Reduction of quaternary ammonium-induced ocular surface toxicity by emulsions: an in vivo study in rabbits

摘要

Purpose: To evaluate and compare thetoxicological profiles of two quaternary ammonium compounds (QAC),benzalkonium chloride (BAK), and cetalkonium chloride (CKC), instandard solution or cationic emulsion formulations in rabbit eyesusing newly developed in vivo and ex vivo experimental approaches. Methods: Seventy eyes of 35 adult maleNew Zealand albino rabbits were used in this study. They were randomlydivided into five groups: 50 μl of phosphate-buffered saline (PBS), PBScontaining 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively),and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em,respectively) were applied to rabbit eyes 15 times at 5-min intervals.The ocular surface changes induced by these eye drops were investigatedusing slit-lamp examination, flow cytometry (FCM), impression cytology(IC) on conjunctiva, and corneal in vivo confocal microscopy (IVCM).Standard immunohistology in cryosections was also examined for clusterof differentiation (CD) 45+ infiltrating and terminal deoxynucleotidyltransferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells. Results: Clinical observations and IVCMshowed that the highest toxicity was induced by BAK Sol, characterizedby damaged corneal epithelium and a high level of inflammatoryinfiltration. BAK Em and CKC Sol presented moderate effects, and CKC Emshowed the lowest toxicity with results similar to those of PBS.Conjunctival imprints analyzed by FCM showed a higher expression ofRLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all othergroups, especially at 4 h. Immunohistology was correlated with invivo and ex vivo findings and confirmed this toxicity profile. A highlevel of infiltration of CD45+ inflammatory cells and TUNEL+ apoptoticcells was observed in limbus and conjunctiva, especially in QACsolution-receiving eyes compared to QAC emulsion-instilled eyes. Conclusions: The acute administration of15 instillations at 5 min intervals was a rapid and efficient model toassess quaternary ammonium toxicity profiles. This model showed thehighest toxicity, induced by the BAK solution, and the lowest level oftoxicity, induced by the CKC emulsion. These in vivo and ex vivoexperimental approaches demonstrated that ocular surface toxicity wasreduced by using an emulsion instead of a traditional solution and thata CKC emulsion was safe for future ocular administration.