Evaluation of an ex vivo model implication for carrier-mediated retinal drug delivery.
OBJECTIVE
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 ([(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
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).


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.
Kansara
Viral
V
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64112, USA.
Mitra
Ashim K
AK
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival; D002829:Choroid / Q000378:metabolism; D003975:Diazepam / Q000378:metabolism; D015194:Diffusion Chambers, Culture; D016503:Drug Delivery Systems; D004553:Electric Conductivity; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D008727:Methotrexate / Q000494:pharmacology; D008954:Models, Biological; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D012590:Sclera / Q000378:metabolism; D013696:Temperature; D013763:Tetrahydrofolates / Q000502:physiology
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; D008353:Mannitol; D005492:Folic Acid; D003975:Diazepam; C005984:5-methyltetrahydrofolate; D008727:Methotrexate