TY - JOUR T1 - In Vitro Methods for Estimating Unbound Drug Concentrations in the Brain Interstitial and Intracellular Fluids JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1711 LP - 1719 DO - 10.1124/dmd.107.015222 VL - 35 IS - 9 AU - Markus Fridén AU - Anubha Gupta AU - Madeleine Antonsson AU - Ulf Bredberg AU - Margareta Hammarlund-Udenaes Y1 - 2007/09/01 UR - http://dmd.aspetjournals.org/content/35/9/1711.abstract N2 - Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unheplful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (Vu,brain). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with Vu,brain microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the Vu,brain of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug. The American Society for Pharmacology and Experimental Therapeutics ER -