| The delivery of anti-human immunodeficiency virus (anti-HIV) agents to the brain and central nervous system is very limited due to their poor transport through the bloodbrain barrier (BBB). Our incomplete knowledge of these agents' biochemical and physiological transport mechanisms precludes development of new methods for improved delivery. Brain microvascular endothelial cells isolated from bovine brain tissue (BBMEC) are one of the most popular in vitro models for studying drug transport across the BBB. However, such primary cells cannot be passaged, and repeated isolation is both labor intensive and expensive. Therefore, immortalized cells are useful alternatives to primary cell models. This dissertation validated a new BBMEC cell line (MYEC-1) immortalized by co-transfecting of Polyoma middle-T protein and pSV2neo plasmid. MYEC-1 cells express endothelial markers (Factor VIII and LDL receptor), drug-resisting transporters (P-glycoprotein, MRP1,MRP4 and MRP5), organic anion transporting polypeptides (OATP-1), and nucleoside transporters (ENT1 and ENT2). Their transport properties were comparable to their primary progenitor cells in passive and active uptake experiments with several different permeants including urea, sucrose, mannitol, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), and vinblastine.; The mechanisms of 2',3'-dideoxyinosine (ddI) and F-ddI uptake by MYEC-1 cells were examined with six different inhibitors including digoxin, indomethacin, p-aminohippurate, nitrobenzylthioinosine, verapamil and probenecid. Results showed that NBMPR, verapamil, probenecid and indomethacin (only partially) inhibited ddI uptake, while indomethacin, verapamil and probencid inhibited F-ddI uptake. PAH and digoxin did not affect the uptake of either drug because MYEC-1 cells do not express organic anion transporters or organic anion transporting polypeptide 2. These results suggested that nucleoside transporters ENT1 and ENT2 facilitated ddI uptake, while OATP-1 facilitated F-ddI uptake. Despite physicochemical similarities between F-ddI and ddI, their transport across the BBB probably utilizes different mechanisms. These studies demonstrated the capability of cultured MYEC-1 cells in probing the mechanisms of important anti-HIV dideoxynucleosides in brain delivery.; Further exploration of F-ddI transport revealed that it is metabolized inside MYEC-1 cells, possibly to F-ddI mono-phosphate (F-ddIMP). Indomethacin can slow the efflux of this metabolite probably by inhibiting MRP4, resulting in a higher measured intracellular F-ddI concentration at steady-state (measured concentration included both FddI and metabolite concentrations). |
