The objective of this research is to characterize the role of the multi-drug resistance-associated protein (MRP) in drug efflux processes from the central nervous system (CNS). NW s a membrane-bound transport protein belonging to the ATP-binding cassette superfamily of transport proteins, and it is overexpressed in several multidrug resistant (MDR) cancer cell lines. MDR cancers represent an important challenge in cancer chemotherapy. Several inhibitors of the glycoprotein (P-gp) -mediated drug efflux are currently in clinical trials, and several more compounds are under development specifically for P-gp and NW modulation. Like P-gp, NW is found in many normal tissues that have secretary and barrier functions, and it is expressed in the brain. However, cell-type localization and functional studies in the CNS have not been reported. The research objective relates to the hypothesis that the transport of NW substrates to the CNS depends on the expression and functional capacity of NW in the blood-brain (BBB) and blood-CSF barriers (BCSFB). The goal will be to characterize the functional capacity of the NW efflux pump in the BBB in both in vitro and in vivo models using a pharmacokinetic analysis of the effect of MRP inhibition on NW substrate distribution to the brain.
The Specific Aims are as follows: 1) to characterize the kinetics of the cellular accumulation and directional flux of selected NW substrates in an in vitro model of the BBB, brain endothelial cell monolayers, 2) to examine the expression and localization of NW on brain endothelial cell monolayers, 3) to characterize the CNS distributional kinetics of NW substrates using in vivo microdialysis in the brain extracellular fluid and the CSF, and 4) to determine if acute or chronic exposure to an NW inhibitor will change the capacity of the transport system due to induction of NW in the BBB. The functional capacity of MRP in the brain will be determined by examining the effect of known inhibitors of the NW transporter (probenecid, LTD4 antagonist (MK-571), and leads from Lilly, e.g., LY-329146) on the CNS kinetics of NW substrates, fluorescein, calcein and LTC4, in an in vitro model of the BBB and utilizing in vivo brain microdialysis. Characterizing the role NW plays in limiting CNS drug exposure will guide the design of therapeutic approaches for the safe pharmacologic modulation of NW-mediated MDR cancer. Moreover, these studies will further understanding of the physiological relevance of NW and its relationship to long-described organic anion transport systems.
