The Science

It is estimated that 26 million Americans between the ages of 20 and 69 have significant sensorineural hearing loss that is likely the result of loud noise exposure (NIDCD 2008). Noise-induced hearing loss (NIHL) is one of the leading causes of hearing impairment in the U.S. and worldwide. While personal hearing protection devices (HPDs), work place engineering measures, and other precautionary actions are key and have been employed for over two decades in hearing conservation programs, these measures are not always adequate to prevent NIHL. Military training and combat environments also pose significant challenges for HPD-mediated protection. Limitations of HPDs include lack of comfort, lack of complete protection, the need for an airtight fit, the element of surprise, and the inability of these devices to protect against damage caused by bone-conducted acoustic energy through the skull. Recent findings in rodents have demonstrated that acute loud noise exposures, which initially caused no apparent permanent hearing loss, may adversely affect how the cochlea ages (Furman 2013; Kujawa 2006) and thus, may be a major underlying factor in presbycusis. Therefore, if these findings are applicable to humans, it may be even more important than previously realized to treat incidents of acute noise trauma that are currently below OSHA safety standards.

The mechanisms of NIHL can be classified into two main categories: (1) direct mechanical trauma (Hamernik 1984) and (2) indirect metabolic damage to the cellular components of the inner ear (Coling 2003; Ohlemiller 2000; Van Campen 2002) due to excessive formation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and other free radical species (Fetoni 2013; Henderson 2006; Hu 2000; Hu 2002; Lamm 2000; Miller 2003; Ohinata 2003; Puel 1996; Puel 1998; Saunders 1985; Yamashita 2004). ROS directly damage cell structures by reacting with proteins, lipids and DNA and can also act as signaling molecules that up-regulate genes responsible for apoptosis (programmed cell death). Acoustic injury can result in the initiation of inflammatory and cell death pathways that remain active for days to weeks and the ensuing permanent neural and hair cell injury can play out over a period of days or weeks. Therefore, a short therapeutic window exists post-injury where an antioxidant, anti-inflammatory or antiapoptotic treatment can enhance recovery and reduce permanent hearing loss. Accordingly, a great deal of research has been dedicated to discover antioxidant compounds that can neutralize ROS with the aim of preventing and/or treating damage to the cochlea (Coling 2003; Hight 2003; Hu 1997; Kopke 2002; Kopke 2000; Ohinata 2000; Seidman 1993; Yamasoba 1999).

N-acetylcysteine (NAC) is a thiol-containing amino acid derivative that acts as a ROS scavenger and a substrate for glutathione (GSH), the major endogenous antioxidant produced by cells. NAC has been shown to reduce NIHL in animal models (Fetoni 2009; Kopke 2000; Lorito 2008; Mortazavi 2010) and correlates of noise-induced cochlear injury in clinical trials (Lin 2010; Lindblad 2011) (Kopke, submitted). When NAC was combined with a nitrone-based spin trapping agent of free radical species, 4-OHPBN, a synergistic effect was observed between the two compounds on reducing acute acoustic trauma (Choi 2008). Recent advancements on this combinatorial treatment regimen have been achieved, using a structural analog of 4-OHPBN, disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07, previously called NXY-059). The improved clinical potential for this approach relies on the fact that HPN-07 has been shown to be: (1) a better neuroprotectant than 4-OHPBN (Clausen 2008; Hainsworth 2008; Kuroda 1999; Sydserff 2002), (2) effective in treating acute acoustic trauma in chinchilla (unpublished data from our lab), and (3) low in toxicity in Phase I , II and III clinical trials for treatment of stroke (Green 2003; Lees 2001).

We have demonstrated that a combinatorial treatment of NAC plus HPN-07 effectively reduced hearing loss and cochlear hair cell death in rats when administered after exposure to blast overpressure (Ewert 2012). Subsequent studies revealed that this novel antioxidant treatment regimen simultaneously reduced blast-induced expression of brain injury biomarkers, indicating that this therapeutic strategy has the capacity to provide a complementary reduction in blast-induced traumatic brain injury in rats (Du 2013). In parallel studies, the combination of NAC plus HPN-07 also effectively reduced hearing loss induced by exposure to extremely high level steady state noise in rats. Using our innovative treatment regimen, noise-induced sensory hair cell death was substantially reduced and auditory nerve function was preserved. Moreover, noise-induced brainstem changes were also reversed by this combinatorial antioxidant treatment (Lu 2014). In summary, a treatment regimen of NAC plus HPN-07 when administered systemically shortly after extreme acute steady state noise or a simulated explosion has the ability to reduce ensuing damage to the entire auditory pathway (i.e. cochlea, brainstem and cortex). Thus, intervention with our combined antioxidant approach during a post-injury therapeutic window has the capacity to curtail the wave of progressive detrimental effects induced by the noise or blast trauma, thus preventing extensive permanent injury.

In light of the potential wide-reaching clinical utility of this therapeutic approach, we have formulated an oral drug combination consisting of encapsulated NAC and HPN-07, henceforth referred to as NHPN-1010. As described above, this drug combination was found to substantially and synergistically reduce both blast-induced cochlear and brain injury in regions associated with tinnitus, memory and anxiety (Du 2013; Ewert 2012). In previous studies, NAC (Lin 2010; Lindblad 2011) and HPN-07 (Hu in preparation) were shown to individually reduce noise-induced cochlear injury and hearing loss. They were also found to independently reduce brain injury in rodent models (Chen 2008; Clausen 2008) and NAC was recently shown to reduce blast-induced mild traumatic brain injury (TBI) in humans (Hoffer 2013). However, high doses of the individual drugs were required to elicit these efficacious outcomes, thus it was significant to find that a combinatorial therapy (NHPN-1010) gave rise to synergistic effects. With injuries involving a myriad of altered pathologic processes, combinatorial therapy with NHPN-1010 may have distinctive advantages over existing approaches (Abdel Baki 2010). NAC concurrently targets oxidative stress, glutathione deficiency, neuroinflammation, mitochondrial injury and cell death (Kopke 2007), while HPN-07 scavenges reactive nitrogen species, inhibits the induction of inducible nitric oxide synthase (iNOS), reduces excitotoxicity and acts as a neuroprotectant (Floyd 2008). Thus, NHPN-1010 simultaneously addresses a multitude of injury mechanisms.

Currently, no FDA approved treatment exists for blast-induced tinnitus or sensory-neural hearing loss (SNHL). NHPN-1010 has patents pending in the U.S., Canada, the European Union, Israel, Japan, and Australia and has received favorable indications for patentability for use and composition of matter in the European Union. It is slated for study this year in Phase I clinical trials as a treatment for acute NIHL.

Future studies will also be aimed at investigating a potential role for NHPN-1010 in the amelioration of off-target ototoxicity associated with the administration of the common anti-cancer drugs, cisplatin and carboplatin, in light of the fact that this undesirable side-effect is also mediated by ROS and RNS. Therefore, we plan to formally evaluate the effects of NHPN-1010 in pre-clinical models of cisplatin-induced ototoxicity and initiate clinical trials based on the results of these investigations.

Selected Publications:

Abdel Baki S, Schwab B, Haber M, Fenton A, Bergold P (2010) Minocycline synergizes with n-acetylcysteine and improves cognition and memory following traumatic brain injury in rats. PLoS One 5(8):12490.

Chen G, Shi J, Hu Z, Hang C (2008) Inhibitory effect on cerebral inflammatory response following traumatic brain injury in rats: A potential neuroprotective mechanism of n-acetylcysteine. Mediators Inflamm 2008:716458.

Choi CH, Chen K, Vasquez-Weldon A, Jackson RL, Floyd RA, Kopke RD (2008) Effectiveness of 4-hydroxy phenyl n-tert-butylnitrone (4-ohpbn) alone and in combination with other antioxidant drugs in the treatment of acute acoustic trauma in chinchilla. Free Radic Biol Med 44(9):1772-1784.

Clausen F, Marklund N, Lewen A, Hillered L (2008) The nitrone free radical scavenger nxy-059 is neuroprotective when administered after traumatic brain injury in the rat. J Neurotrauma 25(12):1449-1457.

Coling DE, Yu KC, Somand D, Satar B, Bai U, Huang TT, Seidman MD, Epstein CJ, Mhatre AN, Lalwani AK (2003) Effect of sod1 overexpression on age- and noise-related hearing loss. Free radical biology & medicine 34(7):873-880.

Du X, Ewert DL, Cheng W, Lu J, Floyd R, R.D. K (2013) Effects of antioxidant treatment on blast-induced brain injury. PLOS ONE 8(11).

Ewert DL, Lu J, Li W, Du X, Floyd R, Kopke R (2012) Antioxidant treatment reduces blast-induced cochlear damage and hearing loss. Hear Res 285(1-2):29-39.

Fetoni AR, Ralli M, Sergi B, Parrilla C, Troiani D, Paludetti G (2009) Protective effects of n-acetylcysteine on noise-induced hearing loss in guinea pigs. Acta Otorhinolaryngol Ital 29(2):70-75.

Fetoni AR, De Bartolo P, Eramo SL, Rolesi R, Paciello F, Bergamini C, Fato R, Paludetti G, Petrosini L, Troiani D (2013) Noise-induced hearing loss (nihl) as a target of oxidative stress-mediated damage: Cochlear and cortical responses after an increase in antioxidant defense. The Journal of neuroscience : the official journal of the Society for Neuroscience 33(9):4011-4023.

Floyd RA, Kopke RD, Choi CH, Foster SB, Doblas S, Towner RA (2008) Nitrones as therapeutics. Free Radic Biol Med 45(10):1361-1374.

Furman AC, Kujawa SG, Liberman MC (2013) Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates. Journal of neurophysiology 110(3):577-586.

Green AR, Ashwood T, Odergren T, Jackson DM (2003) Nitrones as neuroprotective agents in cerebral ischemia, with particular reference to nxy-059. Pharmacol Ther 100(3):195-214.

Hainsworth AH, Bhuiyan N, Green AR (2008) The nitrone disodium 2,4-sulphophenyl-n-tert-butylnitrone is without cytoprotective effect on sodium nitroprusside-induced cell death in n1e-115 neuroblastoma cells in vitro. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 28(1):24-28.

Hamernik RP, Turrentine G, Roberto M, Salvi R, Henderson D (1984) Anatomical correlates of impulse noise-induced mechanical damage in the cochlea. Hearing research 13(3):229-247.

Henderson D, Bielefeld EC, Harris KC, Hu BH (2006) The role of oxidative stress in noise-induced hearing loss. Ear Hear 27(1):1-19.

Hight NG, McFadden SL, Henderson D, Burkard RF, Nicotera T (2003) Noise-induced hearing loss in chinchillas pre-treated with glutathione monoethylester and r-pia. Hearing research 179(1-2):21-32.

Hoffer ME, Balaban C, Slade MD, Tsao JW, Hoffer B (2013) Amelioration of acute sequelae of blast induced mild traumatic brain injury by n-acetyl cysteine: A double-blind, placebo controlled study. PLoS One 8(1):e54163.

Hu BH, Zheng XY, McFadden SL, Kopke RD, Henderson D (1997) R-phenylisopropyladenosine attenuates noise-induced hearing loss in the chinchilla. Hear Res 113(1-2):198-206.

Hu BH, Guo W, Wang PY, Henderson D, Jiang SC (2000) Intense noise-induced apoptosis in hair cells of guinea pig cochleae. Acta oto-laryngologica 120(1):19-24.

Hu BH, Henderson D, Nicotera TM (2002) Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise. Hearing research 166(1-2):62-71.

Hu N, Du X, Li W, Lu J, Ewert DL, Choi C-H, Floyd RA, Kopke RD (in preparation) Effects of treatment with the free radical spin-trapping agent hpn-07 on cellular and functional changes in the cochlea following acute noise injury.

Kopke RD, Weisskopf PA, Boone JL, Jackson RL, Wester DC, Hoffer ME, Lambert DC, Charon CC, Ding DL, McBride D (2000) Reduction of noise-induced hearing loss using l-nac and salicylate in the chinchilla. Hear Res 149(1-2):138-146.

Kopke RD, Coleman JK, Liu J, Campbell KC, Riffenburgh RH (2002) Candidate’s thesis: Enhancing intrinsic cochlear stress defenses to reduce noise-induced hearing loss. Laryngoscope 112(9):1515-1532.

Kopke RD, Jackson RL, Coleman JK, Liu J, Bielefeld EC, Balough BJ (2007) Nac for noise: From the bench top to the clinic. Hear Res 226(1-2):114-125.

Kraus KS, Canlon B (2012) Neuronal connectivity and interactions between the auditory and limbic systems. Effects of noise and tinnitus. Hear Res 288(1-2):34-46.

Kujawa SG, Liberman MC (2006) Acceleration of age-related hearing loss by early noise exposure: Evidence of a misspent youth. The Journal of neuroscience : the official journal of the Society for Neuroscience 26(7):2115-2123.

Kuroda S, Tsuchidate R, Smith ML, Maples KR, Siesjo BK (1999) Neuroprotective effects of a novel nitrone, nxy-059, after transient focal cerebral ischemia in the rat. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 19(7):778-787.

Lamm K, Arnold W (2000) The effect of blood flow promoting drugs on cochlear blood flow, perilymphatic po(2) and auditory function in the normal and noise-damaged hypoxic and ischemic guinea pig inner ear. Hearing research 141(1-2):199-219.

Lanting CP, de Kleine E, van Dijk P (2009) Neural activity underlying tinnitus generation: Results from pet and fmri. Hearing research 255(1-2):1-13.

Lees KR, Sharma AK, Barer D, Ford GA, Kostulas V, Cheng YF, Odergren T (2001) Tolerability and pharmacokinetics of the nitrone nxy-059 in patients with acute stroke. Stroke; a journal of cerebral circulation 32(3):675-680.

Lin CY, Wu JL, Shih TS, Tsai PJ, Sun YM, Ma MC, Guo YL (2010) N-acetyl-cysteine against noise-induced temporary threshold shift in male workers. Hear Res 269(1-2):42-47.

Lindblad AC, Rosenhall U, Olofsson A, Hagerman B (2011) The efficacy of n-acetylcysteine to protect the human cochlea from subclinical hearing loss caused by impulse noise: A controlled trial. Noise Health 13(55):392-401.

Lorito G, Giordano P, Petruccelli J, Martini A, Hatzopoulos S (2008) Different strategies in treating noiseinduced hearing loss with n-acetylcysteine. Med Sci Monit 14(8):BR159-164.

Lu J, Li W, Du X, Ewert DL, West MB, Stewart C, Floyd RA, Kopke RD (2014) Antioxidants reduce cellular and functional changes induced by intense noise in the inner ear and cochlear nucleus. JARO In Press.

Miller JM, Brown JN, Schacht J (2003) 8-iso-prostaglandin f(2alpha), a product of noise exposure, reduces inner ear blood flow. Audiology & neuro-otology 8(4):207-221.

Mortazavi S, Kashani M, Khavanin A, Alameh A, Mirzaee R, Akbari M (2010) Effects of n-acetylcysteine on auditory brainstem response threshold shift in rabbits exposed to noise and carbon monoxide. Am J Applied Sci 7(2):201-207.

Ohinata Y, Yamasoba T, Schacht J, Miller JM (2000) Glutathione limits noise-induced hearing loss. Hearing research 146(1-2):28-34.

Ohinata Y, Miller JM, Schacht J (2003) Protection from noise-induced lipid peroxidation and hair cell loss in the cochlea. Brain Res 966(2):265-273.

Ohlemiller KK, McFadden SL, Ding DL, Lear PM, Ho YS (2000) Targeted mutation of the gene for cellular glutathione peroxidase (gpx1) increases noise-induced hearing loss in mice. Journal of the Association for Research in Otolaryngology : JARO 1(3):243-254.

Puel J, D’Aldin C, Saffiende S, Eybalin M, Pujol R (1996). Excitotoxicity and plasticity of ihc-auditory nerve contributes to both temporary and permanent threshold shift. New York, Thieme.

Puel JL, Ruel J, Gervais d’Aldin C, Pujol R (1998) Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport 9(9):2109-2114.

Rauschecker JP, Leaver AM, Muhlau M (2010) Tuning out the noise: Limbic-auditory interactions in tinnitus. Neuron 66(6):819-826.

Roberts LE, Eggermont JJ, Caspary DM, Shore SE, Melcher JR, Kaltenbach JA (2010) Ringing ears: The neuroscience of tinnitus. J Neurosci 30(45):14972-14979.

Saunders JC, Dear SP, Schneider ME (1985) The anatomical consequences of acoustic injury: A review and tutorial. The Journal of the Acoustical Society of America 78(3):833-860.

Seidman MD, Shivapuja BG, Quirk WS (1993) The protective effects of allopurinol and superoxide dismutase on noise-induced cochlear damage. Otolaryngology–head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery 109(6):1052-1056.

Sydserff SG, Borelli AR, Green AR, Cross AJ (2002) Effect of nxy-059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, plasma concentration and therapeutic time window. British journal of pharmacology 135(1):103-112.

Van Campen LE, Murphy WJ, Franks JR, Mathias PI, Toraason MA (2002) Oxidative DNA damage is associated with intense noise exposure in the rat. Hearing research 164(1-2):29-38.

Yamashita D, Jiang HY, Schacht J, Miller JM (2004) Delayed production of free radicals following noise exposure. Brain Res 1019(1-2):201-209.

Yamasoba T, Schacht J, Shoji F, Miller JM (1999) Attenuation of cochlear damage from noise trauma by an iron chelator, a free radical scavenger and glial cell line-derived neurotrophic factor in vivo. Brain research 815(2):317-325.