Analysis of drug interactions with high-density lipoprotein by high-performance affinity chromatography

Abstract

Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography (HPAC). This approach was evaluated by using it to examine the binding of high-density lipoprotein (HDL) to the drugs propranolol and verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPAC columns with reproducible binding to propranolol over 4-5days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interaction were occurring between R- or S-propranolol and HDL at 37 degrees C: saturable binding with an association equilibrium constant (K(a)) of 1.1-1.9x10(5)M(-1) and nonsaturable binding with an overall affinity constant (n K(a)) of 3.7-4.1x10(4)M(-1). Similar results were found at 4 and 27 degrees C. Verapamil also gave similar behavior, with a K(a) of 6.0x10(4) M(-1) at 37 degrees C for the saturable sites and an n K(a) for the nonsaturable sites of 2.5x10(4)M(-1). These measured affinities gave good agreement with solution phase values. The results indicated that HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body.