Translational Evaluation of In Vitro-to-In Vivo Extrapolation Techniques for Brain Penetration Using Open Flow...

There is a growing need to leverage mathematical models of the central nervous system (CNS) with translational approaches employing in vitro-in vivo extrapolation (IVIVE) approaches. As a benchmark of brain penetration, unbound brain partitioning (K_p,uu), and subsequently a combination of cerebral Open Flow Microperfusion (cOFM) and microdialysis (MD) were evaluated in rats. Additionally, a six-compartment physiologically-based pharmacokinetic (PBPK) model of the CNS was developed in rats using six drugs with varying affinities to the P-glycoprotein (P-gp) transporter to learn and verify the in vivo studies. Active and passive drug efflux and permeability parameters derived from two P-gp overexpressing cell lines (MDCK-I and LLC-PK₁) were scaled using proteomics-informed methods. Model predictions were evaluated with data obtained from terminal brain-to-plasma partition coefficient from both rat cOFM and MD experiments. In correcting for loss of drug in both the cOFM and MD equipment, a relative recovery (RR) showed significantly higher recovery in cOFM compared to MD experiments; for phenytoin, quinidine, and verapamil these were 0.53/0.063, 0.73/0.46, and 0.16/0.04 (cOFM/MD), respectively. Notably, higher P-gp efflux obtained from experiments with MDCK-I-transfected cells resulted in simulated lower drug concentrations in brain compartments. Overall, the CNS-IVIVE-PBPK model tended to overpredict brain drug concentrations, especially using data obtained from transfected LLC-PK₁ cells. However, there is better agreement between the model predictions and the observed data for whole-brain concentrations obtained from terminal studies. We also demonstrated the feasibility of an IVIVE framework that integrates cell line derived passive and active drug parameters to estimate brain concentrations.