Effect of Immunophilin Inhibitors on Cochlear Fibroblasts and Spiral Ganglion Cells.

INTRODUCTION: Loss of hair cells and degeneration of spiral ganglion neurons (SGN) lead to severe hearing loss or deafness. The successful use of a cochlear implant (CI) depends among other factors on the number of surviving SGN. Postoperative formation of fibrous tissue around the electrode array causes an increase in electrical impedances at the stimulating contacts. The use of immunophilin inhibitors may reduce the inflammatory processes without suppressing the immune response. Here, we report on in vitro experiments with different concentrations of immunophilin inhibitors MM284 and compound V20 regarding a possible application of these substances in the inner ear. METHODS: Standard cell lines (NIH/3T3 fibroblasts), freshly isolated SGN, and fibroblasts from neonatal rat cochleae (p3-5) were incubated with different concentrations of immunophilin inhibitors for 48 h. Metabolic activity of fibroblasts was investigated by MTT assay and cell survival by counting of immunochemically stained neurons and compared to controls. RESULTS: MM284 did not affect SGN numbers and neurite growth at concentrations of 4 × 10-5 mol/L and below, whereas V20 had no effect at 8 × 10-6 mol/L and below. Metabolic activity of fibroblasts was unchanged at these concentrations. CONCLUSION: Especially MM284 might be considered as a possible candidate for application within the cochlea.

Development of a drug delivering round window niche implant for cochlear pharmacotherapy.

BACKGROUND: There exists an unfulfilled requirement for effective cochlear pharmacotherapy. Controlled local drug delivery could lead to effective bioavailability. The round window niche (RWN), a cavity in the middle ear, is connected to the cochlea via a membrane through which drug can diffuse. We are developing individualized drug-eluting RWN implants (RNIs). To test their effectiveness in guinea pigs, a commonly used model in cochlear pharmacology studies, it is first necessary to develop guinea pig RNIs (GP-RNI). METHODS: Since guinea pigs do not have a RWN such as it is present in humans and to reduce the variables in in vivo studies, a one-size-fits-all GP-RNI model was designed using 12 data sets of Dunkin-Hartley guinea pigs. The model was 3D-printed using silicone. The accuracy and precision of printing, distribution of the sample ingredient dexamethasone (DEX), biocompatibility, bio-efficacy, implantability and drug release were tested in vitro. The GP-RNI efficacy was validated in cochlear implant-traumatized guinea pigs in vivo. RESULTS: The 3D-printed GP-RNI was precise, accurate and fitted in all tested guinea pig RWNs. DEX was homogeneously included in the silicone. The GP-RNI containing 1% DEX was biocompatible, bio-effective and showed a two-phase and sustained DEX release in vitro, while it reduced fibrous tissue growth around the cochlear implant in vivo. CONCLUSIONS: We developed a GP-RNI that can be used for precise inner ear drug delivery in guinea pigs, providing a reliable platform for testing the RNI's safety and efficacy, with potential implications for future clinical translation.

Micro Injection Molding of Drug-Loaded Round Window Niche Implants for an Animal Model Using 3D-Printed Molds.

A novel approach for the long-term medical treatment of the inner ear is the diffusion of drugs through the round window membrane from a patient-individualized, drug-eluting implant, which is inserted in the middle ear. In this study, drug-loaded (10 wt% Dexamethasone) guinea pig round window niche implants (GP-RNIs, ~1.30 mm × 0.95 mm × 0.60 mm) were manufactured with high precision via micro injection molding (µIM, Tmold = 160 °C, crosslinking time of 120 s). Each implant has a handle (~3.00 mm × 1.00 mm × 0.30 mm) that can be used to hold the implant. A medical-grade silicone elastomer was used as implant material. Molds for µIM were 3D printed from a commercially available resin (TG = 84 °C) via a high-resolution DLP process (xy resolution of 32 µm, z resolution of 10 µm, 3D printing time of about 6 h). Drug release, biocompatibility, and bioefficacy of the GP-RNIs were investigated in vitro. GP-RNIs could be successfully produced. The wear of the molds due to thermal stress was observed. However, the molds are suitable for single use in the µIM process. About 10% of the drug load (8.2 ± 0.6 µg) was released after 6 weeks (medium: isotonic saline). The implants showed high biocompatibility over 28 days (lowest cell viability ~80%). Moreover, we found anti-inflammatory effects over 28 days in a TNF-α-reduction test. These results are promising for the development of long-term drug-releasing implants for human inner ear therapy.

Development and in vivo validation of small interfering RNAs targeting NOX3 to prevent sensorineural hearing loss.

The reactive oxygen species (ROS)-generating enzyme NOX3 has recently been implicated in the pathophysiology of several acquired forms of sensorineural hearing loss, including cisplatin-, noise- and age-related hearing loss. NOX3 is highly and specifically expressed in the inner ear and therefore represents an attractive target for specific intervention aiming at otoprotection. Despite the strong rationale to inhibit NOX3, there is currently no specific pharmacological inhibitor available. Molecular therapy may represent a powerful alternative. In this study, we developed and tested a collection of small interfering (si) RNA constructs to establish a proof of concept of NOX3 inhibition through local delivery in the mouse inner ear. The inhibitory potential of 10 different siRNA constructs was first assessed in three different cells lines expressing the NOX3 complex. Efficacy of the most promising siRNA construct to knock-down NOX3 was then further assessed in vivo, comparing middle ear delivery and direct intracochlear delivery through the posterior semi-circular canal. While hearing was completely preserved through the intervention, a significant downregulation of NOX3 expression in the mouse inner ear and particularly in the spiral ganglion area at clinically relevant levels (>60%) was observed 48 h after treatment. In contrast to successful intracochlear delivery, middle ear administration of siRNA failed to significantly inhibit Nox3 mRNA expression. In conclusion, intracochlear delivery of NOX3-siRNAs induces a robust temporal NOX3 downregulation, which could be of relevance to prevent predictable acute insults such as cisplatin chemotherapy-mediated ototoxicity and other forms of acquired hearing loss, including post-prevention of noise-induced hearing loss immediately after trauma. Successful translation of our concept into an eventual clinical use in humans will depend on the development of atraumatic and efficient delivery routes into the cochlea without a risk to induce hearing loss through the intervention.

Consecutive Treatment with Brain-Derived Neurotrophic Factor and Electrical Stimulation Has a Protective Effect on Primary Auditory Neurons.

Degeneration of neurons, such as the inner ear spiral ganglion neurons (SGN), may be decelerated or even stopped by neurotrophic factor treatment, such as brain-derived neurotrophic factor (BDNF), as well as electrical stimulation (ES). In a clinical setting, drug treatment of the SGN could start directly during implantation of a cochlear implant, whereas electrical stimulation begins days to weeks later. The present study was conducted to determine the effects of consecutive BDNF and ES treatments on SGN density and electrical responsiveness. An electrode drug delivery device was implanted in guinea pigs 3 weeks after deafening and five experimental groups were established: two groups received intracochlear infusion of artificial perilymph (AP) or BDNF; two groups were treated with AP respectively BDNF in addition to ES (AP + ES, BDNF + ES); and one group received BDNF from the day of implantation until day 34 followed by ES (BDNF ⇨ ES). Electrically evoked auditory brainstem responses were recorded. After one month of treatment, the tissue was harvested and the SGN density was assessed. The results show that consecutive treatment with BDNF and ES was as successful as the simultaneous combined treatment in terms of enhanced SGN density compared to the untreated contralateral side but not in regard to the numbers of protected cells.