Development and characterization of an open tubular column containing immobilized P-glycoprotein for rapid on-line screening for P-glycoprotein substrates

Abstract

Cellular membranes from a cell line expressing P-glycoprotein (Pgp(+)) and from a cell line that does not express Pgp (Pgp(−)) were immobilized on the surface of glass capillaries ( 25 cm×100 μm i.d.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns, Pgp(+)-OT and Pgp(−)-OT. Frontal displacement chromatography on the Pgp(+)-OT demonstrated that the immobilized Pgp retained its ability to specifically bind the known Pgp substrates vinblastin and ketoconazole. The calculated affinities, expressed as K d, for vinblastin and ketoconazole were 97 nM and 12.1 μM, which were comparable with previously reported K d values of 37 nM and 8.6 μM, respectively. The results confirm that the Pgp(+)-OT can be used to quantitatively estimate binding affinities for the Pgp. Frontal displacement chromatography on the Pgp(−)-OT demonstrated that the immobilized membranes retained the ability to bind some Pgp substrates, but that the binding was not due to specific binding to Pgp. A cohort of compounds containing high affinity Pgp substrates (vinblastin, prazosin) and moderate-low affinity Pgp substrates (doxorubicin, verapamil, ketoconazole) and a non-substrate (nicotine) were chromatographed on the Pgp(+)-OT and Pgp(−)-OT using fast frontal analysis and mass spectrometric detection. The results demonstrated that when the retention on the Pgp(+)-OT was corrected by subtraction of the retention on the Pgp(−)-OT, the test compounds could be accurately sorted into high, moderate-low and non-substrate categories. The data from the study indicates that a single 30-min parallel chromatographic experiment can be used to rank a compound based upon its relative affinity for the immobilized Pgp.