AbstractThe permeation of drugs across the blood-brain barrier (BBB) is a prerequisite for central nervous system (CNS) drug penetration. The BBB, possessing efflux transporters and tight junctions, limits drug penetration to the brain. Consequently, the discovery of novel drugs to treat CNS diseases remains problematic and is lagging behind other therapeutic areas. In vitro assays have progressed understanding of the factors that govern brain penetration. Central nervous system drug penetration is now thought to be modulated by three main processes, namely BBB permeability, active transport at the BBB and drug binding in blood and brain tissue. A more integrated approach to CNS drug discovery programmes is emerging which encompasses these processes in order to examine the rate and extent of drug brain penetration across species and improve predictions in human.A primary porcine in vitro BBB model was developed and characterised for the prediction of CNS drug permeability in vivo. Characterisation confirmed that the model exhibited physiologically realistic cell architecture, the formation of tight junction protein complexes, transcellular electrical resistance consistently >2000 Ω.cm2, functional expression the P-gp efflux transporter and γ-glutamyl transpeptidase and alkaline phosphatase activities.Transport of 12 centrally acting test drugs was investigated across four in vitro BBB models in order make comparisons between models and to generate in vitro permeability and efflux measurements. Blood-brain barrier permeability and active efflux processes are two major influences on the rate of drug penetration across the BBB. Species differences in fublood and fubrain, two prime influences on the extent of drug penetration, were investigated using equilibrium dialysis. Fraction unbound in brain was shown to be comparable across species suggesting that species differences in brain penetration could be due to variation in fublood for drugs that cross the BBB by passive diffusion, and/or species differences in transporter characteristics for drugs that are subject to active transport processes at the BBB. An in-house hybrid-PBPK rat CNS model was used to predict calculated rat Kp,uu using in vitro permeability, efflux, fublood and fubrain parameters generated during this work. The predicted Kp,uu generated using the rat CNS hybrid-PBPK model were within 3-fold of calculated Kp,uu. The rat CNS hybrid-PBPK model has potential use, as a tool for drug discovery scientists to aid the prediction of the extent of drug penetration in the early stages of drug discovery.This work has demonstrated that in vitro permeability and unbound drug fraction can be used to predict CNS drug penetration.