Drug delivery to brain is limited by the blood-brain barrier (BBB) (i.e., neurovascular unit) which markedly impacts treatment for many central nervous system (CNS) diseases. Of the various BBB factors that limit brain drug delivery, one of the least well understood is the contribution of plasma protein binding, which involves a complex interplay between brain blood flow, the brain capillary glycocalyx and plasma membrane, and the free and bound drug concentrations in the capillary circulation. Most drugs bind significantly to plasma proteins: approximately half bind 90% or more. For some, plasma protein binding dramatically reduces brain uptake and distribution, whereas for others there is little or no effect. The great majority of BBB drug transport studies report greater brain uptake than can be accounted for based upon the free Fraction of drug in plasma and have suggested that special interactions occur in the capillary circulation leading to "enhanced dissociation" in vivo. The net effect is that this has impeded understanding and prediction of CNS drug penetration in drug development and clinical analysis. The primary hypothesis of this grant is that brain uptake for many drugs can be predicted using a modified Crone-Renkin model that incorporates drug dissociation and rebinding to plasma proteins in the brain capillary in addition to free drug uptake and exchange with brain. This hypothesis will be pursued through four specific aims: (1) to confirm using a carefully controlled in situ rat brain perfusion technique that initial, unidirectional drug uptake into brain can be predicted for many drugs with a simple model based upon four readily determined parameters - the arterial input drug concentration, the free fraction (fu) of drug in the arterial input, the apparent BBB permeability-surface area product (PSU) to free (unbound) drug, and the flow rate of fluid through the brain vasculature (F);(2) to evaluate the rates of drug dissociation and rebinding to plasma protein and their influence on initial drug uptake into brain for drugs that exhibit high capillary extraction at defined flow rate;(3) to show using the perfusion technique that plasma protein binding directly affects the steady state distribution of drug in brain;and (4) to validate using the Nagase albumin knockout rat that this same relationship holds in vivo and that brain drug concentration at steady state is driven ultimately by the plasma free drug concentration and the brain drug distribution volume. This research will provide a novel mechanism to distinguish drugs that exhibit restrictive vs nonrestrictive plasma protein binding effects on brain uptake, will provide a rational means upon which to base CNS drug-dosing for agents that bind significantly to plasma proteins, and will assist in selection of agents with optimal brain delivery in CNS drug development. The critical importance of this work to public health is that it will improve our ability to readily predict and identify new drug agents that cross the BBB for the treatment of CNS diseases.