Nicole Thuaud

Separation procedures used to reveal and follow drug-protein binding

The review gives a critical evaluation of the different separation procedures used to study drug-protein interactions and describes their various fields of application. For pharmacological studies, the most widely used methods are dialysis and ultrafiltration, because they allow measurements with solutions of high protein concentrations, such as those found in therapeutic conditions. Both techniques use membrane devices, which may induce additional binding effects. Another drawback of these techniques is the need for radiolabelled compounds. Chromatographic methods, which now take advantage of the technology of high-performance liquid chromatography, are generally faster and do not use drug labelling because of the higher sensitivities of the detectors. Two different approaches are possible: either all the interacting species (protein and drug) are dissolved in the mobile phase, or one of them (protein or drug) is immobilized on the support. Several chromatographic methods are available for studies in solution that differ according to the sample injection mode (frontal or zonal elution) and the nature of the mobile phase used. They include quantitation of the drug-protein complex by zonal elution, the Hummel and Dreyer method, frontal elution, the vacancy peak method, and retention analysis by zonal elution. Frontal elution is the most rigorous method since all the species at equilibrium are present in the mobile phase with known and constant concentrations. The most promising one is the Hummel and Dreyer method, because of the very small amount of protein injected in the mobile phase containing the drug. Drug-protein interactions may be studied by affinity chromatography by immobilizing one of the interacting species on the support. Comparison of the constants obtained with methods when both the drug and the protein are in solution is questionable, since the immobilized species in affinity separations differ in their physical properties from those in solution. The main advantage with studies on immobilized proteins is the easy comparison of the binding properties of various drugs, especially when they are enantiomeric. The results of the binding constants measured by different separation methods are given for the albumin-phenylbutazone and albumin-warfarin systems. Good agreement is generally obtained, which proves the validity of using chromatography as a tool to study drug-protein interactions.

Study of binding of low-molecular-weight lignd to biological macromolecules by high-performance liquid chromatography : Evaluation of binding parameters for two drugs bound to human serum albumin

Abstract The binding to a biological macromolecule (human serum albumin, HSA) of small molecules (two drugs: warfarin and furosemide) has been studied by hig-performance liquid chromatography. Two methods have been used and compared: frontal analysis and the Hummel and Dreyer method. The association parameters of each of the two drugs on HSA were determined. The results obtained are in good agreement with those previously published using the techniques. The competition of these two drugs for the same site on HSA has also been shown.

Equilibrium saturation chromatographic method for studying the binding of ligands to human serum albumin by high-performance liquid chromatography. Influence of fatty acids and sodium dodecyl sulphate on warfarin-human serum albumin binding.

A size exclusion chromatographic method for studying ligand-macromolecule binding parameters is described. This equilibrium saturation method allows the determination of the concentrations of constituents in equilibrium and is specially useful for characterizing ligand--protein binding under conditions that can be compared with physiological conditions. The method has been used for measuring warfarin--human serum albumin (HSA) binding and for studying the influence of free fatty acids (FFA) and sodium dodecyl sulphate on warfarin--HSA binding. Some comparisons with the Hummel and Dreyer method are given. The influence of the FFA is strongly dependent on their chain length, with an inversion of the effect for a 10-carbon chain.

Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids

Abstract Warfarin enantiomerrs that have previously been reported to be difficult to separate on cyclodextrin bonded high-performanceliquid chromatographic supports can be easily and completely resolved on a stationary phase obtained by deposition on silica of an epichlorohydrin-β-cyclodextrin polymer derivative. The separation of other hydroxy-coumarin analogues and Dns-amino acids is also demonstrated. Studying the influence of the pH and methanol content of the aqueous mobile phase allows the conditions required to separate these compounds to be optimized.

Determination of association constants of β-cyclodextrin and β-cyclodextrin-bearing polymers with drugs by high-performance liquid chromatography

Abstract A high-performance liquid chromatographic (HPLC) support was prepared, based on silica beads coated with a β-cyclodextrin-containing polymer, which allows the elution of solutes in order of their affinity for β-cyclodextrin. Binding constants were determined from the retention data for drugs eluted with pure buffer on the new support and good agreement was observed with results obtained by the Hummel and Dreyer method, previously used in HPLC studies of drug—protein interactions.

Enantiomer separations with chromatographic supports based on β-cyclodextrin polymers immobilized on porous silica. Role of the polymer structure in separating ability

SummaryTwo β-cyclodextrin (β-CD)-containing polymers have been prepared either by condensation of β-CD molecules with a bifunctional reagent or by grafting a β-CD derivative on to a linear polymer (polyvinylimidazole). HPLC stationary phases were obtained by adsorption of the β-CD polymers on to silica. The ability of these chromatographic supports to resolve racemic mixtures of organic compounds such as amino acid derivatives, phenylhydantoins, barbiturates, and hydroxycoumarin derivatives has been investigated. Results were found to depend on the chemical structure of the β-CD polymers

Retention data methods for the determination of drug-protein binding parameters by high-performance liquid chromatography

The binding to human serum albumin of some drugs (warfarin, furosemide and phenylbutazone) has been studied by high-performance liquid chromatography. Two methods have been used and compared, based on the measurement of the ligand retention volume under different conditions. The obtained total affinities of the drugs for the protein are in accordance with our previous results. The equilibrium saturation method leads easily to ni and Ki parameters from the retention volume of the ligand.

Determination of Tryptophan-Human Serum Albumin Binding from Retention data and Separation of Tryptophan Enantiomer by High Performance Liquid Chromatography

Abstract The retention volume of a ligand, injected onto a size exclusion chromatographic column and eluted by a macromolecular complexing solution, is theoretically analysed for evaluating the binding between the solute and the macromolecule. The binding of D- and L- tryptophan to human serum albumin is given as an example, and the separation of these enantiomers is then achieved

PREPARATION OF CHIRAL STATIONARY PHASES BY GRAFTING β-CD DERIVATIVES ON POLYVINYLIMIDAZOLE COATED SILICA

Abstract Several β-cyclodextrin (β-CD) chiral stationary phases (CSP) for HPLC were prepared, from silica beads coated with polyvinylimidazole (PVI). The functionalization carried out by reaction with 3-chloro-2-hydroxypropyl β-CD, gave rise to supports with varying content of β-CD cavities. The influence of the amount of β-CD and of the average molecular weight of PVI on the resolution of d,l -methionine β-naphthylamide are studied for optimization. Results are compared to those obtained with a stationary phase prepared by coating silica with a β-CD containing PVI preformed polymer.

Determination by high-performance liquid chromatography of the binding properties of charged β-cyclodextrin derivatives with drugs

Study of the complexing behaviour towards basic, acidic and neutral drugs of two β-cyclodextrin derivatives highly soluble in water and bearing either negative or positive electric charges