Hearing Loss and Otopathology Following Systemic and Intracerebroventricular Delivery of 2-Hydroxypropyl-Beta-Cyclodextrin

Abstract

Cyclodextrins are simple yet powerful molecules widely used in medicinal formulations and industry for their ability to stabilize and solubilize guest compounds. However, recent evidence shows that 2-hydroxypropyl-β-cyclodextrin (HPβCD) causes severe hearing loss in mice, selectively killing outer hair cells (OHC) within 1 week of subcutaneous drug treatment. In the current study, the impact of HPβCD on auditory physiology and pathology was explored further as a function of time and route of administration. When administered subcutaneously or directly into cerebrospinal fluid, single injections of HPβCD caused up to 60 dB threshold shifts and widespread OHC loss in a dose-dependent manner. Combined dosing caused no greater deficit, suggesting a common mode of action. After drug treatment, OHC loss progressed over time, beginning in the base and extending toward the apex, creating a sharp transition between normal and damaged regions of the cochlea. Administration into cerebrospinal fluid caused rapid ototoxicity when compared to subcutaneous delivery. Despite the devastating effect on the cochlea, HPβCD was relatively safe to other peripheral and central organ systems; specifically, it had no notable nephrotoxicity in contrast to other ototoxic compounds like aminoglycosides and platinum-based drugs. As cyclodextrins find expanding medicinal applications, caution should be exercised as these drugs possess a unique, poorly understood, ototoxic mechanism.

Keywords: cochlea; cyclodextrin; drug delivery; hearing loss; ototoxicity.

FIG. 1

ABR thresholds were elevated 1 week after HPβCD treatment by SQ or ICV injection. Mean thresholds of control, SQ, ICV, and combined cohorts for high dose (A) and low dose (B) HPβCD are shown for three test frequencies. Saline vehicle controls for SQ and ICV injections were combined for a single control group since there was no statistical difference between these individual control groups (Student’s two-tailed t test for each frequency: 4 kHz P = 0.27; 16 kHz P = 0.19; 32 kHz P = 0.15). Elevated thresholds were observed for all high dose groups compared to controls, whereas only concurrent administration of SQ low and ICV low resulted in similarly elevated thresholds across all test frequencies. Statistically significant differences in mean ABR thresholds for each frequency were tested across groups using one-way ANOVA (4 kHz: F = 16.85, df = 53, P < 0.001; 16 kHz: F = 26.68, df = 53, P < 0.001; 32 kHz: F = 30.55, df = 53, P < 0.001). Post hoc pairwise comparisons between groups was performed using a Tukey test with significant differences from controls indicated by an asterisk (*P < 0.001). The number of animals per group: control (15), SQ low (6), ICV low (9), SQ + ICV low (6), SQ high (12), ICV high (8), SQ + ICV high (4). Histograms of control ABR thresholds (gray-shaded region) and all HPβCD treatment groups (filled black symbols) are plotted as threshold shift from control means for all test frequencies (C). The HPβCD data was best fit (Simplex algorithm) with a two-component Gaussian function, revealing one component centered with control data and the other component centered at ~57 dB.

FIG. 2

Hair cell loss from HPβCD is primarily restricted to OHCs. Mid-apical segments of the organ of Corti are shown for control (A), SQ high (B), ICV high (C), and combined SQ + ICV low (D) preparations. Percent missing IHCs (E, G, I) and OHCs (F, H, J) are plotted for each treatment condition from animals selected at random, without consideration of ABR response. Control data are re-plotted in each panel for comparison. Legend in E applies to F, G to H, and I to J. The number of animals per group is shown in parentheses alongside each legend. A map of cochlear location to characteristic frequency is illustrated in (E).

FIG. 3

The extent of OHC loss was highly correlated with ABR threshold. The average % hair cell loss is plotted against the mean threshold shift at 16 kHz (relative to control data) for individual animals contributing to data in Fig. 2. Control data (filled symbols) are shown alongside HPβCD treatment groups (open symbols) for percent missing IHCs (circles) and OHCs (triangles). There is a normal complement of IHCs regardless of the impact of HPβCD on hearing sensitivity. In contrast, elevated ABR thresholds were uniformly associated with greater OHC loss (N = 39).

FIG. 4

OHC loss progressed over days for SQ high (A) and hours for ICV high (B). The percent missing OHC were averaged from randomly selected animals in each condition and at each time point. SQ high data are shown for the whole cohort (open triangle) and a subset of affected animals (closed square). Hour time points for ICV high groups were collected over a time range, ±~20 % of the midpoint indicated in the legend. Only positive error of the mean is shown for each point for clarity. The number of animals per group is shown in parentheses alongside legends. The mean composite loss of OHCs throughout the full length of the organ of Corti is shown (C).

FIG. 5

Plastic sections of control and HPβCD treated animals revealed only minor degeneration outside of the organ of Corti. Basal turns are shown for control (A, E), SQ high (B, F), ICV high (C, G), and combined SQ and ICV high (D, H). A–D Drug effects were absent in stria vascularis, but drug-dependent vacuolization of the lateral wall was apparent in some HPβCD treated animals, particularly in the region of type I fibrocytes. E–H SGC shape and density was unaffected by drug treatment. Scale bar in (H) = 50 μm and applies to all panels. Images are representative of the most severe lesions in 6 independent preparations per condition.

FIG. 6

ICV injections in vehicle control mice (A) and HPβCD-treated mice (B) had similar lesions in brain related to injection trauma, including degeneration and necrosis of the neuropil (arrowheads). SQ vehicle administration (C) elicited few inflammatory cells within the skin, whereas SQ administration of HPβCD (D) elicited a regional dermatitis, cellulitis, and myositis. There were no histologic differences between kidneys in mice treated with vehicle (E) compared to mice treated with HPβCD (F). Images are representative of the most severe lesions in 6 independent preparations per condition.