Tim Quinn

Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux.

PURPOSE The purpose of this manuscript is to investigate the presence of nucleoside/nucleotide efflux transporter in cornea and to evaluate the role in ocular drug efflux. METHODS RT-PCR, immunoprecipitation followed by Western blot analysis and immunostaining were employed to establish molecular presence of multidrug resistance associated protein 5 (MRP5) on cornea. Corneal efflux by MRP5 was studied with bis(POM)-PMEA and acyclovir using rabbit and human corneal epithelial cells along with MRP5 over expressing cells (MDCKII-MRP5). Ex vivo studies using excised rabbit cornea and in vivo ocular microdialysis in male New Zealand white rabbits were used to further evaluate the role of MRP5 in conferring ocular drug resistance. RESULTS RT-PCR confirms the expression of MRP5 in both rabbit and human corneal epithelial cells along with MDCKII-MRP5 cells. Immunoprecipitation followed by Western blot analysis using a rat (M511-54) monoclonal antibody that reacts with human epitope confirms the expression of MRP5 protein in human corneal epithelial cells and MDCKII-MRP5 cells. Immunostaining performed on human cornea indicates the localization of this efflux pump on both epithelium and endothelium. Efflux studies reveal that depletion of ATP decreased PMEA efflux significantly. MRP5 inhibitors also diminished PMEA and acyclovir efflux. However, depletion of glutathione did not alter efflux. MDR1 and MRP2 did not contribute to PMEA efflux. However, MRP2 is involved in acyclovir efflux while MDR1 do not participate in this process. TLC/autoradiography suggested the conversion of bis(POM)-PMEA to PMEA in rabbit and human corneal epithelial cells. Two well known antiglaucoma drugs, bimatoprost and latanoprost were rapidly effluxed by MRP5. Ex vivo study on intact rabbit corneas demonstrated accumulation of PMEA in cornea in the presence of ATP-depleting medium. In vivo ocular pharmacokinetics also revealed a significant increase in maximum aqueous humor concentration (C(max)) and area under the aqueous humor time curve (AUC) of acyclovir in the presence of MK-571, a specific MRP inhibitor. CONCLUSIONS Taken together immunolocalization on human cornea, in vitro efflux in human, rabbit corneal and MRP5 over expressing cells, ex vivo and in vivo studies in intact rabbit cornea suggest that MRP5 on cornea can significantly lower the permeability of antiviral and glaucoma drugs. These findings may be valuable in developing formulation strategies to optimize ocular bioavailability of topically administered ocular agents.

Treatment with Ω-3 fatty acids but not exendin-4 improves hepatic steatosis

Background Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the Western world. The aim of this study was to evaluate the biochemical and histological effects of Ω-3 fatty acid and exendin-4 treatment on NAFLD in an animal model. Methods Sixty-three 8-week-old outbred Sprague–Dawley male rats were used for this study. Three animals were used as procedure controls, and 30 rats were fed a methionine and choline deficient (MCD) diet and 30 were fed a regular chow diet. In each group of 30 animals, 10 served as controls, 10 received exendin-4, and 10 received Ω-3 fatty acids. After 75 days of treatment, the animals were euthanized, the tissues and serum were harvested, and the livers were formalin-fixed for histology. Results The MCD diet was exceptionally efficient at producing fatty livers. The MCD control animals had a liver steatosis score of 38±6.7 (of 50 possible); treatment with exendin-4 was not associated with a significant reduction of steatosis (44±5.16, P=0.07) and the Ω-3 fatty acid treatment was associated with a significant decrease in the liver steatosis score (15.6±13.46, P<0.001) compared with both the controls and the exendin-4 groups. The Ω-3 fatty acid treatment increased serum aspartate aminotransferase significantly, whereas exendin-4 had no effect. Conclusion In an animal model of NAFLD, the Ω-3 fatty acid therapy was associated with significant improvement in hepatic steatosis compared with exendin-4. These data suggest that Ω-3 fatty acid supplements may have a potential therapeutic role in patients with NAFLD.