Middle Ear Administration of a Particulate Chitosan Gel in an in vivo Model of Cisplatin Ototoxicity

Abstract

Background: Middle ear (intratympanic, IT) administration is a promising therapeutic method as it offers the possibility of achieving high inner ear drug concentrations with low systemic levels, thus minimizing the risk of systemic side effects and drug-drug interactions. Premature elimination through the Eustachian tube may be reduced by stabilizing drug solutions with a hydrogel, but this raises the secondary issue of conductive hearing loss.

Aim: This study aimed to investigate the properties of a chitosan-based particulate hydrogel formulation when used as a drug carrier for IT administration in an in vivo model of ototoxicity.

Materials and methods: Two particulate chitosan-based IT delivery systems, Thio-25 and Thio-40, were investigated in albino guinea pigs (n = 94). Both contained the hearing protecting drug candidate sodium thiosulfate with different concentrations of chitosan gel particles (25% vs. 40%). The safety of the two systems was explored in vivo. The most promising system was then tested in guinea pigs subjected to a single intravenous injection with the anticancer drug cisplatin (8 mg/kg b.w.), which has ototoxic side effects. Hearing status was evaluated with acoustically evoked frequency-specific auditory brainstem response (ABR) and hair cell counting. Finally, in vivo magnetic resonance imaging was used to study the distribution and elimination of the chitosan-based system from the middle ear cavity in comparison to a hyaluronan-based system.

Results: Both chitosan-based IT delivery systems caused ABR threshold elevations (p < 0.05) that remained after 10 days (p < 0.05) without evidence of hair cell loss, although the elevation induced by Thio-25 was significantly lower than for Thio-40 (p < 0.05). Thio-25 significantly reduced cisplatin-induced ABR threshold elevations (p < 0.05) and outer hair cell loss (p < 0.05). IT injection of the chitosan- and hyaluronan-based systems filled up most of the middle ear space. There were no significant differences between the systems in terms of distribution and elimination.

Conclusion: Particulate chitosan is a promising drug carrier for IT administration. Future studies should assess whether the physical properties of this technique allow for a smaller injection volume that would reduce conductive hearing loss.

Keywords: auditory brainstem response; cisplatin; hair cell; hearing loss; intratympanic administration; magnetic resonance imaging; particulate chitosan; sodium thiosulfate.

FIGURE 1

Guinea pigs were subjected to a single, unilateral intratympanic (IT) injection of sodium thiosulfate (100 mM) in a particulate chitosan gel with a gel particle concentration of 25% (Thio-25 group, n = 15) or 40% (Thio-40 group, n = 15). Electrophysiological hearing thresholds assessed with air-conducted acoustically evoked auditory brainstem response (ABR) were measured at 12.5, 20.0, and 30.0 kHz before (pre) and 7 and 10 days after IT administration. Hearing threshold (decibel sound pressure level, dB SPL) vs. time point stratified by IT administration and frequency is shown. Each color represents one animal. Fewer colors than 15 per graph are due to overlapping lines.

FIGURE 2

Guinea pigs were subjected to a single, unilateral IT injection of a sodium thiosulfate-containing chitosan-based gel (Thio-25, n = 15), while the other ear was left untreated, serving as a control (None). The animals received a single high dose of cisplatin (8 mg/kg b.w., i.v.) 1 h later. Electrophysiological hearing thresholds (in decibel sound pressure level, dB SPL) assessed with air-conducted acoustically evoked auditory brainstem response (ABR) at 12.5, 20, and 30 kHz before and 10 days after IT administration are shown. Each color represents one animal. Fewer colors than 15 per graph are due to overlapping lines.

FIGURE 3

Animals in the Thio-25-cispt group (shown in Figure 2) were euthanized after hearing threshold assessment on day 10. Their cochleae were collected to quantify loss of inner hair cells (IHCs) and outer hair cells (OHCs) in the first (OHC1), second (OHC2), and third (OHC3) rows. Cytocochleogram results from an animal with a loss pattern that was representative of most of the cisplatin-treated guinea pigs subjected to no IT administration (A) and to IT administration of Thio-25 (B). Cytocochleogram results from the only cisplatin-treated animal with a large loss in the untreated ear (C) and the Thio-25-treated ear (D).

FIGURE 4

Animals in the Thio-25-cispt group (presented in Figure 2) were euthanized after hearing threshold assessment on day 10. Their cochleae were collected to quantify hair cell loss. Repeated measures ANOVA of the percentage loss of outer hair cells (OHCs) in the first (OHC1), second (OHC2), and third (OHC3) rows are shown for ears injected with a thiosulfate- containing gel (Thio-25) and for the contralateral, non-injected ear (None). ^§§§ signifies p < 0.001.

FIGURE 5

Guinea pigs (n = 19) were subjected to IT administration of a paramagnetic chitosan-based gel (Chito-Dota) in one ear and a hyaluronan- based gel (Hya-Dota) in the contralateral ear. The gels were then visualized by magnetic resonance imaging. Different orthogonal viewing planes of 3D data from one animal taken immediately after (A–C) and 4 days after (D–F) injection of Chito-Dota into the right ear and Hya-Dota into the left ear. The position of each cochlea is indicated with a yellow arrow in B and E. The sagittal plane in C and F shows the left ear. Keys (in yellow): a, anterior; i, inferior; l, left; p, posterior; r, right; s, superior.

FIGURE 6

Guinea pigs were subjected to intratympanic IT administration of two different paramagnetic gels, one based on chitosan (Chito-Dota) in one ear and one based on hyaluronan (Hya-Dota) in the contralateral ear. Volume and intensity in the middle ear were explored with magnetic resonance imaging performed on three different occasions in each guinea pig. The volume (A,B) and intensity (C,D) in the middle ear vs. time curves for each guinea pig are shown. n = 19 on day 1, n = 5 on day 3, n = 4 on day 5, n = 9 on day 7, n = 8 on day 10, n = 5 on day 12, and n = 5 on day 14 for both Chito-Dota and Hya-Dota.