Conditions: Noise-induced Hearing Loss
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 351
Healthy volunteers: yes

Brief summary:
This prospective study is a randomized, double-blind, placebo-controlled Phase 3 clinical trial of oral D-methionine (D-met) to reduce noise-induced hearing loss (NIHL) and tinnitus. The goal of the study is to develop a safe, oral pharmacological agent to augment physical hearing protectors for noise exposures that exceed the protective capabilities of ear plugs and/or muffs. The study population is a cohort of Drill Sergeant (DS) instructor trainees during and 22 days after their 11 day weapons training. The primary objective of this study is to determine the efficacy of D-met in preventing NIHL or reducing tinnitus secondary to a minimum of 500 rounds of M-16 weapons training occurring over an 11 day period.

Detailed description:
Hearing loss can render a soldier less able to detect and identify the enemy, less able to understand commands, particularly in background noise typical on the battlefield, and may permanently reduce quality of life. In some cases, hearing loss may preclude redeployment or result in less optimal job assignment. Currently, no FDA approved pharmacological prevention exists for noise-induced hearing loss (NIHL). We have documented in animal studies that administration of D-methionine (D-met) can reduce or prevent NIHL. We now need to determine if it has similar efficacy in humans. Although we have not yet tested D-met on protection from noise-induced tinnitus in animals, this clinical trial would provide us the opportunity to also test for protection from noise induced tinnitus simultaneously.

Objective Hypotheses:

Primary Hypothesis: Administration of oral D-methionine prior to, during and 4 days post-weapons training will reduce or prevent permanent noise-induced hearing loss.

Secondary Hypothesis: Administration of oral D-methionine prior to, during and 4 days post-weapons training will reduce or prevent noise-induced tinnitus.

Specific Aims:

To determine whether administering oral D-met can prevent permanent NIHL after weapons training. This aim will be addressed by comparing the results of D-met versus placebo administration starting 3 days prior to, during the 11 day period of weapons training (Monday-Friday for first week, Monday-Thursday for second week), and 4 days after for a total of 18 days. Pure tone hearing thresholds will be assessed before and 22 days after completion of weapons training (ie; 18 days after the last day of study drug/placebo administration).

To determine whether administering oral D-met can prevent tinnitus after weapons training. This aim will be addressed by comparing the results of D-met versus placebo administration starting 3 days prior to, during the 11 day period of weapons training (Monday-Friday for first week, Monday-Thursday for second week), and 4 days after for a total of 18 days. Tinnitus questionnaires will be assessed before and 22 days after completion of weapons training (ie; 18 days after the last day of study drug/placebo administration).

To monitor for any potential side effects of D-met in human subjects. This aim will be accomplished by subject query on each day study drug is dispensed (twice daily) and at final study visit, routing of any adverse event reports to study medical personnel, statisticians and to the Food and Drug Administration (FDA).

Arms:
arm 1: Placebo contains sorbitol, orange flavor and purified water; is flavor and color matched to D-Met. arm 2: D-methionine liquid suspension also contains sorbitol, orange flavor and purified water Intervention: Drug: D-methionine, oral liquid suspension.

Interventions: intervention 1: D-methionine intervention 2: Placebo
Intervention descriptions: intervention 1: D-methionine, oral liquid suspension intervention 2: Placebo contains sorbitol, orange flavor and purified water; is flavor and color matched to D-Met.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
D-methionine for Noise-Induced Hearing Loss 
 
 
 
 
 
 
 
 
             
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss (NIHL): A Phase 3 Clinical Trial 
 
 
Sponsored by:  
Southern Illinois University School of Medicine 
 
IND Sponsor: 
Southern Illinois University School of Medicine 
  
IND #115567 
[D-methionine]  
 
 
Project Director: 
Kathleen C. M. Campbell, Ph.D., CCC-A 
Professor and Director of Audiology Research 
Department of Surgery 
Southern Illinois University School of Medicine 
Springfield, IL  62794 
Phone:  217-545-7310 
Fax:  217-545-7771 
E-mail:  kcampbell@siumed.edu 
 
Principal Investigator: 
CPT William P. Grimes, MD 
 Department of Preventive Medicine 
Moncrief Army Community Hospital  
Fort Jackson, South Carolina  
 
Medical Monitor: 
 
COL Mark D. Packer, MD 
Executive Director, DoD/VA Hearing Center of Excellence 
 
Clinical Assistant Professor, UTSA School of Medicine 
 
Chief of Neurotology & Cranial Base Surgery 
 
 
 
Protocol Version 6.2 
23 March 2016 
 
Confidentiality Statement 
This document is confidential and is to be distributed for review only to investigators, potential 
investigators, consultants, study staff, and applicable independent ethics committees or institutional review 
boards.  The contents of this document shall not be disclosed to others without written authorization from 
SIU School of Medicine, unless it is necessary to obtain informed consent from potential study participants. 


 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
ii 
 
Statement of Compliance 
 
The study will be conducted in accordance with the design and specific provisions of this IRB 
approved protocol, in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki, and that are consistent with Good Clinical Practice (GCP) and the 
applicable regulatory requirement(s). 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 IRB, except where necessary to eliminate an immediate hazard(s) 
to the trial participants. The Principal Investigator will promptly report to the IRB and the sponsor 
any changes in research activity and all unanticipated problems involving risk to human subjects, 
or others. 
 
 
 
_____________________________________________________________________ 
Principal Investigator (Printed Name)   
Principal Investigator (Signature) 
 
___________________________ 
Date


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
iii 
 
Investigational Agent: 
The test drug is D-methionine formulated as an oral suspension.   
 
IND Number:  
 
 
115567 
Protocol History 
 
Version #: 
 
 
 6.2 
 
Version Date:  
 
23 March 2016 
 
Version Number 
Amendment Date 
Reason for Amendment 
2.1 
19April2012 
 
2.2 
10August2012 
Ft Jackson requested changes to the protocol 
 
3.0 
05October2012 
Ft Jackson clarifications to the protocol; change 
in personnel 
3.1 
5Nov2012 
Changes requested per DDEAMC IRB 
3.2 
30Nov2012 
Changes requested per DDEAMC IRB 
3.3 
07Mar2013 
Changes requested per 
DDEAMC IRB 
3.4 
01Apr2013 
Changes requested per DDEAMC IRB 
3.5 
01 May2013 
Changes requested per DDEAMC IRB 
4.0 
01 Aug2013 
Changes in personnel 
4.1 
28 Aug 2013 
Principal Investigator Change 
Ft. Jackson clarifications  
4.2 
08 Jan 2014 
Personnel Change for Medical Monitor 
Added On-site Research Monitor 
Changes to inclusion criteria 
4.3 
25 Feb 2014 
Added Section 15, 16 per DDEAMC IRB 
request 
4.4 
27 June 2014 
Fort Jackson requested changes, personnel 
changes, inclusion/exclusion criteria changes 
5.0 
30 March 2015 
Site PI and other personnel changes 
Changes to Screening Questionnaire per DSMC 
5.1 
15 July 2015 
Add additional Associate Investigator, minor 
edits, change of ombudsmen 
6.0 
15 Dec 2015 
Additional Safety Assessments, DSMC Roster 
Changes, Personnel changes, minor edits 
6.1 
17 February 2016 
Site PI Change 
6.2 
23 March 2016 
Formatting adjustments 
Added Personnel 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
iv 
 
Roles and Responsibilities for Clinical Study 
 
Kathleen C.M. Campbell is the Project Director for this clinical study.  Dr. Campbell is Professor of 
Surgery and Director of Audiology Research at Southern Illinois University School of Medicine.  The 
Principal Investigator for this IND application is CPT William P. Grimes, M.D., Department of 
Preventive Medicine at Moncrief Army Community Hospital, Fort Jackson, South Carolina (SC).  Yale 
University is responsible for data management and statistical analyses. The performance site for this 
study is Fort Jackson, SC. 
 
 
NAME AND TITLE 
 
 
ROLE IN 
PROJECT 
RESPONSIBILITIES 
Kathleen C.M. Campbell, 
CCC-A, PhD/SIU School 
of Medicine 
 Project Director 
Responsible for overall study management and 
oversight.  Responsible for selecting qualified 
investigators, providing them with the information 
needed to conduct investigation properly. 
Responsible for ensuring that the investigation is 
conducted according to the signed investigator 
statement of compliance, investigational plan, and 
applicable regulations; ensuring proper monitoring; 
protecting the rights, safety and welfare of subjects; 
and for the control of clinical supplies under 
investigation.  Maintains records of disposition of 
test articles, accurate case histories of subjects, 
progress reports, and assurance of IRB review.  
Responsible for Final Clinical Study Report. 
CPT William P. Grimes, 
MD, Preventive Medicine 
Moncrief Army 
Community Hospital  
Fort Jackson, SC 
Principal 
Investigator  
All investigator responsibilities noted above, 
including conduct of investigational plan, following 
applicable regulations, maintaining accurate case 
histories of subjects. 
Physician responsible for prescribing study drug 
and will provide medical care, if needed. 
CPT Eric Bunnell, AUD, 
Deputy Chief, Fort 
Jackson Army Hearing 
Program, Moncrief Army 
Community Hospital 
Fort Jackson, SC 
Co-Investigator, 
Audiologist 
Responsible for performing audiologic 
examinations. 
CPT Jenny Davis, AUD, 
Ft Jackson Army Hearing 
Program Manager, 
Moncrief Army 
Community Hospital 
Fort Jackson, SC 
Co-Investigator, 
Supervising 
Audiologist 
Responsible for overseeing all testing and for 
maintaining all audiologic data files for all subjects 
enrolled in the study. Assists with on-site study 
planning and coordination. Responsible for 
supervising staff audiologist. 
Demarcus F. Bush, AUD 
Audiologist 
Responsible for performing audiologic 
examinations as needed. 
 
 
 
Joseph Milbrandt, PhD/ 
SIU School of Medicine 
 
Co-Investigator, 
Clinical Trials 
Monitor 
Duties delegated by Project Director, for instance, 
ensuring the investigation is conducted according to 
the signed investigator statement of compliance, 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
v 
 
investigational plan, and applicable regulations; for 
protecting the rights, safety and welfare of subjects; 
and for the control of clinical supplies under 
investigation.  Maintains records of disposition of 
test articles, accurate case histories of subjects, 
progress reports, and assurance of IRB review. 
Carrie Redlich, MD, 
MPH/Yale  
Epidemiologist 
Responsible for design and oversight of data 
management and statistical analyses. Will prepare 
data for progress reports, FDA submission, 
publication and presentation. 
Martin Slade, MPH 
Yale 
Statistician/Data 
Manager 
Responsible for data management and quality.  Will 
conduct statistical analyses. Will oversee 
programming and data entry.   
Meredith Stowe, PhD 
Yale 
Data Programmer 
Responsible for data programming, entry and 
verification. 
Other Personnel 
 
 
 
COL Mark D. Packer, 
USAF, MD 
Medical Monitor 
(off-site) 
Sponsor liaison for study performance site.  Off-site 
medical monitor.  Will provide clinical perspective 
and oversight.  Will coordinate with Project 
Director and PI to conduct appropriate data and 
safety monitoring.  
Daniel Fox, PhD, MPH 
Off-Site Study 
Coordinator 
Off-site study coordination/regulatory support.  
Duties delegated by Clinical Study Manager. 
1) Elizabeth Bullock, RN 
 
2) Shelley Laird, LPN 
(2) On-site Study 
Coordinators 
Responsible for participant orientation, obtaining 
informed consent, clinical assessments, distribution 
of the study drug, distribution/collection of surveys, 
monitoring of side effects for medical referrals.  
Responsibilities also include monitoring drug and 
placebo shipments, tracking distribution of drug 
and placebo to study participants, maintaining files 
and confidentiality of records, and 
scanning/shipping all study data to Yale for 
statistical analyses. On-site study coordination and 
study assessments.   
 
 
 
Lin Wright 
Roosevelt Barnwell 
Ombudsmen 
Will serve as an independent, neutral and impartial 
mediator of the Soldiers in order to ensure that no 
coercion is observed for the recruiting and 
consenting study activities. 
Rick Lampe 
Regulatory, 
Manufacturing,  
Nonclinical and 
Clinical 
Consultant/ 
GCP Auditing 
Responsible for overall communications with FDA 
and assurance that all components are in place for 
investigator-sponsor and ensure all pertinent 
regulations are being met; also responsible for 
ensuring randomization code is properly handled, 
study balance and SOPs, as needed.  Responsible 
for audit of conduct of the study. Will assist with 
Final Clinical Study Report. 
 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
vi 
 
CLINICAL PROTOCOL  
TABLE OF CONTENTS 
 
CLINICAL SYNOPSIS ....................................................................................................................... ix 
STUDY DESIGN SCHEMA................................................................................................................ xi 
LIST OF ABBREVIATIONS ............................................................................................................. xii 
LIST OF DEFINITIONS ................................................................................................................... xiii 
LIST OF FIGURES ............................................................................................................................ xv 
 
1.0 
OBJECTIVES .......................................................................................................................... 1 
 1.1 
Primary Objectives ............................................................................................................... 1 
 
1.1.1 ............................................................................................................................... 1 
 
1.1.2 ............................................................................................................................... 1 
 1.2 
Secondary Objectives ........................................................................................................... 1 
  
 
1.2.1 ............................................................................................................................... 1 
  
 
 
 1.3 
Study Design ........................................................................................................................ 1 
 
2.0 
BACKGROUND ...................................................................................................................... 1 
 2.1 
Justification for the Clinical Trial .......................................................................................... 1 
 2.2 
Noise-Induced Ototoxicity: Cochlear Organ of Corti Effects   ............................................... 2 
 2.3 
Selection of D-methionine as an Otoprotective Agent against NIHL ...................................... 3 
 2.4 
D-methionine Versus Other Otoprotective Agents ................................................................. 4 
  
 
2.4.1 
N-acetylcysteine (NAC) ..................................................................................... 5 
  
 
2.4.2 
Ebselen .............................................................................................................. 5 
  
 
2.4.3 
ACE Magnesium (ACE-Mg)  ............................................................................. 6 
 2.5 
Distribution of D-methionine  ............................................................................................... 6 
 2.6  D-methionine Safety Factors ................................................................................................. 6 
 2.7 
Methionine: Comparison of the D isomer vs. the L isomer .................................................... 7 
  
 
2.7.1  D versus L Methionine Utilization Varies by Species ......................................... 7 
  
 
2.7.2 
D versus L Methionine Excretion Varies by Species ........................................... 9 
  
 
2.7.3 
D versus L Methionine Safety Comparison ...................................................... 10 
  
 
2.7.4 
Potential Risks in Humans................................................................................ 11 
  
 
 
2.7.4.1 
Potential Increase in Homocysteine Levels for L-methionine 
  
 
 
 
Administration, Not Investigated for D-methionine 
  
 
 
 
Administration ........................................................................... 11 
  
 
 
2.7.4.2 
DL Methionine May Exacerbate Schizophrenia .......................... 11 
  
 
 
2.7.4.3 
Potential Antidote if Needed ...................................................... 12 
 
3.0 
SUMMARY OF PRE-CLINICAL AND CLINICAL STUDIES....................................... 12 
 3.1 
Previous Pre-Clinical Studies .............................................................................................. 12 
 3.2 
D-methionine Protection Against Cisplatin-Induced 
  
Hearing Loss in Humans ..................................................................................................... 13 
 3.3 
D-methionine Protection Against Aminoglycoside-Induced  
  
Ototoxicity ......................................................................................................................... 15 
 3.4 
D-methionine Protection From Noise-Induced Hearing Loss ............................................... 19 
 3.5 
D-methionine Affects The Glutathione Pathway 
  
In Response to Noise Exposure ........................................................................................... 26 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
vii 
 
 3.6 
Summary  ........................................................................................................................... 27 
 
4.0  
STUDY PLAN ........................................................................................................................ 28 
 4.1 
Description of Overall Study Design  .................................................................................. 28 
 4.2 
Study Endpoints.................................................................................................................. 28 
  
 
4.2.1 
Primary Endpoints ........................................................................................... 28 
  
 
4.2.2 
Secondary Endpoint ......................................................................................... 28 
 4.3 
Subject Selection ................................................................................................................ 28 
 4.4 
Inclusion Criteria ................................................................................................................ 29 
 4.5 
Exclusion Criteria ............................................................................................................... 29 
 4.6 
Inclusion of Women and Minorities .................................................................................... 30 
 4.7 
Subject Withdrawals ........................................................................................................... 30 
 4.8 
Subject Replacement ........................................................................................................... 31  
 
5.0 
STUDY PROCEDURES ....................................................................................................... 31 
 5.1 
General Guidelines ............................................................................................................. 31 
 5.2 
Recruitment Procedures ...................................................................................................... 31 
 5.3 
Screening ............................................................................................................................ 32
 5.4 
Randomization .................................................................................................................... 32 
 5.5 
Audiological Assessments: Baseline and End of Study  ....................................................... 32 
  
 
5.5.1 
Tinnitus Assessment ........................................................................................ 33 
  
 
5.5.2 
Otoscopy and Tympanometric Screening ......................................................... 33 
  
 
5.5.3 
Pure Tone Air Conduction Threshold/Bone Conduction Testing ....................... 33 
 
6.0  
INTERVENTION .................................................................................................................. 34 
 6.1 
Study Day  1 ...................................................................................................................... 34 
 6.2 
Study Days 2-18 ................................................................................................................. 34 
 6.3 
Study Day 29-30 ................................................................................................................. 35 
 
7.0  
STUDY DRUG ....................................................................................................................... 35 
 7.1 
Formulation, Packaging, and Labeling ................................................................................ 35 
 7.2 
Dispensing Study Drug ....................................................................................................... 35 
 7.3 
Preparation, Administration, and Dosage of Study Drug ...................................................... 36 
 7.4 
Study Drug Storage ............................................................................................................. 36 
 7.5 
Study Product Accountability Procedures ............................................................................ 36 
 7.6 
Assessment of Participant Compliance with Study Drug ..................................................... 37 
 7.7 
Concomitant Medications and Procedures ........................................................................... 37 
 7.8 
Prohibited Medications and Procedures ............................................................................... 37 
 7.9 
Dietary Information (Daily Protein Intake) .......................................................................... 37 
 7.10   D-methionine Antidote ....................................................................................................... 37 
 
8.0  
ASSESSMENTS OF SAFETY ............................................................................................. 37 
 8.1 
Safety Parameters ............................................................................................................... 37 
 8.2 
Definition of an Adverse Event (AE) .................................................................................. 37 
 8.3 
Definition of a Serious Adverse Event (SAE) ...................................................................... 38 
 8.4 
Methods and Timing for Assessing, Recording, and Analyzing 
  
Managing Safety Parameters ............................................................................................... 38 
 8.5 
Specific Serious Adverse Event Requirements .................................................................... 40 
 8.6 
Safety Monitoring ............................................................................................................... 41 
 8.7 
Stopping Rules Based on Adverse Events ........................................................................... 42 
 8.8 
Data Safety Monitoring Committee ..................................................................................... 42 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
viii 
 
 8.9 
Adverse Events (AE) Reporting .......................................................................................... 43 
 8.10 Reporting Requirements to the Human Research Protection Office (HRPO) ........................ 43 
 8.11 Reporting to FDA ............................................................................................................... 44 
 
 9.0 
DATA MANAGEMENT ....................................................................................................... 44 
  9.1 
Data Quality Assurance ...................................................................................................... 44 
 9.2 
Management of Tympanometry Data .................................................................................. 44 
 9.3 
Management of Pure Tone Audiometric Data ...................................................................... 45 
 
10.0 
STATISTICAL CONSIDERATIONS ................................................................................. 45 
 10.1 Sample Size Justification .................................................................................................... 45 
 10.2 Primary Efficacy Analysis .................................................................................................. 45 
 10.3 Interim Analysis ............................................................................................................. 46 
 
11.0   
ETHICS/PROTECTION OF HUMAN SUBJECTS .......................................................... 46 
 11.1 Risks to Human Subjects .................................................................................................... 46 
 11.2 Adequacy of Protection Against Risks ................................................................................ 49 
 11.3 Clinical Trial Registration ................................................................................................... 50 
 
12.0   
SCHEDULE OF ASSESSMENTS/EVENTS ...................................................................... 51 
 
13.0   
STUDY TIMELINE .............................................................................................................. 52 
 
14.0   
BIBLIOGRAPHY .................................................................................................................. 53 
 
15.0 
SIGNATURES………………………………………………………………………………65 
 
  
 
  
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
ix 
 
CLINICAL SYNOPSIS 
TITLE OF STUDY 
Phase 3 Clinical Trials:  D-methionine to Reduce Noise-Induced Hearing Loss 
(NIHL) 
STUDY DESIGN 
Randomized, Double-Blind, Placebo-Controlled Clinical Trial of  
D-methionine to Reduce Noise-Induced Hearing Loss (NIHL) 
INVESTIGATOR/ 
STUDY CENTER 
Project Director: Kathleen C. Campbell, Ph.D., CCC-A 
Professor and Director of Audiology Research 
Southern Illinois University School of Medicine 
Principal Investigator: CPT William P. Grimes, M.D., Preventive Medicine, 
Moncrief Army Community Hospital, Fort Jackson, South Carolina  
COMPOUND 
D-methionine 
ROA 
Oral, Liquid Suspension 
DURATION OF 
ADMINISTRATION 
18 days 
POPULATION 
U.S. Army Soldiers (Drill Sergeant School Candidates) 
PHASE  
Phase 3 
OBJECTIVES 
Primary: (1) To confirm D-methionine safety and tolerability. 
                 (2) To determine the effect of D-methionine on preventing or reducing 
hearing loss due to impulse noise associated with required weapons training.  
Secondary:  (1) To determine the effect of D-methionine on preventing or 
reducing tinnitus due to impulse noise associated with required weapons 
training.  
DESIGN 
This is a randomized, double-blind, placebo-controlled study to evaluate the 
effect of D-methionine on noise-induced permanent hearing loss and/or tinnitus 
after required weapons training.  Subjects will be randomly assigned in a 1:1 
ratio to receive up to 100mg/kg/day D-methionine or placebo fractionated into 
two daily doses. 
PLANNED SAMPLE 
SIZE 
600 U.S. Army Drill Sergeant School Candidates 
DIAGNOSIS AND 
KEY SUBJECT 
SELECTION 
CRITERIA 
Inclusion criteria: 
1.  Male or female 
2.  21 to 45 years of age 
3.  Negative pregnancy test at enrollment and prior to taking study drug  
4.  Willing to use an effective method of birth control during the study (Both 
male and female participants should avoid pregnancy during study) 
5.  Pure tone air conduction threshold average at 0.5, 1 and 2 kHz of no greater 
than 40 dB HL bilaterally with no air bone gaps greater than 10 dB and normal 
otoscopy and tympanometry screens 
6.  Willing to refrain from using supplements containing or derived from 
protein while participating in this study 
7.  Ability to comply with all study requirements  
Exclusion criteria: 
1.  History of allergic or idiosyncratic reaction to  methionine, amino acid 
mixtures, nutritional supplements, egg white, or other proteins or food additives 
2.  Vegetarian (Individual excludes meat and fish from their diet) 
3.  History of chronic balance disorders 
4.  Abnormal otoscopic findings, otologic surgery, autoimmune inner ear 
disease, significant air-bone gaps, abnormal tympanograms or other indication 
of middle-ear abnormality, history of fluctuant hearing or asymmetric hearing 
worse than 25 dB at any frequency in either ear, perilymphatic fistula, tumor of 
the auditory system, or other CNS disorder that is likely to affect hearing 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
x 
 
5.  Treatment with intravenous (IV) antibiotics within the past 6 months  
6.  Any surgical or medical condition which might significantly alter the 
absorption, distribution, metabolism, or excretion of any drug 
7.  History of abnormal kidney function or kidney impairment 
8.  Treatment for alcohol or substance abuse within past 6 months 
9.  Women of childbearing age who are not using effective contraceptive 
methods and who may become pregnant during the course of the study 
10.  Women who are pregnant or breastfeeding. 
11.  National Guardsmen 
12.  History of psychotic schizophrenia 
13.  Exposure within the previous 6 months to systemic ototoxic substances 
including aminoglycosides or vancomycin 
14.  Body weight exceeding 225 pounds 
15.  Renal impairment measured as eGFR < 50 on screening creatinine 
clearance blood draw 
TREATMENTS 
Two doses per day of an orange-flavored oral suspension (D-methionine or 
placebo) starting 3 days prior to weapons training, 11 days during weapons 
training, and 4 days after completion of weapons training. The total daily dose of 
D-methionine is up to100 mg/kg fractionated into 2 doses of 50 mg/kg each. 
MAIN PARAMETERS 
OF EFFICACY 
Primary endpoint is to confirm safety and tolerability of up to 100 mg/kg daily 
given in divided doses approximately 12 hours apart. 
Primary efficacy endpoint is change from baseline in pure-tone threshold as 
measured by absolute change and frequency of significant noise-induced 
threshold shift (STS). 
Secondary efficacy endpoint is change from baseline scores for the tinnitus 
scales for both loudness and annoyance. 
MAIN PARAMETERS 
OF SAFETY 
Adverse event assessments will be made by study coordinator(s) as study drug is 
dispensed (twice daily) and at the end of the study. 
Adverse events will be summarized by treatment group according to the last 
treatment taken before the Adverse Event (AE) began. 
STOPPING RULES 
Adverse event resulting in death, emergency surgery, or permanent and 
irreversible disability, unless determined unrelated to study participation. If an 
unexpected adverse event occurs in more than 3 subjects, then the risk of the 
active drug will be compared against risk of the placebo.  Significantly higher 
rates in the active study group, based on severity of the adverse event, could 
warrant stopping trial. 
DATA MONITORING  
An on-site research monitor and an off-site medical monitor are assigned to this 
protocol. The project director, principal investigator, medical monitors, 
coordinator(s) and data manager of this protocol will schedule regular meetings 
via video or telephone conference to review study progress and safety data. The 
regulatory representative will also attend as needed, via teleconference, to 
determine if there are regulatory concerns or issues to handle based on the 
meeting content and outcome. 
STATISTICAL 
ANALYSIS 
 
To confirm that the total daily dose of D-methionine 100mg/kg fractionated into 
2 doses of 50 mg/kg each is safe and tolerable.  
To determine if there is significantly reduced change in threshold hearing in 
each ear, adjusting for handedness, for the D-methionine group as compared to 
the placebo group at the tested audiometric frequencies. 
To determine if there is a significantly lower rate of STS (in either ear) for the 
D-methionine group compared to the placebo group. 
To determine if there is a significant difference in reported level of tinnitus 
between the D-methionine and the placebo groups. 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
xi 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
xii 
 
List of Abbreviations 
 
AAA  
 
American Academy of Audiology 
ACE-Mg 
 
ACE-Magnesium 
ADR  
 
Adverse Drug Reaction 
AE 
 
 
Adverse Event/Adverse Experience 
ANSI  
 
American National Standards Institute 
CCR  
 
Center for Clinical Research 
CFR  
 
Code of Federal Regulations 
COI  
 
Conflict of Interest 
CONSORT 
 
Consolidated Standards of Reporting Trials 
CRF  
 
Case Report Form 
DDEAMC 
 
Dwight D. Eisenhower Army Medical Center 
D-met 
 
D-methionine 
DFMO 
 
Difluoromethylornithine 
DHHS 
 
Department of Health and Human Services 
DOEHRSHC 
Defense Occupational Environmental Health Readiness System-Hearing 
Conservation 
DSM  
 
Data and Safety Monitoring 
FDA  
 
Food and Drug Administration 
FWA  
 
Federal Wide Assurance 
GCP  
 
Good Clinical Practice 
GSH  
 
Glutathione 
GSSG 
 
Oxidized Glutathione 
HIPAA 
 
Health Insurance Portability and Accountability Act 
IB 
 
 
Investigator’s Brochure 
ICF 
 
 
Informed Consent Form 
IDE  
 
Investigational Device Exemption 
IND  
 
Investigational New Drug 
IRB  
 
Institutional Review Board 
ISM  
 
Independent Safety Monitor 
JC 
 Joint Commission  
MACH 
 
Moncrief Army Community Hospital 
MCRD 
 
Marine Corps Recruit Depot 
N 
 
 
Number (typically refers to participants) 
NAC  
 
N-acetylcysteine 
NB 
 
 
Narrow Band 
NIHL  
 
Noise Induced Hearing Loss 
NCI  
 
National Cancer Institute, NIH 
NIH  
 
National Institutes of Health 
PI 
 
 
Principal Investigator 
PK 
 
 
Pharmacokinetics 
PTA  
 
Pure Tone Average 
QA 
 
 
Quality Assurance 
QC 
 
 
Quality Control 
SAE  
 
Serious Adverse Event 
SOP  
 
Site Operations Manual 
STS  
 
Significant Noise-Induced Threshold Shifts 
WHO  
 
World Health Organization 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
xiii 
 
 
 
Definitions  
 
Air conduction - AC method of delivering acoustic signals through an earphone; COM: bone conduction 
 
Air-bone gaps – ABG; difference in dB between air-conducted and bone-conducted hearing thresholds for a 
given frequency in the same ear, used to describe the magnitude of conductive hearing loss 
 
Bel – unit expressing the intensity of a sound to a reference intensity; intensity in bels is the logarithm (to 
the base 10) of the ratio of power of a sound to that of a reference sound; after Alexander Graham Bell 
 
Bone conduction – BC; method of delivering acoustic signals through vibration of the skull; COM: air 
conduction 
 
Cochlear hearing loss – hearing sensitivity loss due to hair cell damage or other damage to the cochlea 
 
Decibel (dB) –  A unit of measure of Sound Pressure Level (SPL). When used to measure SPL, a dB is 
equal to 20 times the common logarithm of the ratio of the existing sound pressure to a reference sound 
pressure of 20 micropascals 
 
Effective Methods of Birth Control for this Study –Subject “should either abstain from sexual relations or 
practice a method of birth control while taking part in the study.  Except for surgical removal of the uterus, 
birth control methods such as condoms, a diaphragm or cervical cap, birth control pills, IUD, or sperm 
killing products are not totally effective in preventing pregnancy”  
“Male study participants should refrain from fathering babies while enrolled in this study.”  
 
Hertz (HZ) - A unit of measure of frequency, numerically equivalent to cycles per second 
 
High frequency – HF; audiologically, a nonspecific term referring to frequencies of 2000 Hz or higher. 
 
Impulse Noise – A short burst of acoustic energy consisting of either a single impulse or a series of 
impulses. The pressure-time history of a single impulse includes a rise of 40 dB or more in 1 second or 
faster to a peak pressure, followed by a somewhat slower decay of the pressure envelope to ambient 
pressure, both occurring within 1 second. When the intervals between impulses are less than 500 
milliseconds, the noise is considered continuous, except for short bursts of automatic weapons fire which 
are considered “impulse noise”. 
 
Meniere’s disease – idiopathic endolymphatic hydrops, characterized by fluctuating or episodic vertigo, 
hearing loss, tinnitus, and aural fullness 
 
Narrow Band (NB) Noise - Noise in which the acoustic energy is concentrated in a relatively narrow range 
of frequencies 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
xiv 
 
Otoscope – a speculum-like instrument for visual examination of the external auditory meatus and tympanic 
membrane 
 
Otoscopy – inspection of the external auditory meatus and tympanic membrane with an otoscope 
 
Perilymph – cochlear fluid, found in the scala vestibule, scala tympani, and spaces within the organ of 
Corti, which is high in sodium and calcium and has an ionic composition that resembles cerebrospinal fluid 
Perilymphatic fistula – abnormal passageway between the perilymphatic space and the middle ear, resulting 
perilymph leakage at the oval or round window, usually caused by congenital defects or trauma  
 
Potentially Hazardous Noise  - Exposure to steady-state noise having an 8-hour TWA noise level of > 
85dBA, or exposure to impulse/impact noise levels greater than 140 dB peak SPL, regardless of duration 
 
Pure tone –Sound wave having only one frequency of vibration 
 
Pure-tone air-conduction threshold – lowest level at which a pure-tone stimulus, presented through 
earphones is audible 50% of the time 
 
Pure-tone average – PTA; average of hearing sensitivity thresholds to pure-tone signals at 500, 1000 and 
2000 Hz  
 
Pure-tone bone-conduction threshold – lowest level at which a pure-tone stimulus, presented via a vibrating 
oscillator, usually placed on the forehead or mastoid, is audible 50% of the time 
 
Significant Threshold Shift (STS) -  An average change of plus or minus 10 dB at 2000, 3000, and 4000 Hz, 
relative to the reference audiogram, in either ear, without age corrections (military definition)  
 
Sound Pressure level – SPL; magnitude or quality of sound energy relative to a reference pressure, 
0.0002dyne/cm² or 20µPa 
 
Threshold – level at which a stimulus or change in stimulus is just sufficient to produce a sensation or an 
effect that is perceived 50% of the time 
 
Tinnitus – sensation of ringing or other sound in the head, without an external cause 
 
Tympanogram –graph of the middle ear immittance as a function of the amount of air pressure delivered to 
the ear canal.  Its purpose is to assess tympanic membrane and middle ear function 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
xv 
 
List of Figures 
 
Figure 1/2 
Plasma Concentrations of D and L methionine ...................................................... 9 
Figure 3/4 
Effects of experimental diets on the growth (A) and food consumption (B) 
 
of rats .....................................................................................................................11 
Figure 5 
Protection from cisplatin-induced auditory threshold shift in rats.........................12 
Figure 6 
Protection from cisplatin induced cochlear hair cell loss ......................................13 
Figure 7 
Protection from cisplatin induced hearing loss in humans ....................................14 
Figure 8 
Protection from cisplatin induced hearing loss in humans ....................................15 
Figure 9 
Protection from aminoglycoside auditory threshold shift in guinea pigs:  
 
Twice daily D-methionine dosing ..........................................................................16 
Figure 10 
Protection from aminoglycoside auditory threshold shift in guinea pigs:  
 
Twice daily D-methionine dosing (3 kHz) ............................................................17 
Figure 11 
Protection from aminoglycoside auditory threshold shift in guinea pigs:  
 
Twice daily D-methionine dosing (9 kHz) ............................................................18 
Figure 12 
Protection from aminoglycoside auditory threshold shift in guinea pigs:  
 
Twice daily D-methionine dosing (18 kHz)……………………………………...19 
Figure 13 
D-methionine protection from noise-induced threshold shift in the chinchilla .....20 
Figure 14 
D-methionine protection from noise-induced cochlear inner and outer hair  
 
Cell loss in chinchillas ...........................................................................................21 
Figure 15 
Cochlear outer hair cell protection from noise exposure in chinchillas .................22 
Figure 16/17   Outer hair cell protection in individual chinchilla ..................................................23 
Figure 18 
D-Methionine rescue from noise-induced hearing loss: D-methionine  
 
started 1 hour after Noise cessation........................................................................24 
Figure 19 
D-methionine rescue from noise-induced hearing loss at  
 
various time delays ................................................................................................25 
 
Figure 20 
ABR Threshold Shifts 21 days post noise exposure at 2, 4, 6, and 8 kHz ......................26 
 
Figure 21 
Ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) 
 
(nmol/mg) following noise exposure with and without D-methionine ..................27 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
1 
 
1. 0 
OBJECTIVES 
 
1.1   Primary Objectives 
 
1.1.1 To monitor for any potential side effects of D-methionine in human subjects. This aim will be 
accomplished by assessing for side effects each time study drug is dispensed (twice daily by the study 
coordinator(s) and at the final study visit. 
 
1.1.2 To determine whether administering oral D-methionine can prevent permanent noise-
induced hearing loss (NIHL) due to impulse noise associated with required M-16 weapons training. This 
aim will be addressed by comparing the results of D-methionine versus placebo administration starting 3 
days prior to, during the 11 day period of weapons training (Monday-Friday and Monday -Thursday over 
a 2 week period), and 4 days after weapons training for a total of 18 days of dosing.  Pure tone air 
conduction hearing thresholds will be assessed before and 15-16 days after completion of weapons 
training (i.e., 11-15 days after the last day of study drug/placebo administration). 
 
1.2  Secondary Objective 
 
1.2.1 To determine whether administering oral D-methionine can prevent tinnitus after required 
M-16 weapons training. This aim will be addressed by comparing the results of D-methionine versus 
placebo administration starting 3 days prior to, during the 11 day period of weapons training (over a 2 
week period), and 4 days after weapons training for a total of 18 days of dosing.  Tinnitus questionnaires 
will be completed before weapons training and 11-15 days after the last day of study drug/placebo 
administration.  
 
1.3   Study Design 
 
This is a prospective, randomized, double-blind, placebo-controlled study to evaluate the effect of D-
methionine on permanent NIHL after required M-16 weapons training. The study will include 600 U.S. 
Army personnel enrolled in Drill Sergeant Drill Sergeant School (DSS) scheduled to undergo 9 days of 
M-16 weapons training over an 11 day period at U.S. Army Basic Combat Training Center of Excellence, 
Ft Jackson, South Carolina. This study is funded by the Department of Defense and written permission 
has been obtained from Sonya Cable, LTC, SP, Director, Experimentation and Analysis Element, Office 
of Deputy Commanding General, Initial Military Training, Fort Jackson, South Carolina and 
Commandant COL Mark Higdon, MD, of Moncrief Army Medical Center (MACH) and 2nd Commandant 
CSM Michael McCoy, DSS 
 
2.0 
BACKGROUND 
 
2.1  Justification for the Clinical Trial 
 
Although significant progress has been made in developing physical hearing protectors and in controlling 
work-related noise exposure, permanent noise-induced hearing loss (NIHL) still affects at least 10 million 
Americans (Lang 1994; Alberti et al., 1998). Further, harmful levels of occupational noise exposure may 
affect close to 30 million Americans (Rabinowitz, 2000). NIHL is also an international problem. 
According to the World Health Organization, exposure to excessive noise is the major avoidable cause of 
permanent hearing loss worldwide (Smith 1998).  Recreational activity with firearms, amplified music, 
motorcycles, and power tools also expose millions of people to sound capable of producing permanent 
hearing loss (Metternich and Brusis1999; Axelsson et al., 1991; Rabinowitz 2000). Recently, studies have 
shown that even young children suffer from hearing loss after exposure to sudden noise emitted by toy 
pistols and firecrackers (Hellstrom 1992; Segal et al., 2003). 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
2 
 
Noise is defined as a short burst of acoustic energy comprising either a single impulse or a series of 
impulses. The pressure-time history of a single impulse includes a rise of 40 dB or more in 1 second or 
faster to a peak pressure, followed by a somewhat slower decay of the pressure envelope to ambient 
pressure, both occurring within 1 second. When the intervals between impulses are less than 500 
milliseconds, the noise is considered continuous, except for short bursts of automatic weapons fire which 
are considered “impulse noise” (Stach, 2003). 
 
NIHL generally first affects the high frequency range with a characteristic “notch” at approximately 4 
kHz. As the loss progresses, the patient may have difficulty in all listening environments affecting both 
social and work life; sometimes impacting employability. The financial impact to the government is 
significant. The U.S. Veteran’s Administration alone paid approximately $24 billion dollars in hearing 
loss compensation from 1970-1990 (Wolgemuth et al., 1995). Noise-induced tinnitus is less well studied 
but is a frequently reported consequence of noise exposures (Henry et al., 2005). The world-wide social 
and financial impact of military, industrial, and recreational noise exposure is enormous. 
 
For the military particularly, NIHL has high costs not only in financial but in human terms. Military 
efficacy frequently depends on hearing. For the dismounted Soldier, Letowski (2003) described hearing 
as the most important sense for survival. Further, physical hearing protectors such as earmuffs and plugs 
provide insufficient hearing protection for many military exposures. Taggart 2001 reported that 11% of 
marines had permanent hearing loss following recruit training even with all hearing conservation 
protocols in place.  
 
NIHL appears to be a problem for all branches of the military including the U.S. Army (Helfer et al., 
2005) U.S. Navy, (Bohnker et al., 2002a,b, 2003, 2004) U.S. Marines (Taggart 2001, Bohnker et al., 
2002, Barney and Bohnker 2006) and U.S. Air Force (Ritter and Perkins 2001).  Further, NIHL affects 
not only front line military personnel.  In a study of personnel on the flight deck of a Nimitz class aircraft 
carrier, Rovig et al. (2004) reported high levels of NIHL for engineers (27%) and flight personnel (17%) 
compared with administrative personnel (4%). 
 
Currently, no FDA approved pharmacologic prevention exists for NIHL. Animal studies have shown that 
administration of D-methionine can reduce or prevent NIHL (Kopke et al., 2002, Campbell et al., 2007, 
Campbell et al., 2011). The goal of this IND study is to determine if it has similar efficacy in humans.  
Although testing for protection from noise-induced tinnitus in animals has not yet occurred, this clinical 
trial would provide an opportunity to also simultaneously test for protection from noise-induced tinnitus 
in humans. 
 
The purpose of this specific clinical trial is to determine if impulse-induced permanent NIHL and tinnitus can 
be prevented in a large cohort of Army personnel, during their required weapons training as a part of Drill 
Sergeant Instructor Training School at Ft Jackson.  This is an ideal subject population comprising highly 
disciplined and motivated Soldiers who will fire exactly 500 rounds of M-16 weapon fire (156 dB SPL) 
within a 9 day period of weapons training. Soldiers unavoidably develop permanent NIHL during their 
course of training, even with the best of physical hearing protection and the most proactive training in its use. 
Proof of concept data from several studies demonstrates that D-methionine given before and after noise 
exposure in animals can prevent permanent NIHL. This study is designed to document whether or not it 
prevents permanent NIHL in Soldiers.  
 
2.2   Noise-Induced Ototoxicity: Cochlear Organ of Corti Effects   
 
In general, acoustic stimulation deflects the stereocilia on top of the hair cells. When the shear forces 
stretch tip links between adjacent stereocilia, the stereocilias’ mechanoelectrical transduction channels 
can open. This opening causes excitation by ion influx into the cell, thus depolarizing the plasma 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
3 
 
membrane, which in turn causes neurotransmitter release. Shear forces in the opposite direction can close 
the channels (Saunders et al., 1991, Howard et al., 1988, Roberts et al., 1988). Noise exposure can also 
temporarily decrease cochlear microcirculation (Vertes et al., 1979; Axelsson et al., 1981; Quirk et al., 
1992; Henderson and Hamernik, 1995; Miller et al., 1996).    
 
In excessive noise exposure, a variety of cochlear anatomical changes occur. Outer hair cell (OHC) loss 
and, to a lesser extent, inner hair cell loss, predominantly in the basal portion of the cochlea, are well-
established findings (Henderson and Hamernik, 1995). However, some variability across species has been 
reported (Hamernik et al., 1984).  Bohne and Rabbitt (1983) first reported that phalangeal scars will 
eventually replace degenerated hair cells, but 1 - 2 hours after noise exposure, holes in the reticular 
lamina may be present where the hair cells were, possibly allowing endolymph infiltration. They 
hypothesized that subsequent degeneration of supporting cells, nerve fibers and possibly sensory cells 
may be secondary to the damage caused by potassium rich endolymph contaminating the fluid spaces.   
 
In addition to actual loss of cochlear hair cells, more subtle forms of hair cell damage may include 
swelling and vacuolization of the hair cells, swelling of the supporting cells, fractures or discontinuities of 
the stereocilia rootlets, splaying of the stereocilia, deterioration of the stereocilia shafts’ actin crystals and 
loss of tip links (Engstrom et al., 1983; Slepecky 1986; Thorne et al.,1986; Lim 1986; Liberman and 
Dodds 1987; Liberman 1987; Pickles et al., 1987; Raphael and Altschuler, 1992).  Dendritic swelling of 
the afferent fibers beneath the hair cells may also occur (Robertson 1983; Puel et al.,1996).  
 
Noise induces hearing loss by either mechanically over-stimulating the cochlea or by metabolic processes. 
Mechanical damage, in addition to metabolic damage, generally occurs when noise intensity levels 
exceed 125 dB SPL (Henderson and Hamernik, 1995). For noise exposures under 125 dB SPL, damage is 
for the most part secondary to metabolic processes (Henderson and Hamernik, 1995). For impulse noise 
and continuous noise above 125 dB SPL, both metabolic and mechanical damage can occur however 
protective agents have also shown some promise. Partial but not complete protection from PTS and OHC 
loss have been reported for N-acetyl cysteine (NAC) and acetyl-L-carnitine (ALCAR) (Kopke et al., 
2004). However in other studies D-methionine has shown superior protection to NAC for continuous 
noise and equal or better protection than ALCAR (Kopke et al., 2000, 2002; Campbell et al., 2007, 2011).  
 
2.3   Selection of D-methionine as an Otoprotective Agent Against NIHL 
 
D-methionine was first discovered as an otoprotective agent in 1996 in studies investigating cisplatin-
ototoxicity by Dr. Campbell l at Southern Illinois University School of Medicine (Campbell, et al., 1996). 
Since that time D-methionine has been found to protect hearing from a variety of ototoxins, including noise. 
D-methionine protects against cisplatin-induced (Campbell et al.,1996, 1999, 2007; Kopke et al. 1997; Reser 
et al.,1999) carboplatin-induced (Lockwood et al., 2000), aminoglycoside-induced (Sha and Schacht, 2000) 
and noise-induced hearing loss (Coleman et al., 2002 a,b; Kopke et al., 2002; Campbell et al., 2011).  
 
D-methionine protection from NIHL in animals has been confirmed in other independent labs (Samson et al., 
2008; Cheng et al., 2008). However, this would be the first study in humans for D-methionine protection 
from NIHL and cochlear outer hair cell loss.  In studies to date, D-methionine provides virtually complete 
protection from permanent NIHL and cochlear hair cell loss (Kopke et al., 2002, Campbell et al., 2007; ; 
Samson et al., 2008; Cheng et al., 2008; Campbell et al., 2011).  These studies all consistently report 
protection from NIHL. No studies report a lack of D-methionine protection or exacerbation of permanent 
NIHL. Additionally almost complete protection from permanent NIHL in chinchillas has been obtained even 
when D-methionine is first administered up to 7 hours after noise cessation (Campbell et al., 2010).  
Protection from temporary thresholds shift has been variable but has not been as carefully studied. No agent 
has been found to be more effective in animal studies to date.  It has been studied for decades in multiple 
species as a part of nutrition. 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
4 
 
Noise exposure increases cochlear reactive oxygen species (ROS) levels (Ohlemiller et al., 1999), thus 
placing an oxidative challenge on the cochlea.  ROS levels, exceeding the cochlea’s capability to detoxify 
or eliminate them, can cause cochlear damage (Huang et al., 2000; Clerici et al.,1995; Clerici and Yang 
1996; Seidman et al., 1993). D-methionine may provide prophylactic rescue protection from permanent 
noise-induced hearing loss primarily through two putative mechanisms: 1) acting as a free radical 
scavenger and 2) altering glutathione (GSH) levels. Unlike most amino acids, D-methionine is reversibly 
oxidized (Vogt 1995).  
 
Because noise exposure markedly increases ROS formation (Ohlemiller et al., 1999), D-methionine may 
protect against NIHL by serving as a free radical scavenger. Because the nearly 4-fold noise-induced 
cochlear ROS elevation continues for at least 1-2 hours after noise exposure (Ohlemiller, et al., 1999), 
post-exposure administration of a protective-rescue agent (e.g., D-methionine) can be effective.  
Campbell et al., (2007, 2011), demonstrated that D-methionine first administered 1-7 hours and then 
twice a day for an additional 2 days after a 6 hour 4 kHz octave band noise exposure markedly reduced 
PTS measured 3 weeks following the noise exposure in a chinchilla.  Therefore, it is known that D-
methionine rescue can occur. 
 
The glutathione pathway is one of the body’s major detoxification pathways. Methionine may also act by 
increasing intracellular reduced glutathione (GSH) (Lu, 1998) and particularly mitochondrial glutathione 
(Fernandez-Checa et al., 1998). Hyde and Rubel (1995), reported that mitochondrial function appears to 
regulate the probability of cochlear hair cell survival after noise exposure. Because methionine can 
increase not only mitochondrial GSH levels (Lu,1998) but also can prevent the efflux of cellular GSH 
secondary to injury (Ghibelli et al.,1998), methionine administration may increase overall cochlear GSH 
levels. Further studies are currently being conducted to determine if that is the case. Noise exposure does 
alter cochlear GSH and oxidized glutathione (GSSG) levels (Bobbin et al., 1995, Yamasoba et al., 1998a; 
Campbell et al., 2003) and GSH inhibition exacerbates NIHL (Yamasoba et al., 1998b). Consequently, 
using an agent such as D-methionine that can potentially increase cochlear GSH levels may decrease 
noise-induced hearing loss via the GSH pathway.  
 
Therefore, D-methionine may ameliorate NIHL by direct free radical scavenging, increasing cochlear 
glutathione levels or both. Regardless of the mechanism, it is established that D-methionine is an effective 
agent for NIHL prevention and rescue (Coleman et al., 2002a,b; Kopke et al., 2002; Campbell et al., 
2007, 2011; Cheng et al., 2008; Samson et al., 2008). However further studies need to be conducted to 
move towards clinical trials. 
 
2.4  D-methionine Versus Other Otoprotective Agents 
 
D-methionine can be administered orally as an orange flavored suspension for this clinical study, along 
with a flavor matched placebo. The volume per dose is approximately a teaspoonful depending on subject 
weight. This formulation is stable for at least 18 months at up to 40 degrees centigrade. Further, 
packaging will be in individual doses for easy and contamination free distribution. Animal data for 
multiple applications including prevention of NIHL, cisplatin-induced hearing loss, aminoglycoside-
induced hearing loss and radiation-induced oral mucositis (Campbell et al., 1996, Campbell et al., 1999, 
Campbell et al., 2003, Campbell et al., 2007; Vuyyuri, et al., 2008; Campbell et al., 2009; Campbell et al., 
2011) with this formulation exist and have been reported.  
 
As described above, human safety and efficacy data to prevent cisplatin induced hearing loss and 
radiation induced oral mucositis have been obtained through ex-US studies. (Campbell et al., 2009; 
Hamstra et al., 2010). Findings in animals in D-methionine protection against NIHL have been confirmed 
in independent labs (Campbell, et al., 2007;, Samson et al., 2008; Cheng et al., 2008; Campbell et al., 
2010). However, this would be the first study in humans for protection from NIHL. In studies to date, D-


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
5 
 
Methionine provides virtually complete protection from permanent NIHL and cochlear hair cell loss  in 
animals (Kopke et al., 2002; Campbell et al., 2007;; Samson et al., 2008; Cheng et al., 2008 Campbell et 
al., 2011). These results are consistent.  All studies show D-methionine protection from NIHL. No studies 
show a lack of protection or exacerbation of permanent NIHL. Additionally almost complete protection 
from permanent NIHL has been obtained in chinchillas even when it is first administered up to 7 hours 
after noise cessation (Campbell et al., 2011). Protection from temporary thresholds shift has been variable 
but has not been as carefully studied. No agent has been found to be more effective in animal studies to 
date. It has been studied for decades in multiple species because it is a part of nutrition. 
 
2.4.1  N-acetylcysteine (NAC) - NAC has been the most widely studied agent for protection 
from NIHL, but results have been variable. Most studies show at least partial protection from permanent 
NIHL in animals when administered either before or within 24 hours noise exposure (Ohinata 2003; 
Lorito et al., 2008; Coleman et al., 2007; Bielefeld et al., 2007; Fetoni et al., 2009) but other studies have 
shown that NAC provides no protection or even exacerbation of NIHL (Duan et al., 2004; Hamernik et 
al., 2008). NAC appears to work best in combination with other agents.  Kopke et al. (2000, 2001),  
attributed the protection against NIHL to NAC but only found significant protection from NIHL when 
combined with high dose salicylate. However salicylate itself has otoprotective properties (Yu et al., 
1999) and may have contributed to the results observed. Unfortunately, high dose salicylate (aspirin) is 
probably not advisable for military use because it can increase the risk of bleeding. Some studies do show 
partial protection from permanent NIHL using NAC in isolation however (Kopke et al., 2005, 2007). 
 
Three human clinical trials with NAC have been conducted but none showed significant protection from 
NIHL (Toppila et al., 2002; Kramer et al., 2006, and Kopke et al., unpublished data). Toppila et al. (2002, 
unpublished), used 400 mg NAC per day and Kramer et al. (2006) used 900 mg NAC per day in a double 
blind placebo clinical trial of 31 normal hearing subjects before and after 2 hours of night club noise. 
Neither study showed any otoprotection.  Another study using 900 mg NAC administered 3 times per day 
in 566 U.S. Marine recruits at Camp Pendleton exposed to 300 rounds of M-16 weapon fire reported no 
protection from permanent NIHL. The results have not been published but have been publicly presented.  
D-methionine and NAC are among the most widely studied protective agents for NIHL. In comparative 
studies however, thus far D-methionine seems to provide superior otoprotection. Coleman et al., 2002 
reported that pre-administration of a combination of low-dose D-methionine and NAC markedly reduced 
permanent noise-induced threshold shift in chinchillas, but that the protection afforded by the D-
Methionine component alone was similar to the combined administration of D-methionine and NAC. 
However, when the NAC component was delivered alone, no protection was provided.  Kopke et al., 
(2002) reported that both D-methionine and NAC pre- administration (Kopke et al., 2000) could protect 
against NIHL in the chinchilla. However D-methionine provided superior cochlear outer hair cell 
protection.  Over 90% of outer hair cells were preserved with D-methionine protection as compared to 
only 50-60% with NAC/salicylate pre-administration. The reason they administered NAC with salicylate 
is reportedly to improve the stability of NAC, but as reviewed above, the salicylate may have also served 
as the otoprotective agent.  
 
2.4.2   Ebselen - Ebselen, a selenium containing compound, has also shown some efficacy in 
animals and is reportedly approaching clinical trials at Camp Pendleton. However, even with 14 days of 
administration for a single noise exposure the protection from NIHL appears less than in the D-
methionine studies (Kil et al., 2007; Kopke et al., 2002, Campbell et al., 2007, 2011) although noise 
exposure paradigms were not identical. Partial protection from permanent NIHL has been consistently 
observed across studies in the rat and guinea pig (Pourbakht and Yamasoba 2003; Lynch et al., 2004; 
Lynch and Kil 2005) with no studies reporting a lack of protection or exacerbation of NIHL. Only one 
study has been published addressing temporary threshold shift and that study did show significant 
reduction of temporary threshold shift in the guinea pig (Yamasoba et al., 2005.) Ebselen can be 
administered orally and they are using a dry blend capsule for clinical studies.  


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
6 
 
 
2.4.3   ACE Magnesium (ACE Mg) – Ace-Mg (a combination of beta carotene -the precursor to 
vitamin A, plus vitamins C and E, and magnesium) is another otoprotective agent for NIHL which can be 
delivered as a capsule. This agent combination is currently in clinical trials in Florida through a grant 
from the National Institutes of Health (NIH) grant awarded to Dr. Miller of the University of Michigan. 
Drs. Miller and Le Prell are the inventors on that patent application but Dr. Campbell is the audiology 
clinical trials coordinator for those studies. To date, the animal work with ACE Mg seems promising (Le 
Prell et al., 2007; 2009a,b). While Mg alone or the ACE combination alone did not confer significant 
hearing protection, the combination of ACE Mg provided partial but significant protection from 
permanent NIHL  in the guinea pig (Le Prell et al., 2007, 2011) and the mouse (LePrell et al., 2009a).  It 
also reduced temporary threshold shift in the guinea pig (Le Prell et al., 2009b).  However these findings 
have not yet been confirmed in research labs other than the inventors’ labs.  
 
One limitation of this combination is that it cannot be used in smokers because beta carotene may 
increase the risk of lung cancer and cannot be used in individuals with gastric disorders because of the Mg 
content. The Mg content may increase the risk of loose stools which may limit use in some occupations 
such as the military or certain industries. Thus, even if successful in clinical trials, it cannot be used in all 
patient populations exposed to excessively high noise levels.  
 
2.5   Distribution of D-methionine 
 
Methionine is an amino acid, and both the D and L isomers been studied for several decades (Block and 
Bolling 1945) in a wide variety of animal models and in humans as it is part of normal protein intake in 
the diet with oral intake and also part of parenteral nutrition. The D isomer is more common in fermented 
proteins such as cheese and yogurt. Thus, an extensive literature exists regarding its distribution, 
degradation, catabolism, metabolism in many texts (Linder 1985; Kleinman and Lemann1987; Abelow 
1998; Stipanuk and Watford 2000; Liberman et al., 2006).  
 
The D-isomer of methionine is primarily distributed in plasma and is excreted in the urine unless it is 
transaminated to the L-isomer, which can be incorporated into protein formation. Both isomers of 
methionine are small molecules with wide distribution throughout the body including easily passing 
through the blood brain barrier.  
 
2.6   D-methionine Safety Factors 
 
Methionine is a micronutrient, and thus it is not alien to the human system.  It is present in a wide variety 
of foods (Friedman, 1999b).  Methionine comprises 26 mg/g high quality protein in the diet (National 
Academy of Sciences 1980). Methionine is used for other purposes and at relatively high doses. The 
World Health Organization lists methionine as an essential drug for treating acetaminophen overdose 
(WHO 1997).  As an oral antidote, methionine is administered initially at 2.5 g, followed by three more 
2.5 g doses at four-hour intervals, for a total dose of 10 g over 12 hours.   
Monteagudo et al., (1986) noted that methionine is “remarkably free of side effects” including nausea and 
vomiting.  Di Rocco et al., 1998 administered 3 g L-methionine twice a day for six months to treat 
vacuolar myelopathy in 12 HIV-infected human adults.  Patients tolerated it (6 grams/day) well, other 
than one complaint of some nausea.  DiRocco et al. (1998) further reported that even 20g/day for an adult 
is safe for chronic administration.   
 
Methionine has been available for decades as an over-the-counter orally administered preparation to 
reduce urinary odor and dermatitis.  For that application in adults, the recommended dosing is 200-400 
mg orally three to four times per day (Drug Facts and Comparisons 1991). Most human studies using 
methionine reported no side effects (Kies et al.,1975; Kaji et al., 1987; Stegink et al., 1986).  However, 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
7 
 
methionine toxicity can occur with very high dosing of racemic or L-methionine, particularly in the 
presence of a low protein diet and/or in developing animals as opposed to adults (Benevenga, 1974; 
Klavins and Johansen, 1965; Daniel and Waisman, 1969; Cohen et al., 1958; Muramatsu et al., 1971; 
Klavins, et al., 1963). 
 
To date, no side effects have been encountered greater in D-methionine than in placebo groups in the ex-US 
normal Phase 1 subjects or in Phase 2 clinical trials for either radiation-induced oral mucositis or cisplatin-
induced hearing loss (Hamstra et al., 2010). Because the Phase 2 studies were conducted in cancer patients 
undergoing therapy, some nausea was reported but it was not significantly different between the treated and 
control groups and it is probably not D-methionine related. No side effects occurred in normal subjects in the 
Phase 1 study (Hamstra et al., 2010).   
 
2.7  Methionine: Comparison of the D isomer vs. the L isomer 
 
Methionine is a compound known to have relatively low toxicity in humans (Kaji et al. 1987; Kies et al., 
1975; Stegink et al., 1986).  It is used throughout Europe and India in high doses (total dose of 10 g given 
over 12 hours) to prevent toxicities associated with acetaminophen overdose (WHO, 2011).  It is also 
used in lower doses (200-400 mg 3 to 4 times a day) to reduce urinary odor and dermatitis.  The D-isomer 
appears to be better tolerated than either the L-isomer or the racemic mixture (Monteagudo et al. 1986; 
and Stekol et al, 1962).   
 
2.7.1 D versus L Methionine Utilization Varies by Species 
 
Humans and monkeys utilize only 30% of D-methionine but utilize 100% of L-methionine (Baker 2006). 
This poor utilization by humans and monkeys, specifically of the D isomer of methionine, is in contrast to 
the excellent utilization of D-methionine in the dog, pig, mouse, rat, rabbit, and chick (Stegink et al., 
1980; Cho et al. 1980; Burns and Milner 1981; Baker 2006) [Table 1].  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
8 
 
 
 
 
TABLE 1.  Relative utilization on sulfur amino acid isomers, analogs, and precursors (Baker 2006) 
 
 
 
As shown in Table 1, oral utilization of D-methionine in the pig, dog, and growing kittens is near 100%, 
90 % for the rat and chick, and 75% for the mouse in contrast to the human and the monkey’s utilization 
rate of only 30%.  However for L-methionine, utilization is 100% for all the aforementioned species 
including the human.   
 
Specifically, the utilization of D-methionine in rats is quite different than in humans. In rats, over 90% of 
D-methionine is rapidly converted to the L-isomer (Hasegawa et al., 2005). In rats, D-methionine is the 
most effectively used of all the amino acids and is also toxic but less so than L-methionine when added to 
the diet at excess levels (Sauberlich 1961; Sugiyama and Muramatsu 1987). Interestingly, transport 
characteristics in the intestine are similar in rats and humans (Zheng et al., 1994) thus the differences in 
utilization are not secondary to simple differences in transport across the intestinal epithelium. 
 
D-methionine also has the advantage in humans, in that plasma concentrations of D-methionine peak at a 
higher level (Stegink et al., 1986) and decline at a slower rate than for L-methionine, possibly because of 
the lower utilization(see Figure 1 & 2). Thus the D-isomer may be available in the plasma longer to serve 
as an antioxidant.  Yet, nitrogen balances of human subjects fed D-methionine are no different than 
subjects fed no methionine, although subjects fed L-methionine had significantly higher nitrogen balances 
(Kies et al., 1973; Zezulka and Calloway 1976).  
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
9 
 
Figures 1 & 2.   Plasma concentrations of D and L methionine 
 
 
 
 
 
 
 
 
 
2.7.2  D versus L Methionine excretion varies by species: 
 
Researchers have found that D-methionine, in humans, whether administered orally or parenterally, is 
excreted in large quantities in urine (Stegink et al., 1980; Printen et al., 1979; Stegink et al., 1971; Heller 
et al., 1970; Efron et al., 1969). Some variation exists across human studies, probably secondary to 
administration method and specific human population, but D-methionine is clearly less well utilized in 
humans than in rats, chicks, pigs, rabbits and dogs as indicated by preferential urinary excretion of the D-
isomer in humans. Term infants excreted 34-45% of the D-isomer from DL methionine containing 
formula (Stegink et al., 1971). Adult humans excreted 11-37% of oral D-methionine (Zezulka and 
Calloway 1976 and Boggs 1978). Infused D-methionine in post-surgical patients given complete 
parenteral nutrition containing DL methionine resulted in a 55-70% excretion rate (Printen et al., 1979). 
While Brummel et al. reported 33% of infused D-methionine was excreted in normal adults receiving 
protein sparing regimens. These data are in contrast to the rat in which less than 1% of D-methionine was 
excreted in rats infused with parenteral solutions of DL methionine (Cho and Stegink 1979). Similarly 
low excretion levels of D-methionine urinary excretion have been reported in neonatal pigs infused with 
DL methionine (Cho et al., 1980), and no significant urinary excretion of D-methionine in rabbits or dogs 
and ingesting oral D-methionine loads (Cho et al., 1980). 
 
The only reported animal species somewhat similar to the human appears to be the monkey, although 
reported excretion of the D-isomer is still less than in most human studies (Stegink et al., 1971; Zezulka 
1978 and Boggs 1978; Printen et al., 1979). Monkeys, unlike rats, chicks, pigs, rabbits, and dogs, process 
D-methionine more similarly to humans in that D-methionine is preferentially excreted in the urine while 
L-methionine is not. In 4 normal adult cynomolgus monkeys, Stegink et al., 1980 reported that for an 
ingested average of 488 micromoles DL methionine per day, mean daily urinary excretion was 67.4 
micromoles of which 65.5 micromoles was the D-isomer. Thus, 26.8% of ingested D-methionine was 
excreted but only .008% of L-methionine was excreted versus only .077% of the L-isomer. Based on 
Figure 1. Mean ± SD plasma total free 
methionine (MET) concentrations (μmol/dl) in 4 
normal adults administered 0.0605 mmol/kg 
body wt of D-methionine ( … , D-MET), L-
methionine (     , L-MET) and L-
methionine-dl-sulfoxide (    , L-MSO). 
(Stegink, et.al., 1986) 
Figure 2. Mean ± SD plasma concentrations of 
L-isomer (----) and D-isomer (      ) of 
methionine in 4 normal adults administered 
0.0605 mmol/kg body wt of D-methionine. 
(Stegink, et.al., 1986) 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
10 
 
plasma measurements, the authors also concluded that the excretion did not reflect an “overflow amino 
aciduria”. In an additional experiment, the animals were fasted for 8 hours then administered 150 mg 
(1,006 micromoles) dissolved in 10ml water of D-methionine by feeding tube. They then fasted for an 
additional 24 hours but with water ad libitum. During this 24 hour period, 173 ± 46 micromoles 
methionine of which 97% was of the D configuration. Thus 17% of administered D-methionine was 
excreted. 
 
Methionine loading tests (L or DL methionine) have been performed in over 6000 subjects without any 
serious side effects with one exception (Garlick 2006). Cottington et al., 2002 reported one death that was 
reportedly secondary to a 10 fold overdose (1 g/kg rather than 100mg/kg for a total dose of up to 80 g) of 
L-methionine. Methionine loading tests are usually conducted with 100 mg/kg methionine which is about 
7 times the daily requirement for total sulfur amino acids (methionine plus cysteine) (Garlick 2006). 
However, no study reported in the literature has performed D-methionine loading.  
 
2.7.3  D versus L Methionine Safety Comparisons 
 
In the rat, growth depression by D-methionine is significantly less than with L-met (Sugiyama and 
Muramatsu 1987). However for the chick, Baker and Boebel 1979 reported that L-methionine was 
superior to D-methionine in promoting growth as did Friedman and Gumbmann 1984 in the rat. However, 
adverse effects on growth have not been reported in human adults.   
 
The limited capacity of the transulfuration pathway rather than metabolism of methionine in the 
transamination pathway is primarily responsible for the adverse effects of methionine (Sugiyama and 
Muramatsu 1987). The limited capacity of the transulfuration pathway is primarily responsible for 
methionine toxicity which suggests that L-methionine, primarily metabolized through the transulfuration 
pathway, underlies methionine toxicity. D-methionine is metabolized via the transamination pathway 
which does not appear to play a major role in toxicity (Sugiyama and Muramatsu 1987). See Figure 3 and 
4. D-methionine can contribute to toxicity if converted to L-methionine thus then being metabolized 
through the transsulfuration pathway (Sugiyama and Muramatsu 1987). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
11 
 
 
Figures 3 & 4.  Effects of experimental diets on the growth (A) and food consumption (B) of rats 
 
 
Figures 3 & 4 show the metabolic relationship between D and L methionine in mammals     1   Met 
adenosyltransferase;  2   Met transaminase(s);   3  D-amino acid oxidase (Sugiyama and Muramatsu 1987) 
 
 
However, because D-methionine is so quickly and effectively converted to L-methionine in the rat, unlike 
the human and monkey, the toxicities observed in the rat may be the result of the resultant L-methionine 
rather than specifically the D-isomer (Hasegawa et al., 2005). Similarly, Benevenga 1974 concluded that 
“the apparent toxicity of D-methionine may be simply due to its conversion to L-methionine”.  
 
2.7.4  Potential Risks in Humans 
 
2.7.4.1 Potential Increase in Homocysteine Levels for L-Methionine Administration,     
            Not Investigated for D-Methionine Administration 
 
High dose methionine administration, such as methionine loading, has been reported to increase 
homocysteine levels (See review by Garlick 2006).  However, no studies have reported the relationship of 
D-methionine to homocysteine levels. Considering that D-methionine is highly excreted in humans, 
without conversion to the L isomer, and utilization of the D-isomer is only 30% as opposed to 100% for 
L-methionine it seems unlikely that D-methionine would be metabolized to increase homocysteine levels 
in the same manner as L-methionine. However, even for L-methionine, B-12 and folate can reduce the 
correlation of methionine loading to homocysteine formation (Garlick 2006).  
 
2.7.4.2  D L Methionine May Exacerbate Schizophrenia 
 
Baldessarini et al. reported that large doses of DL methionine (4 to 40 g/d of L or DL methionine for 1 
week to 2 months) in neuropsychiatric disease, such as, schizophrenia, markedly exacerbated 
schizophrenics’ psychotic symptoms (Garlick 2006). Cohen et al. noted large doses of methionine (5-40 
g/d of L or DL methionine) with or without a monoamine oxidase inhibitor, given over periods of 1 week 
to 2 months have resulted in striking exacerbation of psychotic symptoms in chronic schizophrenic 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
12 
 
patients (Garlick 2006). In healthy subjects, doses of 10 g of methionine have been shown to have no 
effect (Baldessarini et al., 1979). 
 
2.7.4.3  Potential Antidote if Needed 
 
The toxicity of methionine can be alleviated by dietary supplementation with glycine (Benevenga and 
Harper 1967; Benevenga 1974). However, these studies were conducted with L-methionine and only in 
animals. 
 
 
 3.0   SUMMARY OF PRE-CLINICAL AND CLINICAL STUDIES 
 
3.1  Previous Pre-Clinical Studies 
 
In 1996, it was first reported that D-methionine protected against cisplatin-induced (CDDP) ototoxicity in 
the rat (Campbell et al., 1996). Figure 5 below shows the change in auditory brainstem response (ABR) 
thresholds for 14 kHz tone bursts in rats receiving saline injection only (untreated control), 16 mg/kg 
CDDP (treated control group), and rats receiving either 75, 150, or 300mg/kg D-methionine prior to the 
16 mg/kg CDDP. 
 
 
Figure 5.  Protection from cisplatin-induced auditory threshold shift in rats. 
 
 
 
Additionally, the literature contains reports that the 300 mg/kg dose of D-methionine prevented 
CDDP-induced outer hair cell loss. Representative photomicrographs from the basal turn are 
presented in Figure 6 below. 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
13 
 
Figure 6.  Protection from cisplatin induced cochlear hair cell loss. 
 
3.2   D-Methionine Protection Against Cisplatin-Induced Hearing Loss in Humans 
 
To date, one study has been conducted in normal human subjects for pharmacokinetics and side effects; 
two studies evaluating the safety and effectiveness of D-methionine for radiation-induced oral mucositis 
in humans. A fourth study evaluated the effectiveness of D-methionine protection in cisplatin-induced 
hearing loss. Findings from the first two studies were recently published (Hamstra, et al., 2010); studies 
#3 and #4 are in preparation. 
 
 
Study #1:  Phase 1a: Pharmacokinetic Evaluation of MRX-1024 included 12 normal human adult 
volunteers.  
 
 
Study #2:  Phase 1b: Open-label, multiple-dose, phase 1study of MRX-1024 concurrent with 
radiation therapy with or without cisplatin.  Purpose of the study was to evaluate the effect of 
MRX-1024 on radiation-induced oral mucositis in head and neck cancer. This study included 25 
adult subjects with head and neck cancer (15 male/10 female; mean age 47 years).  
 
 
Study #3: Phase 2:  Multi-center, randomized, double-blind, placebo-controlled, phase 2 study to 
evaluate the safety and effectiveness of MRX-1024 for mucosal protection in head and neck 
cancer patients. This study included 58 adult subjects (44 male/14 female; mean age 49 years). 
 
 
Study #4:  Phase 2: Randomized, double-blind, placebo-controlled, phase 2 study to evaluate 
MRX-1024 protection in cisplatin-induced hearing loss.  This study included 27 adult subjects (6 
male/21female; mean age 55 years). 
 
Results from the most recent study, provides some evidence of effectiveness in humans for protecting 
hearing from cisplatin chemotherapy.  In this double blind randomized pilot study, 14 adult patients 
received 100 mg/kg dose of an oral orange flavored suspension of D-met (MRX-1024) and 13 subjects 
received flavor matched placebo in equivalent volume prior to each dose of cisplatin.  Mean cumulative 
cisplatin dosing was 263.57 (SD 74.79) in the experimental group and 253.85 (SD 56.94) in the control 
group. Primary tumor sites ranged from genitourinary tract to head and neck cancers.  Six patients in the 
experimental group and four patients in the placebo group also received radiation to the head and/or neck 
area for primary tumors in that region. Auditory thresholds were tested bilaterally at 8, 10, 11.2 and 12.5 
kHz with a GSI 61 audiometer using a modified Hughson-Westlake technique. Significant threshold 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
14 
 
protection was obtained for the frequencies of 10 kHz and 11.2 kHz.  No difference in tumor regression 
was noted between groups. 
 
Although the sample size was small (n = 27), protection from hearing loss at 10 kHz and 11.2 kHz was 
statistically significant as demonstrated in Figures 7 and 8. For the other two frequencies tested, 8 kHz 
and 12.5 kHz, the results were completely consistent with the protection observed at 10 kHz and 12.5 
kHz, but because of slightly greater variability in the relatively small sample size, the results at those two 
frequencies did not reach statistical significance (Campbell et al, 2009). While additional audiometric 
frequency testing would have been desirable; test time was an issue with these patients, so the four most 
sensitive frequencies were chosen for cisplatin ototoxicity which was the higher frequency range. Nausea 
was the most common side effect, but was not significantly different between treatment and placebo 
groups.  
 
Figure 7.  Protection from cisplatin induced hearing loss in humans.      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
15 
 
Figure  8.  Protection from cisplatin induced hearing loss in humans. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3.3   D-methionine Protection Against Aminoglycoside-Induced Ototoxicity  
In another set of experiments (Campbell, 2007), showed that D-methionine partially protected against 
amikacin-induced hearing loss in guinea pigs. Hartley guinea pigs, 5 per group, received either saline 
injections (control) 200 mg/kg subcutaneously (s.c.) or amikacin only for 28 days (treated control) or 300 
mg/kg ip D-methionine 30 minutes prior to each 200 mg/kg s.c. amikacin injection (experimental group).  
ABR results for the 1, 4, 8, and 14 kHz stimuli after 28 days are shown in Figure 9.  


 
 
 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
16 
 
 
Figure 9.  Protection from aminoglycoside-induced auditory threshold shift in guinea pigs. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The left bar in each group is the control, the middle bar amikacin alone and the right bar D-met prior to the amikacin. D-met provided partial 
but incomplete protection against amikacin-induced ototoxicity.  As expected, no threshold shift occurred in the untreated control group, and 
significant (p .05) threshold shift occurred in the treated control group for all stimuli.  For 1, and 4 kHz stimuli, ABR threshold elevation was 
reduced in the D-met treated group with values not significantly different from either the treated or untreated control group, suggesting only 
partial protection. For the 8 kHz stimulus, the average ABR threshold elevation was somewhat less for the D-met protected group than for the 
amikacin only group but was significantly greater than for the saline only control group.  For the 14 kHz stimulus, the D-met protected group 
did show significantly less threshold shift than the amikacin only group but protection was still only partial.  Although a significant difference 
between the amikacin only and D-met protected groups was found only at 14 kHz, there was a definite trend toward otoprotection for all tone-
burst stimulus conditions with average ABR threshold shift from the D-met protected group was consistently lower than for the amikacin only 
group but variability (note error bars) may have precluded statistical significance.


 
 
 
 
 
D-methionine to Reduce Noise-Induced Hearing Loss 
17 
 
Studies have confirmed that aminoglycoside otoprotection in animals with divided daily doses is as 
beneficial as increasing the dose. Sha and Schacht (2000) reported that twice daily injections of D-
methionine markedly improved D-methionine protection from gentamicin aminoglycoside ototoxicity in a 
guinea pig model (Figures 10, 11, & 12 used with permission from Hearing Research and Dr. Schacht) 
show the protection of twice daily administration of 200 mg/kg D-methionine 7 hours apart, using the 
same gentamicin model for the ABR stimulus frequencies of  9, and 18 kHz respectively. ABR thresholds 
(mean plus one SD N=6 per group) were determined from 0 to 6.5 weeks. The top line on each graph 
(filled circles) represents gentamicin alone. The next line (open squares) represents gentamicin plus twice 
daily D-methionine. The bottom line (open circles) represents saline controls. Twice daily D-methionine 
provided significant though incomplete protection at all frequencies. From Sha and Schacht, it is clear 
that D-methionine protection was greater with two as opposed to one daily injection of 200 mg/kg D-
methionine. However, because the D-methionine dose was always 200 mg/kg for each injection, it could 
not be determined whether the additional D-methionine protection was the consequence of the twice daily 
dosing strategy or simply increasing the daily dose of D-methionine from 200 mg/kg to 400 mg/kg.   
 
Figure 10.  Protection from aminoglycoside auditory threshold shift in guinea pigs: twice daily D-
methionine dosing.  
 
Effect of twice-daily treatment with D-methionine on gentamicin-induced threshold shifts. ABR thresholds were 
determined from 0 to 6.5 weeks. Values are means S.D. (n=6). Open circles, controls; filled circles, gentamicin alone; 
open squares. gentamicin plus D-methionine. Gentamicin increased thresholds at 3.5 weeks and later (P<0.05). D-
methionine attenuated these threshold shifts at 3 kHz at 3.5 and 4.5 weeks (0.1>P<0.05). (Sha and Schact, 2000) 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
18 
 
Figure 11.  Protection from aminoglycoside auditory threshold shift in guinea pigs: twice 
daily D-methionine dosing. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Effect of twice-daily treatment with D-methionine on gentamicin-induced threshold shifts. ABR thresholds were 
determined from 0 to 6.5 weeks. Values are means S.D. (n=6). Open circles, controls; filled circles, gentamicin 
alone; open squares, gentamicin plus D-methionine. Gentamicin increased thresholds at 3.5 weeks and later 
(P<0.05).  D-methionine attenuated these threshold shifts at 9 kHz at 3.5 and later (P<0.05). (Sha and Schact, 
2000) 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
19 
 
Figure 12.  Protection from aminoglycoside auditory threshold shift in guinea pigs: twice 
daily D-methionine dosing.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3.4   D-methionine Protection from Noise-Induced Hearing Loss   
 
A number of studies have been conducted investigating D-methionine protection and rescue from NIHL 
(Kopke, 2002; Campbell et al 2007, Campbell et al, 2011).From studies with D-methionine protection 
from cisplatin and aminoglycoside protection and in studies of the mechanisms of D-methionine, it has 
been shown that D-methionine is a potent direct and indirect antioxidant that could protect the auditory 
system from a variety of insults. Campbell previously collaborated with Dr. Kopke in testing D-
methionine protection from noise- induced hearing loss. In 2002,  publication of the data and study 
results, Kopke (2002) showed that D-methionine provided virtually complete protection from noise- 
induced hearing loss in the chinchilla when it was administered 200 mg/kg twice/day starting 2 days 
before and continuing after a 6 hour 105 dB SPL 4 kHz narrow band noise for 6 hours.  Further, the 
protection of outer hair cells was excellent. 
 
Figure 13 shows D-methionine protection from permanent noise-induced hearing loss (ABR threshold 
shifts) in chinchillas 21 days after a 105 dB SPL 4 kHz narrow band (NB) noise exposure for 6 
hours.(Kopke et al., 2002). D-methionine was administered with 200 mg/kg ip injections at 12 hour 
intervals starting 2 days prior to the noise, 1 hour prior to the noise and then at 12 hour intervals for an 
additional 2 days. 
 
 
 
Effect of twice-daily treatment with D-methionine on gentamicin-induced threshold shifts. ABR thresholds were 
determined from 0 to 6.5 weeks. Values are means S.D. (n=6). Open circles, controls; filled circles, gentamicin 
alone; open squares, gentamicin plus D-methionine. Gentamicin increased thresholds at 3.5 weeks and later 
(P<0.05).  D-methionine attenuated these threshold shifts at 18 kHz at 3.5 and later (P<0.05). (Sha and Schact, 
2000) 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
20 
 
Figure 13.  D-methionine protection from noise-induced threshold shift in the chinchilla 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
          Control 
          D-Met 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
21 
 
 
Figure 14. Protection from inner and outer hair cell loss in the same groups of chinchillas as in the 
Figure 13 ABR data. 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
22 
 
Figure 15. Cochlear outer hair cell protection from noise exposure in chinchillas. 
Representative Samples: 
 
A.  D-methionine protection 
 
 
 
 
B.  No D-methionine protection 
 
 
 
 
 
 
 
Figure 15 shows a representative example of the photomicrographs of the basal region of the chinchilla 
inner and outer hair cells, with D-methionine protection (top) and without D-methionine protection 
(bottom) 21 days after the 6 hour, 105 dB SPL 4 kHz NB noise exposure. Further, protection was 
consistent (Figure 16: below) although the outer hair cell loss varied across unprotected animals (Figure 
17: below). 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
23 
 
Figures 16 & 17. Outer hair cell protection in individual chinchillas. 
D-methionine can also protect against permanent noise-induced hearing loss in chinchillas exposed to  a 6 
hour 105 dB SPL continuous noise even when first delivered one hour after noise exposure as 
demonstrated in Figure 18 (Campbell et al., 2007).  Significance is noted in the next four graphs. 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
24 
 
Figure 18.  D-methionine rescue from noise-induced hearing loss: D-methionine started 1 hour after 
noise cessation. 
We have now extended these studies to document that D-methionine (200 mg/kg bid) can protect against 
permanent NIHL even when first administered up to 7 hours after cessation of the 6 hour 105 dB SPL 4 kHz 
NB noise exposure and then continued for another 48 hours. In Figure 19 A-D D-methionine protection from 
ABR threshold shift is shown for the tone-burst center frequencies of 2, 4, 6 and 8 kHz respectively for the 
1,3,5  and 7 hour time delays for D-methionine after the noise cessation (one star indicates p ≤ .05).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
25 
 
Figure 19 A-D.  D-methionine Rescue from Noise-Induced Hearing Loss at Various Time Delays 
 
 
Figure 19. Mean percentage of OHCs present in the 2, 4, 6, and 8 kHz frequency regions. Error bars 
indicate +1 SD per row of hair cells. Values significantly different than the control group at the 0.05 level, 
as determined by Tukey’s tests, are indicated by *. Histology results are also plotted by individual hair 
cell row within each region. 
 
The results of outer hair cell analyses for these same animals are consistent with the ABR findings. D-
methionine provided statistically significant, almost complete outer hair cell protection, when 200 mg/kg 
bid D-methionine was first initiated 1, 3 or even 5 hours after the noise cessation (105 dB SPL 4 kHz NB 
for 6 hours). At 7 hours the protection appeared to be slightly less but was significant at 2, 4, and 8 kHz. 
In the 4 panels of Figure 19 the outer hair cell data corresponding to the same frequency regions as in the 
ABR data of Figure 20 are presented. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
26 
 
Figure 20. ABR Threshold Shifts 21 days post noise exposure at 2, 4, 6, and 8 kHz.  
 
 
ABR Threshold Shifts 
 
Figure 20. Mean post-noise exposure at Day 21 ABR threshold shifts from baseline at 2, 4, 6, and 8 kHz. 
Error bars indicate +1 SD. Mean values that are significantly different from the control group at the 0.05 
level, as determined by Tukey’s test, are indicated by *. No threshold protection was observed 24 hours 
after noise exposure but D-methionine conferred significant threshold protection for 2 and 4 kHz for all 
time delay intervals at 21 days. No significant ABR threshold protection was observed at 6 kHz and only 
for the 3 hour delay at 8 kHz at 21 days possibly because of the minimal threshold shift in the control 
group for those frequencies. 
 
 
3.5   D-methionine Affects the Glutathione Pathway in Response to Noise Exposure   
 
We have been investigating the glutathione pathway in D-methionine protection against NIHL. The 
impact of D-methionine on this pathway may explain, at least in part, why D-methionine protects against 
so many types of ototoxicity including noise. Ten groups of 3 chinchillas Laniger were exposed to 105 dB 
SPL NB noise for either 0.5, 2, 4, 6, or 8 hours of noise with either 200 mg/kg ip D-methionine BID or 
equivalent volume saline delivered for 2 days prior to the noise exposure. Animals were sacrificed 
immediately after the noise exposure and cochlear tissues were harvested for HPLC analysis of oxidized 
(GSSG) and reduced (GSH) glutathione levels. Although only 3 animals per cell were used the 
GSH/GSSG ratio was significantly increased (p≤ .05) as demonstrated in (Figure 21). The change in the 
GSH/GSSG ratio resulted from both a significant increase in GSH and a significant decrease in GSSG.   
 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
27 
 
Figure 21.  Ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (nmol/mg) following 
noise exposure with and without D-methionine. 
 
 
 
3.6  Summary 
 
In conclusion, D-methionine can protect against a variety of cochlear insults including noise-induced 
hearing loss and the concomitant outer hair cell loss.  
 
The purpose of this study is to determine if permanent noise-induced hearing loss (NIHL) and tinnitus can 
be prevented or reduced in a large cohort of Army personnel during their weapons training as a part of 
Drill Sergeant Instructor Training School at Ft Jackson.  Soldiers at Ft Jackson are required to fire a 
minimum of 500 rounds of M-16 weapons fire (156 dB SPL) within a 9 day weapons training period., 
The proof of concept data has been published in several studies demonstrating that D-methionine, given 
before and after noise exposure in animals, can prevent permanent NIHL. The current goal is to document 
whether or not it prevents permanent NIHL in a clinical population of Soldiers.  
 
If an oral preparation of D-methionine could prevent noise induced hearing loss and/or tinnitus, 
potentially millions of Americans and people world-wide could have an improved quality of life by 
retaining their hearing. Further the financial impact of preventing permanent NIHL and/or tinnitus could 
provide tremendous cost savings to the military and to industry. This Phase 3 study will enable us to 
determine the efficacy of oral D-methionine in preventing permanent NIHL and tinnitus in humans. 
Additionally they will allow us to ascertain any potential side effects. Ultimately these studies are 
expected to support an FDA approved pharmacologic intervention to prevent permanent NIHL and 
tinnitus due to impulse noise. 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
28 
 
 
4.0 
STUDY PLAN 
 
4.1  Description of Overall Study Design 
 
This is a prospective, randomized, double-blind, placebo-controlled study to evaluate the effect of D-
methionine (D-met) on permanent NIHL after required weapons training. The study will include 600 
Army personnel enrolled in Drill Sergeant School (DSS) scheduled to undergo 9 days of weapons training 
over an 11 day period at Ft Jackson, South Carolina. This training requires that they each fire 
approximately 500 rounds of M-16 weapons fire (156 dB SPL) within a 9-11 day period. Spent cartridges 
are collected and tallied by DSS leaders to document rounds fired. All subjects will be randomized to two 
equal arms; one to oral D-methionine (test drug) and the other to flavor-matched placebo. In both arms, 
there will be no other change in scheduled treatment. This facility trains approximately 2000 drill sergeant 
instructors annually.  
 
4.2   Study Endpoints 
 
 
4.2.1   Primary Endpoints 
 
 
The first primary endpoint is to confirm safety and tolerability of administering up to 
100 mg/kg daily, given in divided doses,  twice daily (with morning and evening 
meal). The other primary efficacy endpoint is change from baseline in pure-tone 
thresholds as measured by absolute change and frequency of significant noise-
induced threshold shift (STS). 
 
 
4.2.2   Secondary Endpoint 
 
Secondary efficacy endpoint is change from baseline in scores for the tinnitus scales 
for both loudness and annoyance. 
 
4.3  Subject Selection 
 
Six hundred military personnel enrolled in the Drill Sergeant School (DSS Candidates) at Ft. Jackson will 
be recruited for the study. This training requires that each Soldier fire approximately 500 rounds of M-16 
weapons fire (156 dB SPL) within a 9-11 day period of weapons training with a possible additional 
makeup day if necessary to meet training requirements. Ft Jackson makes great efforts to prevent noise-
induced hearing loss by means of physical hearing protection for all Soldiers.  Every DSS Candidate in 
training at Ft Jackson is issued physical hearing protection, trained and required to use them at all times 
on the weapons range.  Spent magazines are collected and tallied to document rounds fired.   
 
Study subjects will be randomized to two equal arms: oral D-methionine (test drug) or flavor-matched 
placebo. All 600 subjects will receive either D-methionine (n = 300) or placebo (n = 300) twice daily for 
18 days starting 3 days prior to the weapons training and ending 4 days after cessation of weapons 
training.  Drug will be dispensed in the morning and in the evening at meal times. Drug logs will be kept 
for every subject, recording every dose taken or missed and whether or not battle plugs were used. Side 
effects will be assessed each time study drug is dispensed.  
 
Soldiers routinely undergo pre-training audiology assessments that include pure tone threshold, otoscopy 
and typanometry testing. Study participation will include the following additional audiology assessments: 
pre-post training pure tone threshold and tympanometry assessments. Pre- post training tinnitus 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
29 
 
assessments are not routinely done and are a required assessment for this study which will be conducted 
by the audiologist and/or study coordinators.  
 
In order to be a Drill Sergeant Instructor, the Soldier must have achieved a rank of E5 (Sergeant) or 
higher, which generally takes between 4 to 5 years to obtain. Soldiers in DSS training are generally male 
(87%), between 21 – 45 years of age and racially diverse.  In addition, Soldiers are routinely excluded 
from participation in DSS training if they have any driving under the influence convictions; domestic 
violence accusations or if they are under any Uniform Code of Military Justice (UCMJ) action or have any 
felony convictions.  Soldiers are routinely screened for drug use by urinalysis and are excluded if test 
results are positive. They are also excluded if they fail to pass the Physical Training (PT) test, are 
restricted from any PT activity, or fail to meet weight, height or pregnancy restrictions. 
 
4.4   Inclusion Criteria:       
 
1.   Male or female 
 
2.   21 to 45 years of age 
 
3.   Negative pregnancy test confirmed by urine sample at enrollment and prior to taking first 
study drug dose.  
 
4.   Willing to use an effective method of birth control during the study (Female participants 
should avoid pregnancy and male participants should avoid fathering children during study). 
Subject should either abstain from sexual relations or practice a method of birth control 
while taking part in the study.  Except for surgical removal of the uterus, birth control 
methods such as condoms, a diaphragm or cervical cap, birth control pills, IUD, or sperm 
killing products are not totally effective in preventing pregnancy. Male study participants 
should refrain from fathering babies while enrolled in this study. 
 
5.   Pure tone air conduction threshold average at 0.5, 1 and 2 kHz of no greater than 40 dB HL 
bilaterally with no air bone gaps greater than 10 dB and normal otoscopy and tympanometry 
screens. 
 
6.   Willing to limit the use of nutritional supplements containing or derived from protein to 50 
grams of protein per day while participating in this study. 
 
7.   Ability to comply with all study requirements  
 
4.5   Exclusion Criteria: 
 
Most of these exclusion criteria apply to DSS candidates, but also apply in this study: 
 
1.   History of allergic or idiosyncratic reaction to methionine, amino acid mixtures, nutritional 
supplements, egg white or other proteins or food additives 
 
2.   Vegetarian (Individual excludes meat and fish from their diet) 
 
3.   History of chronic balance disorders  
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
30 
 
4.   Abnormal otoscopic findings, otologic surgery, autoimmune inner ear disease, significant air 
bone gaps, abnormal tympanograms, or other indication of middle-ear abnormality, history of 
fluctuant hearing or asymmetry in hearing worse than 25 dB at any test frequency, or central 
       nervous system disorder that is likely to affect hearing  
 
5.   Treatment with intravenous (IV) antibiotics or Carboplatin/Cisplatin within the past 6 months 
 
6.   Any surgical or medical condition which might significantly alter the absorption, distribution,   
      metabolism, or excretion of any drug  
 
7. History of abnormal kidney function or kidney impairment 
 
8.   Treatment for alcohol or substance abuse within past 6 months  
 
9. Women of childbearing age who are not using effective contraceptive methods and who may    
become pregnant during the course of the study  
 
10. Women who are pregnant or breastfeeding  
 
11.  National Guardsmen 
 
12.  History of psychotic schizophrenia 
 
13.  Exposure within the previous 6 months to systemic ototoxic substances including 
aminoglycosides or vancomycin 
 
14.  Body weight exceeding 225 pounds 
 
15.  Renal impairment measured as eGFR < 50 on screening creatinine clearance blood draw 
 
 
4.6   Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible to participate in this study.  
No exclusion based on race or ethnicity will be used. Although all eligible and consenting women and 
minorities will be included in this study, the demographic information for the study site suggests that this 
population is typically 87% male and 13% female.  Approximately, 14% are Hispanic or Latino, 1 % 
American Indian or Native Alaskan, 3 % Asian, 1 % Native Hawaiian or other Pacific Islander, 17% 
Black or African American and 74 % White. 
 
4.7   Subject Withdrawals 
 
Every effort will be made to ensure that subjects complete the study through the 30 day study period, 
consistent with provisions of informed consent and good clinical judgment with respect to safety.  The 
following are potential reasons to terminate the participation of a subject in the study: 
 
1. The subject’s health would be jeopardized. 
 
2. Lost to follow-up:  the subject fails to return to the site for scheduled visits and does not respond 
to reminders or attempts to contact. 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
31 
 
 
3. Withdrawal of consent:  subject decides to stop participation for any reason or is unable to 
complete the study as described in the protocol. The investigator will make a reasonable effort by 
person-to-person or telephone interview if allowed by the withdrawn volunteer, to ascertain the 
subject’s reason(s) for withdrawal. 
 
The reason for withdrawal will be captured on the appropriate case report form (CRF) and the medical 
monitor will be informed of removal or early withdrawal of a subject from the study.  
 
4.8    Subject Replacement 
 
If a subject receives any part of the investigation treatment, and must discontinue treatment for any 
reason, they will not be replaced. 
 
 
5.0 
STUDY PROCEDURES 
 
5.1   General Guidelines 
 
To be eligible for this study, the DSS Candidate must meet each inclusion criteria listed on the eligibility 
checklist and none of the exclusion criteria should apply.  To be enrolled, the DSS candidate must have 
the ability to understand and the willingness to sign the informed consent form.  An ombudsman must be 
present during the recruiting and consenting process. 
 
The Schedule of Assessments/Events (Section 12.0) summarizes the frequency and timing of the required 
study assessments.   
 
5.2   Recruitment Procedures 
 
All DSS Candidates will be invited to participate in the proposed study. An information session will be 
scheduled at the beginning of each training period to provide an overview of study purpose and 
procedures and to give Candidates s an opportunity to ask questions. Written informed consent will be 
obtained prior to initiating any study procedures.  Consent will be obtained by study coordinator(s) not 
affiliated with the DSS program.   
 
Fort Jackson has approximately 16 classes per year for DSS Candidates. These classes start every 2-3 
weeks with a maximum enrollment for the year of 2,040 Soldiers or approximately 100 enrollees per 
class. The M-16 weapons training takes place on study days 4-14, with no training on Saturdays and 
Sundays. Thus, the weapons training occurs on days 4-8 and days 11-14.  
 
Candidates will attend a recruitment briefing to introduce this research study during the first 14 days of 
class.  The presentation will be conducted by study coordinator(s) not affiliated with the DSS training 
program. During recruitment briefings to a unit where a percentage of the unit is being recruited to 
participate as a group, an ombudsman not connected in any way with proposed research or the unit, shall 
be present to monitor that the voluntary nature of individual participants is adequately stressed and that 
the information provided about the research is adequate and accurate (DoDD 3216.02, March 25, 2002). 
Written informed consent will be obtained before any study assessments are performed. 
 
Candidates will be required to use issued personal hearing protection. Every Soldier in training at Ft 
Jackson is issued physical hearing protection, is trained and required to use them at all times on the 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
32 
 
weapons range.  Fort Jackson makes great efforts to prevent noise-induced hearing loss by means of 
physical hearing protection for all Soldiers.  According to the Department of Defense Instruction, 
6055.12, Hearing Conservation Program (HCP), December 3, 2010, the DoD Components shall issue 
personal hearing protectors at no cost to all personnel working or training in hazardous noise 
environments. All DoD Components shall ensure proper initial fitting and supervise the correct use of all 
hearing protection.   
 
We anticipate that all subjects will be using Battle Plugs provided by the Fort Jackson audiologists.  
Compliance with hearing protection use will be assessed and recorded each day by the study 
coordinator(s).  
 
5.3   Screening 
 
Once written informed consent has been obtained, the following assessments will be conducted. 
 
 
Review of inclusion/exclusion criteria 
 
 
Review of relevant medical history 
 
 
Review of current medication history 
 
 
Collect demographic data 
 
 
Body weight  – Body weight obtained by the study coordinator(s) at  enrollment will be used to 
determine dosing of the Study Drug.  
 
 
Urine sample to be collected from all subjects for routine urinalysis.  A urine pregnancy test will 
be performed for all women of child bearing potential by the study coordinator(s).  Pregnancy test 
results will be recorded.  
 
 
A blood draw for serum creatinine will be conducted prior to enrollment in order to calculate 
estimated GFR by the Cockroft-Gault equation. Those subjects with an estimated eGFR < 50 will 
be excluded from enrollment. 
 
 
Two additional samples of blood (20-25 ml) will be collected for chemistry tests, complete blood 
counts, and tests of liver function, and homocysteine level.  
 
5.4  Randomization   
 
Enrolled subjects will be randomly assigned to receive study drug or placebo in a 1:1 ratio.   
Randomization scheme was developed by study statisticians and provided to KP Pharmaceutical 
Technology.  
 
 
5.5  Audiological Assessments: Baseline and End of Study     
 
Subjects will be given oral and written instructions by the study coordinator(s) to avoid noise exposure 
for at least 24 hours prior to all audiological assessments (hearing tests). Audiological assessments will be 
performed using the following standardized procedures. CPT Jenny Davis, AuD, a licensed audiologist 
and Army Hearing Program Chief at Fort Jackson, will be responsible for overseeing all testing and for 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
33 
 
maintaining all audiologic data files for all subjects enrolled in the study. Audiological data is collected 
electronically via Interacoustic 629 clinical audiometers with insert earphones and TDH 39 earphones..  
The threshold data will be directly exported to an Excel file and copied into the research chart.  
 
5.5.1  Tinnitus Assessments  
 
All subjects will be asked to complete the questionnaires at baseline and at the end of the study to 
determine the extent to which tinnitus has been induced by the noise exposure. The tinnitus questionnaire 
is modified from the Tinnitus Ototoxicity Monitoring Interview (TOMI) and the Tinnitus Handicap Index 
and the Tinnitus Loudness Index.  The Tinnitus Handicap Inventory (THI), the most widely validated 
assessment tool for tinnitus, will also be used. 
 
The TOMI (Fausti et al., 2007) and THI questionnaires, developed at the Portland, Oregon, Veteran’s 
Administration and the Tinnitus Clinic of the Oregon Hearing Research Center have been documented in 
several ways. The THI was derived from detailed evaluation of over 2000 patients (Meikle 1992; Meikle 
et al., 1995).  It is a twelve-item scale that quantifies the magnitude of tinnitus-related impairment, 
disability, and handicap according to guidelines established by the World Health Organization (WHO 
2011; Meikle and Griest 2002). Tinnitus Handicap Inventory (THI) is the most widely validated and used 
tinnitus instrument (Newman et al., 1996, 2008). The THI has high internal consistency and reliability 
(Cronbach’s alpha=.93) and test-retest stability (r=.92) Newman et al 2008).   
 
Subjects will be administered the tinnitus questionnaires at the time of their initial and final audiometric 
evaluations. Differences in the questionnaire scores will be compared pre- and post-noise in the same 
subject and between treatment groups. In addition to the THI, an important measure for use in evaluating 
potential preventive or treatment effects for tinnitus is that of magnitude scaling for the subjective 
loudness dimension of tinnitus.  Subjective ratings for tinnitus, using a scale from 0 – 10, are well 
documented by a number of investigators (Meikle, 1992; Levine, 1999; Bauman and Jastreboff, 1999; 
Newman et al., 1996). Such ratings are quickly and easily administered, and can be efficient measures for 
quantifying tinnitus treatment effects. The subjective loudness rating for tinnitus captures the aversive 
aspect of tinnitus, in a similar manner to that of pain rating scales, which are widely used by pain experts 
to quantify the aversive aspect of chronic pain. It has the advantage that it does not require the subject to 
try and recall his or her tinnitus problems during a preceding time interval (such as a week or more), but 
instead can be given as an immediate and current rating of the tinnitus magnitude. 
 
5.5.2   Otoscopy and Tympanometric Screening  
 
Prior to each hearing test, each Soldier’s ear will be examined with a Welch Allyn 3.5v MacroView 
otoscope to ensure that the ear canals are clear and the tympanic membranes are normal in appearance.  
Tympanometry will be screened in each ear using pass/fail criteria of middle ear pressure: (pass is 
between +110 daPa and -160 daPa) and compliance:(Pass is between .2 and 1.4 ml) using  the automated 
Interacoustics Handheld Middle Ear Analyzer MT10. 
 
 
5.5.3   Pure Tone Air Conduction Threshold/Bone Conduction Testing 
 
Pure-tone air conduction threshold testing both at baseline and post-treatment (11-15 days after the last 
day of study drug/placebo administration) will be conducted utilizing the modified Hughson-Westlake 
procedure.  Pure-tone air-conduction testing will be conducted at .5, 1, 2, 3, 4, 6, 8 kHz.  Bone conduction 
testing will be conducted at .5, 1, 2, 3, 4 kHz if the pure tone air-conduction threshold at that frequency is 
greater than or equal to 15 dB HL. All testing will be conducted using standard Army test procedures 
using Interacoustic 629 clinical audiometers meeting all Defense Occupational Environmental Health 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
34 
 
Readiness System – Hearing Conservation (DOEHRS-HC) and American National Standards Institute 
(ANSI) criteria. All testing will be conducted in a single -walled sound booth with doors closed, meeting 
ANSI specifications. Test equipment, test environment, procedures, and personnel will meet all relevant 
DOEHRS-HC, ASHA, AAA, and ANSI standards and guidelines.  
 
Pure-tone threshold testing will be conducted using the modified Hughson Westlake procedure as 
follows: Initial descent towards threshold is accomplished in 10-dB steps. Beginning with the first non-
response, level is increased by 5-dB for each non-response, and decreased by 10-dB after each correct 
detection response. Threshold is defined as the lowest level at which two responses are obtained out of 
three presentations on an ascending run.   
 
Threshold shifts will also be evaluated using DoD significant change criteria and ASHA early detection 
criteria (1994). Significant noise-induced threshold shifts (STS) in either ear are defined by the 
DOEHRS-HC as an increase of 10 dB or greater change (decrease in hearing) for the average of 2, 3 
AND 4 kHz in either ear. A significant negative STS (improved hearing) is defined as a decrease of 10 dB 
or greater change (improvement in hearing) for the average of 2, 3 AND 4 kHz in either ear. An early 
warning shift STS (decrease in hearing) is defined as a 15 dB or greater change at 1, 2, 3 OR 4 kHz in 
either ear. Subjects with an STS will undergo a final tympanogram screen to rule out middle ear 
pathology as a cause of the hearing loss.  If they pass the tympanogram screen in both ears, they will be 
referred for a diagnostic audiologic follow-up exam. If they do not pass the tympanogram screen they will 
be referred for otologic check and then diagnostic audiological assessment. 
 
Experimental design consists of within-subjects serial testing in which baseline standard frequency (.5, 1, 
2, 3, 4, 6, and 8 kHz) audiograms are initially acquired.  By comparing similar measures obtained at the 
end of the study period to the relevant pre-exposure measure, reliable noise-induced changes in pure-tone 
hearing threshold can be identified. Thus, subjects will serve as their own control for identifying hearing 
change, relative to their baseline evaluation.  
 
6.0 
INTERVENTION 
 
6.1   Study Day 1 
 
 
Second urine pregnancy test will be obtained by the study coordinator(s) for women of child-
bearing potential.  Pregnancy test results will be obtained and recorded by the study 
coordinator(s) immediately prior to study drug administration. 
 
 
Assess noise exposure compliance 
 
 
Assess for medication compliance/change 
 
 
Study Drug to be dispensed and recorded at morning and evening meals by the on-site study 
coordinator(s). 
 
6.2   Study Days 2-18 
 
 
Assess for Adverse Events and complete AE Log 
 
 
Assess for medication compliance/change 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
35 
 
 
Assess for hearing protection compliance (Days 4-14 only) 
 
 
Study Drug to be dispensed and recorded at morning and evening meals by on-site study 
coordinator(s). 
 
 
Urine and blood samples will be collected while on treatment (during second week). Two 
additional samples of blood (20-25 ml) will be collected for chemistry tests, complete blood 
counts, and tests of liver function, and homocysteine level. Urine sample will be collected for 
urinalysis. 
 
6.3   Study Days 29-32 
 
At the end of the study, Study Days 29-32, subjects will return to complete post-study audiologic exams 
including pure-tone thresholds, screening tympanometry and tinnitus questionnaires. Subjects will be 
contacted by telephone or text message to remind them to avoid noise exposure for at least 24 hours prior 
to the audiological assessments (hearing tests) by the on-site study coordinator(s). Concomitant 
medication usage, adverse events and study procedures/ compliance will be reviewed and confirmed. 
 
 
Urine and blood samples will also be collected post treatment. Two additional samples of blood 
(20-25 ml) will be collected for chemistry tests, complete blood counts, and tests of liver 
function, and homocysteine level. Urine sample will be collected for urinalysis. 
 
7.0 
STUDY DRUG 
 
Study drug will be shipped to the study site as blind-labeled, ready-to-use bottles containing D-
methionine or placebo ready for administration.  
 
7.1   Formulation, Packaging, and Labeling 
 
Study drug and Placebo will be formulated, shipped and packaged by KP Pharmaceutical Technology 
(KPT), Bloomington, Indiana. The suspension is provided in small vials at a D-methionine concentration 
of 200 mg/mL.  Study drug will be prepared and labeled for each subject in individual vials by KPT.  The 
formulation is comprised of the common excipients polysorbate 80 (Tween), methylparaben, 
propylparaben, sorbitol and orange extract added for taste and palatability.  The formulation is stable 
when stored at controlled room temperature for at least 3 months.  For longer storage, suspension must be 
maintained under refrigeration. 
 
The placebo is flavor and color matched and delivered in equivalent volume to the active compound.  The 
test drug D-methionine and the placebo are both formulated according to Good Manufacturing Practice 
(GMP) standards by KP Pharmaceutical Technology (KPT) as an oral suspension.  KPT prepared these 
same formulations for the ex-US clinical trials for prevention of radiation-induced oral mucositis and 
cisplatin-induced hearing loss. 
 
7.2   Dispensing Study Drug 
 
Study drug will be dispensed by MACH Pharmacy to on-site study coordinator(s), who are qualified 
medical personnel (RN or LPN).  The study drug will be distributed to the study participants as labeled in 
accordance with the study protocol.   CPT William Grimes, M.D. will supervise the activities of the study 
coordinator(s) and audiologists. 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
36 
 
 
The formulated drug product (D-methionine) will be provided for clinical use as an oral suspension. Each 
dose will be packaged in individually labeled vials.  Each vial will have a subject identification number 
on the label (Section 15.1 Master Label).  The label will include instructions to shake the suspension prior 
to administration.  
 
7.3   Preparation, Administration, and Dosage of Study Drug 
 
Subjects will be given two doses per day of an oral orange flavored suspension of either D-methionine or 
placebo daily starting three days prior to weapons training, for 11 days during weapons training, and an 
additional 4 days after completion of weapons training for total of 18 days administration. The total daily 
dose of D-methionine will not exceed 100 mg/kg per day.  
 
Each subject’s dose of D-methionine will be individually determined based on actual subject weight. For 
ease of administration and packaging, dosing is broken down by weight categories with dosing based on 
the lowest weight in that category as follows:  
 
Body Weight lbs (kg) 
Total Daily Dose (Up to 100 mg/kg/day) 
100-125 lbs  (45 - 57 kg) 
4.6 grams (4600 mg) 
126-150 lbs  (57 - 68 kg) 
5.6 grams (5600 mg) 
151-175 lbs  (68 - 80 kg) 
6.8 grams (6800 mg) 
176-200 lbs  (80 - 91 kg) 
8.0 grams ( 8000 mg) 
201-225 lbs  (91 - 102 kg) 
9.2 grams (9200 mg) 
 
For example, if subject’s body weight is 155 lbs (70.4 kg) then the subject’s total daily dose equals 6.8 
grams per day.  This dose will be divided into two equal doses (3.4 grams) and administered prior to 
morning and evening meal (approximately 12 hours apart).  Each dose (AM and PM) will be packaged in 
individually labeled vials by KP Pharmaceuticals. 
 
KPT will assign the appropriate randomization code and prepare individualized doses of study drug prior 
to shipping to the Moncrief Army Community Hospital (MACH) Pharmacy at FT Jackson. 
 
7.4  Study Drug Storage 
 
All study drug will be stored under conditions consistent with package labeling (15-30 ° C; 59-86 ° F).  
All study drug will be stored in a secure limited-access area under controlled temperature in accordance 
with labeled storage requirements.  Refrigeration is not required.  Room temperature will be monitored 
and recorded daily, including weekends and holidays. 
 
Shelf life at Ft. Jackson is not an issue because the study drug will arrive one week prior to the 
administration to each study participant and will only be dispensed over an 18 day period thereafter. 
 
7.5  Study Product Accountability Procedures 
 
Upon receipt of study drug, the MACH Pharmacist will ensure that the information on the packing slips 
matches exactly with information sent to the site, including content, amount, lot numbers, quantity, and 
expiration date.  
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
37 
 
The study coordinator(s) will also maintain accurate records of all study drug. In addition, accurate 
records will be kept regarding when and how much of each study drug is dispensed and used by each 
subject.  This information will be recorded in each subject’s EHR and on their medication log.  
 
7.6  Assessment of Participant Compliance with Study Drug 
 
Subject compliance will be determined by reconciliation of the subject’s daily dosing regimen against the 
medication log. 
 
7.7  Concomitant Medications and Procedures 
 
All other medications taken by the subject during the study period will be recorded. Information will 
include:  drug, dose, route of administration, start and end dates and indication. 
 
7.8  Prohibited Medications and Procedures 
 
Treatment with intravenous (IV) antibiotics, such as Amikacin: Amikin®, Gentamicin, Netilmicin: 
Netromycin®, Streptomycin, Tobramycin: Nebcin® or any other aminoglycosides and/or vancomycin.  
Also, any surgical or medical condition that might significantly alter the absorption, distribution, 
metabolism, or excretion of any drug.  Subjects who received treatment for drug or alcohol abuse within 
the previous 6 months are prohibited from  participation in this study. 
 
7.9  Dietary Information (Daily Protein Intake) 
 
Daily dietary information is available from Ft Jackson to ensure that adequate protein is available in the 
participant’s daily diet. 
 
7.10  D-methionine Antidote 
 
The toxicity of methionine can be alleviated by dietary supplementation with glycine (Benevega and 
Harper 1967; Benevega 1974). However, these studies were conducted with L-methionine in animals.  
 
8.0 
ASSESSMENTS OF SAFETY  
 
8.1  Safety Parameters 
 
Safety and tolerability will be determined by evaluating any treatment emergent adverse events or change in 
baseline concomitant medications.  
 
8.2  Definition of an Adverse Event (AE) 
 
An adverse event (AE) is any unanticipated or unintended medical occurrence or worsening of a sign or 
symptom (including an abnormal laboratory finding) or disease in a study participant, which does not 
necessarily have a causal relationship with the study condition, procedures or study agent(s), that occurs 
after the informed consent is obtained. 
 
Pre-existing conditions or illnesses, which are expected to exacerbate or worsen, are not considered 
adverse events and will be accounted for in the subject’s medical history.  
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
38 
 
8.3  Definition of a Serious Adverse Event (SAE) 
 
A Serious Adverse Event (SAE) is defined as an AE meeting one of the following outcomes: 
 
Death during the period of protocol defined surveillance 
 
Life Threatening Event (defined as a participant at immediate risk of death at the time of the 
event) 
 
Inpatient hospitalization or prolongation of existing hospitalization during the period of protocol 
defined surveillance  
 
Results in congenital anomaly or birth defect 
 
Results in a persistent or significant disability/incapacity 
 
Any other important medical event that may not result in one of the above outcomes, may be considered a 
serious adverse event when, based upon appropriate medical judgment, the event may jeopardize the 
participant and may require medical or surgical intervention to prevent one of the outcomes listed above. 
Examples of such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse.  
 
8.4  Methods and Timing for Assessing, Recording, Analyzing, and Managing Safety Parameters 
 
 
8.4.1   Assessment of Safety  
 
General health status and symptom assessments performed at Baseline/Eligibility Visit and Study Visit 1 
will serve as baseline for any new signs or symptoms that arise during the study.  All such changes 
occurring after Study Visit 1 will be evaluated as possible adverse events and appropriately recorded on 
the Adverse Event Case Report Form (CRF). 
 
Baseline and Study Visit 1 assessments are limited to general health history information, pregnancy 
testing and laboratory assessment for kidney function. Additional safety assessments have been added to 
include: laboratory evaluations (hematology, blood chemistry, and urinalysis) at pre-specified time points 
(e.g., pre-treatment, during treatment, and post-treatment). Expected adverse events include possible 
gastrointestinal (GI) symptoms (nausea, vomiting, constipation, diarrhea, dysphagia).  
 
Subjects will be questioned prior to receiving each study dose and on the last study day for the presence  
of GI side effects. *See Estimating Severity Grade and method for classifying GI adverse events.  
Other subject reported symptoms that occur between Study Day 1 and Study Day 18, including the follow up 
visit at Study Day 29 - 32, will be evaluated as a possible adverse event. If a subject experiences a side effect 
that requires additional medical attention, the subject will seek medical attention through appropriate 
standard operating sick call procedures and the medical monitor will be contacted.   
  
Adverse events will be evaluated according to the Estimating Severity Grade and will be recorded on the 
Adverse Event Log and the Adverse Event CRF.  Adverse events deemed greater than Grade 2 will be 
considered a Serious Adverse Event (SAE). All SAEs will be recorded on the SAE CRF and reported 
according to FDA and institutional requirements (See Section 8.5 – Specific Serious Adverse Event 
Reporting Requirements). 
 
Compliance and adverse event reports will be obtained each day that study drug is administered (Study 
Days 1-18) and at the end of the study (Study Day 29 -32) by the Study Coordinator(s).  Each adverse 
event will be graded according to the Table for Estimating Severity Grade. 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
39 
 
For all subjects receiving at least one dose of study drug, the number and percentage of subjects reporting 
adverse events will be tabulated by cohort and overall by severity and body system. 
ESTIMATING SEVERITY GRADE 
Grade 1 
(Mild) 
Event requires minimal or no treatment and does not interfere with the subject’s daily 
activities 
Grade 2 
(Moderate) 
Event results in a low level of inconvenience or concern with the therapeutic measures.  
Moderate events may cause some interference with functioning. 
Grade 3  
(Severe) 
Event interrupts a patient’s usual daily activity and may require systemic drug therapy or 
other treatment.  Severe events are usually incapacitating. 
Grade 4 
(Life-
Threatening 
Any adverse drug experience that places the patient or participant, in the view of the 
investigator, at immediate risk of death from the reaction as it occurred, i.e., it does not 
include a reaction that had it occurred in a more severe form, might have caused death. 
Grade 5 
Death 
GASTROINTESTINAL 
 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Nausea 
Mild or transient; 
maintains 
reasonable intake 
Moderate 
discomfort; intake 
decreased 
significantly; 
some activity 
limited 
No significant 
intake; requires 
IV fluids 
Hospitalization 
required 
Vomiting 
1 episode in 24 
hours 
2-5 episodes in 24 
hours 
> 6 episodes in 24 
hours or needing 
IV fluids 
Physiological 
consequences 
requiring 
hospitalization or 
requiring 
parenteral 
nutrition 
Constipation 
Requiring stool 
softener or dietary 
modification 
Requiring 
laxatives 
Obstipation 
requiring manual 
evacuation or 
enema 
Obstruction or 
toxic megacolon 
Diarrhea 
Mild or transient; 
3-4 loose 
stools/day or mild 
diarrhea lasting 
for less than 1 
week 
Moderate or 
persistent; 5-7 
loose stools/day or 
diarrhea lasting > 
1 week 
> 7 loose 
stools/day or 
bloody diarrhea; 
or orthostatic 
hypotension or 
electrolyte 
imbalance or > 2L 
IV fluids required 
Hypotensive 
shock or 
physiological 
consequences 
requiring 
hospitalization 
Oral 
discomfort/dysphagia 
Mild discomfort; 
no difficulty 
swallowing 
Some limits on 
eating/drinking 
Eating/talking 
very limited; 
unable to swallow 
solid foods 
Unable to drink 
fluids; requires IV 
fluids 
 
All AEs will be reviewed by the Medical Monitor (see Section 8.6) and the Data Safety Monitoring 
Committee (DSMC). The medical monitor will discuss each adverse event and determine causality in 
consultation with the Principal Investigator. The degree of certainty about causality will be graded using 
the categories below: 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
40 
 
 
 
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event occurs in a plausible time relationship to 
drug administration and cannot be explained by concurrent disease or other drugs or chemicals. The 
response to withdrawal of the drug should be clinically plausible.  
 
Probably Related: There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event occurs within a reasonable time sequence to administration of 
the drug, is unlikely to be attributed to concurrent disease or other drugs or chemicals. 
 
 Possibly Related: There is some evidence to suggest a causal relationship (e.g., the event occurred 
within a reasonable time after administration of the trial medication).  However, the influence of 
other factors may have contributed to the event (e.g., the subject’s clinical condition, other 
concomitant events). Although an adverse drug event may rate only as “possibly related” soon after 
discovery, it can be flagged as requiring more information and later be upgraded to “probably 
related” or “definitely related”, as appropriate. 
 
Unlikely: A clinical event whose temporal relationship to drug administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time after administration 
of the trial medication) and in which other drugs or chemicals or underlying disease provides 
plausible explanations (e.g., the subject’s clinical condition, other concomitant treatments).  
 
Unrelated: The AE is completely independent of study drug administration, and/or evidence exists 
that the event is definitely related to another etiology. There must be an alternative, definitive 
etiology documented by the clinician.  
 
Expected Events Related to Disease Process: Expectedness refers to the awareness of adverse 
events previously observed, not on the basis of what might be anticipated from the pharmacological 
properties of the study agent. 
 
Subjects will be questioned by the on-site study coordinator(s) prior to receiving each study dose and on the 
last study day for the presence of side effects or adverse experiences.  If an event is reported, the following 
questions will be asked of the subjects and recorded by the on-site study coordinator(s): 
 
 
Onset of event 
 
Duration of event 
 
Intensity of event 
 
Relationship to study medication 
 
Each subject’s baseline data (pre-noise exposure) will act as their own control.  Any side effects reported 
after receiving the first dose will be recorded (with answers to the aforementioned questions).  At study 
completion, any new occurrence of a side effect or an increase in intensity of a pre-existing event will be 
recorded. If a subject experiences a side effect that warrants additional medical attention, the subject will 
seek medical attention through appropriate standard operating sick call procedures and the medical 
monitor will be contacted and it will be entered on the CRF.  
 
Side effects will be compared within subjects, as well as between treatment groups (D-methionine and 
placebo) for onset, duration, frequency and relationship to treatment.  These comparisons will be made for 
side effects present at baseline, and for those that occurred during and after completion of treatment.  
 
8.5  Specific Serious Adverse Event Requirements 
 
The Southern Illinois University School of Medicine is the sponsor for the Investigational New Drug 
application (IND) filed with the U.S. Food and Drug Administration (FDA). In the interest of subject 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
41 
 
safety and to fulfill regulatory requirements, all deaths and life-threatening SAEs due to any cause, which 
occur during the course of the study must be reported to the Institutional Review Board (IRB) and the 
Regulatory Affairs Representative within 24 hours after the clinical site becomes aware of the event, and 
all other SAEs must be reported as soon as possible, but no later than 5 business days.  Serious adverse 
events will be reported to: Dwight D. Eisenhower Army Medical Center (DDEAMC) IRB and to Rick 
Lampe at 609-636-9909. See SAE Reporting procedures, regulatory authority reporting and follow-up 
under Section 8.6. 
 
In accordance with the FDA Code of Federal Regulations (CFR), the sponsor (Southern Illinois 
University) must report SAEs that are serious, unexpected, and related to the study intervention to the 
FDA in the form of a written IND Safety Report.  Deaths and life-threatening events with any possible 
relationship to a study intervention must be reported to the FDA by telephone or fax as soon as possible 
but within 7 calendar days of IRB awareness. This initial report must be followed by as complete a 
written report as possible within 8 additional calendar days. All other IND Safety Reports must be 
submitted to the FDA as soon as possible, but no later than 15 calendar days after IRB is notified of the 
SAE. SAEs that do not meet the requirements for expedited reporting and all documented adverse events 
will be reported to the FDA in the IND annual report by the sponsor.  
 
8.6    Safety Monitoring 
 
Role of Research  Monitors 
 
For research involving greater than minimal risk to volunteers, the DoD requires that an independent 
research  monitor must be appointed by name. Research monitors can be physicians, dentists, 
psychologists, nurses, or other healthcare providers capable of overseeing the progress of research 
protocols, especially issues of individual volunteer/patient management and safety. Research monitors 
must be independent of the investigative team and must possess sufficient educational and professional 
experience to serve as the volunteer/patient advocate. Depending on the nature of the study, the research  
monitor may be asked to assess one or more of the following phases of a research: volunteer recruitment, 
volunteer enrollment, data collection, or data storage and analysis. 
 
At the discretion of the IRB or the Human Research Protection Office (HRPO), the research monitor may 
be asked to discuss research progress with the principal investigator, interview volunteers, consult on 
individual cases, or evaluate adverse event reports for the safety and protection of the volunteers. 
Research monitors shall promptly report discrepancies or problems to the IRB and the HRPO. They shall 
have the authority to stop a research study in progress, remove individual volunteers from a study, and 
take whatever steps are necessary to protect the safety and well-being of research volunteers until the IRB 
can assess the research  monitor’s report. At a minimum the HRPO requires that the research  monitor 
provide a written opinion regarding the relationship and outcome of any unanticipated problems related to 
participation, serious adverse events, and subject deaths. 
 
COL Mark D. Packer MD will serve as the medical monitor and CPT William P. Grimes, MD will serve 
as the site Principal Investigator and will oversee all study drug administration by the on-site study 
coordinator(s). Col Packer is Clinical Assistant Professor UTSA School of Medicine.  CPT Grimes is a 
family physician in the Department of Preventive Medicine at Moncrief Army Community Hospital, Fort 
Jackson. David B. Pavlakovich, PA-C will serve as the independent research monitor on site. 
 
Adverse event data will be reviewed by the research monitor at the end of each class.  The research 
monitor will discuss each adverse event and determine causality in consultation with the Principal 
Investigator.  Study personnel will immediately alert the research monitor to any unanticipated problems 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
42 
 
or serious adverse events. Unanticipated problems and adverse events will be reported to the IRB in 
accordance with institutional policy and to the FDA as required. Data and safety monitoring reports will 
be provided to the IRB at the time of continuing review per DDEAMC policy. 
 
The investigators, research monitors, coordinator(s) and data manager of this protocol will meet at least 
once per month via videoconference or teleconference to review the following: study progress (including 
data quality and timeliness, recruitment, accrual and retention), study procedures (including subject 
privacy and data confidentiality protection), outcomes of adverse events and new literature reviews to 
assess change to the risk/benefit ratio and to determine if modifications need to be made or the study 
terminated.  The Regulatory Representative (Rick Lampe) will be included via teleconference to assess 
the regulatory impact of outcomes of adverse events and new literature reviews to assess change to the 
risk/benefit ratio and to determine if modifications need to be made or the study terminated.. 
 
 
8.7     Stopping Rules Based on Adverse Events 
 
The safety of individual research subjects is of primary importance in this study as hearing loss does not 
result in serious morbidity or mortality and the active drug is considered relatively benign.  Thus, for this 
study, the following stopping rules for safety will be utilized: 
 
1. The study will be stopped if any subject dies, if any subject requires emergency surgery, or if any 
subject suffers a permanent and irreversible disability, unless it can be shown that this event was 
unrelated to participation in the clinical trial. 
 
2. Should an adverse event occur that is not mentioned in the consent form, then the study will be 
stopped until both the consent form has been revised to include this adverse event and the 
institutional review board (IRB) gives re-approval of the study. The adverse event will also be 
reported to the FDA as it impacts safety. 
 
3. If an adverse event that is not covered under item 1 occurs in more than three subjects, then the 
risk of the adverse event associated with the active drug group will be compared to the risk of the 
adverse event associated with the placebo group.  If the rate is significantly higher for the active 
drug group at an unadjusted confidence level of 95% (i.e., alpha level of 0.05), then the data 
safety monitoring committee (DSMC) will be required to determine, based on the severity of the 
adverse event, if the trial should be stopped for safety.  
 
8.8     Data Safety Monitoring Committee 
 
 
The data safety monitoring committee (DSMC) evaluates study data on an ongoing basis to assure 
participant safety and study integrity. The DSMC will review study data and unanticipated problems 
and make recommendations based on their reviews. The DSMC will review the protocol prior to study 
initiation and meet no less than once per year and more frequently if needed to review enrollment and 
safety data and after the planned interim analysis. Evaluation will also occur if three subjects have met 
the stopping criteria. If all three are determined to be in the D-methionine-treated group, the DSMC will 
consider a lower dose as a safe dose with which to continue the study.  The DSMC Chair may call an 
emergency meeting at any time should issues of patient safety arise. The DSMC will be composed of at 
least four voting members. The current DSMC includes: DSMC Chair (Patrick J. Antonelli, MD, Chair 
of Otolaryngology, University of Florida, Gainesville, the Medical Monitor for the Sponsor (Mark D. 
Packer, MD), the Research Monitor, David B. Pavlakovich, PA-C, D. Bradley Welling, MD, PhD, Chair 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
43 
 
of Otolaryngology, Harvard University, Boston, the Regulatory Representative (Rick Lampe), and the 
Yale University epidemiologist/statistician (Carrie Redlich, MD, MPH).  Only the Yale University 
statistician will be unblinded and will endeavor to keep the committee blinded during discussion of the 
stopping criteria, adverse events, and any study adjustments recommended by the committee. 
 
8.9     Adverse Events (AE) Reporting  
 
We will follow 1996 and 2000 International Conferences on Harmonization, Sections E2 and E6 Good 
Clinical Practice, HHS and FDA regulations.  An adverse event (AE) is defined as any untoward medical 
occurrence that does not necessarily have a causal relationship with this treatment. 
 
A Serious Adverse Event (SAE) is defined as any untoward medical occurrence that results in death, is 
life-threatening, requires hospitalization or prolongation of hospitalization, results in persistent or 
significant disability/incapacity, or is a congenital anomaly or birth defect. 
 
8.10   Reporting Requirements to the Human Research Protection Office (HRPO) 
 
The HRPO requires approval of protocol amendments, acknowledgement or acceptance of continuing 
review documents, reporting of protocol deviations, serious adverse advents, and unanticipated problems. 
The following is a synopsis of what must be reported. 
 
 
Major amendments and amendments that increase risk to subjects must be pre-approved. 
 
 
All other amendments must be submitted for acceptance with the continuing review report. 
 
 
Unanticipated problems involving risks to subjects or others, serious adverse events related to 
participation, and deaths related to participation must be promptly reported. 
 
 
Suspensions, voluntary/involuntary clinical holds, or any terminations of the research must be 
promptly reported to the HRPO. 
 
 
Deviations that affect the safety or right of subjects or the integrity of the study must be promptly 
reported; exceptions (from approved inclusion/exclusion criteria or stopping criteria) must be pre-
approved. 
 
 
Continuing Review reports and IRB approval documentation must be submitted as soon as the 
documentation is available. All amendments that occurred during the reporting period must be 
submitted at this time. 
 
 
The final study report and any supporting documents must be submitted to the HRPO as soon as 
all documents become available. 
 
 
The knowledge of any pending compliance inspection/visit by the FDA, OHRP, or other 
government agency concerning the DOD supported research, the issuance of Inspection Reports, 
FDA Form 483, warning letters, or actions taken by any Regulatory Agencies including legal or 
medial actions and any instances of serious or continuing noncompliance with the regulations or 
requirements must be reported immediately to the HRPO. 
 
 
Accurate and complete study records must be maintained and made available to representatives of 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
44 
 
the USAMRMC as a part of their responsibility to protect human subjects in research. 
 
8.11     Reporting to FDA 
 
Reporting to FDA will be accomplished within the regulatory time frame by the Regulatory 
Representative. For each adverse event observed, the Principal Investigator must decide whether it meets 
the definition of a “serious” adverse event.  The regulatory definition of a serious adverse event is an 
event that is fatal or life threatening, results in persistent or significant disability, requires intervention to 
prevent permanent impairment/damage, or an event that results in congenital anomaly, hospital admission 
or prolongation of hospitalization.  These will be reported within 24 hours by phone and by fax to the 
Regulatory Representative and submitted to FDA within the regulatory timeframes, and followed up in a 
timely manner. Regulatory Representative contact information is provided below. 
 
 
Rick Lampe 
 
Phone:  609-636-9909 
 
Email:  rick_lampe@comcast.net 
 
 
rlampe@metarmor.com 
 
The Regulatory Representative will confer with the sponsor’s medical monitor prior to submission. The 
sponsor has not given up the SAE reporting responsibility and will remain responsible for such. 
 
9.0 
DATA MANAGEMENT 
 
9.1   Data Quality Assurance 
 
To ensure the integrity of the data collected from study participants several procedures will be 
implemented. All personnel involved in data collection will be thoroughly trained in the assessment 
methods thus ensuring consistent applications of procedures and measurement consistency across 
participants. All required data for this study are to be collected on case report forms (CRFs).  Data will be 
recorded on appropriate CRFs at the time of each assessment or as soon as possible after the results of the 
hearing tests are available. 
 
Data collection will occur at the study site through the Research Electronic Data Capture (REDCap) 
program (Harris et al., 2009) created and supported by Vanderbilt University and all study data will be 
transferred to Martin Slade, MS of the data management unit at Yale University according to standard 
operating procedures developed for this study.  All data will be automatically saved to a computer hard 
drive and stored on a secure server (i.e., password protected) that is backed up daily.  All demographic 
and medical history forms will be kept secured in locked cabinets.  All informed consent documents will 
be kept at the study site in a separate locked file cabinet with restricted access. Issues related to data 
integrity will be discussed as a recurring agenda item in the bi-monthly study team meetings.  
 
The data manager is responsible for data security.  Data management activities include: source 
verification of audiology data and monitoring outliers.  Data security measures include keeping hard 
copies of data double-locked at all times with access limited to investigators, the coordinator(s) and data 
manager, as well as using password protection and secure socket layer for the database. 
 
9.2  Management of Tympanometry Data: 
 
Subjects who do not pass the tympanometry screening on initial hearing testing will be noted and 
excluded from the study. The results of the tympanometry screen will be recorded for each subject. 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
45 
 
Tympanometry data will be imported to the master data base so that subjects with an abnormal final 
tympanogram can be excluded from the final data analysis since the abnormal tympanometry may 
indicate that the threshold shift is due to middle ear pathology and not noise damage.  
 
9.3  Management of Pure Tone Audiometric Data: 
 
On days of pure tone air conduction threshold testing, data will be stored for each ear of every subject in 
the computerized audiometer and will be exported to a centralized Microsoft Access database. These data 
will also be imported into an Excel spreadsheet for storage and input into a statistical package for offline 
data analysis. The identifiers in the computer databases will consist solely of the subject identification 
number (PIN).  
 
10.0 
STATISTICAL CONSIDERATIONS 
 
10.1  Sample Size Justification 
 
Based upon the results of the 2004 study at the Marine Corps Recruit Depot (MCRD), it is estimated that 
the rate of significant threshold shift (STS) will be approximately 38% for the control (placebo) group.  A 
power analysis incorporating a 95% confidence level (alpha = 0.05), 80% power, a 1:1 ratio (placebo to 
D-methionine), and a detectable difference in threshold shift rates of 30% yields a required sample size of 
252 subjects for each of the two arms (placebo and D-methionine) of the study.  Thus a total of 504 
subjects need to complete the study.  Assuming an attrition rate of 20% in each arm over the course of the 
study, a total of 600 subjects will need to be enrolled.  
 
10.2  Primary Efficacy Analysis 
 
Two groups are planned in the randomized prospective design: the D-met and the placebo groups.  The 
primary objectives, and their associated analyses, for this study are as follows: 
 
1. To determine if there is a significant difference between the D-methionine and placebo treated 
groups in the average severity of documented side effects where severity is defined as the 
frequency of side effect multiplied by the intensity of the side effect.  Repeated measures analysis 
of variance (ANOVA) will be utilized to determine significance. 
 
2. To determine if there is a significantly reduced change in threshold hearing in each ear, adjusting 
for handedness, for the D-methionine group as compared to the placebo group at the tested 
audiometric frequencies.  The tested audiometric frequencies will be 0.5, 1, 2, 3, 4, 6 and 8 kHz.  
Multi-way (2 groups x 7 frequencies) analysis of variance (ANOVA) with interaction effects and 
correction for multiple comparisons (Newman-Keuls) will be utilized to determine significance. 
Determination for early detection will be according to the American Speech-Language Hearing 
Association (ASHA, 1994) criteria of: 1) greater than or equal to a 20 dB change at any 
frequency, 2) greater than or equal to a 10 dB change at any 2 adjacent frequencies, or 3) loss of 
response at 3 consecutive frequencies where responses were obtained at baseline.  To be 
considered significant ototoxicity, these changes must replicate within 24 hours (may be 
immediate) with no indication of middle ear abnormality.  Subjects serve as their own controls 
for audiometric change, which is computed relative to baseline measures. 
 
3. To determine if there is a significantly lower rate of STS (in either ear) for the D-methionine group 
as compared to the placebo group where STS is defined according to DOEHRSHC criteria as an 
increase of 10 dB or greater change (decrease in hearing) for the average of 2, 3 AND 4 kHz in 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
46 
 
either ear. A significant negative STS (improved hearing) is defined as a decrease of 10 dB or 
greater change  for the average of 2, 3 AND 4 kHz in either ear. An early warning shift STS 
(decrease in hearing) is defined as a 15 dB or greater change at 1, 2, 3 OR 4 kHz in either ear.  
Fisher’s exact test will be utilized to determine statistical significance. 
  
Secondary objective of the study is as follows: 
 
1.   To determine if there is a significant difference in reported level of tinnitus between the D-
methionine and the placebo groups where the level (loudness) of tinnitus will be scored on an 
eleven point scale with a value of 0 associated with very quiet and a value of 10 associated with 
very loud and on an eleven point scale where the degree of annoyance secondary to the tinnitus 
will also be scored on an eleven point scale with a value of  zero indicating that it is not 
bothersome and a value of 10 indicating that it is unbearable. Repeated measures analysis of 
variance (ANOVA) will be utilized to determine significance. 
 
The 95% confidence intervals will be estimated for all D-methionine vs. placebo differences.  
 
10.3   Interim Analysis  
 
Subjects are required to fire a minimum of 500 rounds of M-16 weapons fire as opposed to the 300 rounds 
for the Camp Pendleton study over the same period of time. Therefore a NIHL as great or greater in this 
study population is anticipated as opposed to the Camp Pendleton sample. However, no comparative data 
exist for NIHL for 500 rounds as opposed to 300 rounds, so this sample size will be adequate.  
 
To determine if the study data indicate greater NIHL for the 500 rounds could potentially support 
reduction of the sample size, therefore an interim analysis will be conducted once half the subjects have 
been enrolled.  This analysis will be limited to verification of the assumptions made for the determination 
of the sample size requirements, be handled by an outside statistician with no communication of the data 
or results to FDA, specifically, on the observed shift rate among the placebo arm.  The final sample size 
will be adjusted based upon the results of this analysis.   
 
Thus, if the shift rate of the placebo arm is greater than originally assumed, then the total number of 
subjects will be decreased accordingly based on the outside body’s statistician’s determination.  If, on the 
other hand, the shift rate of the placebo arm is less than originally assumed, then the total number of 
subjects will be increased, also based on the outside body statistician’s pronouncement. No data or results 
will be communicated to anyone involved in the study; only a determination of the increase or decrease in 
sample size will be determined and the study enrollment only will be adjusted accordingly.   
 
 
11.0 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
11.1   Risks to Human Subjects 
 
a. Human Subjects Involvement and Characteristics 
 
This is a randomized, double-blind, placebo-controlled study to determine the effect of D-methionine on 
preventing or reducing noise-induced permanent hearing loss (NIHL) before, during, and after military 
weapons training. The study drug, D-methionine is an oral, liquid suspension to be administered twice 
daily for 18 days at a dose not to exceed 100 mg/kg/day.  Six hundred subjects will be enrolled in this 
study and will be randomly assigned 1:1 to receive study drug or placebo.  


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
47 
 
 
The proposed study includes U.S. Army personnel enrolled in Drill Sergeant School (DSS) at the Combat 
Training Center of Excellence in Fort Jackson, South Carolina.  Approximately 2000 Army personnel 
participate in 17 DSS training classes each year and each class includes about 100 candidates. The 
training period is 16 days and includes 9 days of required M-16 weapons training (~500 rounds @ 156 dB 
SPL).  Subjects will be recruited during the first week of training and will be enrolled in the study for up 
to 30 days.   
 
All Soldiers are required to pass a physical training (PT) class to qualify for DSS training. There are 
additional restrictions based on weight, height and pregnancy. All Candidates are routinely screened for 
drug use by urinalysis and are excluded from DSS if any are found. DSS candidates are generally between 
21 – 45 years of age, therefore the study sample will include subjects between 21-45 years of age. 
Approximately 75% of the candidates are male and the study sample is expected to be similar. 
 
Inclusion Criteria:       
 
1.   Male or female 
 
2.   21 to 45 years of age 
 
3.   Negative pregnancy test confirmed by urine test at enrollment and prior to taking first study 
drug dose 
 
4.   Willing to use an effective method of birth control during the study (Female 
 participants should avoid pregnancy and male participants should avoid fathering children 
during study). Subject should either abstain from sexual relations or practice a method of birth 
control while taking part in the study.  Except for surgical removal of the uterus, birth control 
methods such as condoms, a diaphragm or cervical cap, birth control pills, IUD, or sperm 
killing products are not totally effective in preventing pregnancy. Male study participants 
should refrain from fathering babies while enrolled in this study. 
 
5.   Pure tone air conduction threshold average at 0.5, 1 and 2 kHz of no greater than 40 dB HL 
bilaterally with no air bone gaps greater than 10 dB and normal otoscopy and tympanometry 
screens 
 
6.    Willing to limit the use of nutritional supplements containing or derived from protein to 50 
grams of protein per day while participating in this study. 
 
7.   Ability to comply with all study requirements  
 
Exclusion Criteria: 
 
Some of these exclusion criteria already apply to DSS Candidates, but also apply in this study: 
 
1.    History of allergic or idiosyncratic reaction to methionine, amino acid mixtures, nutritional 
supplements, egg white or other proteins or food additives 
 
2.    Vegetarian (Individual excludes meat and fish from their diet) 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
48 
 
 
3.    History of chronic balance disorders  
 
4.   Abnormal otoscopic findings, otologic surgery, autoimmune inner ear disease, significant air-
bone gaps, abnormal tympanograms, or other indication of middle-ear abnormality, history of 
fluctuant hearing or asymmetry in hearing worse than 25 dB at any test frequency, or central 
nervous system disorder that is likely to affect hearing  
 
5.   Treatment with intravenous (IV) antibiotics within the past 6 months 
 
6.   Any surgical or medical condition which might significantly alter the absorption, distribution, 
metabolism, or excretion of any drug  
 
7.    History of abnormal kidney function or kidney impairment 
 
8.    Treatment for alcohol or substance abuse within last 6 months 
 
9.   Women of childbearing age who are not using effective contraceptive methods and who may 
become pregnant during the course of the study  
 
         10.  Women who are pregnant or breastfeeding  
 
  11.  National Guardsmen 
 
  12.  History of psychotic schizophrenia 
 
         13.  Exposure within the previous 6 months to systemic ototoxic substances including   
                aminoglycosides or vancomycin 
 
         14.  Body weight exceeding 225 pounds 
 
         15.  Renal impairment measured as eGFR < 50 on screening creatinine clearance blood draw  
 
b. Source of Materials 
 
1.) Demographic, health and medication history information will be collected at baseline.  Weight will be 
determined from the medical record and pregnancy testing will be performed at baseline.  Pregnancy 
testing is not routinely performed and is considered to be a study-related procedure. 
 
Audiologic assessments such as Threshold Testing, Otoscopy and Tympanometry Testing are routinely 
performed on all DSS Candidates within 3-6 months of the training period.  Repeat audiological 
assessments will be performed at the study baseline and are considered study-related procedures.  
Tinnitus assessments will be performed at the study baseline and are considered to be study-related 
procedures. All audiologic assessments will be performed using the following standardized procedures.  
CPT Jenny Davis, AuD, a licensed audiologist from the Army Hearing Program at Ft Jackson, will be 
responsible for overseeing all testing and for maintaining all audiologic data files for all subjects enrolled 
in the study.  
 
2.)  Otoscopy and Tympanometric Screening: Prior to each hearing test each Subject’s ear will be 
examined with a Welch Allyn 3.5v Macroview otoscope to ensure that the ear canals are clear and the 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
49 
 
tympanic membranes are normal in appearance. Tympanometry will be screened in each ear using pass- 
fail criteria of middle ear pressure: (pass is between +110 daPa and -160 daPa) and compliance: (Pass is 
between .2 and 1.4 ml) using the automated Interacoustics  Handheld Middle Ear Analyzer MT10. 
 
3.)  Threshold Testing:  Pure-tone threshold testing both at baseline and post-test (15-16 days after 
completion of weapons training) will be conducted utilizing the modified Hughson -Westlake procedure.  
Pure-tone air-conduction testing will be conducted at .5, 1, 2, 3, 4, 6, 8 kHz.  Bone conduction testing will 
be conducted at 5, 1, 2, 3, 4 kHz if the air-conduction threshold at that frequency is 15 dB HL or greater.   
Audiological data is collected electronically via Interacoustic 629 clinical audiometers with insert 
earphones and TDH 39 earphones.  Pure-tone threshold testing is conducted by the audiologists using 
DOEHRSHC standard test procedures using the modified Hughson Westlake procedure as follows: Initial 
descent towards threshold is accomplished in 10-dB steps. Beginning with the first non-response, level is 
increased by 5-dB for each non-response, and decreased by 10-dB after each correct detection response. 
Threshold is defined as the lowest level at which two responses are obtained out of three presentations on 
an ascending run.  Test equipment, test environment, procedures, and personnel will meet all relevant 
DOEHRS, ASHA, AAA, ANSI and JC standards and guidelines.   
 
4.)  Tinnitus Measures: Tinnitus measures: A tinnitus questionnaire modified from the Tinnitus 
Ototoxicity Monitoring Interview and the Tinnitus Handicap Index and the Tinnitus Loudness Index will 
be used on each visit to assess tinnitus.  
 
c.  Potential Risks 
 
Methionine is a micronutrient, and thus it is not alien to the human system.  Both the D- and  L- 
methionine isomers are present in a wide variety of foods.  Methionine comprises 26 mg/g high quality 
protein in the diet (National Academy of Sciences 1980). Methionine is used therapeutically for other 
purposes and at relatively high doses. Previous studies of D-methionine have had few, minor side effects, 
such as nausea and vomiting. The recommended dosing is 200-400mg orally three to four times per day. 
 
There is the potential for loss of confidentiality; however safeguards have been made to protect individual 
the Candidates’ identity. 
 
11.2   Adequacy of Protection Against Risks 
 
a.  Recruitment and Informed Consent 
 
Recruitment procedures will be finalized during the one year planning period with input from the military 
to ensure appropriateness and to enhance effectiveness. CPT Jenny Davis, AuD, Army Hearing Program, 
will provide guidance with study recruitment and orientation procedures. At a minimum, all DSS 
Candidates will be introduced to the study within the first 14 days of class and given the opportunity to 
participate.  These recruitment briefings will be coordinated and presented by non-military study 
personnel who will explain the purpose of the study, the procedures, the risks and benefits, voluntary 
nature of study participation and answer all questions prior to obtaining written informed consent. During 
recruitment briefings to a unit where a percentage of the unit is being recruited to participate as a group, 
an ombudsman not connected in any way with proposed research or the unit shall be present to monitor 
that the voluntary nature of individual participants is adequately stressed and that the information 
provided about the research is adequate and accurate (DoDD 3216.02, March 25, 2002) Written informed 
consent will be obtained before any study assessments are performed. 
 
 


 
 
 
 
 
 
 
 
 
          
           
 
D-methionine to Reduce Noise-Induced Hearing Loss 
 
 
 
50 
 
b.  Protections Against Risk 
 
All participants will be carefully screened for eligibility.  All doses of study drug will be based on subject 
weight and will be distributed with verbal and written instructions for administration by study personnel.  
Subjects will be required to use assigned military hearing protection during weapons training.  Adverse 
events will be carefully assessed and monitored throughout this study.  Should any subject experience a 
side effect or event that warrants additional medical attention, he/she will be provided medical care 
through appropriate standard operating sick call procedures at Fort Jackson.  
 
Every effort will be made to protect the confidentiality of the data obtained from each subject. Each 
subject will be assigned a study identification code number. The list of subject names and code numbers 
will be accessible to study personnel only.  This list will be kept in a locked file cabinet in the on-site 
study coordinators’ office. This code number will be used on all data collection forms.  All of the data 
will be collated into a database that will be coded so that individual subject identities will be known only 
to the investigators.  A separate code-breaking file will be kept in written form separate from the 
electronic database.  This method will be used to protect individual privacy.  Only aggregate data will be 
reported in publications with no reference to individual subjects. 
 
c.  Potential Benefits of the Proposed Research to Human Subjects and Others 
 
No promise of a direct benefit will be made to the participants. There is currently no available treatment 
or procedure that can fully protect Soldiers against permanent noise-induced hearing loss.  Results from 
this study will improve the understanding of NIHL. 
 
d.  Importance of the Knowledge to be Gained 
 
If an oral preparation of D-methionine could prevent noise-induced hearing loss and/or tinnitus, 
potentially millions of Americans and people world-wide could have an improved quality of life by 
retaining their hearing. Further the financial impact of preventing permanent NIHL and/or tinnitus could 
provide tremendous cost savings to the military and to industry.  
 
11.3   Clinical Trial Registration:  Registered on April 27, 2011 (NCT01345474), and currently 
listed as “Recruiting”. 
 
 
 
 
 
 
 
 
 
 
 
 


D-methionine to Reduce Noise-Induced Hearing Loss 
51 
 
12.0 SCHEDULE OF ASSESSMENTS/EVENTS 
a =  No noise exposure for 24 hours prior to hearing testing 
b=   Modified Hughson-Westlake procedure (0.5, 1, 2, 3, 4, 6, 8 kHz) re: DOEHRS-HC and ANSI criteria  
c=   Only if Pure Tone air conduction threshold is ≥ 15 dB HL at any frequency from .5 -4 kHz 
d=  Total Daily Dose:  Up to 100mg/kg/day x 18 days (to be administered twice daily) 
e=  Weapons training takes place on Training Day 23-32 
f=  Drug Compliance and Adverse Event Assessment 
g=  Serum creatinine at baseline; Additional safety assessments at baseline, during and after treatment (Hematology, Chemistry, Homocysteine, 
Urinalysis) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Day 
Day --30 
to --1  
Day 
1 
Day 
2 
Day 
3 
Day  
4 –14 
Day 
15-18 
Day 
19-28 
Day 
29-32 
Orientation/Recruitment 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
 
 
 
 
 
 
 
 
Screen/Eligibility  
 
Demographic 
 
Health/Medication Survey 
 
Weight  
 
Pregnancy Test 
 
Blood and Urine Samplesg 
 
X 
X 
  X 
X 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 X 
Hearing Testsa 
 
Tinnitus Questionnaires 
 
Otoscopy 
 
Tympanometry 
 
Pure tone air conduction 
thresholdb 
 
Bone conductionc 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
Randomization 
X 
 
 
 
 
 
 
 
 
Oral D-methionined 
 
 
X 
X 
X 
X 
X 
 
 
Weapons traininge 
 
 
 
 
 
X 
 
 
 
Hearing 
noise 
exposure 
compliancea 
 
 
X 
 
 
 
 
 
X 
AEsf 
 
 
X 
X 
X 
X 
X 
 
X 


D-methionine to Reduce Noise-Induced Hearing Loss 
52 
 
13.0  STUDY TIMELINE 
Up to 16 Drill Sergeant School (DSS) classes are scheduled each year and each class includes approximately 100 
candidates. To meet the enrollment goal of 600 subjects it is anticipated that approximately 50 subjects per class will 
enroll. An estimated 504 subjects are needed to complete all follow-up assessments (252 subjects per group) to detect 
significant differences for the primary outcome measures. The five year proposed timeline is provided in the table below. 
Recruitment will begin by the fourth quarter of Year 3. New DSS Training Sessions begin approximately every two-three 
weeks. Approximately 50 subjects are expected to enroll per session X 13 sessions. Please note that that the protocol 
includes enrolling the first group and completing all procedures and data collection on that group prior to additional 
treatment groups. The study team will then review that process before proceeding to the next group. After all procedures 
are documented to be flowing smoothly recruitment will proceed from each group of DSS students with classes starting 
every 2-3 weeks and thus, recruitment and data collection will overlap. 
* Subject Enrollment is continuing beyond March 2015.  
 
 
 
 
 
 
Mar 1 2011 
 
 
Mar 1 2012 
 
 
 
Mar 1 2013 
 
 
 
Mar 1 2014 
 
 
 
Mar 1 
2015* 
 
 
 
Years 
1 
 
 
2 
 
 
 
3 
 
 
 
4 
 
 
 
5 
 
 
 
Months 
1 
3 6 
9 
12 15 18 
21 24 
27 
30 
33 
36 
39 
42 45 
48 
51 54 57 
Site Visit 
x 
x 
 
 
x 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
Study Team 
Meetings 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
Prepare and 
File IND 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
IRB 
Submission/ 
Approval 
 
 
 
 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
Order Study 
Drug 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Formulate 
Study Drug 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Finalize 
Recruitment 
Plan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hire Study 
Staff 
x 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
Site Training 
 
 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
Finalize 
MOP 
 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
Pilot  
 
 
 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
Recruit/Enrol
l Subjects 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
 
 
Data 
Collection 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
Data 
Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FDA Reports 
 
 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
Publications / 
Presentations 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
Final Reports 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 


D-methionine to Reduce Noise-Induced Hearing Loss 
53 
 
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D-methionine to Reduce Noise-Induced Hearing Loss 
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D-methionine to Reduce Noise-Induced Hearing Loss 
65 
 
 
 
15.0 SIGNATURES 
 
15.1 “As the Principal Investigator, I confirm that I have read and will comply with the DDEAMC Human Research 
Protection Program (HRPP), Chapter 13, Investigator Responsibilities as available at, 
http://www.ddeamc.amedd.army.mil/clinical/investigation/documents/HRPP%20Version%20Date%20August%202010.p
df.  The protection of research subjects is the shared responsibility of Principal Investigator (PI), Associate Investigators 
(AIs), members of the research team, and the DDEAMC Institutional Review Board (IRB). However, the ultimate 
responsibility for the safety and welfare of research subjects lies with the PI." 
 
15.2 “I am aware that I am not authorized to accept any funds or other form of compensation for conducting this 
research." 
 
15.3  Date prepared:    23 March 2016 
 
 
 
 
 
 
___________________________________________________________ 
PRINCIPAL INVESTIGATOR: 
 
 
 
          
CPT William P. Grimes, M.D. 
Department of Preventive Medicine 
Moncrief Army Community Hospital 
 
 
__________________________________________________________ 
COL Traci E. Crawford, Commander  
Moncrief Army Community Hospital 
 
 


D-methionine to Reduce Noise-Induced Hearing Loss 
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Section 
Version 6.1   17 February 2016 
Version 6.2   23 March 2016 
Title 
Page 
Version 6.1    17 February 2016 
Version 6.2 
23 March 2016 
Page iii 
17 February 2016 
Version 6.1 
23 March 2016 
Version 6.2 
Page v 
TBA (Data Programmer) 
Meredith Stowe, PhD Yale 
 
 
Corrected Table of Contents Page Numbers 
Page xv 
Removed “Outer hair cell quantification” in Fig 20 label 
Corrected List of Figures Page Numbers 
Corrected Fig 20 label to “ ABR Threshold Shifts” 
Page 8 
Recent Microsoft WORD Software upgrades caused formatting 
and image errors in newer versions of the protocol. Several 
adjustments have been made to align the tables, images and 
figures. 
Adjusted Table 1 formatting 
Page 13 
Same as above 
Adjusted spacing around Fig 6 
Page 14 
Same as above 
Adjusted spacing around Fig 7 
Page 18 
Same as above 
Adjusted spacing between Fig 11 and 12 
Page 20 
Same as above 
Adjusted Fig 13 
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Same as above 
Adjusted Fig 14 
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Adjusted Fig 15 
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Same as above 
Adjusted Fig 16 and 17 
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Same as above 
Adjusted Fig 19 
Page 25 
284 kHz 
2, 4, 8 kHz 
Page 25 
 
Corrected Figure 19 and 20 alignment 
Page 26 
Removed Figure 20 label “Outer hair cell quantification….” 
 Corrected Fig 20 label to “ABR Threshold Shifts” 
Page 27 
 
Adjusted Fig 21 
Page 51  
 
Adjusted Schedule of Assessments/Events Table 
Page 52 
 
Adjusted Timeline formatting and added asterisk next to date. 
Subject enrollment is continuing beyond March 2015. 
Page 65 
 
17 February 2016 
CPT William P. Grimes, MD 
Department of Preventive Medicine 
 
 


D-methionine to Reduce Noise-Induced Hearing Loss 
67 
 
 
Section 
Version 6.0 15 December 2015 
Version 6.1  17 February 2016 
Title 
Page 
MAJ Christopher Wilson, MD, MPH 
Chief, Preventive Medicine 
15 December 2015 
CPT William P. Grimes, MD 
Department of Preventive Medicine 
17 February 2016 
Page iii 
15 December 2015 
Version 6.0 
17 February 2016 
Version 6.1 
Page iv 
MAJ Christopher Wilson, MD, MPH 
MAJ Christopher Wilson, MD, MPH 
Chief, Preventive Medicine 
CPT William P. Grimes, MD 
CPT William P. Grimes, MD 
Department of Preventive Medicine 
Page ix 
MAJ Christopher Wilson, MD, MPH 
CPT William P. Grimes, MD 
Page 33 
MAJ Christopher Wilson, MD, MPH 
CPT William P. Grimes, MD 
Page 39 
MAJ Christopher Wilson, MD, MPH 
MAJ Wilson is Chief of Preventive Medicine 
CPT William P. Grimes, MD 
CPT Grimes is a Family Physician in the Department of 
Preventive Medicine 
Page 63 
15 December 2015 
MAJ Christopher Wilson, MD, MPH 
Chief, Preventive Medicine 
17 February 2016 
CPT William P. Grimes, MD 
Department of Preventive Medicine 
 
Section 
Version 5.1 15 July 2015 
Version 6.0  15 December 2015 
Title Page 
15 July 2015 
December 2015 
Page iii 
 
Version 6.0, 15 December 2015 
Page iv 
Removed: Jill Anderson, PhD, AUD, Off-site research 
coordinator 
Add: CPT Eric Bunnell, AUD, Deputy Chief, Fort Jackson 
Army Hearing Program, Responsible for performing 
audiology testing 
 
Add: Daniel Fox, PhD, MPH, Off-site research coordinator 
Page v 
Judi Weissinger, PhD/Weissinger Solutions, Inc. 
Rick Lampe 
 
 
Page v 
 
Add:  Roosevelt Barnwell, Ombudsmen 
Page ix. 
 
Insert Exclusion Criterion:  7. History of abnormal kidney 
function or kidney impairment 
Page x 
12 hours 
approximately 12 hours 
 
Page x 
Two medical monitors will be assigned to this protocol.  One 
off site (SIU School of Medicine) and one on site to be named. 
An on-site research monitor and an off-site medical monitor 
are assigned to this protocol. 
Section 1.1 
Page 1 
11-12 days (typo) 
11-15 days 
Section 4.1 
Page 26 
fire 500 rounds of M-16 weapons fire (156 dB SPL) within a 9-
11 day period. Spent magazines 
each fire approximately 500 rounds of M-16 weapons fire 
(156 dB SPL) within a 9-11 day period. Spent cartridges 
Section 4.5 
Page 27 
 
Insert Exclusion Criterion: 7. History of abnormal kidney 
function or kidney impairment 
Section 5.2  
Page 29 
Combat plugs 
Battle plugs 
Section 5.2 
Page 29 
Deleted: a 50% study enrollment is anticipated 
 
Section 5.3 
Page 30 
A urine pregnancy test will be performed for all women of child 
bearing potential by the study coordinator(s).  Pregnancy test 
results will be recorded. 
Screening Assessments -  
Urine sample to be collected from all subjects for routine 
urinalysis.  A urine pregnancy test will be performed for all 
women of child bearing potential by the study coordinator(s).  
Pregnancy test results will be recorded. 
 
Two additional samples of blood (20-25 ml) will be 
collected for chemistry tests, complete blood 
counts, and tests of liver function, and 
homocysteine level.  
Section 6.2 
Page 33 
 
 
Urine and blood samples will be collected while on 
treatment (during second week). Two additional 
samples of blood (20-25 ml) will be collected for 
chemistry tests, complete blood counts, and tests of 
liver function, and homocysteine level. Urine 
sample will be collected for urinalysis. 


D-methionine to Reduce Noise-Induced Hearing Loss 
68 
 
Section 6.3 
Page 33 
 
 
Urine and blood samples will also be collected 
post-treatment. Two additional samples of blood 
(20-25 ml) will be collected for chemistry tests, 
complete blood counts, and tests of liver function, 
and homocysteine level. Urine sample will be 
collected for urinalysis. 
Section 8.4 
Page 36 
 
Additional safety assessments have been added to include: 
laboratory evaluations (hematology, blood chemistry, and 
urinalysis) at pre-specified time points (e.g., pre-treatment, 
during treatment, and post-treatment). 
Section 8.6 
Page 40 
Judi Weissinger, PhD 
Rick Lampe 
Section 8.8 
Page 40-41 
CDR Royce Clifford MD,  
Revised DSMC Roster: The DSMC will be composed of at 
least four voting members. The current DSMC includes: 
DSMC Chair (Patrick J. Antonelli, MD, Chair of 
Otolaryngology, University of Florida, Gainesville, the 
Medical Monitor for the Sponsor (Mark D. Packer, MD), the 
Research Monitor, David B. Pavlakovich, PA-C,  
D. Bradley Welling, MD, PhD, Chair of Otolaryngology, 
Harvard University, Boston, the Regulatory Representative 
(Rick Lampe), 
Section 
8.11 
Page 42 
 Judi  Weissinger, PhD 
Rick Lampe 
Section 
10.3 
Page 44 
Removed following statement that was inadvertently included 
in par 3: 95% confidence intervals will be used to estimate D-
methionine vs. placebo differences 
 
Section 
11.0 
Page 46 
 
Insert Exclusion Criterion: 7.  History of abnormal kidney 
function or kidney impairment 
Section 
12.0 
Page 49 
Schedule of Assessments Table 
Added additional urine and blood testing to the Schedule of 
Assessments Table 
Section 
15.0 
Page 63  
15 July 2015 
15 December 2015; Removed reference to IRBNet (no longer 
being used by DDEAMC IRB) 
 
Section 
Version 5.0 30 March 2015 
Version 5.1 15 July 2015 
Title 
Page 
30 March 2015 
15 July 2015 
Page iii 
 
Version 5.1,  15 July 2015, add associate investigator, minor 
edits 
Page iv 
 
Demarcus F. Bush, AUD 
Responsible for performing audiologic testing. 
Page v 
 
Add/remove ombudsmen, minor edits 
 
 
 
Section 
Version 4.4  June 27, 2014 
Version 5.0  March 30 2015 
Title 
Page 
Protocol Version 4.4 
Serial #0003 
LTC William Bimson, D.O. 
Deputy Commander for Clinical Services 
27 June 2014 
Protocol Version 5.0 
Serial #0005 
MAJ Christopher E. Wilson, MD, MPH, Chief, Preventive 
Medicine 
30 March 2015 
 
iii 
Version #:  4.4 
Version #:  5.0 
iii 
Version Date: 27 June 2014 
Version Date:  30 March 2015 
iii 
 
Site PI and other personnel changes 
iv 
CPT Rebecca Ludwig, AUD 
CPT Jenny Davis 
v 
MAJ Christopher Wilson MD, MPH 
David B. Pavlakovich, PA-C 
 
Linda Poole, Jeffrey Bullock 
Lin Wright 
5.5 
CPT Rebecca Ludwig AUD 
CPT Jenny Davis AUD 
7.2 
LTC William Bimson, D.O. 
Major Christopher Wilson, M.D. 
8.6 
LTC William Bimson D.O. 
MAJ Christopher Wilson, MD, MPH 
MAJ Christopher Wilson, MD, MPH 
David B. Pavlakovich, PA-C 


D-methionine to Reduce Noise-Induced Hearing Loss 
69 
 
8.8 
MAJ Christopher Wilson, MD, MPH 
David B. Pavlakovich, PA-C 
11.1 
CPT Rebecca Ludwig, AUD, Chief 
CPT Jenny Davis, AUD 
11.2 
CPT Rebecca Ludwig AUD, Chief 
CPT Jenny Davis AUD 
16.0 
June 27, 2014 
March 30, 2015 
16.0 
LTC William Bimson, D.O. 
MAJ Christopher E. Wilson MD, MPH 
 
Section 
Version 4.3  February 25, 2014 
Version 4.4 June 27, 2014 
Title 
Page 
Version 4.3 
February 25, 2014 
Version 4.4  
June 27, 2014 
iii 
Version #:  4.3 
Version #:  4.4 
iii 
Version Date:  25 February 2014 
Version Date:  27 June 2014 
iii 
 
Fort Jackson requested changes, personnel changes 
iv 
CPT Virginia Bailey 
CPT Jenny Davis 
v 
MAJ Matthew Hanna, PA-C 
MAJ Christopher Wilson, M.D. 
v 
Marilynn Bailey, Yvette Bennett, Jeffrey Bullock, Kendra Neely 
Jeffrey Bullock, Ciera Nicholson, Sarah Sims, Gregory Bullock 
x 
10.  Army Reservists or National Guardsmen 
10. National Guardsmen 
4.3 
. . .exactly 500 rounds 
. . . to meet the 500 round requirements . 
. . .approximately 500 rounds 
. . . to meet training requirements. 
4.4 
2.  21 to 40 years of age. 
2.  21 to 45 years of age. 
4.4 
6.  Willingness to refrain from using nutritional supplements 
containing or derived from protein while participating in this 
study. 
6.  Willing to limit the use of nutritional supplements containing 
or derived from protein to 50 grams per day while participating 
in this study 
4.5 
10. Army Reservists or National Guardsman 
10.  National Guardsmen 
8.6 
MAJ Matthew Hanna, PA-C, MAPS 
MAJ Christopher Wilson, M.D. 
8.8 
MAJ Matthew Hanna PA-C, MAPS 
MAJ Christopher Wilson  M.D. 
11.1 
. . . generally between 21 - 40 years of age. 
. . .generally between 21- 45 years of age. 
11.1 
.. . subjects between 21-40 years of age. 
. . . subjects between 21 - 45 years of age. 
11.1 
2.  21 to 40 years of age 
2.  21 to 45 years of age 
11.1 
6.  Willingness to refrain from taking nutritional supplements 
containing or derived from protein while participating in this 
study. 
6.  Willing to limit the use of supplements containing or derived 
from protein to 50 grams per day while participating in this 
study. 
 
Section 
Version 4.2 January 8, 2014 
Version 4.3 February 25, 2014 
15.0 
 
Added Signature Page:  
As the Principal Investigator, I confirm that I have read and 
will comply with the DDEAMC Human Research Protection 
Program (HRPP), Chapter 13, Investigator Responsibilities as 
available at, 
http://www.ddeamc.amedd.army.mil/clinical/investigation/do
cuments/HRPP%20Version%20Date%20August%202010.pdf
.  The protection of research subjects is the shared 
responsibility of Principal Investigator (PI), Associate 
Investigators (AIs), Careline/Department Chief, members of 
the research team, and the DDEAMC Institutional Review 
Board (IRB). However, the ultimate responsibility for the 
safety and welfare of research subjects lies with the PI." 
15.2 “  I am aware that I am not authorized to accept any funds 
or other form of compensation for conducting this research.  
16.0 Date prepared:   25 February 2014 
 
PRINCIPAL INVESTIGATOR: 
 
 
 
          
LTC William Bimson, D.O. 
Deputy Commander of Clinical Services 
Moncrief Army Medical Center 
 
DEPT CHIEF 
 
COL Mark L. Higdon, Commander 
 


D-methionine to Reduce Noise-Induced Hearing Loss 
70 
 
 
 
Section 
Version 4.1 August 28, 2013 
Version 4.2 January 8, 2014 
 
Title 
page 
Leonard Rybak, M.D., Ph.D 
Professor of Surgery 
Southern Illinois University Medical School 
Col Mark D. Packer, MD 
Executive Director, DoD/VA hearing Center of Excellence 
Clinical Assistant Professor, UTSA school of Medicine 
Chief of Neurology & Cranial Base Surgery 
Title 
page 
Protocol Version 4.1 
28 August 2013 
Protocol Version 4.2 
8 January 2014 
Page iii 
 
Added version change, date change and personnel change 
Page iv 
Leonard Rybak, MD, PhD 
Col Mark D. Packer, MD 
Page v 
 
Added: Research Monitor- 
MAJ Matthew Hanna, PA-C, MAPS 
Page v 
 
Added: Marilynn Bailey, Yvette Bennett, Jeffrey Bullock and 
Kendra Neely as Ombudsmen 
Page ix 
6.  Willing to refrain from using multivitamins, herbals or other 
nutritional supplements while participating in this study 
6.  Willing to refrain from using supplements containing or 
derived from protein while participating in this study 
Page xi 
 
Modified schema to reflect Post audiological assessments on 
Study Days 29-32 
Page 1 
(Monday-Friday and Monday-Thursday over a 2 week period), 
and 4 days after weapons training for a total of 18 days of dosing.  
Tinnitus questionnaires will be assessed before weapons training  
and 15-16 days after completion of weapons training (i.e. 11days 
(over a 2 week period), and 4 days after weapons training for a 
total of 18 days of dosing.  Tinnitus questionnaires will be 
completed before weapons training  begins (i.e. 11-15days 
Page 26 
The first primary endpoint is to confirm safety and tolerability of 
administering up to 100 mg/kg daily, given in divided doses, 12 
hours apart 
The first primary endpoint is to confirm safety and tolerability 
of administering up to 100 mg/kg daily, given in divided doses, 
twice daily (with morning and evening meal). 
Page 27 
6. Willingness to refrain from taking multivitamins, herbals, or 
other  nutritional supplements while participating in this study. 
 
6. Willingness to refrain from using  nutritional supplements 
containing or derived from protein while participating in this 
study. 
 
Page 33 
Study Days 28-30 
Study Days 29-32 
Page 29 
 
Deleted: with weapons training occurring Monday through 
Friday of the first week and Monday through Thursday of the 
second week. 
Page 29 
Candidates will be invited, but not required, to attend a 
recruitment briefing to introduce this research study during the 
first 14 days of class 
Candidates will attend a recruitment briefing to introduce this 
research study during the first 14 days of class 
 
 
Deleted:  The type of personal protection will be captured on 
the Candidate’s Reference Audiogram, Form DD2215 and 
Hearing Conservation, Form DD 2216 that is completed after 
noise exposure. 
Page 30 
We anticipate that all subjects will be using Combat Plugs 
provided by the Ft Jackson audiologists.  Compliance with hearing 
protection use will be assessed each day by the study 
coordinator(s). and recorded on the Case Report Form (CRF). 
We anticipate that all subjects will be using Combat Plugs 
provided by the Ft Jackson audiologists.  Compliance with 
hearing protection use will be assessed and recorded each day 
by the study coordinator(s). 
Page 30 
Audiological data will be recorded on the standard military 
audiologic assessment collection form, Department of Defense 
(DD) Form 2215/2216 modified to include 8 kHz data.   
Deleted: Audiological data will be recorded on the standard 
military audiologic assessment collection form, Department of 
Defense (DD) Form 2215/2216 modified to include 8 kHz data.  
The threshold data will also be directly exported to an Excel 
file and copied into the research chart. onto case report forms. 


D-methionine to Reduce Noise-Induced Hearing Loss 
71 
 
 
 
 
 
Section 
Version 4.0 August 1, 2013 
Version 4.1 August 28, 2013 
Title page 
  
Changed serial number, Protocol Version, Date and 
Principal Investigator 
Page iii 
  
Changed Version, Date 
  
  
Added Version, Date and Reason for Revision 
Page iv 
  
Changed Principal Investigator 
Page v 
  
Corrected name of Study Coordinator, added second Study 
Coordinator 
Page ix 
  
Changed Principal Investigator 
  
 
3.  History of balance disorders 
 
3. History of chronic balance disorders 
Page 32 
The threshold data will also be directly exported 
to an Excel file and copied onto case report forms. 
The threshold data will also be directly exported to an Excel 
file and copied into the research chart. 
Page 32 
 
Deleted: The computerized database will be configured so 
that when the post-noise audiogram is entered in the 
database, criteria for hearing change will automatically be 
calculated. and flagged, so that the examiner can recheck the 
frequencies in question 
Page 33 
Evening formations 
Evening meals 
Page 33 
Study Day 28- 30 
Study Day 29 - 32 
Page 33 
Subjects will be contacted by to remind them to 
avoid noise exposure for at least 24 hours prior to 
the audiological assessments (hearing tests) by the 
on-site study coordinator(s). 
Subjects will be contacted by telephone or text message to 
remind them to avoid noise exposure for at least 24 hours 
prior to the audiological assessments (hearing tests) by the 
on-site study coordinator(s). 
Page 36 
Baseline and Study Visit 1 assessments are 
limited to general health history information and 
pregnancy testing.   
Baseline and Study Visit 1 assessments are limited to general 
health history information, pregnancy testing and laboratory 
assessment for kidney function.   
Page 37 
(Study Day 29 or 30) 
(Study Day 29 -32) 
Page 39 
Dr. Leonard Rybak 
Col Mark D. packer 
Page 39  
An on-site medical monitor will be identified 
prior to study enrollment 
Deleted 
Page 40  
 
Added MAJ Matthew Hanna, PA-C, MAPS will serve as the 
independent research monitor on site 
Page 40 
The monitor will review data after each cohort has 
completed the study because all of the 
investigators are blind to the study arm 
assignment 
Sentence Deleted 
Page 41  
Leonard Rybak, MD, PhD 
Mark D. Packer, MD 
Page 41 
 
Added:  the Research Monitor, MAJ Matthew Hanna PA-C, 
MAPS 
Page 43 
Data management activities include: using double 
data entry and monitoring outliers 
Data management activities include: source verification of 
audiology data and monitoring outliers 
Page 46 
6. Willingness to refrain from taking 
multivitamins, herbals, or other  nutritional 
supplements while participating in this study. 
 
6.  Willing to refrain from using supplements containing or 
derived from protein while participating in this study 
Page 49 
Day -14 to -10 and Day 29-30 
Day -30—10 and Day 29-32 


D-methionine to Reduce Noise-Induced Hearing Loss 
72 
 
Section 4.1 
This is a prospective, randomized, double-blind, 
placebo-controlled study to evaluate the effect of 
D-methionine (D-met) on permanent NIHL after 
required weapons training. The study will 
include 600 Army personnel enrolled in Drill 
Sergeant School (DSS) scheduled to undergo 9 
days of weapons training over an 11 day period 
at Ft Jackson, South Carolina. This training 
requires that they fire 500 rounds of M-16 
weapon fire (156 dB SPL) in a 9-11 day period. 
Spent magazines are collected and tallied by 
DSS leaders  to document rounds fired. All 
subjects will be randomized to two equal arms; 
one to oral D-methionine (test drug) and the 
other to flavor-matched placebo. In both arms, 
there will be no other change in scheduled 
treatment. This facility trains 1600- 2040 drill 
sergeant instructors annually. 
This is a prospective, randomized, double-blind, placebo-
controlled study to evaluate the effect of D-methionine (D-
met) on permanent NIHL after required weapons training. 
The study will include 600 Army personnel enrolled in Drill 
Sergeant School (DSS) scheduled to undergo 9 days of 
weapons training over an 11 day period at Ft Jackson, South 
Carolina. This training requires that they fire 500 rounds of 
M-16 weapon fire (156 dB SPL) within a 9-11 day period. 
Spent magazines are collected and tallied by DSS leaders  to 
document rounds fired. All subjects will be randomized to 
two equal arms; one to oral D-methionine (test drug) and the 
other to flavor-matched placebo. In both arms, there will be 
no other change in scheduled treatment. This facility trains 
approximately 2000 drill sergeant instructors annually. 
Section 4.5 
3.   History of balance disorders  
 
3.   History of chronic balance disorders  
 
Section 5.2 
Candidates will be invited, but not required, to 
attend a recruitment briefing to introduce this 
research study during the first week of class. 
(Days 1 - 5)  
Candidates will be invited, but not required, to attend a 
recruitment briefing to introduce this research study during 
the first 14 days of class.   
Section 7.2 
  
Changed Principal Investigator 
Section 8.6 
  
Changed Principal Investigator 
Section 8.8 
The DSMC will meet prior to study initiation- 
no less than once per year and more frequently if 
needed to review enrollment and safety data and 
after the planned interim analysis.  
The DSMC will review the protocol prior to study initiation 
and meet no less than once per year and more frequently if 
needed to review enrollment and safety data and after the 
planned interim analysis.  
Section 11.1  
Exclusion 
Criteria 
3. History of balance disorders. 
3. History of chronic balance disorders 
Section 11.2  
a.  Recruitment and Informed Consent 
Recruitment procedures will be finalized during 
the one year planning period with input from the 
military to ensure appropriateness and to 
enhance effectiveness. CPT Rebecca Ludwig, 
AuD, Army Hearing Program Manager, will 
provide guidance with study recruitment and 
orientation procedures. At a minimum, all DSS 
Candidates will be introduced to the study 
within the first 1-4 days on class Day 11 of 
training and given the opportunity to participate.   
a.  Recruitment and Informed Consent Recruitment 
procedures will be finalized during the one year planning 
period with input from the military to ensure appropriateness 
and to enhance effectiveness. CPT Rebecca Ludwig, AuD, 
Army Hearing Program Manager, will provide guidance 
with study recruitment and orientation procedures. At a 
minimum, all DSS Candidates will be introduced to the 
study within the first 14 days of class and given the 
opportunity to participate.   
Section 13.0  
Study Timeline 
  
Dates added to reflect current status.  Extended to 57 
months. 


D-methionine to Reduce Noise-Induced Hearing Loss 
73 
 
Page 63  Product 
Label 
  
  
 
 
"Placebo to  D-methionine" 
Removed and submitted as "Revised Product Label."  
 
"D-Methionine/Placebo" 
Pages 64 - 91  
 Case Report Forms listed 
Removed and submitted as Case Report Forms (separate 
docs)_ 
Section 
Version 3.5 May 1, 2013 
Version 4.0 August 1, 2013 
Page iv and 
throughout the 
document 
PI: LTC Neil Page, M.D. 
PI: LTC William Bimson, M.D. 
 
Page vi 
On site Study Coordinator: TBA 
Beth Bullock, R.N. : On-site Study Coordinator 
 
Page 47 
within the first 1-4 days 
on class Day 11 
Page 49 
19-23 and 26-29 
23-32 
Section 
Version 3.4 March 7, 2013 
Version 3.5 May 1, 2013 
Cover Page 
Protocol Version 3.4  
Protocol Version 3.5 
Cover Page 
April, 2013, 2012  
May 1, 2013 
DIAGNOSIS 
AND KEY 
SUBJECT 
SELECTION 
CRITERIA 
 
Added:  14.  Renal impairment measured as eGFR < 50 on 
screening creatinine clearance blood draw 
 
Deleted: History of renal impairment 
Section 4.5  
 
Added:  14.  Renal impairment measured as eGFR < 50 on 
screening creatinine clearance blood draw 
 
Deleted: History of renal impairment 
Section 5.3  
 
Added:  A blood draw for serum creatinine will be conducted 
prior to enrollment in order to calculate estimated GFR by the 
Cockroft-Gault equation. Those subjects with an estimated 
eGFR < 50 will be excluded from enrollment. 
Section 11.1  
 
Added:  A blood draw for serum creatinine will be conducted 
prior to enrollment in order to calculate estimated GFR by the 
Cockroft-Gault equation. Those subjects with an estimated 
eGFR < 50 will be excluded from enrollment 
 
Deleted: History of kidney impairment 
 
 
Section 12.0  
 
Added:  Serum Creatinine 
Section 
Version 3.3 March 7, 2013 
Version 3.4 March 7, 2013 
Cover Page 
Protocol Version 3.3  
Protocol Version 3.4 
Cover Page 
March  7, 2013, 2012  
April 2013 
Page v 
 
Added: Marilynn Bailey, Sarah Dishon, and Deb Dunkelberg--
-Ombudsmen---Will serve as an independent, neutral and 
impartial mediator of the Soldiers in order to ensure that no 
coercion is observed for the recruiting and consenting study 
activities. 
Section 7.3 
The formulated drug product (D-methionine) will 
be provided for clinical use as an oral suspension. 
Each dose will be packaged in individually labeled 
vials.  The label will include instructions to shake 
the suspension prior to administration. 
The formulated drug product (D-methionine) will be provided 
for clinical use as an oral suspension. Each dose will be 
packaged in individually labeled vials.  Each vial will have a 
subject identification number on the label (Section 15.1 Master 
Label).  The label will include instructions to shake the 
suspension prior to administration. 
Section 7.4 
 
Added:  Refrigeration is not required.  Room temperature 
will be monitored and recorded daily, including weekends 
and holidays. 
 
Section 15.1 
 
Replaced Master Label 


D-methionine to Reduce Noise-Induced Hearing Loss 
74 
 
 
Section 
November 30, 2012 Version 3.2 
Version 3.3 March 7, 2013 
Cover Page 
Protocol Version 3.2  
Protocol Version 3.3 
Cover Page 
November 30, 2012  
March 07 2013 
Page iii 
Version # 3.2 
Version # 3.3 
Page iii 
Version Date: 30November2012 
Version Date: 07March2012 
Page v 
TBA 
Marilyn Bailey and staff 
DIAGNOSIS 
AND KEY 
SUBJECT 
SELECTION 
CRITERIA 
(Exclusion 
criteria) 
 
Added: 12.  History of psychotic schizophrenia 
13.  Exposure within the previous 6 months to systemic 
ototoxic substances including aminoglycosides or vancomycin 
14  Body weight exceeding 225 pounds 
Section 4.5 
 
Added: 12.  History of psychotic schizophrenia 
13.  Exposure within the previous 6 months to systemic 
ototoxic substances including aminoglycosides or vancomycin 
14.  Body weight exceeding 225 pounds 
Section 7.2  
Study drug will be dispensed  by on-site study 
coordinator(s), who are qualified medical 
personnel (RN or LPN).  The study drug will be 
dispensed as labeled in accordance with the study 
protocol.  LTC Neil Page, M.D., will supervise the 
activities of the study coordinator(s) and 
audiologists. 
Study drug will be dispensed  by MACH Pharmacy to on-site 
study coordinator(s), who are qualified medical personnel (RN 
or LPN).  The study drug will be distributed to the study 
participants as labeled in accordance with the study protocol.  
LTC Neil Page, M.D., will supervise the activities of the study 
coordinator(s) and audiologists. 
Section 7.3 
Each subject’s dose of D-methionine will be 
individually determined based on actual subject 
weight and assigned dose level. For ease of 
administration and packaging, dosing is broken 
down by weight categories with dosing based on 
the lowest weight in that category as follows: 
Each subject’s dose of D-methionine will be individually 
determined based on actual subject weight. For ease of 
administration and packaging, dosing is broken down by 
weight categories with dosing based on the lowest weight in 
that category as follows: 
Section 7.3 
Body Weight 
lbs (kg) 
Total Daily Dose (Up 
to 100 mg/kg/day) 
100-125 lbs  
(45 - 57 kg) 
4.6 grams (4600 mg) 
126-150 lbs  
(57 - 68 kg) 
5.6 grams (5600 mg) 
151-175 lbs  
(68 - 80 kg) 
6.8 grams (6800 mg) 
176-200 lbs  
(80 - 91 kg) 
8.0 grams ( 8000 mg) 
201-225 lbs  
(91 - 102 kg) 
9.2 grams (9200 mg) 
226-250 lbs 
(103 -114 kg) 
10.3 grams (10,300 
mg) 
Body Weight lbs (kg) 
Total Daily Dose (Up 
to 100 mg/kg/day) 
 
100-125 lbs  (45 - 57 kg) 
4.6 grams (4600 mg) 
126-150 lbs  (57 - 68 kg) 
5.6 grams (5600 mg) 
151-175 lbs  (68 - 80 kg) 
6.8 grams (6800 mg) 
176-200 lbs  (80 - 91 kg) 
8.0 grams ( 8000 mg) 
201-225 lbs  (91 - 102 kg) 
9.2 grams (9200 mg) 
 
Section 7.3 
For example, if subject’s weight is 155 lbs (70.4 
kg) at baseline, then the subject will receive 6.8 
grams of study drug per day.  This dose will be 
divided into two equal doses (3.4 grams) and 
administered prior to morning and evening meal 
(approximately 12 hours apart).   
For example, if subject’s body weight is 155 lbs (70.4 kg) then 
the subject’s total daily dose equals 6.8 grams per day.  This 
dose will be divided into two equal doses (3.4 grams) and 
administered prior to morning and evening meal 
(approximately 12 hours apart).  Each dose (AM and PM) will 
be packaged in individually labeled vials by KP 
Pharmaceuticals. 
 
Section 7.4  
All study drug will be stored under conditions 
consistent with package labeling.  All study drug 
will be stored in a secure limited-access area under 
controlled temperature in accordance with labeled 
storage requirements.  Room temperature will be 
monitored and recorded daily 
All study drug will be stored under conditions consistent with 
package labeling.  All study drug will be stored in a secure 
limited-access area under controlled temperature in accordance 
with labeled storage requirements.  Room temperature will be 
monitored and recorded daily including weekends and 
holidays. 


D-methionine to Reduce Noise-Induced Hearing Loss 
75 
 
Section 7.8  
Treatment with intravenous (IV) antibiotics, such 
as Amikacin: Amikin®, Gentamicin, Netilmicin: 
Netromycin®, Streptomycin, Tobramycin: 
Nebcin® or any surgical or medical condition 
which might significantly alter the absorption, 
distribution, metabolism, or excretion of any drug.  
Subjects who received treatment for drug or 
alcohol abuse within the previous 6 months are 
prohibited from  participation in this study. 
Treatment with intravenous (IV) antibiotics, such as 
Amikacin: Amikin®, Gentamicin, Netilmicin: Netromycin®, 
Streptomycin, Tobramycin: Nebcin® or any other 
aminoglycosides and/or vancomycin.  Also, any surgical or 
medical condition which might significantly alter the 
absorption, distribution, metabolism, or excretion of any drug.  
Subjects who received treatment for drug or alcohol abuse 
within the previous 6 months are prohibited from  participation 
in this study. 
Section 11.1 
Exclusion Criteria 
 
Added:  12.  History of psychotic schizophrenia 
13.  Exposure within the previous 6 months to systemic 
ototoxic substances including   aminoglycosides or 
vancomycin 
 
Section 
November 5, 2012 Version 3.1 
November 30, 2012 Version 3.2  
Cover Page 
Protocol Version 3.1  
Protocol Version 3.2 
Page iii 
Version # 3.1 
Version # 3.2 
Page iii 
Version Date: 5November2012 
Version Date: 30November2012 
Page 28 
5.  Treatment with intravenous (IV) antibiotics 
within the past 6 months 
 
5.  Treatment with intravenous (IV) antibiotics or 
Carboplatin/Cisplatin within the past 6 months 
 
Section 5.1 
 
Added:  An ombudsman must be present during the recruiting 
and consenting process. 
Section 5.2 
 
Added: Consent will be obtained by study coordinator(s) not 
affiliated with the DSS program. 
Section 8.6  
The Regulatory  Representative  will   be included 
via teleconference to assess the regulatory impact 
of outcomes of adverse events and new literature 
reviews to assess change to the risk/benefit ratio 
and to determine if modifications need to be made 
or the study terminated 
The Regulatory  Representative (Dr. Weissinger) will   be 
included via teleconference to assess the regulatory impact of 
outcomes of adverse events and new literature reviews to 
assess change to the risk/benefit ratio and to determine if 
modifications need to be made or the study terminated 
Section 8.8 
The data safety monitoring committee (DSMC) 
evaluates study data on an ongoing basis to assure 
participant safety and study integrity. The DSMC 
will review study data and unanticipated problems 
and make recommendations based on their 
reviews. The DSMC will meet prior to study 
initiation, no less than once per year and more 
frequently if needed to review enrollment and 
safety data and after the planned interim analysis. 
Evaluation will also occur if three subjects have 
met the stopping criteria. If all three are 
determined to be in the D-methionine-treated 
group, the DSMC will consider a lower dose as a 
safe dose with which to continue the study.  The 
DSMC Chair may call an emergency meeting at 
any time should issues of patient safety arise. The 
DSMC will be composed of at least three voting 
members. The current DSMC includes: DSMC 
Chair (CDR Royce Clifford, MD, Officer-in-
charge, Marine Air Group 39 Medical Clinic  
Camp Pendleton, CA), the medical monitor for the 
Sponsor (Leonard Rybak, MD, PhD), the 
Regulatory Representative (Judi Weissinger, 
PhD), and the Yale University 
epidemiologist/statistician (Carrie Redlich, MD, 
MPH).  Only the Yale University statistician will 
be unblinded and will endeavor to keep the 
committee blinded during discussion of the 
stopping criteria, adverse events, and any study 
adjustments recommended by the committee. 
The data safety monitoring committee (DSMC) evaluates 
study data on an ongoing basis to assure participant safety and 
study integrity. The DSMC will review study data and 
unanticipated problems and make recommendations based on 
their reviews. The DSMC will meet prior to study initiation, 
no less than once per year and more frequently if needed to 
review enrollment and safety data and after the planned 
interim analysis. Evaluation will also occur if three subjects 
have met the stopping criteria. If all three are determined to be 
in the D-methionine-treated group, the DSMC will consider a 
lower dose as a safe dose with which to continue the study.  
The DSMC Chair may call an emergency meeting at any time 
should issues of patient safety arise. The DSMC will be 
composed of at least three voting members. The current 
DSMC includes: DSMC Chair (CDR Royce Clifford, MD, 
Officer-in-charge, Marine Air Group 39 Medical Clinic  Camp 
Pendleton, CA), the medical monitor for the Sponsor (Leonard 
Rybak, MD, PhD), the Regulatory Representative (Judi 
Weissinger, PhD), and the Yale University 
epidemiologist/statistician (Carrie Redlich, MD, MPH).  Only 
the Yale University statistician will be unblinded and will 
endeavor to keep the committee blinded during discussion of 
the stopping criteria, adverse events, and any study 
adjustments recommended by the committee. 


D-methionine to Reduce Noise-Induced Hearing Loss 
76 
 
Section 11.1 
3.   
Negative pregnancy test confirmed by 
urine test at enrollment and prior to taking first 
study drug dose 
3.   
Negative pregnancy test confirmed by urine dipstick 
test at enrollment and prior to taking first study drug dose 
 
 
 
Section 
October 5, 2012 Version 3.0 
November 5, 2012 Version 3.1 
Section 4.4 
 
Added an Inclusion Criterion: Willingness to refrain from 
taking multivitamins, herbals, or other nutritional supplements 
while participating in this study. 
Section 4.5  
Exclusion Criterion #5:Exposure in the previous 6 
months to systemic ototoxic substances including 
aminoglycoside   antibiotics, or chemotherapy 
with carboplatin, cisplatin, vincristine, vinblastine 
or difluoromethylornithine 
Exclusion Criterion #5:  Treatment with intravenous (IV) 
antibiotics or Carboplatin/Cisplatin within the past 6 months 
Section 7.8 
Exposure in the previous 6 months to systemic 
ototoxic substances including aminoglycoside 
antibiotics, or chemotherapy with carboplatin, 
cisplatin, vincristine, vinblastine or 
difluoromethylornithine will exclude a subject. In 
addition any surgical or medical condition which 
might significantly alter the absorption, 
distribution, metabolism, or excretion of any drug 
and a recent history of drug or alcohol abuse are 
prohibitive of participation in this study. 
 Treatment with intravenous (IV) antibiotics, such as 
Amikacin: Amikin®, Gentamicin, Netilmicin: Netromycin®, 
Streptomycin, Tobramycin: Nebcin® or any surgical or 
medical condition which might significantly alter the 
absorption, distribution, metabolism, or excretion of any drug.  
Subjects who received treatment for drug or alcohol abuse 
within the previous 6 months are prohibited from  participation 
in this study. 
Section 11.3 
April 27, 2001 
April 27, 2011  
Section 
August 10, 2012 Version 
2.2 
October 5, 2012 Version 3.0 
Title Page 
 
Added IND #, changed serial number, Protocol Version and Principal Investigator 
Page iii 
 
Added IND# , changed version date and version# 
Roles and 
Responsibilities 
for Clinical Study 
 
The Principal Investigator 
for this IND application is 
CPT William G. Callis, 
M.D., M.P.H., Preventive 
Medicine Physician 
 
CPT Rebecca Ludwig-
Supervising Audiologist 
 
1LT Virginia Best Bailey, 
Audiologist 
The Principal Investigator for this IND application is, LTC Neil Page, MD, Deputy 
Commander for Clinical Services 
 
 
 
 
CPT Rebecca Ludwig, Co-Investigatory, Supervising Audiologist 
 
 
CPT Virginia Best Bailey, Co-Investigator, Audiologist 


D-methionine to Reduce Noise-Induced Hearing Loss 
77 
 
Roles and 
Responsibilities 
for Clinical Study 
 
Jose Cruz, PAC, MPAS/FT 
Jackson 
 
 
TBA (non-military)  On-site 
Study Coordinator 
DELETED 
 
 
 
(2) TBA (non-military) (2) On-site Study Coordinators 
 
Added: Marilyn Bailey  Responsible for assigning an ombudsman for the  
Army Corp Coordinator study-related recruiting activities 
 
Added: TBA  Ombudsman  Will serve as an independent neutral and impartial  
mediator of the Soldiers in order to ensure that no coercion is observed for the 
recruiting and consenting study activities. 
Table of Contents 
Study Day 33-36 
Study Day 29-30 
Table of Contents 
15.3  CRF-Follow-Up-Study 
Visit 2 (19-22 Days Post 
Weapons Training 
15.3  CRF – Follow Up – Study Visit 2 (15-16 Days Post Weapons Training) 
Clinical Synopsis 
 
Changed Phase 2 to Phase 3 Clinical Trials, updated PI, changed trainees to 
Candidates 
Study Design 
Schema 
Study Day 36 
Study Days 29-30 
List of 
Abbreviations 
 
Added: MACH and SOP 
Section 1.0 
19-22 days 
 
Drill Sergeant Instructor 
Training School 
15-16 days 
 
Drill Sergeant School (DSS) 
 
Added: … commandant COL Mark Higdon, MD, of Moncrief Army Medical Center 
(MACH) and 2nd Commandant CSM Michael McCoy, DSS. 
Section 3.6 
Phase II 
Phase 3 
Section 4.3 
This training requires that 
each Soldier fire exactly 500 
rounds of M-16 weapons fire 
(156 dB SPL) in a 9 day 
period of weapons training 
with a possible additional 
makeup day. 
 
Every Soldier 
 
Drug logs will be kept for 
every subject, recording 
every dose taken or missed. 
  
Pre and post training tinnitus 
assessments are not routinely 
done and are a required 
assessment for this study 
which will be conducted by 
the audiologist.  
 
This training requires that each Soldier fire exactly 500 rounds of M-16 weapons fire 
(156 dB SPL) within a 9-11 day period of weapons training with a possible additional 
makeup day if necessary to meet the 500 round requirements 
 
 
 
 
Every DSS Candidate 
 
Drug logs will be kept for every subject, recording every dose taken or missed or 
whether or not battle plugs were used.  
 
 
Tinnitus assessments are not routinely done and are a required assessment for this 
study which will be conducted by the audiologist and/or study coordinators.  
 
 
 
 
Section 4.7 
…the 36 day study period 
…the 30 day study period 
Section 5.0 
 
Section 5.1 
 
 
Section 5.2 
 
 
 
 
 
Ft Jackson has 17 classes per 
year for Drill Sergeant 
Instructor Trainees. 
All instances of “Trainees” changed to “Candidates” 
 
Added:  An ombudsman must be present during the recruiting and consenting process. 
 
Ft Jackson has 16 classes per year for DSS Candidates 
 


D-methionine to Reduce Noise-Induced Hearing Loss 
78 
 
Section 5.3 
 
 
 
 
 
 
 
Section 5.5 
Body weight and height- 
Weight will be measured on 
a calibrated scale with the 
subject wearing usual 
military clothing and no 
shoes by the study 
coordinators (s)  
 
Audiological data will be 
recorded on the standard 
military audiologic 
assessment collection form, 
Department of Defense (DD) 
Form 2215/2216 modified to 
include 8 kHz data. The 
thresholds will also be 
copied onto case report 
forms. 
Body weight  – Body weight obtained at DSS entry and recorded in the candidate 
electronic health record  will be used to determine dosing of the Investigational Drug.  
 
 
 
 
Audiological data is collected electronically via XXXX audiometers and recorded on 
the standard military audiologic assessment collection form, Department of Defense 
(DD) Form 2215/2216 modified to include 8 kHz data. The thresholds will also be 
copied onto case report forms. 
Section 5.5.3 
Pure-tone air conduction 
threshold testing both at 
baseline and post-test (19-22 
days after completion of 
weapons training and 15-18 
days after the last day of 
study drug/placebo 
administration) will be 
conducted utilizing the 
modified Hughson-Westlake 
procedure.19-22 days 
Pure-tone air conduction threshold testing both at baseline and post-test (15-16 days 
after completion of weapons training and 11-12 days after the last day of study 
drug/placebo administration) will be conducted utilizing the modified Hughson-
Westlake procedure. 
Section 6.2 
 
 
Study Days 1-18 
Added: Assess for hearing protection compliance 
 
Study Days 2-18 
Section 6.3 
Study Days 33-36 
Study Days 28-30 
Section 7.1 
bottles 
Small vials 
Section 7.2  
CPT William C. Callis M.D., 
M.P.H. will supervise the 
activities of the study 
coordinator(s). 
 
 
LTC Neil Page, M.D., will supervise the activities of the study coordinator(s) and 
audiologists. 
 
 
Section 7.3 
The study coordinator(s) will 
be responsible for recording 
the weight of each Soldier. 
DELETED 
Section 7.3 
KPT will assign the 
appropriate randomization 
code and prepare 
individualized doses of study 
drug prior to shipping. 
KPT will assign the appropriate randomization code and prepare individualized doses 
of study drug prior to shipping to the Moncrief Army Community Hospital (MACH) 
Pharmacy at FT Jackson 
Section 7.5  
Upon receipt of study drug, 
the MACH Pharmacist will 
ensure that the information 
on the packing slips matches 
exactly with information sent 
to the site, including content, 
amount, lot numbers, 
quantity and expiration date.  
 
Upon receipt of study drug, the MACH Pharmacist will ensure that the information on 
the packing slips matches exactly with information sent to the site, including content, 
amount, lot numbers, quantity and expiration date.  
 
Added:  This information will be recorded in each subject’s EHR and on their 
medication log (CRF). 
Section 7.6  
All doses of study drug will 
be recorded on each 
subject’s medication log. 
DELETED 
Section 8.4.1  
Study Day 36 
Study Day 29 or 30 
Section 8.6 
Two medical monitors will 
be assigned to this study.  
Dr. Leonard Rybak will 
Dr. Leonard Rybak will serve as the off-site medical monitor and LTC Neil Page, MD 
will serve as the Principal Investigator and will oversee all study drug administration 
by the on-site study coordinator(s). Dr. Rybak is Professor of Surgery and a licensed 


D-methionine to Reduce Noise-Induced Hearing Loss 
79 
 
serve as the off-site 
medical monitor and Jose 
Cruz, PA C, MPAS will 
serve as the onsite medical 
monitor.  CPT William G. 
Callis, MD, MPH  will 
serve as the Principal 
Investigator and will 
oversee all study drug 
administration by the on-
site study coordinator(s). 
Dr. Rybak is Professor of 
Surgery and a licensed 
otolaryngologist at SIU 
School of Medicine.  Dr. 
Callis is Preventative 
Medicine Physician at 
Moncrief Army 
Community Hospital, Fort 
Jackson.  
otolaryngologist at SIU School of Medicine.  Dr. Page is  the Deputy Commander for 
Clinical Services at Moncrief Army Community Hospital, Fort Jackson.   
 
 
Section 9.1 
Data collection will occur 
at the study site and all 
study data will be 
transferred to the data 
management unit at Yale 
University according to 
standard operating 
procedures developed for 
this study. 
Data collection will occur at the study site through the Research Electronic Data 
Capture (REDCap) program created and supported by Vanderbilt University and all 
study data will be transferred to Martin Slade, MS of the data management unit at 
Yale University according to standard operating procedures developed for this study. 
Section 9.3 
Subject name and social 
security number will not 
appear in the computer 
databases 
 
DELETED 
Section 10.3 
…handled by and outside 
body 
…handled by an outside statistician 
Section 11.1 
The training period is 55 
days… 
 
…will be enrolled in the 
study for up to 36 days 
 
Audiologic assessments 
such as Threshold Testing, 
Otoscopy and 
Tympanometry Testing are 
routinely performed on all 
DSS trainees. 
 
3)….(19-22 days) 
The training period is 16 days 
 
 
 
…will be enrolled in the study for up to 30 days 
 
 
Audiologic assessments such as Threshold Testing, Otoscopy and Tympanometry 
Testing are routinely performed on all DSS candidates within 3-6 months of the 
training period.  Repeat audiological assessments will be performed at the study 
baseline and are considered study-related procedures. 
 
 
 
 
3) 
Section 12.0 
Schedule of 
Assessments 
Weight & Height 
 
Day 19-32 
 
Day 33-36 
Weight 
 
Day 29-30 
 
Day 29-30 


D-methionine to Reduce Noise-Induced Hearing Loss 
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Section 13.0 
…17 Drill Sergeant School 
classes 
 
…every three weeks 
…16 Drill Sergeant School classes 
 
 
….every two-three weeks 
Appendices 
Version 30March2012 
 
Phase 2 
 
15.1 CASE REPORT 
FORMS 
 
15.1 CASE REPORT 
FORMS 
Version 15August2012 
 
DELETED 
 
15.1  MASTER LABEL 
 
 
15.2  CASE REPORT FORMS 
 
 
Section 
April 19. 2012 Version 2.1 
August 10, 2012 Version 2.2 
 
Minor typographical errors, date
errors and personnel changes 
Minor typographical, date and personnel name corrections 
Section 
April 18, 2012 Version 2.0 
April 19th, 2012 Version 2.1 
Minor typographical errors 
Minor typographical errors corrected 
Section 
December 21, 2011 Version 
1.0 
April 18th, 2012 Version 2.0 
2.7.1 
TABLE 1.  Relative utilization 
on sulfur amino acid 
isomers, analogs, and 
precursors 
TABLE 1.  Relative utilization on sulfur amino acid isomers, analogs, and 
precursors (Baker 2006) 
2.7.3 
Figures 3 & 4.  Effects of 
experimental diets on the 
growth (A) and food 
consumption (B) of rats is 
followed by incorrect 
diagrams 
Figures 3 & 4.  Effects of experimental diets on the growth (A) and food 
consumption (B) of rats, is followed by the correct diagrams 
4.4 
4.  Willing to use an effective 
method of birth control during 
the study (Both male and 
female participants should 
avoid pregnancy during study).
Subject should either abstain 
from sexual relations or 
practice a method of birth 
control while taking part in the
study.  Except for surgical 
removal of the uterus, birth 
control methods such as 
condoms, a diaphragm or 
cervical cap, birth control 
pills, IUD, or sperm killing 
products are not totally 
effective in preventing 
pregnancy. Male study 
participants should refrain 
from fathering babies while 
enrolled in this study. 
4.  Willing to use an effective method of birth control during the study (Female 
participants should avoid pregnancy and male participants should avoid fathering 
children during the study.)  Subject should either abstain from sexual relations or 
practice a method of birth control while taking part in the study.  Except for surgical 
removal of the uterus, birth control methods such as condoms, a diaphragm or cervical 
cap, birth control pills, IUD, or sperm killing products are not totally effective in 
preventing pregnancy. Male study participants should refrain from fathering babies 
while enrolled in this study. 
5.3 
• Body weight and height - 
Weight will be recorded and 
used for ordering study drug 
for each individual subject.   
• Body weight and height - Weight will be measured on a calibrated scale with the 
subject wearing usual military clothing and no shoes by the study coordinator(s).   
Screening weight will be recorded on the Case Report Form by the study coordinator(s) 
and used to determine dosing of the Investigational Drug.  


D-methionine to Reduce Noise-Induced Hearing Loss 
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8.4.1 
Compliance and adverse event 
reports will be obtained each 
day that study drug is 
administered (Study Days 1-
18) and at the end of the study 
(Study Day 36).  Each adverse 
event will be graded according 
to the Table for Grading 
Severity of Adverse Events 
(AEs). All other laboratory 
and clinical AEs that occur in 
a subject will be assessed for 
severity and classified into one 
the categories below. 
 Grade 1 (Mild): event 
requires minimal or no 
treatment and do not interfere 
with the patient’s daily 
activities. 
 Grade 2 (Moderate): event 
results in a low level of 
inconvenience or concern with 
the therapeutic measures.  
Moderate events may cause 
some interference with 
functioning. 
 Grade 3 (Severe): event 
interrupts a patient’s usual 
daily activity and may require 
systemic drug therapy or other 
treatment.  Severe events are 
usually incapacitating. 
 Grade 4 (Life threatening):  
Any adverse drug experience 
that places the patient or 
participant, in the view of the 
investigator, at immediate risk 
of death from the reaction as it 
occurred, i.e., it does not 
include a reaction that had it 
occurred in a more severe 
form, might have caused 
death. 
 Grade 5 (Death) 
8.4.1   Assessment of Safety  
General health status and symptom assessments performed at Baseline/Eligibility Visit 
and Study Visit 1 will serve as baseline for any new signs or symptoms that arise 
during the study.  All such changes occurring after Study Visit 1 will be evaluated as 
possible adverse events and appropriately recorded on the Adverse Event Case Report 
Form (CRF). 
Baseline and Study Visit 1 assessments are limited to general health history information 
and pregnancy testing.  No additional physical exam, clinical and/or laboratory 
assessments are included in the protocol. Expected adverse events include possible 
gastrointestinal (GI) symptoms (nausea, vomiting, constipation, diarrhea, dysphagia).  
Subjects will be questioned prior to receiving each study dose and on the last study day 
for the presence  
of GI side effects. *See Estimating Severity Grade and method for classifying GI adverse 
events.  
Other subject reported symptoms that occur between Study Day 1 and Study Day 18, 
including the follow up visit at Study Day 36, will be evaluated as a possible adverse 
event. If a subject experiences a side effect that requires additional medical attention, 
the subject will seek medical attention through appropriate standard operating sick call 
procedures and the medical monitor will be contacted.   
 
Adverse events will be evaluated according to the Estimating Severity Grade and will be 
recorded on the Adverse Event Log and the Adverse Event CRF.  Adverse events 
deemed greater than Grade 2 will be considered a Serious Adverse Event (SAE). All 
SAEs will be recorded on the SAE CRF and reported according to FDA and institutional
requirements (See Section 8.5 – Specific Serious Adverse Event Reporting 
Requirements). 
Compliance and adverse event reports will be obtained each day that study drug is 
administered (Study Days 1-18) and at the end of the study (Study Day 36) by the Study 
Coordinator(s).  Each adverse event will be graded according to the Table for 
Estimating Severity Grade. 
For all subjects receiving at least one dose of study drug, the number and percentage of 
subjects reporting adverse events will be tabulated by cohort and overall by severity and 
body system.   
ESTIMATING SEVERITY GRADE 
Grade 1 
(Mild) 
Event requires minimal or no treatment and does not interfere with the subject’s  
daily activities 
Grade 2 
(Moderate) 
Event results in a low level of inconvenience or concern with the therapeutic  
measures.  Moderate events may cause some interference with functioning. 
Grade 3  
(Severe) 
Event interrupts a patient’s usual daily activity and may require systemic drug  
therapy or other treatment.  Severe events are usually incapacitating. 
Grade 4 
(Life 
Threatening) 
Any adverse drug experience that places the patient or participant, in the view of the 
investigator, at immediate risk of death from the reaction as it occurred, i.e., it  
does not include a reaction that had it occurred in a more severe form, might have  
caused death. 
Grade 5 
Death 
GASTROINTESTINAL 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Nausea Mild or transient; 
maintains 
reasonable intake 
Moderate discomfort; 
intake decreased 
significantly; some 
activity limited 
No significant intake; 
requires IV fluids 
Hospitalization  
required 
Vomitin
g 
1 episode in 24 
hours 
2-5 episodes in 24 
hours 
> 6 episodes in 24 
hours or needing IV 
fluids 
Physiological 
consequences  
requiring hospitalization 
or requiring parenteral 
nutrition 


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Constipa
tion 
Requiring stool 
softener or dietary 
modification 
Requiring laxatives 
Obstipation requiring 
manual evacuation 
or enema 
Obstruction or toxic 
megacolon 
Diarrhea Mild or transient; 3-
4 loose stools/day 
or mild diarrhea 
lasting for less than 
1 week 
Moderate or 
persistent; 5-7 loose 
stools/day or 
diarrhea lasting > 1 
week 
> 7 loose stools/day 
or bloody diarrhea; 
or orthostatic 
hypotension or 
electrolyte 
imbalance or > 2L 
IV fluids required 
Hypotensive shock or 
physiological 
consequences 
requiring hospitalization 
Oral 
discomf
ort/dys
phagia 
Mild discomfort; no 
difficulty 
swallowing 
Some limits on 
eating/drinking 
Eating/talking very 
limited; unable to 
swallow solid foods 
Unable to drink fluids; 
requires IV fluids 
Bibliography 
List 
None 
Added pages 50-61 listing references 
Appendices list 
None 
Added 6 CRFs including Baseline Eligibility Visit, Study Visit 1, Study Visit 2, 
Medication Log/AE Assessment,  Adverse Events, Serious Adverse Events 
Baseline eligibility 
protocol 
Inadvertently left out page 4 of 
5 in Baseline Eligibility Visit 
Page 4 of 5 in Baseline Eligibility Visit inserted