Evaluation of an immortalized retinal endothelial cell line as an in vitro model for drug transport studies across the blood-retinal barrier.
OBJECTIVE
To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies.


METHODS
TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [14C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (Papp). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075.


RESULTS
TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between Papp and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1.


CONCLUSIONS
TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.
Department of Pharmacokinetics and Drug Metabolism, Allergan, Inc., Irvine, California 92612, USA. shen_jie@allergan.com
Jie
J
Shen
Stacy T
ST
Cross
Diane D S
DD
Tang-Liu
Devin F
DF
Welty
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor / Q000378:metabolism*; D004337:Drug Carriers; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D008954:Models, Biological; D010539:Permeability; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010599:Pharmacokinetics; D051381:Rats; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004337:Drug Carriers; D004364:Pharmaceutical Preparations