Xiaobo Wang

Cu/Zn superoxide dismutase and age-related hearing loss.

Mice, in which the genetics can be manipulated and the life span is relatively short, enable evaluation of the effects of specific gene expression on cochlear degeneration over time. Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. The effects of SOD1 deletion and over-expression on the cochlea were examined in Sod1-null mice, Sod1 transgenic mice and in age- and genetics-matched controls. Auditory brainstem responses (ABR) were measured and cochleae were histologically examined. The absence of SOD1 resulted in hearing loss at an earlier age than in wildtype or heterozygous mice. The cochleae of the null mice had severe spiral ganglion cell degeneration at 7-9 months of age. The stria vascularis in the aged, null mice was thinner than in the heterozygous or wildtype mice. Over-expression of SOD1 did not protect against hearing loss except at 24 months of age. In conclusion, SOD1 seems important for survival of cochlear neurons and the stria vascularis, however even half the amount is sufficient and an over abundance does not provide much protection from age-related hearing loss.

Blockage of immune-mediated inner ear damage by etanercept.

Hypothesis Etanercept will be able to reduce the inflammation and hearing loss associated with experimentally induced labyrinthitis. Background Inner ear immune responses cause hearing loss that may be reversible with pharmacologic treatment. Etanercept, tumor necrosis factor receptor blocker, was investigated in a guinea pig model of immune-mediated hearing loss. Sterile labyrinthitis was created by injection of keyhole limpet hemocyanin into the inner ear after systemic sensitization to keyhole limpet hemocyanin with adjuvant. Labyrinthitis involves infiltration of inflammatory cells and hearing loss detectable 3 to 5 days after challenge with keyhole limpet hemocyanin. Methods Etanercept was administered either systemically (2.5 mg) 30 minutes before intracochlear challenge with keyhole limpet hemocyanin, with a second intraperitoneal dose (2.5 mg) 3 days later or locally by long-term infusion into the scala tympani with an osmotic pump (5.0 &mgr;g/h for 7 days). Auditory evoked brainstem response thresholds were measured before and after treatment to determine hearing loss. Cochleas were evaluated for the amount of inflammation. Results Hearing loss in the untreated systemic group averaged 71 ± 21 dB versus 37 ± 32 dB in the etanercept-treated animals (t test, P < 0.001). There was also less inflammation in the cochleas from etanercept-treated animals (t test, P < 0.01). Hearing loss with local administration of etanercept was 59 ± 31 dB in the nontreated ears and 18 ± 8 dB in the treated ears (t test P < 0.02). Inflammation was also less (t test, P < 0.01). Etanercept was not ototoxic. Conclusion Prompt intervention with the anti-inflammatory drug etanercept significantly reduces inflammation sufficient for substantive hearing preservation.

Dose-Dependent Sustained Release of Dexamethasone in Inner Ear Cochlear Fluids Using a Novel Local Delivery Approach

The thermo-reversible triblock copolymer poloxamer 407 was investigated as a drug delivery vehicle for micronized dexamethasone into the middle and inner ears of guinea pigs. The study characterized the gelation and in vitro release kinetics of poloxamer formulations. In vivo, the pharmacokinetic profile of formulations containing varying concentrations of poloxamer and dexamethasone was examined following intratympanic administration. Significant drug levels within the perilymph were observed for at least 10 days, while systemic exposure was minimal. The sustained-release kinetics profile could be significantly modulated by varying the concentrations of both poloxamer and dexamethasone. Assessment of auditory function revealed a small transient shift in hearing threshold, most probably of conductive nature, which resolved itself within a week. No significant histological changes of the round window membrane or cochlea could be noted. Poloxamer 407 thus represents an effective and safe delivery system to achieve sustained release of dexamethasone to the inner ear.

Tumor Necrosis Factor α Can Induce Recruitment of Inflammatory Cells to the Cochlea

Hypothesis: Leukocyte recruitment to the cochlea can be induced by tumor necrosis factor &agr; (TNF-&agr;) at concentrations that are not cytotoxic to sensory cells in the organ of Corti. Background: Leukocytes participating in inflammation enter the inner ear via the spiral modiolar vein and its tributaries. Many of the infiltrated leukocytes express TNF-&agr; 3 hours after cochlear antigen challenge of systemically antigen-sensitized animals. Competitive inhibition of TNF-&agr; receptors reduces inflammation and hearing loss in experimentally induced labyrinthitis in guinea pigs and mice. However, TNF-&agr; is also potentially cytotoxic, acting through the external apoptotic pathway and TNF-&agr; transmembrane, cell surface receptors. It may therefore also participate in the sensory cell degeneration resulting from inflammation. Methods: To test for recruitment potential, TNF-&agr; or phosphate-buffered saline was infused into the guinea pig inner ear for 2 to 4 days through a cochleostomy using an osmotic pump (0.2 or 2.0 &mgr;g/mL; 1 &mgr;L/h) or a bolus injection (50 &mgr;g/mL; 10 &mgr;L). Auditory evoked brainstem response thresholds were measured before and after challenge, and cochleas were evaluated for the presence of leukocytes. To test for toxicity, organ of Corti explants were subjected to 3 concentrations of TNF-&agr; (0.1, 10, and 1,000 ng/mL) for 96 hours, and the number of hair cell places was counted. Results: Tumor necrosis factor &agr; infused into the guinea pig cochlear scala tympani resulted in infiltration of leukocytes around the venules and within scala tympani. There was no associated hearing loss as measured with a click stimulus. Tumor necrosis factor &agr; applied directly to organ of Corti explants caused minimal hair cell death at concentrations used in the in vivo experiments. At higher concentrations, there was 15 to 20% loss of cells. Conclusion: Tumor necrosis factor &agr; is sufficient to recruit inflammatory cells to the cochlea but is not likely to be directly responsible for the hearing loss that follows immune-mediated labyrinthitis.

OTO-104: a sustained-release dexamethasone hydrogel for the treatment of otic disorders.

Hypothesis: To investigate whether OTO-104, a poloxamer-based hydrogel containing micronized dexamethasone for intratympanic delivery, can provide long-lasting inner ear exposure and be well tolerated. Methods: OTO-104 was administered intratympanically to guinea pigs and sheep, and its pharmacokinetic and toxicity profiles were examined. Results: After a single intratympanic injection of OTO-104 (from 0.6% to 20%, w/w), significant and prolonged exposure to dexamethasone in the inner ear was observed. Increasing the concentration of OTO-104 resulted in higher perilymph drug levels as well as a more prolonged duration of exposure. At the highest dose, therapeutic perilymph levels of dexamethasone could be sustained over 3 months in guinea pigs and more than 1 month in sheep. A toxicologic evaluation was conducted, including assessments of middle and inner ear function and physiology, as well as appraisal of local and systemic toxicity. A small and transient shift in hearing threshold was observed, most probably conductive in nature. No significant histologic changes in middle or inner ear tissues were noted. Although macroscopically mild erythema/inflammation was documented in a subset of guinea pigs treated with 20% OTO-104, the nature and the severity of these changes were not different between the poloxamer vehicle, saline, and 20% OTO-104 groups. No evidence of acute dermal toxicity, delayed hypersensitivity, or systemic adverse effects was found. Conclusion: OTO-104 is a novel proprietary therapeutic delivery system that can achieve prolonged, sustained release of dexamethasone within the inner ear fluids. The administration of this clinical candidate formulation via intratympanic injection is expected to be well tolerated both locally and systemically.

A natural allele of Nxf1 suppresses retrovirus insertional mutations

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The modifier-of-vibrator-1 locus (Mvb1) controls levels of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the Pitpnvb tremor mutation and the Eya1BOR model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between the mRNA export receptor and pre-mRNA processing. Population structure of the suppressive allele in wild Mus musculus castaneus suggests selective advantage. A congenic Mvb1CAST allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements.

Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development.

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant.

Principles of inner ear sustained release following intratympanic administration

Previous studies revealed that intratympanic administration of the steroid dexamethasone in poloxamer 407 hydrogel, a class of thermoreversible polymers, resulted in significant and durable exposure in the inner ear. Interestingly, varying the concentrations of the poloxamer vehicle and of the steroid impacted the pharmacokinetic profile of dexamethasone in the perilymphatic compartment. Here, the respective contributions of different vehicles (aqueous solution, poloxamer hydrogel) and steroid drugs (dexamethasone, methylprednisolone) were investigated. In particular, various forms of the steroids, discriminated by their aqueous solubility, were compared.

Pharmacokinetics of Dexamethasone Solution following Intratympanic Injection in Guinea Pig and Sheep

Information on inner ear pharmacokinetics is limited in the literature, especially in large animals and in humans. A preliminary study was designed to explore the differences in inner ear exposure between guinea pigs and sheep following a single intratympanic injection of a 2% dexamethasone sodium phosphate solution. In both species, significant levels of dexamethasone were observed in the perilymph within 1 h, and decreasing by 50- to 100-fold within 12 h. Overall, the exposure to dexamethasone in the inner ear was significantly lower in sheep by 17- to 27-fold than in guinea pigs. Systemic and CNS exposure were minimal in both species as indicated by the low drug levels observed in plasma and CSF. Altogether, the preliminary evidence presented herein suggests the sheep as a practical and acceptable animal model to study the inner ear pharmacokinetics of drug candidates in large mammals and its potential towards extrapolation to human exposure.

OTO-201: Nonclinical Assessment of a Sustained-Release Ciprofloxacin Hydrogel for the Treatment of Otitis Media

Hypothesis OTO-201 can provide sustained release to the middle ear and effectively treat otitis media, when compared with FDA-approved ciprofloxacin otic drop formulations. Background There is an unmet medical need for antibiotic therapy that can provide a full course of treatment from a single administration by an otolaryngologist at the time of tympanostomy tube placement, obviating the need for twice daily multiday treatment with short-acting otic drops. Methods Studies in guinea pigs and chinchillas were conducted. OTO-201 was administered as a single intratympanic injection and compared with the twice daily multi-day treatment with Ciprodex or Cetraxal otic drops. Results OTO-201 demonstrated sustained release of ciprofloxacin in the middle ear compartment for days to approximately 2 weeks depending on the dose. The substantial Cmax values and steady drug exposure yielded by OTO-201 were in contrast to the pulsatile short lasting exposure seen with Ciprodex and Cetraxal. OTO-201 was also effective in a preclinical chinchilla model of Streptococcus pneumoniae–induced otitis media. The degree of cure was comparable to that afforded by Ciprodex and Cetraxal. There was no evidence of middle or inner ear pathology in guinea pigs treated with OTO-201, unlike Ciprodex and Cetraxal, which both demonstrated mild cochlear ototoxicity. No adverse effects of the poloxamer 407 vehicle were noted. Conclusion Intratympanic injection of OTO-201 constitutes an attractive treatment option to twice daily multiday dosing with ciprofloxacin ear drops for the treatment of otitis media, as evidenced by superior middle ear drug exposure, efficacy in an acute otitis media model, safety of administration, and convenience of a single dose regimen.