a le prell cg, b boxer pa, b bennett dc, c ohlemiller kk, d dolan df, and b,d miller jm
DESCRIPTION
From Bench to Bedside and Back Again: Development and Translation of an Otoprotective Agent. a Le Prell CG, b Boxer PA, b Bennett DC, c Ohlemiller KK, d Dolan DF, and b,d Miller JM a Department of Communicative Disorders, University of Florida b OtoMedicine, Inc., Ann Arbor, MI - PowerPoint PPT PresentationTRANSCRIPT
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)