Abstract
The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain; ml·g brain-1). Vu,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (Kp,uu,cell) and the unbound brain-to-plasma concentration ratio (Kp,uu,brain) is discussed.
Highlights
It is generally accepted that the cerebral concentration of unbound drug is the main pharmacokinetic determinant of Central nervous system (CNS) activity for neurotherapeutics [1,2,3]
Preliminary assessment of the clinically relevant pharmacokinetic parameters required for approximation of the unbounddrug concentration in the brain interstitial fluid is pivotal in guiding early drug discovery research [4]
The reasonable correspondence between the cerebral microdialysis and brain slice method results in this study indicates that the brain slice method is the choice of preference [17]
Summary
5 h incubation-equilibration period and mixed with 200 μl blank aECF. For evaluation of the effects of changes in viability of the brain slices during the incubation, aECF samples can be taken at different time points (after 1, 2, 3, etc. hours). For evaluation of the effects of changes in viability of the brain slices during the incubation, aECF samples can be taken at different time points Once the absorbance of the control and experimental samples has been obtained (Table 3) the relevant viability (%) of the brain slices can be calculated according to Equations 1 and 2: Cytotoxicityð%Þ. Viability values lower than 50% after the 5 h incubation period are associated with dramatic changes in the estimation of Vu,brain, especially for weak bases and results from the experiments should be discarded. The concentration in each brain slice sample is multiplied by 10 to account for the dilution during preparation of the homogenate. The concentration in aECF is multiplied by 2 to account for the dilution during 1:1 mixing of the aECF sample with blank brain homogenate (in 4 volumes (w/v) of aECF).
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