Interaction of nonpeptidic δ agonists with P-glycoprotein by in situ mouse brain perfusion: Liquid chromatography–mass spectrometry analysis and internal standard strategy

Abstract Many opioids are substrates of the efflux transporter P ‐glycoprotein (P‐gp) in the blood–brain barrier (BBB). In situ brain perfusion in wild‐type and mdr 1a (−/−) P‐gp‐deficient mice was utilized to investigate potential P‐gp‐mediated transport of novel nonpeptidic δ agonists (AR‐M δ compounds). Because radioactive compounds were not available for this series, liquid chromatography–mass spectrometric detection (LC–MS) was the assay methodology of choice. Verapamil in the perfusion buffer (0.5 μM) served as a positive control for P‐gp‐mediated efflux and as an experimental internal standard for P‐gp modulation by AR‐M δ compounds. LC–MS provided excellent assay sensitivity with no significant interferences. In P‐gp‐competent mice, the brain extraction of AR‐M δ compounds ranged from 1.1 to 96%. The ratio of initial brain uptake clearances ( Cl up ) in P‐gp‐deficient and wild‐type mice (P‐gp effect) ranged from 0.96 to 4.91. Some compounds increased the Cl up of verapamil in P‐gp‐competent mice, consistent with P‐gp inhibition. These results demonstrate that LC–MS is an appropriate assay methodology for mouse brain perfusion samples, that AR‐M δ compounds may interact with P‐gp in the BBB, and that the internal strategy can provide useful information concerning P‐gp modulation by compounds of interest. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:244–252, 2002