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. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/Spropranolol and HDL at 37°C: saturable binding with an association equilibrium constant (K a ) of 1.1-1.9 × 10 5 M −1 , and non-saturable binding with an overall affinity constant (n K a ) of 3.7-4.1 × 10 4 M −1 . Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a K a of 6.0 × 10 4 M −1 at 37°C for the saturable sites and a n K a value for the non-saturable sites of 2.5 × 10 4 M −1 . These measured affinities gave good agreement with solution-phase values. The results indicated 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.

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

“…Lipoproteins like VLDL are also known to interact with and transport several types of basic or neutral and hydrophobic drugs in the bloodstream [7,23-30]. For instance, it has recently been proposed in work with HDL and LDL that several binding mechanisms, including both saturable interactions with apolipoproteins and non-saturable interactions with the hydrophobic core, may occur between drugs and these lipoproteins [10,14,31]. …”

“…These techniques have included equilibrium dialysis and capillary electrophoresis (CE) using high-performance frontal analysis [14-17]; however, only equilibrium dialysis has been used in studying drug interactions with VLDL [14]. High-performance affinity chromatography (HPAC) is an alternative to these techniques and has been used in various studies to examine the interactions of drugs with proteins and other binding agents [3-6,10,31-38]. …”

“…Evaluation of the subsequent elution profiles can provide data on the equilibrium constants, binding mechanism and number of interaction sites that are present between the drug and immobilized agent. Advantages of this approach include its speed, precision, ease of automation, and good correlation versus solution-phase results and reference methods [3-6,10,31-38]. …”

“…HPAC has been utilized in previous reports to examine the binding of drugs such as propranolol with HDL and LDL [10,31]. The use of HPAC to perform binding studies of drugs with HDL and LDL has led to the confirmation of a non-saturable binding mechanism, as has been proposed in work with other techniques [14-17], as well as the identification of high affinity binding between propranolol and some apolipoproteins [10,31].…”

“…The use of HPAC to perform binding studies of drugs with HDL and LDL has led to the confirmation of a non-saturable binding mechanism, as has been proposed in work with other techniques [14-17], as well as the identification of high affinity binding between propranolol and some apolipoproteins [10,31]. In the case of LDL, this latter process has been found to be stereoselective, with the R- enantiomer but not the S -enantiomer of propranolol undergoing this type of interaction [10].…”