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

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<INF>up</INF>) in P-gp-deficient and wild-type mice (P-gp effect) ranged from 0.96 to 4.91. Some compounds increased the Cl<INF>up</INF> 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