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a Le Prell CG, b Boxer PA, b Bennett DC, c Ohlemiller KK, d Dolan DF, and b,d Miller JM

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

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a Le Prell CG, b Boxer PA, b Bennett DC, c Ohlemiller KK, d Dolan DF, and b,d Miller JM

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  1. From Bench to Bedside and Back Again: Development and Translation of an Otoprotective Agent aLe Prell CG, bBoxer PA, bBennett DC, cOhlemiller KK, dDolan DF, and b,dMiller JM aDepartment of Communicative Disorders, University of Florida bOtoMedicine, Inc., Ann Arbor, MI cDepartment of Otolaryngology, Washington University Medical School dDepartment of Otolaryngology, University of Michigan

  2. Overview: Translational Research From the Bedside Clinical Observations of NIHL in patient populations to the Bench 20+ years of work by many groups Identification of metabolic insult as major contributor to NIHL Therapeutic intervention with free radical scavengers Back to the Bedside Critical challenge is translation of academic data into clinically useful therapeutic agents On-going clinical trials are most recent step in translation from animal models to human patients

  3. NIHL: The Clinical Problem 2nd most common occupational disease Affects workers in numerous industries Hazardous noise exposure prevalent for military personnel US VA spent almost $1B in fiscal year 2005 Recreational activities hunting, concert/nightclub attendance, sporting events MP3 player use? Emerging evidence in animal models suggests sub-clinical neural damage after noise insult may interact with age-related deficits (Kujawa and Liberman)

  4. Historic Thinking: Noise exposure causes hearing loss strictly via mechanical trauma Intact Sensory Cells Noise-Damaged Cells 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.

  5. TRANSDUCTION BY HAIR CELLS Tectorial membrane OHCs IHC Basilar membrane http://www.iurc.montp.inserm.fr/cric/audition/english/ear/fear.htm

  6. H2O2 ●OH O2●¯ Molecular events leading to cell death • Excess free radicals • Damage cellular lipids, proteins, and DNA • Upregulate apoptotic pathways • Manipulation of endogenous antioxidant system influences NIHL Adapted fromLe Prell et al., Hearing Research. 226:22-43,2007

  7. Free Radicals Highly reactive clusters of atoms Produced during cellular respiration, essential for normal cell physiology Intense metabolic activity increases their formation Oxygen-based: Reactive Oxygen Species superoxide (O2-); hydroxyl (OH); peroxyl (RO2); alkoxyl (RO); hydroperoxyl, (HO2) Other oxygen-species that readily generate radical species: hydrogen peroxide (H2O2); hypochlorous acid (HOCl); Ozone (O3); singlet oxygen (102) Nitrogen-based: Reactive Nitrogen Species Nitric oxide (NO) and its by-products Nitrate (NO3-); nitrite (NO2-); peroxynitrite (ONOO-); 3-nitrotyrosine (3-NT)

  8. Persistent Stress Confirmed Post-Noise ROS RNS No noise No noise 3D post 7D post Lateral wall OHCs 7D post 10D post OHCs Supporting cells 4-HNE and Nitrotyrosine, Adapted fromYamashita et al., Brain Research. 1019(1-2):201-9,2004

  9. Salicylate plus vitamin E reduces noise-induced hearing loss Saline Pre 1H 1D 3D 5D 60 50 * 40 * * * Threshold Shift (dB) * * 30 * * * 20 * * * 10 0 4 kHz 8 kHz 16 kHz Frequency Adapted fromYamashita D, Jiang HY, Le Prell CG, Schacht J, Miller JM. Post-exposure treatment attenuates noise-induced hearing loss. Neuroscience, 134:633-42, 2005.

  10. Salicylate plus vitamin E reduces noise-induced outer hair cell death 100 Saline Pre 1H 80 60 40 20 Percent Missing Hair Cells 0 100 1D 3D 5D 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 Distance from Apex (mm) Adapted fromYamashita et al. Neuroscience, 134:633-42, 2005.

  11. Salicylate plus vitamin E reduces noise-induced outer hair cell death 100 80 60 Percent Missing Outer Hair Cells 40 * * * 20 * 0 Saline Pre 1H 1D 3D 5D Group Adapted fromYamashita et al. Neuroscience, 134:633-42, 2005.

  12. Reduced RNS formation at 10 days post-noise Pre-noise, Sal+E Nitrotyrosine 10 days post-noise Saline 1H Post, Sal+E Adapted fromYamashita et al., Neuroscience. 134(2):633-42, 2005.

  13. Reduced ROS formation at 10 days post-noise Pre-noise, Sal+E 4-HNE 10 days post-noise Saline 3D Post, Sal+E Adapted fromYamashita et al., Neuroscience. 134(2):633-42, 2005.

  14. Many Antioxidants Reduce NIHL Salicylate w/NAC, Kopke et al., 2000 w/vitE, Yamashita et al., 2005 resveratrol Seidman et al., 2003 allopurinol Cassandro et al., 2003 R-phenylisopropyladenosine (R-PIA) Hu et al., 1997 Hight et al., 2003 superoxide dismutase-polyethylene glycol Seidman et al., 1993 U74389F Quirk et al., 1994 D-methionine Campbell et al., 2007 Kopke et al., 2002 Ebselen Pourbakht and Yamasoba, 2003 Lynch et al., 2004 Lynch and Kil, 2005 Yamasoba et al., 2005 N-acetylcystein Ohinata et al., 2003; Duan et al., 2004 w/salicylate, Kopke et al., 2000 no effect on TTS in man (Kramer et al., 2006) or rodents (Kopke et al., 2000; Duan et al., 2004)

  15. Translation to humans? Antioxidants and vasodilators likely to be a potent combination For human clinical application, agents must be safe for daily use We evaluated the efficacy of three antioxidant vitamins, magnesium (which acts in part as a vasodilator), and a combination of these agents

  16. Dietary Micronutrients Beta-carotene, Vitamins C and E, Magnesium Beta-carotene: scavenges singlet oxygen, prevents lipid peroxidation Vitamin E: reduces peroxyl radicals in lipid layer Vitamin C: scavenges free radicals in aqueous phase Magnesium: reduces noise-induced vasoconstriction, blocks NMDA receptors, prevents calcium influx and neural excitotoxicity, also has antioxidant properties Patent pending, University of Michigan Inventors: Josef Miller, Colleen Le Prell, Jochen Schacht, Diane Prieskorn Option to license by OtoMedicine, Inc. Human clinical trials in progress

  17. Antioxidants plus magnesium reduce noise-induced hearing loss: additive effects 60 Saline ACE 50 Mg 40 ACEMg Threshold Shift (dB) * 30 * 20 * 10 0 4 kHz 8 kHz 16 kHz Frequency Mean + S. E., Le Prell et al. 2007, Free Rad. Med. Biol., 42,1454-1463.

  18. Antioxidants plus magnesium reduce outer hair cell death: additive effects CharacteristicFrequency (kHz) 0.1 1 10 1 10 0.1 80 Saline Mg 60 40 20 Outer Hair Cell Loss (Percent) 0 80 ACE ACEMg 60 40 20 0 0 5 10 15 0 5 10 15 20 Cochlear Position (Distance from Apex, mm) Mean + S. E., Le Prell et al., 2007, Free Rad. Med. Biol., 42,1454-1463.

  19. Antioxidants & magnesium reduce sensory cell death: effects are additive 60 Saline 50 ACE Mg 40 ACEMg OHC Loss (percent) 30 20 * 10 * 0 10-14.99 15-20 Distance from Apex (mm) Mean + S. E., Le Prell et al., 2007, Free Rad. Med. Biol., 42,1454-1463.

  20. RNS Byproducts Post-Noise in First Turn of Guinea Pig Cochlea • Saline-control • Nutrient treatment starting one day prior to noise Outer hair cells Outer hair cells Octave band noise centered at 4 kHz, 115-dB SPL x 4 hours

  21. EndoG Post-Noise in First Turn of Guinea Pig Cochlea • Saline-control • Nutrient treatment starting one day prior to noise Outer hair cells Outer hair cells Octave band noise centered at 4 kHz, 115-dB SPL x 4 hours

  22. 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.

  23. Oral Treatment Prevents NIHL in Mice: Dose-Dependent Effects Control: N=16 Diet A: N=8 Diet B: N=7 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.

  24. 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.

  25. Mix Matters! Previous studies show any of these nutrients when delivered for days-weeks pre-noise With dosing beginning 1 hour pre-noise, the combination of vitamins and Mg was essential for functional protection

  26. Human Trials with Nutrients Safe dosing limits well-characterized US Recommended Daily Allowance No RDA for beta-carotene Pre-formed Vitamin A: 1.5 mg ~18 mg beta-carotene using retinol activity equivalents Vitamin C: 60 mg Vitamin E: 15 mg Magnesium: 300-400 mg US Upper Limit (Institute of Medicine) No UL for beta-carotene Pre-formed Vitamin A: 3 mg ~36 mg beta-carotene using retinol activity equivalents Vitamin C: 2,000 mg Vitamin E: 1,000 mg Magnesium: 350 mg

  27. Translation to humans • The Agents Must be Safe • AREDS study provides 7-year safety data for the vitamins 1 Based on retinol activity equivalents

  28. Human Clinical Trials: 2008-2013 NIH-funded U01, NIH-funded R44 (Phase II SBIR) Temporary Threshold Shift Model US students listening to music with insert earphones US students exposed to video game impulse noise Soldiers exposed to automatic weapons fire (Sweden) Permanent Threshold Shift Model NATO soldiers at Los Llanos airbase (Spain) Employees at cutlery stamping factories (Spain) Multi-site collaboration: Josef Miller, Glenn Green, and Ken Guire (University of Michigan) Kathleen Campbell (SIU), Sharon Kujawa (Harvard) University of Florida: James W. Hall III, Pat Antonelli Spain: Jose Juiz (UCLM), Julio Carbayo (NATO), Luis Gonzalez (Solemat) Sweden: Ulf, Rosenhall, Ann-Christin Johnson and Mats Ulfendahl (Karolinska Institutet)

  29. Summary In most cases, mechanical hearing protection will be the best option, and drugs/dietary supplements will provide additional benefit for those cases in which mechanical protection is inadequate. Sometimes hearing protection is not an option, sometimes it just isn’t good enough, and sometimes noise is unexpected. We need an arsenal of new drugs and/or other agents. There is no “silver bullet” -- different people will need different treatments. We are getting there! There is an ongoing revolution in hearing science, in which for the first time, we have been able to effectively influence hearing outcomes after noise, drugs, or other chemicals.

  30. Acknowledgments Collaborators Josef Miller (UM) Larry Hughes (SIU) Daisuke Yamashita Tatsuya Yamasoba Funding Agencies University of Florida Office of Research NIH GM/UAW Ruth & Lynn Townsend Endowment Students Ashley Weimer Johnson (UF) Jason Schmitt (UF) Mike Goodson (UF) Bianca Gomez (UF) Dustin Lang (UF) Debbie Joseph (UF) Research Technicians Amanda Dossatt (UF) Diane Prieskorn (UM) Alice Mitchell (UM) Susan DeRemer (UM)

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