aaLe Prell CG, Le Prell CG, bbBoxer PA, Boxer PA, bbBennett DC, Bennett DC, ccOhlemiller KK, Ohlemiller KK, ddDolan DF, and Dolan DF, and b,db,dMiller JMMiller JM

aaDepartment of Communicative Disorders, University of Department of Communicative Disorders, University of FloridaFloridabbOtoMedicine, Inc., Ann Arbor, MIOtoMedicine, Inc., Ann Arbor, MIccDepartment of Otolaryngology, Washington University Department of Otolaryngology, Washington University Medical SchoolMedical SchoolddDepartment of Otolaryngology, University of MichiganDepartment of Otolaryngology, University of Michigan

From Bench to Bedside and From Bench to Bedside and Back Again: Development Back Again: Development

and Translation of an and Translation of an Otoprotective AgentOtoprotective Agent

Overview: Translational Overview: Translational ResearchResearch

From the BedsideFrom the Bedside Clinical Observations of NIHL in patient populationsClinical Observations of NIHL in patient populations

to the Benchto the Bench 20+ years of work by many groups20+ years of work by many groups Identification of metabolic insult as major contributor Identification of metabolic insult as major contributor

to NIHL to NIHL Therapeutic intervention with free radical scavengers Therapeutic intervention with free radical scavengers

Back to the BedsideBack to the Bedside Critical challenge is translation of academic data into Critical challenge is translation of academic data into

clinically useful therapeutic agents clinically useful therapeutic agents On-going clinical trials are most recent step in On-going clinical trials are most recent step in

translation from animal models to human patientstranslation from animal models to human patients

NIHL: The Clinical ProblemNIHL: The Clinical Problem 22ndnd most common occupational disease most common occupational disease Affects workers in numerous industries Affects workers in numerous industries Hazardous noise exposure prevalent for military Hazardous noise exposure prevalent for military

personnelpersonnel US VA spent almost $1B in fiscal year 2005US VA spent almost $1B in fiscal year 2005

Recreational activitiesRecreational activities hunting, concert/nightclub attendance, sporting hunting, concert/nightclub attendance, sporting

events events MP3 player use?MP3 player use?

Emerging evidence in animal models Emerging evidence in animal models suggests sub-clinical neural damage after suggests sub-clinical neural damage after noise insult may interact with age-related noise insult may interact with age-related deficits (Kujawa and Liberman)deficits (Kujawa and Liberman)

Intact Sensory Cells Intact Sensory Cells Noise-Damaged CellsNoise-Damaged Cells

Historic Thinking: Noise exposure causes hearing loss strictly via mechanical trauma

Raphael Y, Lenoir M, Wroblewski R, Pujol R. The sensory epithelium and its innervation in the mole rat cochlea. J Comp Neurol. 314:367-82, 1991.

TRANSDUCTION BY HAIR TRANSDUCTION BY HAIR CELLSCELLS

Tectorial membrane

Basilar membrane

IHC

OHCs

http://www.iurc.montp.inserm.fr/cric/audition/english/ear/fear.htm

Molecular events leading to cell deathMolecular events leading to cell death

O2●¯

●OH

H2O2

Excess free radicals Damage

cellular lipids, proteins, and DNA

Upregulate apoptotic pathways

Manipulation of endogenous antioxidant system influences NIHL

Adapted from Le Prell et al., Hearing Research. 226:22-43, 2007

Free RadicalsFree Radicals Highly reactive clusters of atomsHighly reactive clusters of atoms Produced during cellular respiration, essential for normal Produced during cellular respiration, essential for normal

cell physiologycell physiology Intense metabolic activity increases their formationIntense metabolic activity increases their formation Oxygen-based: Reactive Oxygen SpeciesOxygen-based: Reactive Oxygen Species

superoxide (Osuperoxide (O22--); hydroxyl (OH); peroxyl (RO); hydroxyl (OH); peroxyl (RO22); alkoxyl (RO); ); alkoxyl (RO);

hydroperoxyl, (HOhydroperoxyl, (HO22) ) Other oxygen-species that readily generate radical species: Other oxygen-species that readily generate radical species:

hydrogen peroxide (Hhydrogen peroxide (H22OO22); hypochlorous acid (HOCl); Ozone ); hypochlorous acid (HOCl); Ozone (O(O33); singlet oxygen (); singlet oxygen (110022) )

Nitrogen-based: Reactive Nitrogen Species Nitrogen-based: Reactive Nitrogen Species Nitric oxide (NO) and its by-productsNitric oxide (NO) and its by-products Nitrate (NONitrate (NO33

--); nitrite (NO); nitrite (NO22--); peroxynitrite (ONOO); peroxynitrite (ONOO--); 3-); 3-

nitrotyrosine (3-NT)nitrotyrosine (3-NT)

4-HNE and Nitrotyrosine, Adapted from Yamashita et al., Brain Research. 1019(1-2):201-9, 2004

No noise

7D post

10D post

Persistent Stress Confirmed Post-Noise Persistent Stress Confirmed Post-Noise ROS RNSROS RNS

No noise

3D post

7D post

Lateral wall

OHCs

OHCsSupporting cells

*

*

Salicylate plus vitamin E reduces noise-Salicylate plus vitamin E reduces noise-induced hearing lossinduced hearing loss

Adapted from Yamashita D, Jiang HY, Le Prell CG, Schacht J, Miller JM. Post-exposure treatment attenuates noise-induced hearing loss. Neuroscience, 134:633-42, 2005.

* * * *

8 kHz 16 kHz

Frequency

4 kHz

Th

resh

old

Sh

ift (

dB

)

0

10

20

30

40

50

60 Saline Pre 1H 1D 3D 5D * *

*

* * *

Salicylate plus vitamin E reduces noise-Salicylate plus vitamin E reduces noise-induced outer hair cell deathinduced outer hair cell death

Saline

Pe

rce

nt M

issi

ng H

air

Cel

ls

0

20

40

60

80

100 Pre 1H

1D

0 2 4 6 8 10 12 14 16 18 20

0

20

40

60

80

100 3D

Distance from Apex (mm)2 4 6 8 10 12 14 16 18 20

5D

2 4 6 8 10 12 14 16 18 20

Adapted from Yamashita et al. Neuroscience, 134:633-42, 2005.

*

Salicylate plus vitamin E reduces noise-Salicylate plus vitamin E reduces noise-induced outer hair cell deathinduced outer hair cell death

Group

Saline Pre 1H 1D 3D 5D

Per

cent

Mis

sing

Out

er H

air

Cel

ls

0

20

40

60

80

100

* **

Adapted from Yamashita et al. Neuroscience, 134:633-42, 2005.

Nitrotyrosine 10 days post-noise

Saline

Reduced RNS formation at 10 days post-noise

1H Post, Sal+E

Pre-noise, Sal+E

Adapted from Yamashita et al., Neuroscience. 134(2):633-42, 2005.

4-HNE 10 days post-noise

Saline

Adapted from Yamashita et al., Neuroscience. 134(2):633-42, 2005.

Reduced ROS formation at 10 days post-noise

3D Post, Sal+E

Pre-noise, Sal+E

Many Antioxidants Reduce NIHLMany Antioxidants Reduce NIHL SalicylateSalicylate

w/NAC, Kopke et al., 2000w/NAC, Kopke et al., 2000 w/vitE, Yamashita et al., 2005w/vitE, Yamashita et al., 2005

resveratrol resveratrol Seidman et al., 2003Seidman et al., 2003

allopurinolallopurinol Cassandro et al., 2003Cassandro et al., 2003

R-phenylisopropyladenosine R-phenylisopropyladenosine (R-PIA)(R-PIA) Hu et al., 1997 Hu et al., 1997 Hight et al., 2003Hight et al., 2003

superoxide dismutase-superoxide dismutase-polyethylene glycolpolyethylene glycol Seidman et al., 1993Seidman et al., 1993

U74389FU74389F Quirk et al., 1994Quirk et al., 1994

D-methionine D-methionine Campbell et al., 2007Campbell et al., 2007 Kopke et al., 2002Kopke et al., 2002

Ebselen Ebselen Pourbakht and Yamasoba, Pourbakht and Yamasoba,

20032003 Lynch et al., 2004Lynch et al., 2004 Lynch and Kil, 2005Lynch and Kil, 2005 Yamasoba et al., 2005Yamasoba et al., 2005

N-acetylcystein N-acetylcystein Ohinata et al., 2003; Ohinata et al., 2003; Duan et al., 2004Duan et al., 2004 w/salicylate, Kopke et al., w/salicylate, Kopke et al.,

20002000 no effect on TTS in man no effect on TTS in man

(Kramer et al., 2006) or (Kramer et al., 2006) or rodents (Kopke et al., 2000; rodents (Kopke et al., 2000; Duan et al., 2004)Duan et al., 2004)

Translation to humans?Translation to humans?

Antioxidants and vasodilators likely to be a Antioxidants and vasodilators likely to be a potent combinationpotent combination

For human clinical application, agents For human clinical application, agents must be safe for daily usemust be safe for daily use

We evaluated the efficacy of three We evaluated the efficacy of three antioxidant vitamins, magnesium (which antioxidant vitamins, magnesium (which acts in part as a vasodilator), and a acts in part as a vasodilator), and a combination of these agentscombination of these agents

Dietary MicronutrientsDietary Micronutrients Beta-carotene, Vitamins C and E, MagnesiumBeta-carotene, Vitamins C and E, Magnesium

Beta-carotene: scavenges singlet oxygen, prevents lipid Beta-carotene: scavenges singlet oxygen, prevents lipid peroxidation peroxidation

Vitamin E: reduces peroxyl radicals in lipid layer Vitamin E: reduces peroxyl radicals in lipid layer Vitamin C: scavenges free radicals in aqueous phaseVitamin C: scavenges free radicals in aqueous phase Magnesium: reduces noise-induced vasoconstriction, blocks Magnesium: reduces noise-induced vasoconstriction, blocks

NMDA receptors, prevents calcium influx and neural NMDA receptors, prevents calcium influx and neural excitotoxicity, also has antioxidant propertiesexcitotoxicity, also has antioxidant properties

Patent pending, University of MichiganPatent pending, University of Michigan Inventors: Josef Miller, Colleen Le Prell, Jochen Schacht, Diane Inventors: Josef Miller, Colleen Le Prell, Jochen Schacht, Diane

PrieskornPrieskorn Option to license by OtoMedicine, Inc.Option to license by OtoMedicine, Inc. Human clinical trials in progressHuman clinical trials in progress

Antioxidants plus magnesium reduce noise-Antioxidants plus magnesium reduce noise-induced hearing loss: additive effectsinduced hearing loss: additive effects

8 kHz 16 kHz

Frequency

**

4 kHz

Thr

esho

ld S

hift

(dB

)

0

10

20

30

40

50

60

*

SalineACEMgACEMg

Mean + S. E., Le Prell et al. 2007, Free Rad. Med. Biol., 42,1454-1463.

Antioxidants plus magnesium reduce Antioxidants plus magnesium reduce outer hair cell death: additive effectsouter hair cell death: additive effects

0

Saline

Out

er H

air

Cel

l Los

s (P

erce

nt)

0

20

40

60

800.1 1 10

ACE

Cochlear Position (Distance from Apex, mm)0 5 10 15

0

20

40

60

80

Mg

Characteristic Frequency (kHz)0.1 1 10

ACEMg

5 10 15 20

Mean + S. E., Le Prell et al., 2007, Free Rad. Med. Biol., 42,1454-1463.

Antioxidants & magnesium reduce Antioxidants & magnesium reduce sensory cell death: effects are additivesensory cell death: effects are additive

15-20

*

10-14.99

Distance from Apex (mm)

OH

C L

oss

(per

cent

)

0

10

20

30

40

50

60

*

Saline

ACE

Mg

ACEMg

Mean + S. E., Le Prell et al., 2007, Free Rad. Med. Biol., 42,1454-1463.

RNS Byproducts Post-Noise in First Turn of Guinea Pig Cochlea

Saline-control

Nutrient treatment starting one day prior to noise

Octave band noise centered at 4 kHz, 115-dB SPL x 4 hours

Outer hair cells

Outer hair cells

EndoG Post-Noise in First Turn of Guinea Pig Cochlea

Octave band noise centered at 4 kHz, 115-dB SPL x 4 hours

Outer hair cells

Saline-control

Nutrient treatment starting one day prior to noise

Outer hair cells

Nutrients Prevent NIHL After Less Intense Noise

Adapted from Le Prell, C.G., Schmitt J., Dolan D.F., Boxer P.A., et al. (2009). Prevention of temporary noise-induced threshold deficits using dietary agents. Abs Assoc Res Otolaryngol 32, 280.

Oral Treatment Prevents NIHL in Mice: Dose-Dependent Effects

Adapted from Le Prell, C.G., Ohlemiller, K.K., Gagnon, P.M., and Bennett, D.C., 2009. Reduction in permanent noise-induced threshold deficits in mice fed a combination of dietary agents. Abs. Assoc. Res. Otolaryngol. 32, 280.

Control: N=16Diet A: N=8Diet B: N=7

Functional Protection Explained by Preservation of Cells in Lateral Wall

Cell density in animals fed Diet B was equivalent to that in normal animals without history of noise exposure

Adapted from Le Prell, C.G., Ohlemiller, K.K., Gagnon, P.M., and Bennett, D.C., 2009. Reduction in permanent noise-induced threshold deficits in mice fed a combination of dietary agents. Abs. Assoc. Res. Otolaryngol. 32, 280.

Mix Matters!Mix Matters!

Previous studies Previous studies show any of these show any of these nutrients when nutrients when delivered for days-delivered for days-weeks pre-noiseweeks pre-noise

With dosing beginning With dosing beginning 1 hour1 hour pre-noise, the pre-noise, the combination of combination of vitamins and Mg was vitamins and Mg was essential for essential for functional protectionfunctional protection

Human Trials with NutrientsHuman Trials with Nutrients Safe dosing limits well-characterizedSafe dosing limits well-characterized US Recommended Daily AllowanceUS Recommended Daily Allowance

No RDA for beta-caroteneNo RDA for beta-carotene Pre-formed Vitamin A: 1.5 mg Pre-formed Vitamin A: 1.5 mg ~18 mg beta-carotene using retinol activity equivalents~18 mg beta-carotene using retinol activity equivalents

Vitamin C: 60 mgVitamin C: 60 mg Vitamin E: 15 mgVitamin E: 15 mg Magnesium: 300-400 mgMagnesium: 300-400 mg

US Upper Limit (Institute of Medicine)US Upper Limit (Institute of Medicine) No UL for beta-caroteneNo UL for beta-carotene

Pre-formed Vitamin A: 3 mg Pre-formed Vitamin A: 3 mg ~36 mg beta-carotene using retinol activity equivalents~36 mg beta-carotene using retinol activity equivalents

Vitamin C: 2,000 mgVitamin C: 2,000 mg Vitamin E: 1,000 mgVitamin E: 1,000 mg Magnesium: 350 mgMagnesium: 350 mg

The Agents Must be Safe

AREDS study provides 7-year safety data for the vitamins

Translation to humans

Daily Dose Upper Limit Percent of UL

B-Carotene 18 mg1 36 mg (US)

20 mg (EU)

50 (US)

90 (EU)

Ascorbic Acid 500 mg 2000 mg 25

A-tocopherol 270 mg 1000 mg 27

Magnesium 315 mg 350 mg 90

1 Based on retinol activity equivalents

Human Clinical Trials: 2008-2013Human Clinical Trials: 2008-2013 NIH-funded U01, NIH-funded R44 (Phase II SBIR)NIH-funded U01, NIH-funded R44 (Phase II SBIR) Temporary Threshold Shift ModelTemporary Threshold Shift Model

US students listening to music with insert earphonesUS students listening to music with insert earphones US students exposed to video game impulse noiseUS students exposed to video game impulse noise Soldiers exposed to automatic weapons fire (Sweden)Soldiers exposed to automatic weapons fire (Sweden)

Permanent Threshold Shift ModelPermanent Threshold Shift Model NATO soldiers at Los Llanos airbase (Spain)NATO soldiers at Los Llanos airbase (Spain) Employees at cutlery stamping factories (Spain)Employees at cutlery stamping factories (Spain)

Multi-site collaboration: Multi-site collaboration: Josef Miller, Glenn Green, and Ken Guire (University of Josef Miller, Glenn Green, and Ken Guire (University of

Michigan)Michigan) Kathleen Campbell (SIU), Sharon Kujawa (Harvard) Kathleen Campbell (SIU), Sharon Kujawa (Harvard) University of Florida: James W. Hall III, Pat AntonelliUniversity of Florida: James W. Hall III, Pat Antonelli Spain: Jose Juiz (UCLM), Julio Carbayo (NATO), Luis Gonzalez Spain: Jose Juiz (UCLM), Julio Carbayo (NATO), Luis Gonzalez

(Solemat)(Solemat) Sweden: Ulf, Rosenhall, Ann-Christin Johnson and Mats Sweden: Ulf, Rosenhall, Ann-Christin Johnson and Mats

Ulfendahl (Karolinska Institutet)Ulfendahl (Karolinska Institutet)

Summary

In most cases, mechanical hearing protection will be the In most cases, mechanical hearing protection will be the best option, and drugs/dietary supplements will provide best option, and drugs/dietary supplements will provide additional benefit for those cases in which mechanical additional benefit for those cases in which mechanical protection is inadequate. protection is inadequate. Sometimes hearing protection is not an option, sometimes it just Sometimes hearing protection is not an option, sometimes it just

isn’t good enough, and sometimes noise is unexpected.isn’t good enough, and sometimes noise is unexpected. We need an arsenal of new drugs and/or other agents.We need an arsenal of new drugs and/or other agents. There is no “silver bullet” -- different people will need There is no “silver bullet” -- different people will need

different treatments. different treatments. We are getting there!We are getting there!

There is an ongoing revolution in hearing science, in There is an ongoing revolution in hearing science, in which for the first time, we have been able to effectively which for the first time, we have been able to effectively influence hearing outcomes after noise, drugs, or other influence hearing outcomes after noise, drugs, or other chemicals.chemicals.

AcknowledgmentsAcknowledgments CollaboratorsCollaborators

Josef Miller (UM)Josef Miller (UM)

Larry Hughes (SIU)Larry Hughes (SIU)

Daisuke YamashitaDaisuke Yamashita

Tatsuya YamasobaTatsuya Yamasoba

Funding AgenciesFunding Agencies University of Florida Office of University of Florida Office of

ResearchResearch

NIHNIH

GM/UAWGM/UAW

Ruth & Lynn Townsend Ruth & Lynn Townsend EndowmentEndowment

StudentsStudents Ashley Weimer Johnson (UF)Ashley Weimer Johnson (UF) Jason Schmitt (UF)Jason Schmitt (UF) Mike Goodson (UF)Mike Goodson (UF) Bianca Gomez (UF)Bianca Gomez (UF) Dustin Lang (UF)Dustin Lang (UF) Debbie Joseph (UF)Debbie Joseph (UF)

Research TechniciansResearch Technicians Amanda Dossatt (UF)Amanda Dossatt (UF)

Diane Prieskorn (UM)Diane Prieskorn (UM) Alice Mitchell (UM)Alice Mitchell (UM) Susan DeRemer (UM)Susan DeRemer (UM)