Tightly regulated cellular barriers limit delivery of pharmacological agents to children with primary tumors in the central nervous system (CNS). The physiological regulation of the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCB) and the specificity for excluding or allowing drug transport are not well understood. Camptothecin analogs such as topotecan and irinotecan are used to treat children with primary CNS tumors, but the CNS distribution of these drugs is widely different. The discovery of Pglycoprotein (P-gp) and other ATP-binding cassette (ABC) transport proteins has challenged the view that only passive diffusion and physicochemical properties control drug distribution. The major hypothesis of this proposal is that the distribution of camptothecin analogs between the blood, brain tissue, and the CSF is largely controlled by drug efflux transporters, including P-pg, multi-drug resistance protein 4 (MRP4), and the breast cancer resistance protein (BCRP or ABCG2). We seek to understand how drug distribution is controlled at the BBB and the BCB.
In Aim 1 we will develop tissue engineered models of the BBB and BCB to investigate drug transport in vitro. Cells transfected with different ABC transporters will be used to determine the specificity of transporters for camptothecin analogs. Rat brain endothelial cells and astrocytes will be co-cultured on filter inserts and in hollow fiber cartridges to examine vectorial drug transport in the absence and presence of shear stress. Mathematical models will be used to evaluate transport parameters from experimental data.
In Aim 2 we will determine the mechanisms of camptothecin analog transport in vivo using immunohistochemistry to determine the anatomical distribution of ABC transporters, and microdialysis measurements of brain and CSF drug concentrations in knockout mice deficient in MRP4 and ABCG2.
In Aim 3 we will determine if ancillary drug therapy alters expression of drug metabolizing enzymes or ABC transporters in the CNS.
In Aim 4 we will determine the expression and location of ABC transporters in primary CNS tumors and relate that to intracranial penetration of campothecin analogs and antitumor response. Our long-term goals are to understand the regulation of drug transport in the CNS, to develop in vitro and in vivo models to test camptothecin analogs, and to define and quantify parameters that can be used to compare drugs and ultimately improve therapy for children with primary CNS tumors.
