Received for publication February 20, 2007.
Revised June 15, 2007.
Accepted for publication June 18, 2007.

In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids

Abstract

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 brain has been a common but unfortunate 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 (V_u,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 V_u,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 V_u,brain of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this paper will 1) allow quantification of active transport at the BBB in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on CNS targets, and 3) provide information on intracellular concentrations of unbound drug.

Key words: active transport, blood-CNS transport, CNS pharmacokinetics, drug discovery, drug distribution