Modulation Of Erythromycin Efflux On Rabbit Primary Corneal Epithelial Cells With Corticosteroids And Fluoroquinolones: A Strategy To Improve Ocular Bioavailability Of Efflux Pump Substrates

Abstract

Purpose: : Macrolides like Erythromycin are used to treat bacterial keratitis, but their ocular bio–availability is limited by the presence of efflux pumps localized in cornea. Hence, the objective was to see the interactions of Erythromycin with corticosteroids (anti–inflammatory agents which are P–gp substrates) and fluoroquinolones (antibiotics which are MRP substrates) which act as inhibitors at high concentrations thereby increasing its ocular bioavailability.

Methods: : Cultured rabbit primary corneal epithelial cells (rPCECs) were employed as models for in vitro studies. Uptake of [¹⁴C] Erythromycin was done in the presence of Ketoconazole 50uM and MK–571 50uM (a specific inhibitor for P–gp and MRP respectively) for a time when maximum rate of uptake was obtained. Uptake of [¹⁴C] Erythromycin was carried out in the presence of corticosteroids like Prednisone, Prednisolone, Methyl prednisolone (500uM each) and fluoroquinolones like Levofloxacin and Ofloxacin (1mM each) which act as P–gp and MRP inhibitors respectively. Dose dependent inhibition studies were carried out for the inhibitors and IC₅₀ values were calculated (concentration at which 50% inhibition is obtained). Cytotoxicity studies for these inhibitors were conducted with CellTiter 96 AQ_ueous nonradioactive cell proliferation assay kit.

Results: : All uptake experiments were carried out for 10 minutes as maximum rate of uptake was obtained till 10 minutes. The macrolide to be chosen requires being a substrate of both P–gp and MRP. Since, the uptake of [¹⁴C] Erythromycin increased by 3 and 4 folds in the presence of Ketoconazole and MK–571 respectively, it satisfies the rationale to be used as a model substrate. Uptake was found to increase by more than two folds for all the inhibitors under study. Also, results from cytotoxicity studies confirm that these inhibitors are not cytotoxic at the concentrations used.

Conclusions: : Corticosteroids and Fluoroquinolones are found to interact and modulate the efflux of [¹⁴C] Erythromycin thereby increasing its intracellular concentrations in rPCECs cultures. Similar studies are being carried out in vivo utilizing the ocular microdialysis technique.

Keywords: pump/barrier function • cornea: epithelium • anterior chamber