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 (K-d 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 K-d values obtained on the PGP-IAM for cyclosporin A and verapamil were 492+/-21 and 172+/-29 mu M, respectively. These results were higher than the corresponding K-d 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. (C) 2000 Elsevier Science BN. All rights reserved.