In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells. Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Aggregate dose-response curve when exposed to increasing doses of AZM in AB (A) and myo6b::gfp (B) fish.Significant hair cell damage is observed at high doses (Approximately 65% RFU remaining on average in 380µM AZM (p < 1 x 10 -10 )).

Figure
Figure S1 Neomycin dose response in myo6b::gfp fish.(A) Representative dose-response curve when exposed to increasing doses of NEO, as quantified by counting individual hair cells imaged by confocal microscopy.(B) Representative image of an undamaged neuromast.(C) Representative image of a damaged neuromast, resulting from exposure to 20 μM NEO.(D) Representative dose-response curve when exposed to increasing doses of NEO, as quantified by PEPITA.(E,F) Representative images of whole fish, like those PEPITA takes as input.E is untreated; F is treated with 20 μM NEO.The cluster of fluorescent inner-ear HCs in myo6b::gfp larvae requires manual masking for proper quantification with PEPITA.

Figure S2 .
Figure S2.Overview of the variability in neomycin dose-response curves obtained by PEPITA.Points are measurements, lines are fitted log-logistic models; circles and solid lines represent AB fish stained with YO-PRO-1, stars and dashed lines represent myo6b::gfp fish.Colors represent separate experiments performed, categorized by strain: blues and greens represent AB fish, purples and reds represent myo6b::gfp fish.

Figure S4 .
Figure S4.Comparison of aggregate wEOB value between strains.The aggregate wEOB for each combination tested in both strains shows strong antagonism and does not significantly differ between strains (AZM/GEN, p=0.75;AZM/NEO, p=0.35;ERY/NEO, p=0.44).

Figure S5 .
Figure S5.Overview of interaction scores observed between neomycin and various other drugs screened for significant interactions.Increasingly negative wEOB scores indicate increasing antagonism between NEO and the listed drug; increasingly positive wEOB scores indicate increasing synergy.Most drugs lie near zero, as would be expected for noninteractive compounds.The only listed compounds that deviate toward antagonism are the three macrolides tested, plus benzamil, a potent MET channel inhibitor well established in antagonizing NEO uptake.

Figure S6 .
Figure S6.Overview of interaction scores between macrolide antibiotics and ototoxic drugs tested.

Figure S9 .
Figure S9.Quantification of mitochondrial (left) and cytoplasmic (right) Ca 2+ levels in individual hair cells in response to AZM treatment (blue), NEO treatment (orange), or the two co-administered (green), as measured by mitoGCaMP3 and cytoRGECO fluorescence signal.Drugs were administered at t = 10 min.A majority of cells exposed to NEO alone went on to die (14/21 cells from 9 neuromasts in 4 fish), while most exposed to AZM alone (24/25 cells from 11 neuromasts in 5 fish) and all treated with both drugs (19/19 cells from 10 neuromasts in 5 fish) survived.

Figure S10 .
Figure S10.Hair cell damage dose response of azithromycin individual drug treatment.Aggregate dose-response curve when exposed to increasing doses of AZM in AB (A) and myo6b::gfp (B) fish.Significant hair cell damage is observed at high doses (Approximately 65% RFU remaining on average in 380µM AZM (p < 1 x 10 -10 )).

Table S1 . Comparison between macrolides and a range of known MET channel blockers.
Molecules were visualized using ChemDraw 22.2.