Evaluation of an ex vivo model implication for carrier-mediated retinal drug delivery.

Purpose: The purpose of this study was to evaluate the implication of an ex vivo model for carrier-mediated retinal drug delivery using an Ussing chamber system. Methods: Dutch Belted Pigmented rabbits weighing 2-2.5 kg were used in these studies. Excised posterior segment tissues (RPE-choroid-sclera and sclera), mounted on the Ussing chamber, were used as an ex vivo model. Transport studies were carried out across sclera and RPE-choroid-sclera (RCS) tissue preparations in the sclera to retina (S -> R) and retina to sclera (R -> S) directions for 3 hr at 37 degrees C. The model was validated by permeability studies with paracellular and transcellular markers ([H-3]mannitol and [C-14]diazepam, respectively), tissue viability studies (bioelectrical and biochemical assays), and tissue histology and electron microscopy studies. Functional presence of a carrier-mediated transport system for folic acid (folate receptor alpha) was investigated on the basolateral side of the rabbit retina. Results: Results from bioelectrical, biochemical, and histological evaluation of tissue provide evidence that the RCS tissue preparation remains viable during the period of transport study. Permeability values of [H-3]mannitol across sclera were 4.18 +/- 1.09 x 10(-5) cm/s (R -> S) and 4.11 +/- 1.09 x 10(-5) cm/s (S -> R) and across RCS were 1.77 +/- 0.31 x 10(-5) cm/s (S -> R) and 1.60 +/- 0.19 x 10(-5) cm/s (R -> S). Permeability values of [C-14]diazepam across sclera were 2.37 +/- 0.38 x 10(-5) cm/s (R -> S) and 2.70 +/- 0.70 x 10(-5) cm/s (S -> R) and across RCS were 3.12 +/- 0.12 x 10(-5) cm/s (R -> S) and 2.77 +/- 0.25 x 10(-5) cm/s (S -> R). The rate of [H-3]folic acid transport across RCS was found to be significantly higher in the S -> R direction (16.34 +/- 0.94 fmoles min(-1) cm(-2) ) as compared with R -> S direction (9.38 +/- 1.44 fmoles min(-1) cm(-2)) and nearly 10-fold higher across sclera as compared with RCS in both directions. Transport of [H-3]folic acid was found to be pH and temperature dependent and was inhibited by 44.5%, 35.1%, and 50.3% in the presence of unlabeled folic acid, 5-methyltetrahydrofolate (MTF), and Methotrexate (MTX). Conclusions: The RCS tissue preparation mounted on the Ussing chamber system, an ex vivo model, can be a useful tool for identification and characterization of carrier-mediated systems present on RPE (a major barrier for retinal drug delivery) and to study carrier-mediated retinal drug delivery via prodrug derivatization.