Evaluation of anEx VivoModel Implication for Carrier-Mediated Retinal Drug Delivery

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Abstract
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. 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 ([(3)H]mannitol and [(14)C]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 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 [(3)H]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 [(14)C]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 [(3)H]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 [(3)H]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). 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.