CHROMATOGRAPHIC MEASUREMENT OF DRUG-PROTEIN INTERACTION - DETERMINATION OF HIV PROTEASE INHIBITOR-SERUM ALBUMIN ASSOCIATION

A chromatographic method for evaluation of the serum protein binding o f a large number of non-peptide human immunodeficiency virus (HIV) pro tease inhibitors in short analysis time and automated fashion was deve loped. The method utilizes a size exclusion HPLC column. Bovine or hum an serum albumin is added to the mobile-phase running buffer, Qualitat ively, a shift to shorter drug retention time in the presence of prote in in the mobile phase is indicative of binding interaction of the dru g and protein. The extent of binding of the drug to the protein is qua ntitated by comparison of the shift in retention time in the presence of protein to the retention time of the drug in the same buffer in the absence of protein (i.e., the drug's ''intrinsic'' retention time on the column), Binding measurements were carried out at 37 degrees C wit h a temperature-controlled autosampler and column oven. Results were c ompared with those obtained by ultrafiltration. The method yields ther modynamically valid binding measurements and is capable of directly de tecting differences in the protein binding of individual stereoisomers present in mixtures (either enantiomers or diastereomers) without pri or purification of the individual stereoisomers. The method measures o verall binding to albumin and is not binding-site specific. Because of this, quantitative comparison of the extent of albumin binding of dru gs which bind to the same site, different sites, or nonspecifically (i .e., not at discrete sites) is possible. (C) 1996 Academic Press, Inc.