Chromatographic measurement of drug-protein interaction: determination of HIV protease inhibitor-serum albumin association.
A chromatographic method for evaluation of the serum protein binding of a large number of non-peptide human immunodeficiency virus (HIV) protease inhibitors in short analysis time and automated fashion was developed. The method utilizes a size exclusion HPLC column. Bovine or human serum albumin is added to the mobile-phase running buffer. Qualitatively, a shift to shorter drug retention time in the presence of protein in the mobile phase is indicative of binding interaction of the drug and protein. The extent of binding of the drug to the protein is quantitated 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 a 37 degrees C with a temperature-controlled autosampler and column oven. Results were compared with those obtained by ultrafiltration. The method yields thermodynamically valid binding measurements and is capable of directly detecting differences in the protein binding of individual stereoisomers present in mixtures (either enantiomers or diastereomers) without prior purification of the individual stereoisomers. The method measures overall binding to albumin and is not binding-site specific. Because of this, quantitative comparison of the extent of albumin binding of drugs which bind to the same site, different sites, or nonspecifically (i.e., not at discrete sites) is possible.
Koeplinger
K A
KA
Drug Metabolism Research, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49001, USA.
Zhao
Z
Z
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017320:HIV Protease Inhibitors / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D012709:Serum Albumin; D013237:Stereoisomerism
D017320:HIV Protease Inhibitors; D012709:Serum Albumin