Sample pooling to enhance throughput of brain penetration study

The advent of combinatorial synthesis and high throughput screening in pharmaceutical research has inevitably given rise to a large number of interesting prelead compounds that requires rapid analytical throughput for kinetic characterization. The traditional approach of one-compound-at-a-time bioanalysis has not been able to meet the demand for high productivity of pharmacokinetic screening. This report demonstrates the application of sample pooling in expediting the pharmacokinetic screening, including assessment of brain penetration, of six NK1 receptor antagonists in rats: CAM 6108 (C1), CAM 6122 (C2), CAM 6178 (C3), CAM 5825 (C4), CAM 6182 (C5), and CAM 6121 (C6). The approach was adopted to avoid complications associated with cocktail dosing where multiple compounds are administered to one animal. The present investigation features individualized dosing (one compound per animal), followed by sample pooling of brain and plasma and bioanalysis via a conventional LC-fluorescence method. Rats were dosed intravenously with each of the six NK1 receptor antagonists and blood and brain samples were harvested at suitable post-dose time intervals. Plasma or brain homogenate samples from the same time points were pooled into two groups (C1-C3 and C4-C6) for assay. Drug compounds in plasma or brain were extracted by protein precipitation and quantitated using a validated gradient HPLC/fluorescence method, which was made feasible for both groups of compounds with a modification in gradient scheme. Plasma assay precision and accuracy for C1-C6 were < or =4.7% and within +/-9.8%, respectively. Brain homogenate assay accuracy for C1-C6 was within +/-7.0%. Brain penetration of these compounds was evaluated as the AUC of brain and plasma and their respective brain/plasma AUC ratio. The sample pooling approach helped to quickly identify C1 as the NK1 receptor antagonist with the greatest extent of brain penetration, followed by C2, C6, C4, C5, and C3 in that order. By employing sample pooling approach, pharmacokinetic parameters and brain penetration of all six compounds were obtained in a fraction of the time required by conventional single compound dosing and analysis.