Abstract

Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f_u,brain), determined using the brain homogenate method, with the unbound volume of distribution in brain (V_u,brain), determined using the brain slice method. Discrepancies were frequent and primarily related to drug pH partitioning, due to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V_u,brain from measurements of f_u,brain and drug pKa. This model allowed prediction of V_u,brain from f_u,brain within a 2.2-fold error range for 95% of the drugs, as compared to a 4.5-fold error range using the brain homogenate f_u,brain method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin and prototypic organic cation transporter substrates. It is concluded that intra-brain drug distribution is governed by several diverse mechanisms in addition to non-specific binding and that the slice method is therefore more reliable than the homogenate method. Alternatively, predictions of V_u,brain can be made from homogenate f_u,brain using the presented pH partition model, although this model does not take into consideration possible active brain cell uptake.