Development of an in vitro model of the blood-brain barrier: elucidation of the transport and enzymatic components governing the central nervous system uptake of dideoxynucleosides and related prodrugs

The central nervous system (CNS) uptake of dideoxynucleoside reverse transcriptase inhibitors currently used to treat HIV-1 infection in humans is severely limited and the biochemical and physiological mechanisms governing their uptake are poorly understood. The complexity of the in vivo CNS uptake kinetics of purine and pyrimidine dideoxynucleosides suggests that further mechanistic studies of these processes, presumably regulated at the level of the blood-brain barrier (BBB), are essential for developing rational strategies for enhancing the CNS penetrability of this important class of anti-HIV agents. In vitro models of the BBB offer a promising alternative to in vivo or in situ animal studies for studying the transport properties of this complex microsystem. However, such models must be sufficiently validated to establish that they truly mimic the in vivo BBB. Endothelial cells isolated from bovine cerebral microvasculature were analyzed for the enzymatic activity of two key purine metabolizing enzymes, purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA). Elevated activities of these enzymes in brain capillary endothelial cells may have important implications in the CNS delivery of a number of purine dideoxynucleosides. In addition, uptake studies into monolayers of primary cultured endothelial cells were utilized to quantify other relevant BBB transport mechanisms (i.e., passive diffusion and carrier-mediation) governing dideoxynucleoside transport as well. It was discovered that isolated bovine brain capillaries contain significantly higher activities of PNP and ADA, on a per mg protein basis, than the brain parenchyma itself reinforcing the concept of an enzymatic BBB. Quantitative results obtained from the monolayer uptake kinetic model strongly suggested that PNP mediated metabolism of 2',3'- dideoxyinosine (ddI), a model dideoxynucleoside, can serve as an enzymatic BBB. Moreover, these studies revealed for the first time in vitro, the presence of carrier-mediation in ddI transport across the BBB. Studies such as these also demonstrated the versatility of the monolayer uptake model for probing the relative contributions of passive diffusion, and metabolism in regulating the BBB transport and bioconversion of 2'-?-fluoro-2',3'-dideoxyadenosine (F-ddA), a prototypical ADA-activated prodrug of 2'-?-fluoro-2',3'-dideoxyinosine (F-ddI). Consistent with in vivo observations, simulations based on in vitro parameter estimates derived in these studies have confirmed that ADA in the capillary endothelial cells of the BBB may be exploited in the design of prodrugs with improved delivery to the brain due to their selective bioconversion in the BBB.