Development of an immobilized P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities.

The membrane transporter P-glycoprotein (PGP) has been immobilized on an immobilized artificial membrane (IAM) LC stationary phase. The resulting PGP-IAM phase retained the ability of the native PGP to bind the known PGP-ligand vinblastine. Displacement studies using other known PGP ligands, verapamil and cyclosporin A, demonstrated that there was selective binding between vinblastine and the immobilized PGP transporter. The binding affinity (Kd value) of vinblastine for the PGP-IAM was determined to be 19+/-20 and 71+/-11 nM on two separate columns. These values are consistent with previously reported values of 9+/-2, 8+/-2, and 37+/-10 nM, which were obtained using native membranes. The Kd values obtained on the PGP-IAM for cyclosporin A and verapamil were 492+/-21 and 172+/-29 microM, respectively. These results were higher than the corresponding Kd values obtained using native membranes, but the relative affinities vinblastine > cyclosporin A >> verapamil are consistent in both approaches. During several months of experiments and storage, the PGP-IAM was found to be reproducible and stable. The stationary phase appears to be useful in the on-line screening for PGP ligands.