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

High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein's non-polar core. This partitioning was described by overall affinity constants of 1.2 (±0.3) × 10(6) M(-1) for R-propranolol and 2.4 (±0.6) × 10(6) M(-1) for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (±2.3) × 10(4) M(-1) for R-propranolol and 9.6 (±2.2) × 10(4) M(-1) for S-propranolol. Comparable results were obtained at 20 and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes.

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography.

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK(a)) of 1.9 (±0.1) × 10(5) M(-1) for R-propranolol and 2.7 (±0.2) × 10(5) M(-1) for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K(a)) of 5.2 (±2.3) × 10(5) M(-1) at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.

Analysis of Drug Interactions with Lipoproteins by High-Performance Affinity Chromatography.

Lipoproteins such as high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are known to interact with drugs and other solutes in blood. These interactions have been examined in the past by methods such as equilibrium dialysis and capillary electrophoresis. This chapter describes an alternative approach that has recently been developed for examining these interactions by using high-performance affinity chromatography. In this method, lipoproteins are covalently immobilized to a solid support and used within a column as a stationary phase for binding studies. This approach allows the same lipoprotein preparation to be used for a large number of binding studies, leading to precise estimates of binding parameters. This chapter will discuss how this technique can be applied to the identification of interaction models and be used to differentiate between systems that have interactions based on partitioning, adsorption or mixed-mode interactions. It is also shown how this approach can then be used for the measurement of binding parameters for HDL and LDL with drugs. Examples of these studies are provided, with particular attention being given to the use of frontal analysis to examine the interactions of R- and S-propranolol with HDL and LDL. The advantages and possible limitations of this method are described. The extension of this approach to other types of drug-lipoprotein interactions is also considered.

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

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.

Characterization of drug-protein interactions in blood using high-performance affinity chromatography.

The binding of drugs with proteins in blood, serum, or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug-protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including HSA and alpha(1)-acid glycoprotein (AGP). Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug-protein binding will be discussed.