by visit will be computed as the number of recognized words 
divided by 70 and multiplied by 100, separately for each ear. 
• 
Analysis of the Endpoint 
7.4.2. The percentage of recognized words at each signal-to-noise ratio and overall 
will be summarized descriptively for treated and untreated ears separately by visit 


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and treatment group for subjects in the FAS.Change from baseline in percentage of 
recognized words 
• 
Computation of the Endpoint 
The change from baseline to each post-baseline visit in percentage of recognized words at 
each signal-to-noise ratio will be computed as 100*[(w/10) – (b/10)] where “w” is the 
frequency of words recognized at the post-baseline visit and “b” is the frequency of words 
recognized at baseline.  The change from baseline to each post-baseline visit in percentage 
of recognized words overall will be computed using the same algorithm, but with 70 words 
in the denominator instead of 10.  Missing data will not be imputed. 
• 
Analysis of the Endpoint 
The change from baseline in percentage of recognized words at each signal-to-noise ratio 
and overall will be summarized descriptively for treated and untreated ears separately by 
visit and treatment group for subjects in the FAS. 
The primary analysis for the key secondary endpoint, WIN improvement based on mean 
change from baseline in percentage of recognized words overall at Day 90, will be based on 
the following MMRM model.  In treated ears only for subjects in the FAS, post-baseline 
treatment group comparisons through day 90 will be performed using a MMRM model for 
change from baseline in percentage of recognized words overall with fixed covariates for 
treatment group, visit, interaction between visit and treatment group, baseline WIN score 
overall, the interaction between baseline WIN score overall and visit, and randomization 
stratification factors. If the model fails to converge, the randomization stratification factors 
will be removed from the model.  An unstructured covariance will be used to model the 
within-subject errors. Linear contrasts will be constructed to estimate the treatment effect 
for the mean change from baseline in percentage of recognized words overall by visit (e.g., 
estimated Least squares (LS) Mean differences by visit). 
Line graphs (with standard error bars) will be created for the adjusted (least squares) mean 
change from baseline in percentage of recognized words overall estimates for treated ears 
by post-baseline visit and treatment group for subjects in the FAS.  Post-baseline visit will be 
plotted on the x-axis and the adjusted (least squares) mean change from baseline in 
percentage of recognized words overall estimates for treated ears will be plotted on the y-
axis.  Each treatment group will be presented as a separate line in the plot.   
• 
Sensitivity Analyses of the Endpoint 
The MMRM will be repeated using treated ears for subjects in the PPAS.   
7.4.3. Improvement ≥10% (7 words) from baseline in percentage of recognized words 
overall 
• 
Computation of the Endpoint 


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Improvement ≥10% from baseline in percentage of recognized words overall at each post-
baseline visit for treated ears will be computed as 1 if the change from baseline in 
percentage of recognized words overall ≥10% and as 0, otherwise where the change is not 
missing.  Missing data will not be imputed.  Improvement ≥10% (7 words) from baseline in 
percentage of recognized words will not be computed for each signal-to-noise ratio. 
• 
Analysis of the Endpoint 
The incidence of improvement ≥10% from baseline in percentage of recognized words 
overall at each post-baseline visit for treated ears will be summarized using frequencies and 
percentages by visit and treatment group for subjects in the FAS. 
In treated ears only for subjects in the FAS, post-baseline treatment group comparisons for 
incidence of improvement ≥10% from baseline in percentage of recognized words overall 
will be analyzed using a GEE model.  With the binomial distribution assumption and logit 
link, the model will include fixed effects of treatment group, visit, interaction between visit 
and treatment group, baseline WIN score, interaction between baseline WIN score and visit, 
and randomization stratification factors.  If the model fails to converge, the randomization 
stratification factors will be excluded from the model.  The odds ratios along with 95% CIs 
and p-values between treatment groups will be reported by visit.   
7.4.4. Spearman-Kärber estimates of dB thresholds for word recognition (25th, 50th 
(median) and 75th percentiles) 
• 
Computation of the Endpoint 
The Spearman-Kärber estimates (25th, 50th (median) and 75th percentiles) of dB thresholds 
for word recognition (Wilson et al., 1973) at each visit for treated ears will be computed as  
𝑑𝐵= 𝑖+ (𝑝𝑐∗𝑑) −(𝑑∗𝑤)/𝑐 
 
where “pc” is the percentile of interest, “i” is the initial dB presentation level (fixed at 24), 
“d” is the attenuation dB step size (fixed at 4), “c” is the number of words per decrement 
(fixed at 10), and “w” is the frequency of words recognized overall at each visit. Missing 
data will not be imputed. 
• 
Analysis of the Endpoint 
Spearman-Kärber estimates (25th, 50th (median) and 75th percentiles) will be summarized 
descriptively for treated ears by visit and treatment group for subjects in the FAS. 
For each post-baseline visit, scatter plots of the median Spearman-Kärber estimates at 
baseline versus the post-baseline visit will be produced for treated ears by treatment group 
for subjects in the FAS.  


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7.4.5. Change from baseline in median Spearman-Kärber estimates 
• 
Computation of the Endpoint 
Change from baseline to each post-baseline visit in median Spearman-Kärber estimates will 
be computed as dB post-baseline – dB at baseline.  Missing data will not be imputed. 
• 
Analysis of the Endpoint 
The change from baseline in median Spearman-Kärber estimates will be summarized 
descriptively for treated ears by visit and treatment group for subjects in the FAS. 
In treated ears only for subjects in the FAS, post-baseline treatment group comparisons 
through day 90 for the change from baseline in median Spearman-Kärber estimate will be 
analyzed using a MMRM model. The model will include fixed covariates of treatment group, 
visit, interaction between visit and treatment group, baseline median Spearman-Kärber 
estimate, interaction between baseline median Spearman-Kärber estimate and visit, and 
randomization stratification factors.  If the model fails to converge, the randomization 
stratification factors will be dropped from the model. An unstructured covariance will be 
used to model the within-subject errors.  Linear contrasts will be constructed to estimate 
the treatment effect on the mean change from baseline in median Spearsman-Kärber 
estimate by visit (e.g., estimated LS Mean differences by visit).   
Line graphs (with standard error bars) will be created for the adjusted (least squares) mean 
change from baseline in median Spearman-Kärber estimates for treated ears by post-
baseline visit and treatment group for subjects in the FAS.  Post-baseline visit will be plotted 
on the x-axis and the adjusted (least squares) mean change from baseline in median 
Spearman-Kärber estimates for treated ears will be plotted on the y-axis.  Each treatment 
group will be presented as a separate line in the plot.   
7.4.6. Improvement ≥3 dB shifts in median Spearman-Kärber estimates from baseline 
• 
Computation of the Endpoint 
Improvement ≥ 3 dB shifts in median Spearman-Kärber estimates at each post-baseline visit 
for treated ears will be computed as 1 if the post-baseline Spearman-Kärber estimate – 
baseline Spearman-Kärber estimate is ≥3 and as 0, otherwise where the baseline and post-
baseline Spearman-Kärber estimates are not missing.  Missing data will not be imputed. 
• 
Analysis of the Endpoint 
The incidence of improvement ≥3 dB shifts in median Spearman-Kärber estimates at each 
post-baseline visit for treated ears will be summarized using frequencies and percentages 
by visit and treatment group for subjects in the FAS. 
Post-baseline treatment group comparisons will be performed using the GEE model 
described in Section 7.4.3 for subjects in the FAS. 


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Patient Global Impression of Severity/Patient Global Impression of Change  
PGI-S Hearing Loss Scale, administered at the End of Lead-In/Treatment visit, is a patient 
reported outcome (PRO) measure for subjects to best describe the severity of their hearing 
loss over the past week.   
PGI-C Hearing Loss Scale, administered at the Day 90 visit, is a PRO measure to describe the 
overall change in their hearing loss after study drug administration.   
PGI-S Daily Impacts Scale, administered at the End of Lead-In/Treatment visit, is a PRO 
measure for subjects to best describe the impact of their hearing loss on daily activities.   
PGI-C Daily Impacts Scale, administered at the Day 90 visit, is a PRO measure to describe the 
overall change in the impact of their hearing loss on daily activities since study drug 
administration.   
7.5.1. PGI-S hearing loss scale responses 
• Computation of the Endpoint 
Categories will be assigned to numeric values as follows: “None” = 1, “Mild” = 2, 
“Moderate” = 3, “Severe” = 4, and “Very Severe” = 5. 
• Analysis of the Endpoint 
The number and of percentage of subjects in each category (“None”, “Mild”, “Moderate”, 
“Severe”, “Very Severe”) in PGI-S hearing loss scale will be summarized descriptively by 
treatment group for subjects in the FAS.  The numeric values will be used as a covariate for 
analyzing the PGI-C hearing loss scale. 
7.5.2. PGI-C hearing loss scale responses 
• Computation of the Endpoint 
Categories will be assigned to numeric values as follows: “Much better” = 1, “A little better” 
= 2, “No change” = 3, “A little worse” = 4, and “Much worse” = 5. 
• Analysis of the Endpoint 
The number and of percentage of subjects in each category (“Much better”, “A little 
better”, “No change”, “A little worse”, “Much worse”) in PGI-C hearing loss scale will be 
summarized descriptively by treatment group for subjects in the FAS. 
Additionally, as a key secondary efficacy analysis, the average response in PGI-C hearing loss 
scale, using the assigned numeric values of 1-5, will be compared between treatment 
groups at Day 90.  An ANCOVA model, adjusting for the subject’s baseline PGI-S hearing loss 


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scale category as a quantitative covariate using the assigned numeric values of 1-5, will be 
used.  Subjects with missing data at Day 90 will be excluded from analysis. 
7.5.3. PGI-S daily impacts scale responses 
• Computation of the Endpoint 
Categories will be assigned to numeric values as follows: “None” = 1, “Mild” = 2, 
“Moderate” = 3, “Severe” = 4, and “Very Severe” = 5. 
• Analysis of the Endpoint 
The number and of percentage of subjects in each category (“None”, “Mild”, “Moderate”, 
“Severe”, “Very Severe”) in PGI-S daily impacts scale will be summarized descriptively by 
treatment group for subjects in the FAS.  The numeric values will be used as a covariate for 
analyzing PGI-C daily impacts scale. 
7.5.4. PGI-C daily impacts scale responses 
• Computation of the Endpoint 
Categories will be assigned to numeric values as follows: “Much better” = 1, “A little better” 
= 2, “No change” = 3, “A little worse” = 4, and “Much worse” = 5. 
• Analysis of the Endpoint 
The number and of percentage of subjects in each category (“Much better”, “A little 
better”, “No change”, “A little worse”, “Much worse”) in PGI-C daily impacts will be 
summarized descriptively by treatment group for subjects in the FAS. 
Additionally, as a key secondary efficacy analysis, the average response in PGI-C daily 
impacts scale, using the assigned numeric values of 1-5, will be compared between 
treatment groups at Day 90. An ANCOVA model, adjusting for the subject’s baseline PGI-S 
daily impacts scale category as a quantitative covariate using the assigned numeric values of 
1-5, will be used.  Subjects with missing data at Day 90 will be excluded from analysis. 
 
Pure Tone Audiometry 
Standard Pure Tone Audiometry will be measured to determine a subject’s threshold for 
hearing at various frequencies and will be performed at the Screening/Start of Lead-In, 
Lead-In, End of Lead-In/Treatment, Day 30, Day 60, Day 90, Day 180, and Day 270 visits.  
This will be performed on a calibrated audiometer by a licensed audiologist.  The following 
frequencies will be obtained:  Air: 0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz; Bone: 0.5, 1, 2, 3, and 4 
kHz.  If both masked and unmasked results are available for the same visit, date, ear, and 
frequency, the masked results will be used for analysis.   


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Post-Screening PTA thresholds with missing response values, specifically, no response due 
to a subject’s audiometric threshold greater than the maximum calibrated dB of the 
equipment at a given frequency, will be imputed using the maximum dB for the audiometry 
equipment plus 5 dB.  The value, maximum dB for the audiometry equipment plus 5 dB, was 
collected on the CRF as audiometer upper limit and the result will be classified as no signal 
at that frequency.  Missing response values at Screening will not be imputed and remain 
missing.   
Pure tone audiometry results will be presented in a data listing for the FAS.   
7.6.1. Air and Bone Conduction audiometry dB HL thresholds by frequency  
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the AC and BC audiometry dB HL thresholds will be 
recorded for each frequency (Air:  0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz; Bone:  0.5, 1, 2, 3, and 4 
kHz).  Left and right ears will be mapped to treated and untreated for analysis.  Missing 
values will be imputed per Section 7.6. 
• 
Analysis of the Endpoint 
Separately for AC and BC, the hearing threshold at each frequency will be summarized 
descriptively for treated and untreated ears separately by visit and treatment group for 
subjects in the FAS. 
A line plot of the mean AC audiometry thresholds by frequency will be prepared for treated 
and untreated ears for each treatment group with lines connecting each point. 
7.6.2. Mean overall PTA (AC over 0.5, 1, 2, and 4 kHz) and BCA (BC over 0.5, 1, 2 and 
4 kHz) 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the mean overall PTA will be derived by averaging the 
AC audiometry dB HL thresholds over 0.5, 1, 2, and 4 kHz frequencies.  Left and right ears 
will be mapped to treated and untreated for analysis.  Missing values will be imputed per 
Section 7.6.  The BCA will be derived using the same approach.  If hearing thresholds are 
missing for any of the 0.5, 1, 2, and 4 kHz frequencies (after imputation of records with 
classification of “NR”), PTA or BCA will be missing, respectively. 
• 
Analysis of the Endpoint 
The mean overall PTA will be summarized descriptively for treated ears by visit and 
treatment group for subjects in the FAS.  The summary will be repeated for BCA. 


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7.6.3. AC and BC improvement ≥10 and ≥15 dB HL from baseline, separately, for two 
contiguous frequencies 
• 
Computation of the Endpoint 
AC improvement ≥10 dB HL from baseline for two contiguous frequencies at each post-
baseline visit for treated ears will be computed as 1 if the post-baseline threshold – baseline 
threshold is ≥10 dB HL for two contiguous frequencies and as 0, otherwise.  The endpoint 
will utilize imputed values per Section 7.6.  The AC improvement ≥15 dB HL, BC 
improvement ≥10 dB HL, and BC improvement ≥15 dB HL will be derived using the same 
approach. 
• 
Analysis of the Endpoint 
The incidence of AC improvement ≥10 dB HL from baseline for two contiguous frequencies 
at each post-baseline visit for treated ears will be summarized using frequencies and 
percentages by visit and treatment group for subjects in the FAS.  The summary will be 
repeated for ≥15 dB HL and for BC improvement, ≥10 and ≥15 dB HL. 
7.6.4. AC and BC improvement ≥15 and ≥20 dB HL from baseline, separately, for any 
frequency 
• 
Computation of the Endpoint 
AC improvement ≥15 dB HL from baseline for any frequency at each post-baseline visit for 
treated ears will be computed as 1 if the post-baseline threshold – baseline threshold is ≥15 
dB HL for any frequency and as 0, otherwise.  The endpoint will utilize imputed values per 
Section 7.6.  The AC improvement ≥ 20 dB HL, BC improvement ≥15 dB HL, and BC 
improvement ≥20 dB HL will be derived using the same approach. 
• 
Analysis of the Endpoint 
The incidence of AC improvement ≥15 dB HL from baseline for any frequency at each post-
baseline visit for treated ears will be summarized using frequencies and percentages by visit 
and treatment group for subjects in the FAS.  The summary will be repeated for ≥20 dB HL 
and for BC improvement, ≥15 dB HL and ≥20 dB HL. 
7.6.5. AC and BC composite improvement from baseline 
• 
Computation of the Endpoint 
The first AC composite improvement endpoint is defined as the occurrence of either or both 
of the following conditions for a given subject’s ear for a given visit: 1) AC improvement ≥10 
dB HL from baseline for two contiguous frequencies as described in Section 7.6.2 or 2) AC 
improvement ≥15 dB HL from baseline for any frequency as defined in Section 7.6.4.  The 
first BC composite improvement endpoint is defined using the same approach.  
The second AC composite improvement endpoint is defined as the occurrence of either or 
both of the following conditions for a given subject’s ear for a given visit: 1) AC 


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improvement ≥15 dB HL from baseline for two contiguous frequencies as described in 
Section 7.6.2 or 2) AC improvement ≥20 dB HL from baseline for any frequency as defined in 
Section 7.6.4.  The second BC composite improvement endpoint is defined using the same 
approach.  
• 
Analysis of the Endpoint 
The incidence of each AC composite improvement endpoint at each post-baseline visit for 
treated ears will be summarized using frequencies and percentages by visit and treatment 
group for subjects in the FAS.  The summary will be repeated for each BC composite 
improvement endpoints. 
In treated ears only for subjects in the FAS, post-baseline treatment group comparisons 
through day 90 for the incidence of AC composite improvement from baseline may be 
analyzed using a GEE model.  With the binomial distribution assumption and logit link, the 
model will include fixed effects of treatment group, visit, interaction between visit and 
treatment group, baseline mean overall PTA, interaction between baseline mean overall 
PTA and visit, and randomization stratification factors.  If the model fails to converge, the 
randomization stratification factors will be excluded from the model.  The odds ratios along 
with 95% CIs and p-values between treatment groups will be reported by post-baseline visit.  
The analysis may be repeated for the incidence of BC composite improvement using a 
similar approach replacing the baseline mean overall PTA with the baseline mean overall 
BCA. 
7.6.6. Change from baseline in Mean overall PTA and BCA  
• 
Computation of the Endpoint 
Using the mean overall PTA from Section 7.6.2, change from baseline in PTA will be 
computed as w – b where “w” is the post-baseline PTA and “b” is the PTA at baseline for 
each visit by ear (left, right).  Left and right ears will be mapped to treated and untreated for 
analysis.  The change from baseline in BCA will be derived using the same approach.  
• 
Analysis of the Endpoint 
The change from baseline in mean overall PTA will be summarized descriptively for treated 
ears by visit and treatment group for subjects in the FAS.  The summary will be repeated for 
BCA. 
7.6.7. Shifts from baseline in Mean overall PTA Categories 
• 
Computation of the Endpoint 
Mean overall PTA for each ear will be categorized into <26, 26-40, 41-55, 56-70, and >70 for 
each visit and shifts from baseline to each post-baseline visit will be computed (e.g., 26-40 
to 41-55). 
• 
Analysis of the Endpoint 


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The distribution in the shift in mean overall PTA categories for treated ears from baseline to 
each post-baseline visit will be summarized using frequencies and percentages by visit and 
treatment group for subjects in the FAS.   
7.6.8. Mean low AC dB HL threshold (AC over 0.25, 0.5, 1, and 2 kHz) 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the mean low AC dB HL threshold will be derived by 
averaging the AC audiometry dB HL thresholds over 0.25, 0.5, 1, and 2 kHz frequencies.  Left 
and right ears will be mapped to treated and untreated for analysis.  Missing values will be 
imputed per Section 7.6. 
• 
Analysis of the Endpoint 
The mean low AC dB HL threshold will be summarized descriptively for treated ears by visit 
and treatment group for subjects in the FAS.   
7.6.9. Mean high AC dB HL threshold (AC over 3, 4, 6 and 8 kHz) 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the mean high AC dB HL threshold will be derived by 
averaging the AC audiometry dB HL thresholds over 3, 4, 6 and 8 kHz frequencies.  Left and 
right ears will be mapped to treated and untreated for analysis.  Missing values will be 
imputed per Section 7.6. 
• 
Analysis of the Endpoint 
The mean high AC dB HL threshold will be summarized descriptively for treated ears by visit 
and treatment group for subjects in the FAS.   
7.6.10. Air-Bone Imbalance at 0.5, 1, 2, 3, and 4 kHz 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), Air-Bone imbalance is computed as AC-BC dB HL 
thresholds at 0.5, 1, 2, 3, and 4 kHz.  Left and right ears will be mapped to treated and 
untreated for analysis.  Missing threshold values will be imputed per Section 7.6 prior to the 
derivation. 
• 
Analysis of the Endpoint 
The Air-Bone imbalance at each frequency (0.5, 1, 2, 3, and 4 kHz) will be summarized 
descriptively for treated ears by visit and treatment group for subjects in the FAS. 
7.6.11. Shift from baseline in Air-Bone Imbalance at 0.5, 1, 2, 3, and 4 kHz 
• 
Computation of the Endpoint 


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Air-bone imbalance at each frequency will be categorized into ≤10 dB HL versus >10 dB HL 
for each visit and shifts from baseline to each post-baseline visit will be computed (e.g., ≤10 
dB HL to >10 dB HL). 
• 
Analysis of the Endpoint 
The distribution in the shift in air-bone imbalance for treated ears at each frequency from 
baseline to each post-baseline visit will be summarized using frequencies and percentages 
by visit and treatment group for subjects in the FAS.   
 
Extended High Frequency Audiometry  
Extended high frequency audiometry will be performed to determine a subject’s threshold 
for hearing at frequencies beyond those in standard pure tone audiometry at the 
Screening/Start of Lead-In, Lead-In, End of Lead-In/Treatment, Day 30, Day 60, Day 90, Day 
180, and Day 270 visits.  This will be performed on a calibrated audiometer by a licensed 
audiologist.  The following frequencies will be obtained: Air: 9, 10, 11.2, 12.4, 14, and 16 
kHz.  Because some audiometers use center frequencies that vary by small amounts, sites 
should use the threshold at the closest listed center frequency (e.g., 11 kHz may be used for 
11.2 kHz; 12.5 kHz may be used for 12.4 kHz).  If both masked and unmasked results are 
available for the same visit, date, ear, and frequency, the masked results will be used for 
analysis.   
Post-Screening PTA thresholds with missing response values, specifically, no response due 
to a subject’s audiometric threshold greater than the maximum calibrated dB of the 
equipment at a given frequency, will be imputed using the maximum dB for the audiometry 
equipment plus 5 dB.  The value, maximum dB for the audiometry equipment plus 5 dB, was 
collected on the CRF as audiometer upper limit and the result will be classified as no signal 
at that frequency.  Missing response values at Screening will not be imputed and remain 
missing.   
Extended high frequency audiometry results will be presented in a data listing for subjects 
in the FAS.   
7.7.1. Air audiometry dB HL thresholds by frequency  
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the AC audiometry dB HL thresholds will be recorded for 
each frequency (9, 10, 11.2, 12.4, 14, and 16 kHz).  Left and right ears will be mapped to 
treated and untreated for analysis.  Missing values will be imputed per Section 7.7. 
• 
Analysis of the Endpoint 
Descriptive summaries and the line plot will be performed for AC audiometry dB HL 
thresholds as described in Section 7.6.1. 


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7.7.2. Mean dB HL threshold (AC over 9-16 kHz) 
• 
Computation of the Endpoint 
At each visit and by ear (left, right), the mean dB HL threshold will be derived by averaging 
the AC audiometry dB HL thresholds over 9-16 kHz frequencies.  Left and right ears will be 
mapped to treated and untreated for analysis.  Missing values will be imputed per Section 
7.7. 
• 
Analysis of the Endpoint 
The mean dB HL threshold will be summarized descriptively for treated ears by visit and 
treatment group for subjects in the FAS.  
7.7.3. AC improvement ≥10 dB HL and ≥15 dB HL from baseline, separately, for two 
contiguous frequencies 
• 
Computation of the Endpoint 
AC improvement ≥10 dB HL from baseline for two contiguous frequencies at each post-
baseline visit for treated ears will be computed as 1 if the post-baseline threshold – baseline 
threshold is ≥10 dB HL for two contiguous frequencies and as 0, otherwise.  The endpoint 
will utilize imputed values per Section 7.7.  The AC improvement ≥15 dB HL will be derived 
using the same approach.  
• 
Analysis of the Endpoint 
AC improvement ≥10 dB HL from baseline for two contiguous frequencies will be 
summarized using frequencies and percentages for treated ears by visit and treatment 
group for subjects in the FAS.  The summary will be repeated for AC improvement ≥15 dB 
HL. 
7.7.4. AC improvement ≥15 and ≥20 dB HL from baseline, separately, for any 
frequency 
• 
Computation of the Endpoint 
AC improvement ≥15 dB HL from baseline for any frequency at each post-baseline visit for 
treated ears will be computed as 1 if the post-baseline threshold – baseline threshold is ≥15 
dB HL for any frequency and as 0, otherwise.  The endpoint will utilize imputed values per 
Section 7.7.  The AC improvement ≥20 dB HL will be derived using the same approach. 
• 
Analysis of the Endpoint 
AC improvement ≥15 dB HL from baseline for any frequency will be summarized using 
frequencies and percentages for treated ears by visit and treatment group for subjects in 
the FAS.  The summary will be repeated for AC improvement ≥20 dB HL. 


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7.7.5. AC composite improvement from baseline 
• 
Computation of the Endpoint 
The first AC composite improvement endpoint is defined as the occurrence of either or both 
of the following conditions for a given subject’s ear for a given visit: 1) AC improvement ≥10 
dB HL from baseline for two contiguous frequencies as described in Section 7.7.3  or 2) AC 
improvement ≥15 dB HL from baseline for any frequency as defined in Section 7.7.4.   
The second AC composite improvement endpoint is defined as the occurrence of either or 
both of the following conditions for a given subject’s ear for a given visit: 1) AC 
improvement ≥15 dB HL from baseline for two contiguous frequencies as described in 
Section 7.7.3  or 2) AC improvement ≥20 dB HL from baseline for any frequency as defined 
in Section 7.7.4.   
• 
Analysis of the Endpoint 
The incidence of each AC composite improvement endpoint at each post-baseline visit for 
treated ears will be summarized using frequencies and percentages for treated ears by visit 
and treatment group for subjects in the FAS.   
In treated ears only for subjects in the FAS, post-baseline treatment group comparisons 
through day 90 for incidence of AC composite improvement from baseline may be analyzed 
using a GEE model.  With the binomial distribution assumption and logit link, the model will 
include fixed effects of treatment group, visit, interaction between visit and treatment 
group, baseline mean dB HL threshold (AC over 9-16 kHz), interaction between baseline 
mean dB HL threshold (AC over 9-16 kHz) and visit, and randomization stratification factors.  
If the model fails to converge, the randomization stratification factors will be excluded from 
the model.  The odds ratios along with 95% CIs and p-values between treatment groups will 
be reported by post-baseline visit.   
7.7.6. Shift from baseline of no signal to any signal by frequency 
• 
Computation of the Endpoint 
For each frequency, shifts from no signal to any signal will be computed in subjects in the 
FAS with no signal (missing/imputed hearing response) at baseline in the treated ear.  At 
each post-baseline visit, subjects with any signal (any non-missing/not imputed hearing 
response) in the treated ear will be flagged as 1 and as 0, otherwise.  The endpoint will 
utilize imputed values per Section 7.7. 
• 
Analysis of the Endpoint 
For each frequency, the incidence of shifts from no signal at baseline to any signal at each 
post-baseline visit for treated ears will be summarized using frequencies and percentages 
by visit and treatment group for subjects with no signal at baseline in the FAS. 


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7.7.7. Shift from baseline of no signal to any signal in two contiguous frequencies 
• 
Computation of the Endpoint 
Shifts from no signal in two contiguous frequencies at baseline to any signal in two 
contiguous frequencies at each post-baseline visit will be computed in subjects in the FAS 
with no signal (missing/imputed hearing response) at baseline in the treated ear for any two 
contiguous frequencies.  At each post-baseline visit, subjects with any signal (any non-
missing/not imputed hearing response) in the treated ear at the same two contiguous 
frequencies will be flagged as 1 and as 0, otherwise.  The endpoint will utilize imputed 
values per Section 7.7. 
• 
Analysis of the Endpoint 
The incidence of shifts from no signal in two contiguous frequencies at baseline to any 
signal in two contiguous frequencies at each post-baseline visit for treated ears will be 
summarized using frequencies and percentages by visit and treatment group for subjects in 
the FAS with no signal in any two contiguous frequencies at baseline in the treated ear. 
7.7.8. Shift from baseline of no signal to any signal in three contiguous frequencies 
• 
Computation of the Endpoint 
Shifts from no signal in three contiguous frequencies at baseline to any signal in three 
contiguous frequencies at each post-baseline visit will be computed in subjects in the FAS 
with no signal (missing/imputed hearing response) at baseline in the treated ear for any 
three contiguous frequencies.  At each post-baseline visit, subjects with any signal (any non-
missing/not imputed hearing response) in the treated ear in the same three contiguous 
frequencies will be flagged as 1 and as 0, otherwise.  The endpoint will utilize imputed 
values per Section 7.7. 
• 
Analysis of the Endpoint 
The incidence of shifts from no signal in three contiguous frequencies at baseline to any 
signal in three contiguous frequencies at each post-baseline visit for treated ears will be 
summarized using frequencies and percentages by visit and treatment group for subjects in 
the FAS with no signal in three contiguous frequencies at baseline in the treated ear. 
 
Examination of Subgroups 
Non-stratified treatment differences between treatment groups in the incidence of WR 
Improvement at Day 90, as defined in section 7.3.1, for WR in Quiet improvement 
(Maryland CNC) in the FAS will be summarized within the subgroups listed below along with 
95% CIs in a forest plot.  Missing data will be imputed using LOCF.  In the case a subgroup 
includes less than 20 subjects, the analysis for the given subgroup will not be carried out or 
combining subgroups may be considered.  
• Subject Demographics 


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o Sex 
o Race 
o Ethnicity 
o Age (18 to <35, 35 to <50, 50 to 65) 
• Baseline Disease Characteristics 
o Duration of hearing loss (≤1, >1 to 5, >5 to 10, >10 years from screening) 
o Etiology of hearing loss 
o Individual randomization stratification factors: 
▪  Visit 2 (Day -15) Maryland CNC Word Recognition Score (above or equal 
to 28% words in quiet correct, below 28% words in quiet correct)  
▪ Hearing loss laterality (unilateral, bilateral) 
8. 
SAFETY EVALUATION 
 
Overview of Safety Analysis Methods 
The assessment of safety of single dose of FX-322 or Placebo in subjects with acquired 
sensorineural hearing loss is an objective of this study.  The safety analyses will be 
performed for the SfAS and will include treatment-emergent adverse events (TEAEs), 
serious adverse events (SAEs), vital signs, physical examination, otoscopic examination, 
tympanometry, concomitant medications, and C-SSRS assessments.  Tabular summaries of 
descriptive statistics will be presented for all subjects included in the SfAS and subjects will 
be classified into actual treatment groups.  Where appropriate, the actual treated ear will 
be distinguished from the actual untreated ear.   
Safety data will generally not be imputed, except for partial and missing dates, which will be 
imputed only for defining TEAEs and concomitant medications for analysis and reporting 
purposes.  The detailed imputation rules are specified in section 8.3.1.  Imputed dates will 
not be presented in data listings.  
 
Extent of Exposure 
Exposure will be summarized descriptively by actual treatment group and overall for 
subjects in the SfAS for the following parameters: 
o Number and percentage of subjects receiving study treatment in the study ear 
o Number and percentage of subjects with a full dose administered 
o Number and percentage of subjects receiving incorrect study treatment in the study 
ear to be treated 
o Number and percentage of subjects treated in the non-study ear 
 
An exposure listing will be provided, including all available exposure data. 


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Adverse Events 
All AEs will be recorded from the time of the treatment through the end of the 
observational period.  Adverse events onset or worsening, on or after the study drug 
administration are considered TEAEs.  TEAEs will be tabulated by treatment group for all 
subjects in the SfAS and will be flagged in the “all AEs” listing.  Adverse events will be coded 
using Medical Dictionary for Regulatory Activities (MedDRA).  Any ear-related AE, whether 
occurring in 1 or both ears, will only be counted at most once in subject-level counts per 
system organ class (SOC) and preferred term (PT). 
The following analyses will be conducted: 
o Overall summary of TEAEs 
o Overall summary of TEAEs by SOC and PT 
o Overall summary of TEAEs presented by maximum severity (Mild, Moderate, or 
Severe), by SOC and PT 
o Overall summary of TEAEs presented by maximum relationship (Related, Possible, 
Unlikely or Not Related) to study drug, by SOC and PT 
 
For ear-related TEAE analyses where the ear is the unit of analysis, an event will be 
attributed its occurrence to either the treated ear, the untreated ear, or both ears.  The ear-
related TEAE analyses will be conducted by treatment group for the following: 
o Overall summary of ear-related TEAEs presented by ear attribution, by SOC and PT    
o Overall summary of ear-related TEAEs presented by ear attribution, by phase (up to 
Day 30, Day 30-90, and after Day 90), SOC and PT 
8.3.1. Imputation Rules for Missing AE Date/Time of Onset/Resolution 
Partial dates will be imputed for the purposes of defining TEAEs as follows:  
• For a missing start day where the month and year are present, the start day will be 
set to the first day of the month, unless the following two conditions are met: 
1. the first day of the month is before the date of administration of study drug and 
the month and year are the same as the month and year of the date of 
administration of study drug, and  
2. the end date is on or after the date of administration of study drug or the end 
date is completely missing.  
If the two above conditions are met, the start day will be set to the day of 
administration of study drug.  
• For a missing start day and month where the year is present, the start day and 
month will be set to January 1st, unless 1) January 1st is before the date of 


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administration of study drug and the year is the same as the year of the date of 
administration of study drug, and 2) the end date is on or after the date of 
administration of study drug or the end date is completely missing, in which case the 
start day and month will be set to that of the date of administration of study drug. 
• For a missing end day where the month and year are present, the end day will be set 
to the last day of the month, unless the month and year are the same as the month 
and year of the last contact date for the subject, in which case the end day will be 
set to that of the subject's last contact date, which is captured in SDTM as 
DM.RFPENDTC (Date/Time of End of Participation) with derivation rule specified in 
the study SDTM specifications.  
• For a missing end day and month where the year is present, the end day and month 
will be set to the subject's last contact date, unless the year of the subject's last 
contact date is greater than the end year, in which case the end day and month will 
be set to December 31st.  
Completely missing dates will be imputed for the purposes of classifying TEAEs as follows:  
• For an entirely missing start date (i.e., day, month, and year are missing), the start 
date will be set to the date of administration of study drug unless the end date is 
prior to the date of administration of study drug, in which case the start date will be 
set to the end date.  
 
Deaths, Serious Adverse Events, and Other Significant Adverse Events 
Treatment-emergent SAEs, TEAEs leading to study withdrawal, and fatal TEAEs will be 
summarized for all subjects in the SfAS by treatment group for the following: 
o Overall summary of SAE by SOC and PT 
o Overall summary of TEAE leading to study withdrawal by SOC and PT 
o Overall summary of fatal TEAE by SOC and PT 
 
SAEs, adverse events leading to study withdrawal, and fatal AEs will be presented in data 
listings for subjects in the SfAS. 
Adverse Events of Special Interest (AESI), defined in the Protocol, will be summarized by 
treatment group, SOC, and PT for all subjects in the SfAS.  AESIs that occur after study 
treatment injection will be presented in a data listing for subjects in the SfAS. 
 
Urine Pregnancy Tests 
Urine pregnancy test results will be included in a listing for subjects in the SfAS. 


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Vital Signs, Physical Findings, and Other Observations Related to Safety 
8.6.1. Vital Signs 
Vital signs collected on the CRF will be provided in a listing for subjects in the SfAS.  The 
number and percentage of subjects with clinically notable vital signs as defined below will 
be summarized for post-baseline visits by treatment group for subjects in the SfAS. 
o Systolic blood pressure (BP)  
o ≥180 mmHg and an increase ≥20 mmHg from baseline 
o ≤90 mmHg and a decrease ≥20 mmHg from baseline 
o Diastolic BP   
o ≥105 mmHg and an increase ≥15 mmHg from baseline 
o ≤50 mmHg and a decrease ≥15 mmHg from baseline 
o Heart rate   
o ≥120 bpm with an increase from baseline of ≥15 bpm 
o ≤50 bpm with a decrease from baseline of ≥15 bpm 
o Temperature 
o >38 oC 
o <35 oC 
 
Unscheduled visit and early termination visit data will not be mapped to scheduled visits per 
Section 7.1.2.  Unscheduled visit and early termination visit data will be excluded from 
summary tables and included in listings. 
8.6.2. Physical Examinations 
Physical examination results of Normal, Abnormal and Not Done will be included in listings 
for subjects in the SfAS. 
8.6.3. Otoscopic Examinations 
Otoscopic examinations including external inspection and internal ear examination, will be 
performed at the Screening/Start of Lead-In, End of Lead-In/Treatment, Day 30, Day 60, Day 
90, Day 180, and Day 270 visits.  Otoscopic examination data will be summarized 
descriptively by visit for treated ears by treatment group for subjects in the SfAS.  An 
otoscopic findings shift table for treated ears from baseline to post-baseline visits will also 
be produced by treatment group.  Unscheduled visit and early termination visit data will not 
be mapped to scheduled visits per Section 7.1.2.  Unscheduled visit and early termination 
visit data will be excluded from summary tables and included in listings.   
Otoscopic examination data for both treated and untreated ears, scheduled and 
unscheduled, will be presented in a data listing for subjects in the SfAS. 
8.6.4. Tympanometry 


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Tympanometry will be performed at the Screening/Start of Lead-In, End of Lead-
In/Treatment, Day 30, Day 60, Day 90, Day 180, and Day 270 visits.  Tympanometry data 
including type of tympanogram, ear canal volume, peak admittance, and peak pressure will 
be summarized descriptively for treated ears by visit and treatment group for subjects in 
the SfAS.  A tympanogram classification shift table for treated ears from baseline to post-
baseline visits will also be produced by treatment group.  Unscheduled visit and early 
termination visit data will not be mapped to scheduled visits per Section 7.1.2.  
Unscheduled visit and early termination visit data will be excluded from summary tables 
and included in listings.   
All tympanogram data for both treated and untreated ears, scheduled and unscheduled, will 
be presented in a data listing for subjects in the SfAS. 
8.6.5. Columbia-Suicide Severity Rating Scale Assessments 
The C-SSRS will be completed at the End of Lead-In/Treatment (Baseline/Screening version), 
Day 30, Day 60, Day 90, Day 180, and Day 270 visits.  All suicidal ideation and behavioral 
variables collected will be summarized descriptively for each visit by treatment group for 
subjects in the SfAS.  
All C-SSRS data, scheduled and unscheduled, will be included in data listings for all subjects 
in the SfAS. 
8.6.6. 
Concomitant Medications 
All medications taken within 14 days prior to the Screening/Start of Lead-In visit through the 
end of the follow-up period will be recorded on the Concomitant Medications log.  Prior and 
concomitant medications will be coded using World Health Organization Drug Dictionary 
Enhanced.  
Concomitant medications will include all non-trial medications that are taken on or after 
administration of study drug.  Prior medications will include all non-trial medications that 
are started prior to the day of administration of the study drug.   
The number and percentage of subjects using concomitant medications will be tabulated by 
Anatomical Therapeutic Chemical (ATC) and PT for all subjects in the SfAS by treatment 
group.  For subject-level analysis, a subject will be counted at most once per ATC and per 
PT.   
Prior and concomitant medication data will also be presented in a data listing for subjects in 
the SfAS.  
8.6.6.1. 
Imputation Rules for Missing Concomitant Medication Start/Stop Date 
Partial and completely missing dates will be imputed for the purposes of classifying 
concomitant medications as follows:  


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• Partial dates will be imputed following the same algorithm as for TEAEs.  
• For an entirely missing start date (i.e., day, month, and year are missing), the start 
date will be set to the date of administration of study drug unless the stop date is 
prior to the date of administration of study drug, in which case the start date will be 
set to the stop date.  
9. 
OTHER ANALYSES 
 
Qualitative Questionnaire  
This questionnaire records subject experience 
 
 
9.1.1. Qualitative questionnaire responses 
• 
Analysis of the Endpoint 
Responses to the 
 questionnaire will be summarized with 
frequencies and percentages by item for subjects in the SfAS. 
 
Tinnitus Functional Index 
The Tinnitus Functional Index (Meikle et al 2012) has eight subscales that address the 
intrusiveness of tinnitus, the sense of control the patient has, cognitive interference, sleep 
disturbance, auditory issues, relaxation issues, quality of life, and emotional distress.  The 
subject will report answers to each of the 25 questions using a scale of 0-10.  The TFI will be 
given to the subject to complete at the End of Lead-In/Treatment, Day 90, Day 180, and Day 
270 visits. 
All instrument items, except items 1 and 3, are on an inverted 0-10 scale with 0 being most 
favorable and 10 being least favorable.  Items 1 and 3 are on an inverted 0% to 100% scale 
with 0% being most favorable and 100% being least favorable.  The total score and 
individual subscale scores are the average item response (all subscales consist of 3 or 4 
items) that is then scaled to 0-100.  Detailed scoring instructions are in Section 14.3. 
No imputation will be performed for missing data and Section 14.3 provides further detail 
regarding the impact of missing data on TFI scores.  No sensitivity analyses will be 
performed for the TFI endpoints. 
9.2.1. Mean total score, individual subscale scores, and loudness score 
• 
Computation of the Endpoint 
At each visit, the mean total TFI score and individual subscale scores will be computed per 
Section 14.3.  The loudness score will be the response from item 2. 


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• 
Analysis of the Endpoint 
The mean total TFI score, individual subscale scores, and loudness score will be summarized 
descriptively by visit and treatment group for subjects in the FAS. 
9.2.2. Change from baseline in mean total score, individual subscale scores, and 
loudness score 
• 
Computation of the Endpoint 
The change from baseline to each post-baseline visit will be computed as w – b where “w” is 
the post-baseline score and “b” is the score at baseline. 
• 
Analysis of the Endpoint 
The change from baseline to each post-baseline visit in mean total TFI score, individual 
subscale scores, and loudness score will be summarized descriptively by visit and treatment 
group for subjects in the FAS. 
 
Research Assessment on 
Quality of Life 
 
is a 
 PRO questionnaire 
 
 
 
 
The subject will complete 
the instrument at the End of Lead-In/Treatment and Day 90 visits. 
 
 
   
9.3.1. Mean total score, mean domain scores, and individual scores 
• 
Computation of the Endpoint 
At each visit, the mean 
 score and mean domain scores will be computed per 
  No computation is required for individual scores.  
• 
Analysis of the Endpoint 
The mean 
 score, mean domain scores, and individual scores will be 
summarized descriptively by visit and treatment group for subjects in the FAS. 
9.3.2. Change from baseline in mean total score, mean domain scores, and individual 
scores 
• 
Computation of the Endpoint 


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Using the mean 
 score, mean domain scores, and individual scores 
 
 
   
• 
Analysis of the Endpoint 
The change from baseline to each post-baseline visit in 
 score, mean 
domain scores, and individual scores will be summarized descriptively by visit and 
treatment group for subjects in the FAS. 
9.3.3. Domain level compliance 
• 
Analysis of the Endpoint 
 
 
 
 
 
 
 
 
 
Speech Reception Threshold (SRT)  
Speech reception threshold is being used as a cross-check measure for the results of pure-
tone audiometry.  The PTA shall be compared to the SRT to alert the tester to possible 
concerns about the accuracy of the pure tone results. Left and right ears will be mapped to 
treated and untreated for analysis.   
The qualitative assessment for whether the SRT results suggest hearing thresholds are 
reliable will be summarized descriptively for treated and untreated ears, separately, for 
subjects in the SfAS.  All data, including the SRT value, will be listed for subjects in the SfAS.  
10. 
INTERIM ANALYSIS AND DATA MONITORING 
The blinded data will be continuously monitored for safety throughout the study.  No interim 
analysis is planned. 
11. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL 
Not applicable. 
12. 
REFERENCES 
Carney, E., & Schlauch, R. S. (2007). Critical Difference Table for Word Recognition Testing 
Derived Using Computer Simulation. Journal of Speech, Language, and Hearing Research, 
50(5), 1203-1209. 


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Causey, G. D., Hood, L. J., Hermanson, C. L., & Bowling, L. S. (1984). The Maryland CNC test: 
normative studies. Audiology, 23(6), 552-568. 
DerSimonian R, Laird N (1986). Meta-analysis in clinical trials. Controlled Clinical Trials, 7, 177-
188. 
FDA (2020). Statistical Considerations for Clinical Trials During the COVID-19 Public Health 
Emergency. Guidance for Industry. Published June 2020. Available from: 
https://www.fda.gov/media/139145/download.  
Studebaker, G. A. (1985). A "rationalized" arcsine transform. Journal of Speech, Language, 
and Hearing Research, 28(3), 455-462. 
Thornton, A. R., & Raffin, M. J. (1978). Speech-discrimination scores modeled as a binomial 
variable. Journal of speech and hearing research, 21(3), 507-518. 
Wilson, R. H., Morgan, D. E., & Dirks, D. D. (1973). A proposed SRT procedure and its 
statistical precedent. Journal of Speech and Hearing Disorders, 38(2), 184-191. 
Wilson RH, Abrams, HB, Pillion AL. (2003). A word-recognition task in multitalker babble 
using a descending presentation mode from 24 dB to 0 dB signal to babble. J Rehabil Res 
Dev. 2003 Jul-Aug; 40(4):321-7. 
 


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13. 
APPENDICES 
 Study Flow Chart 
 
 
 


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 Schedule of Assessments 
Visit  
Screening/ 
Start of 
Lead-In 
Lead-In 
Visit 
End of 
Lead-In/ 
Treatment 
In-Clinic 
Follow-up  
In-Clinic 
Follow up   
In-Clinic 
Follow-up / 
ETe 
In-Clinic 
Observation 
In-Clinic 
Observation Un-scheduled 
Visit 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
UNS 
Assessment/Day 
Day -40 to 
-30 
Visit 1 + 
16 days 
Visit 1 + 35 
days 
Day 30 
Day 60 
Day 90 
Day 180 
Day 270 
 
Visit Window 
N/A 
+/- 2 
days 
+/- 5 days +/- 5 days +/- 5 days +/- 5 days 
+/- 7 days 
+/- 7 days 
N/A 
Informed Consent 
X 
 
 
 
 
 
 
 
 
Inclusion/ Exclusion Criteria 
Xa 
Xa 
Xb 
 
 
 
 
 
 
Eligibility Assessment by EDC 
X 
X 
Xb 
 
 
 
 
 
 
Demographics 
Xa 
 
 
 
 
 
 
 
 
Medical History  
Xa 
Xa 
Xb 
 
 
 
 
 
 
Concomitant Medication  
Xa 
Xa 
Xb, c 
X 
X 
X 
X 
X 
X 
Physical Examination including weight and 
height 
X 
 
 
 
 
 
 
 
Xd 
Vital Signs (body temperature, pulse rate, bp) 
X 
X 
Xb 
X 
X 
X 
 
 
Xd 
Tympanometry  
X 
 
Xb 
X 
X 
X 
X 
X 
Xd 
Standard Pure Tone Audiometry 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 
Speech Reception Threshold (SRT) 
X 
X 
X 
X 
X 
X 
X 
X 
Xd 
Extended High Frequency Audiometry 
X 
X 
 
Xb  
X 
X 
X 
X 
X 
Xd 
Word recognition, quiet (Maryland CNC) 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 
Word recognition (W-22) 
X 
X 
Xb 
X 
X 
X 
X 
X 
Xd 
Words in noise (WIN) 
X 
X 
Xb  
X 
X 
X 
X 
X 
Xd 
Tinnitus Functional Index 
 
 
Xb 
 
 
X 
X 
X 
Xd 
 


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Visit  
Screening/ 
Start of 
Lead-In 
Lead-In 
Visit 
End of 
Lead-In/ 
Treatment 
In-Clinic 
Follow-up  
In-Clinic 
Follow up   
In-Clinic 
Follow-up / 
ETe 
In-Clinic 
Observation 
In-Clinic 
Observation 
Un-scheduled 
Visit 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
UNS 
Assessment/Day 
Day -40 to 
-30 
Visit 1 + 
16 days 
Visit 1 + 35 
days 
Day 30 
Day 60 
Day 90 
Day 180 
Day 270 
 
Visit Window 
N/A 
+/- 2 
days 
+/- 5 days +/- 5 days +/- 5 days +/- 5 days 
+/- 7 days 
+/- 7 days 
N/A 
Qualitative 
Questionnaire 
Columbia Suicide Severity Rating Scale 
 
 
 Xb 
X 
X 
X 
X 
X 
Xd 
PGI-S Hearing Loss Scale 
 
 
Xb 
 
 
 
 
 
 
PGI-S Daily Impacts Scale 
 
 
Xb 
 
 
 
 
 
 
PGI-C Hearing Loss Scale 
 
 
 
 
 
X 
 
 
 
PGI-C Daily Impacts Scale 
 
 
 
 
 
X 
 
 
 
Otoscopy 
X 
 
Xb 
X 
X 
X 
X 
X 
Xd 
Urine Pregnancy Test (women of childbearing 
potential only) 
X 
 
Xb 
 
 
X 
 
 
Xd 
Randomization by EDC 
 
 
Xb 
 
 
 
 
 
 
Study Medication (FX-322 or placebo) 
 
 
X 
 
 
 
 
 
 
Adverse Events 
 
 
Xc 
X 
X 
X 
X 
X 
X 
 
a- Performed during the Screening Visit and again if a subject notes a change in their medical status during the lead in period 
b – Assessment performed prior to injection  
c- Assessments performed after injection 
d- Perform at Investigator Discretion 
e- If a subject discontinues prior to Visit 6 (Day 90), all efforts should be made to complete the Visit 6 (Day 90) In-Clinic Follow-up/Early Termination (ET) Visit as soon as 
possible and, whenever possible, prior to starting any new medication or treatment. 


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14. 
ATTACHMENTS 
 Thornton and Raffin (1978) Word Recognition Scoring Chart 
 


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19 January 2023
 
 
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 Carney-Schlauch (2007) Word Recognition Scoring Chart 
 


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19 January 2023
 
 
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 TFI Calculations 
 


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19 January 2023
 
 
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The possible responses to items except 1 and 3 are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.  The 
possible responses to items 1 and 3 are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 
90%, and 100%.  Prior to computing the TFI total score, the responses for items 1 and 3 are 
transformed from a percentage scale to a 0 to 10 scale, by dividing the values by 10.   
The TFI total score is then defined as a sum of the 25 items, after items 1 and 3 are 
transformed.  The total TFI score is calculated as the sum of the 25 scores, divided by the 
number of non-missing scores, multiplied by 10.  The total score ranges from 0 to 100, with 
higher scores representing greater perceived handicap.   
The TFI total score is not valid if 7 or more items are omitted.  To be valid as a measure of 
tinnitus severity, at least 19 items must be completed. 
TFI Subscales 
The 25 items can also be grouped into 8 subscales: intrusive, sense of control, cognitive, 
sleep, auditory, relaxation, quality of life, and emotional. 
• The intrusive group includes items 1, 2, and 3. 
• The sense of control group includes items 4, 5, and 6. 
• The cognitive group includes items 7, 8, and 9. 
• The sleep group includes items 10, 11, and 12. 
• The auditory group includes items 13, 14, and 15. 
• The relaxation group includes items 16, 17, and 18. 
• The quality of life group includes items 19, 20, 21, and 22. 
• The emotional group includes items 23, 24, and 25. 
The subscale scores will be computed by summing the points for each question included in 
the relevant subscale, dividing by the number of non-missing scores within the subscale, 
multiplied by 10.  Each subscale score has a range of 0 to 100.  Each TFI subscale score is not 
valid if 2 or more items are omitted.  To be valid subscale scores, no more than 1 item can 
be omitted. 
 
 


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Protocol No. FX-322-208 
19 January 2023
 
 
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Calculations 
Research Assessment on 
 Quality of 
Life 
 


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19 January 2023
 
 
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Protocol No. FX-322-208 
19 January 2023
 
 
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