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Implantation of an osmotic or digital mini pump to provide sustained delivery of various antioxidants to the RWM in guinea pigs (Wimmer et al., 2004).

The development and utilization of OTO-104 which contains micronized dexamethasone 160 in poloxamer gel. Significant levels of dexamethasone were maintained in perilymph for 3 months in guinea pigs and more than 1 month in sheep (Piu et al., 2011).

The intratympanic injection of a hyaluronic acid liposomal gel for sustained delivery of dexamethasone was tested in the guinea pig. The gel remained for a long time in the middle ear cavity and in the RWM after intra-tympanic injection without any evidence of ototoxicity. This resulted in sustained release of dexamethasone in perilymph for 1 month (El Kechai et al., 2016). This appears to be a promising way to deliver corticosteroids to the inner ear to provide sustained protection against cochlear insults.

Dormer et al. have developed an innovative system to provide extended release of drugs for intra-tympanic injection (Dormer et al., 2019). It contains a film forming agent (FFA) and microspheres to provide prolonged delivery of betamethasone in a formulation called ORB-202 to the round window and inner ear in mice. This technology has shown that corticosteroids contained in microspheres with FFA were retained on the RWM for up to 5 weeks on necropsy examination. A recent review discusses various approaches to nanotechnology for inner ear applications. Only a few examples will be presented below.

Li et al. have developed a nanohydrogel delivery system, which combines nanotechnology with a chitosan-glycerophosphate hydrogel delivery system. These nanoparticles could be delivered across the mouse RWM to reach structures in the scala media (Li et al., 2017).

Nanoparticles are translocated across the RWM by endocytosis. This process follows three different mechanisms: macropinocytosis, caveolin-mediated endocytosis, and clathrin-mediated endocytosis (Zhang et al., 2018).

A study of RW permeation enhancers was carried out using fluorescein tagged dexamethasone applied to the RW niche in guinea pigs. DMSO, N-methylpyrrolidone and benzyl alcohol provided significantly higher entry than that observed in controls (Li et al., 2018).

Adjuvants were used to enhance the permeation of dexamethasone through the RWM. Application of dexamethasone on a hyaluronic acid sponge with or without histamine or dexamethasone with histamine provided greater penetration into perilymph in guinea pigs than did dexamethasone alone (Creber et al., 2019).

A novel method to enhance the delivery of siRNA to the cochlea was developed using a recombination protein, double-stranded RNA-binding domains (TAT-DRBDs). The authors showed efficient siRNA transfection to the cochlea of the chinchilla with this delivery system. They were able to demonstrate successful transfection of Cy3-labeled siRNA into cells of the inner ear through the intact RWM, including the IHCs, OHCs, and vestibular cells in the crista ampullaris, macula utriculi, and macula sacculi (Qi et al., 2014).

Kenpaullone is an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2). Significant otoprotection was demonstrated in both mice and rats. Mice receiving intra-tympanic kenpaullone demonstrated significant reductions of ABR threshold elevation, at frequencies of 16 and 32 kHz. Morphology of OHCs in the 32 kHz region showed significant protection in kenpaullone treated mice. Even more robust findings were demonstrated in rats. Intra-tympanic kenpaullone provided complete protection against cisplatin ototoxicity in the rat. These findings support the hypothesis that CDK2 inhibition by kenpaullone ameliorates cisplatin ototoxicity by inhibiting mitochondrial ROS production and also preventing cochlear cell death mediated by caspase-3/7 (Teitz et al., 2018).

Copper sulfate, a copper transporter-1 inhibitor, when administered intra-tympanically 30 min prior to intraperitoneal cisplatin in mice showed significant protection against threshold shifts in ABR using click stimuli and pure tones at 8, 16, and 32 kHz. However, concerns were expressed about the toxicity of copper sulfate. This led to the suggestion that other less toxic inhibitors of CTR1 should be developed and tested (More et al., 2010).

Thiosulfate, an antioxidant was administered as thiosulphate-hyaluronan gel into the tympanic cavity of guinea pigs 3 h prior to intravenous cisplatin injection. This resulted in high concentrations of thiosulfate in the perilymph of scala tympani and it protected against cochlear hair cell loss from cisplatin. Levels of thiosulfate in blood were kept low, avoiding potential chelation of cisplatin in the blood that could interfere with the anti-tumor efficacy of cisplatin (Berglin et al., 2011).

KR-22332 (3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2, 4-dione) is a novel compound that suppresses ROS. Intra-tympanic administration of KR-22332 in rats protected against cisplatin induced ABR threshold shift to click stimuli. This compound inhibited cisplatin-induced up regulation of NOX3 in the cochlea and reduced the activation of p53, MAP kinases, caspase 3 and tumor necrosis factor-α (TNF-alpha), and TUNEL expression in rat cochlea. KR-22332 may ameliorate cisplatin ototoxicity by reducing the generation of ROS and by preventing mitochondrial dysfunction (Shin et al., 2013).

Antioxidant vitamins such as vitamin E and vitamin C have been tested for protection against cisplatin ototoxicity. Trolox, a water-soluble form of alpha-tocopherol is an antioxidant. It was applied locally on the round window of guinea pigs treated with cisplatin. Trolox administered in combination with cisplatin prevented ABR threshold elevations and protected against the loss of hair cells (Teranishi and Nakashima, 2003). Another study in rats looked at the effect of intra-tympanic application of vitamin E solution followed by cisplatin administration 30 min later. Significant protection against cisplatin induced ABR threshold shifts was seen in rats (Paksoy et al., 2011). Another strategy employed the intra-tympanic administration of vitamin E polymeric nanoparticles in rats treated with cisplatin. Rats pretreated with vitamin E nanoparticles had significant protection against cisplatin-induced ABR threshold shifts at 12, 20, and 32 (Martin-Saldana et al., 2017). Vitamin C administered by intra-tympanic injection protected against cisplatin-induced decrease in DPOAE amplitudes in rats treated with cisplatin (Celebi et al., 2013).

Melatonin is a hormone secreted by the pineal gland that has antioxidant properties. It has both indirect antioxidant and direct free radical scavenger activity. Rats treated with intra-tympanic melatonin showed improved ABR thresholds for clicks, 4, 6, and 8 kHz and threshold shifts for DPOAE. Staining for TNF-alpha was diminished in melatonin treated rats receiving cisplatin (Demir et al., 2015).

Capsaicin is a spicy capsaicinoid, a natural product produced by hot chili peppers, Capsicum fruits. This alkaloid has been used for its analgesic and anti-inflammatory actions (Lavorgna et al., 2019), Capsaicin activates TRPV1 pain receptors, and can produce rapid desensitization of TRPV1. The intra-tympanic administration of capsaicin in rats 24 h prior to cisplatin reduced ABR threshold shifts. Capsaicin appears to prevent cisplatin ototoxicity by increasing the expression of cannabinoid 2 receptors (CB2R) in the cochlea leading to increased activation of pro-survival transcription factor signal transducer and activator of transcription (STAT3) (Bhatta et al., 2019).

JWH-015 (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) is a cannabinoid receptor 2 (CB2) agonist. Pretreatment with intra-tympanic JWH-015, 30 min prior to cisplatin reduced ABR threshold shifts at 8, 16, and 32 kHz and also protected against the loss of OHCs in rats. In addition, this CB2R agonist prevented cisplatin-induced loss of ribbon synapses on inner hair cells (IHCs) and prevented loss of Na⁺/K⁺-ATPase immunoreactivity in the stria vascularis (Ghosh et al., 2018).

Pifithrin-alpha is an inhibitor of p53. Pifithrin-alpha was applied on the RWM of the chinchilla prior to the local application of cisplatin. The cochleae that were pretreated with pifithrin were significantly protected from cisplatin-induced increase in ABR threshold shifts at 1,2,4,8, and 16 kHz (Parhizkar and Rybak, 2003).

R-PIA (R-phenylisopropyladenosine) is an adenosine A1 receptor agonist. Intra-tympanic administration of R-PIA in rats prior to cisplatin reduced cisplatin-induced ABR threshold elevation and OHCs were preserved. This protection was associated with reduced NOX3 expression, STAT1 activation, TNF-α levels, and apoptosis in the cochlea (Kaur et al., 2016).

D-methionine and L-methionine are amino acids with antioxidant properties and both of these compounds have been shown to protect against cisplatin ototoxicity in preclinical studies. D-methionine applied to the RWM provided complete protection against cisplatin applied to the round window in chinchillas. ABR thresholds and OHCs were completely preserved in animals pretreated with D-methionine (Korver et al., 2002). In a study using guinea pigs, osmotic pumps were implanted to provide continuous administration of D-methionine, sodium thiosulfate, fibroblast growth factor-2, or brain-derived neurotrophic factor in animals treated with cisplatin. Guinea pigs receiving D-methionine demonstrated better OAEs on the 3th and 5th day of a 5 day regimen of cisplatin administration. On 5th and 6th day of the treatment, D-methionine failed to provide protection. It appears that the additional dosing of cisplatin overpowered the effectiveness of D-methionine on those later 2 days. The other agents provided no significant protection (Wimmer et al., 2004). The efficacy of L-methionine against cisplatin ototoxicity was investigated in rats. Local application of L-methionine prior to cisplatin provided complete protection against cisplatin-induced ABR threshold shifts and preserved the integrity of OHCs against damage by cisplatin (Li et al., 2001).

L-N-acetylcysteine (L-NAC) is a sulfhydryl compound that can neutralize cisplatin and function as an antioxidant. A 2% solution of L-NAC was administered by intra-tympanic injection in guinea pigs treated with cisplatin. Pretreatment with L-NAC preserved DPOAEs that were otherwise severely affected by cisplatin. This same study successfully utilized lactated Ringer’s solution by intra-tympanic injection prior to cisplatin administration. The latter solution was also effective in preserving DPOAEs in cisplatin treated guinea pigs (Choe et al., 2004). A later study showed that intra-tympanic administration of L-NAC was harmful and exacerbated cisplatin ototoxicity in the guinea pig. However, this latter study utilized extremely high concentrations of L-NAC (20%) and this proved to be toxic. Animals receiving this high dose of L-NAC showed severe disruption of OHC stereocilia (Nader et al., 2010). It appears that a more dilute solution of L-NAC is a better preparation to use for intra-tympanic injection to ameliorate cisplatin ototoxicity.

TNF-alpha antagonist, etanercept, when administered intra-tympanically in rats protected against OHC damage and cisplatin-induced hearing loss. ABR threshold shifts were significantly reduced in rats treated with etanercept 30 min prior to cisplatin. Scanning electron microscopy of etanercept pre-treated animals showed significant protection against cisplatin induced OHC damage (Kaur et al., 2011).

RNA silencing has been successfully employed using intra-tympanic delivery for protection against cisplatin ototoxicity. In a rat model of cisplatin ototoxicity, it was shown than intra-tympanic administration of siRNA to knock down TRPV1 protected against cisplatin-induced hearing loss and damage to outer hair cells in the cochlea (Mukherjea et al., 2008). It was hypothesized that protection was afforded by reducing down-stream targets, such as the cochlear specific NADPH oxidase -3 (NOX3) enzyme, and STAT-1. Indeed, the intra-tympanic injection of siRNA directed against NOX3 (Mukherjea et al., 2011) or STAT-1 siRNA (Kaur et al., 2011) were each protective against cisplatin induced hearing loss and outer hair cell damage. NOX3 activation results in reactive oxygen species upregulation and STAT-1 can promote inflammation and apoptosis in the cochlea as a result of cisplatin’s ototoxic effect. Such deleterious effects can be prevented by the use of these siRNAs.

Dexamethasone is a glucocorticoid that appears to offer protection against cisplatin ototoxicity by several mechanisms. These include the down-regulation of pro-inflammatory genes that regulate the expression of cytokines; the inhibition of apoptosis; the up-regulation of antioxidant enzymes that could antagonize the effects of ROS (Hazlitt et al., 2018).

Prednisolone was found to reduce cisplatin induced ABR threshold elevations in mice. Intra-tympanic magnetically delivered prednisolone-loaded nanoparticles resulted in significantly lower elevations of ABR threshold, particularly at the higher frequencies (16 and 32 kHz) compared with intra-tympanic methylprednisolone solution or empty magnetic nanoparticles (Ramaswamy et al., 2017).

A cell-permeable inhibitor of JNK mediated apoptosis, AM-111, was administered on the RWM (in a hyaluronic acid gel formulation or osmotic mini-pump) 1 or 4 h after impulse noise exposure in chinchillas. Three weeks after traumatic noise exposure the PTS were significantly less in animals receiving AM-111 even when it was administered 4 h after noise exposure (Coleman et al., 2007). D-JNKI-1 was found to block the mitogen-activated protein kinase/JNK-mediated activation of a mitochondrial death pathway. D-JNKi-1 administered intra-tympanically to guinea pigs exposed to acoustic trauma also provided excellent protection. The majority of hair cells were preserved in the area of maximum noise damage and resulted in almost no permanent hearing loss. Treatment was effective even when administered up to 12 h after noise exposure. These findings strongly suggest that the mitogen-activated protein kinase/JNK signaling pathway plays a critical role in producing hair cell death from acoustic trauma (Wang et al., 2007).

Rosmarinic acid is a polyphenol that is found in aqueous extracts of spearmint. It has demonstrated antioxidant, anti-inflammatory and neuroprotective properties (Falcone et al., 2019). Rats were exposed to acoustic trauma and underwent ABR measurements before and up to 30 days afterward. One group was treated with systemic rosmarinic acid and a second treatment group was administered intra-tympanic rosmarinic acid. Significant protection against ABR threshold shifts was seen in both treatment groups compared with controls. Less OHC loss and decreased evidence of superoxide production and lipid peroxidation was ascertained using dihydroethidium and 8-isoprostane immunostaining, respectively. These findings strongly suggest that adminstration of rosmarinic acid by both routes of administration protected the hearing and preserved the cochlea of rats exposed to noise trauma (Fetoni et al., 2018).

Peroxisome proliferator-activated receptors (PPARs) function as lipid sensors and help to regulate redox balance by inhibiting ROS and upregulating antioxidant genes. Pioglitazone is a PPAR-gamma agonist that has been shown to reduce inflammation in patients with type two diabetes and coronary artery disease. This drug seemed to have favorable properties to test as a protective agent against noise trauma. Rats were administered pioglitazone in a temperature sensitive gel intra-tympanically 1 h after acoustic trauma. Pioglitazone significantly protected against threshold shifts in the ABR and significantly reduced the loss of OHCs. These findings were associated with a reduction in superoxide anion expression and lipid peroxidation (8-isoprostane). Anti-inflammatory effects of pioglitazone were demonstrated by its blockade of noise induced upregulation of pNFkB and interleukin 1b (IL-1b). Thus, pioglitazone protection against traumatic noise injury to the cochlea by both anti-oxidant and anti-inflammatory effects (Paciello et al., 2018).

Caroverine is an antagonist of two glutamate receptors, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Caroverine was applied onto the RWM with gelfoam in guinea pigs, followed by noise exposure. ABR threshold shifts were significantly lower in caroverine treated animals (Chen et al., 2004).

Edaravone is a free radical scavenger and antioxidant. Edaravone solid lipid nanoparticles, were delivered to guinea pigs by intra-tympanic injection. Noise exposure resulted in ABR threshold shifts and induced ROS formation. Edaravone reduced the ABR threshold shift and ROS production in noise-exposed animals compared with controls. Edaravone solid lipid nanoparticles show protective effects against noise-induced hearing loss. However, guinea pigs treated with edaravone had no significant protection of OHCs. More experiments will be needed to see if edaravone could be useful in protecting cochlear tissues from noise injury (Gao et al., 2015).

Kenpaullone is an inhibitor of CDK2. When kenpaullone was injected intra-tympanically in mice had significantly better ABR thresholds and wave 1 amplitudes than controls. In animals treated with this agent, the presynaptic ribbon density at D14 after the acoustic damage was diminished. These data support the hypothesis that kenpaullone protects against noise-induced hearing loss in mice. It is interesting to note that kenpaullone also protected against cisplatin (see above) (Teitz et al., 2018).

RNA silencing: Noise exposed mice suffered permanent ABR threshold shifts, loss of OHCs and cochlear synapses. G9a (KMT1C, EHMT2) is an important histone lysine methyltransferase encoded by the human EHMT2 gene and responsible for histone H3 lysine 9 dimethylation (H3K9me2). The intra-tympanic administration of siRNA against G9a to silence the EHMT2 gene 72 h prior to noise exposure significantly reduced ABR threshold shifts and resulted in greater survival of OHCs compared to treatment with the control siRNA. These data suggest that pretreatment with siG9a partially ameliorates noise-induced permanent hearing loss via the inhibition of G9a (Xiong et al., 2019).

Neurotrophins have been used successfully for preservation of IHC pre and post-synaptic ribbon synapses: Guinea pigs were exposed for 2 h to 4 to 8 kHz noise at 95 dB. Auditory brainstem responses to pure-tone pips were acquired preoperatively, and at 1 and 2 weeks’ post exposure. Immediately after noise exposure neurotrophins (brain-derived neurotrophic factor and neurotrophin-3) were applied to the RWM. ABR amplitude growth recovered in the ears of neurotrophin-treated guinea pigs using 16 kHz tones. Significantly more presynaptic ribbons, post-synaptic glutamate receptors, and co-localized ribbon synapse were seen after neurotrophin treatment. These findings supported the hypothesis that the local application of neurotrophins to the round window immediately after noise exposure will prevent noise-induced “hidden hearing loss” (Sly et al., 2016).

Dexamethasone is the most frequently tested glucocorticosteroid by intra-tympanic injection to protect against noise-induced hearing loss. Rats were exposed to noise at 110 dB for 25 min and DPOAE measurements were performed before and after noise exposure. DPOAE measurements were performed before and 7 and 10 days after noise trauma. Rats treated with intra-tympanic dexamethasone had significantly better hearing than controls (Gumrukcu et al., 2018). Guinea pigs receiving intra-tympanic dexamethasone demonstrated significantly smaller ABR threshold shifts and decreased OHC loss compared with controls. They also had significant reduction in malondialdehyde concentration in the cochlea. This suggests that dexamethasone provided antioxidant effects in the treated ears (Chi et al., 2011). Another study showed short-term protection against hearing loss in guinea pigs with intra-tympanic dexamethasone. Animals received dexamethasone by intra-tympanic injection 2 h prior to white noise exposure. ABR thresholds were better and hair cell loss was reduced by this treatment. However, the major flaw of this study is that ABR thresholds and cochlear histology were performed only 2 h after noise exposure (Heinrich et al., 2016). Another study using guinea pigs tested the efficacy of dexamethasone administered intra-tympanically 2 h prior to white noise exposure. One group received dexamethasone solution and a second group was provided dexamethasone microbubbles with ultrasound irradiation. Compared with controls, dexamethasone in both groups provided protection against hair cell loss and auditory threshold shifts. However, significantly greater protection was afforded to the guinea pigs pretreated with ultrasound delivered microbubbles (Shih et al., 2018).

Interesting findings were reported in a study of noise exposed mice. Animals were exposed to 110 db white noise for 60 min in a single exposure. One group received intraperitoneal dexamethasone injection (IP) daily for five consecutive days, while another cohort was given intra-tympanic injection of dexamethasone on days one and four after noise exposure. Mice in both treatment groups showed improved ABR thresholds but no apparent improvement in DPOAEs. Interestingly, better preservation of organ of Corti ultrastructure was observed in mice receiving IP drug than in those who were administered dexamethasone by intra-tympanic injection. On the other hand, efferent synapses were damaged in control (noise only), and in both groups treated with dexamethasone. However, there was better preservation of synapses of efferent terminals on OHCs in the group treated with intra-tympanic steroids (Han et al., 2015).

In efforts to provide sustained release of dexamethasone for prolonged otoprotection against noise the efficacy of OTO-104 was investigated both prior to and following acute acoustic trauma. OTO-104 is a poloxamer-based hydrogel containing micronized dexamethasone. Guinea pigs received a single intra-tympanic injection of OTO-104 and were assessed in a model of acute acoustic trauma. Doses of at least 2.0% OTO-104 offered significant protection against hearing loss induced by noise exposure when administered 1 day prior to trauma and up to 3 days afterward. Otoprotection remained effective even with higher degrees of trauma. In contrast, the administration of a dexamethasone sodium phosphate solution did not protect against noise-induced hearing loss. Activation of the classical nuclear glucocorticoid and mineralocorticoid receptor pathways was required for otoprotection by OTO-104. The sustained release features of OTO-104 provided greater protection than the solution (Harrop-Jones et al., 2016).

Methylprednisolone was administered intra-tympanically to guinea pigs exposed to impulse noise. Animals receiving this treatment had significantly better ABR thresholds at 4 weeks compared with those treated with saline. Significantly better preservation of hair cells was observed in the cochleae of guinea pigs receiving intra-tympanic methylprednisolone compared to those treated with saline (Zhou et al., 2009). Intra-tympanic methylprednisolone injection in rats administered following acoustic trauma was shown to reduce OHC loss. Although DPOAE measurement within the first week demonstrated significantly better amplitudes in the treated rats compared to controls at 2 weeks, there was no significant difference in DPOAE amplitudes between the treated and control group (Ozdogan et al., 2012).