Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions

CHROMATOGRAPHIC ANALYSIS OF ACETOHEXAMIDE BINDING TO GLYCATED HUMAN SERUM ALBUMIN

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used highperformance affinity chromatography to examine the changes in acetohexamide-HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (Ka) for the high affinity interactions was in the range of 1.2–2.0 × 105 M−1 and increased in moving from normal to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The Ka for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the Ka for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA

Chromatographic analysis of drug interactions in the serum proteome

The binding of drugs with serum proteins and binding agents such as human serum albumin, a1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drugprotein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems