Bernard Sebille

Preparation and characterization of water soluble high molecular weight β-cyclodextrin-epichlorohydrin polymers

Abstract Water soluble β-cyclodextrin polymers were prepared from β-cyclodextrin crosslinked with epichlorohydrin under basic conditions. The influence of preparation conditions on the molecular weight distribution has been investigated. The knowledge of this relationship allows for the choice of reaction parameters according to the desired weight distribution of the β-cyclodextrin polymer. A procedure was developed for the preparation of high molecular weight water soluble polymer ( M w higher than 10 4 ).

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.

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.

Evidence for a concentration-dependent polymerization of a commercial human serum albumin

Polymerization of a commercial human serum albumin (Sigma A-1887) was investigated by two different techniques, high-performance liquid chromatography and gel electrophoresis. The chromatographic technique was based on the frontal analysis principle using a column which excludes polymers but retains monomers. The results allowed the determination of the monomer--polymer affinity constant, X = 526 +/- 100. The electrophoresis technique was performed with a polyacrylamide gel containing sodium dodecyl sulphate in order to separate the different polymer species according to their molecular weights. The two techniques gave results in good accordance and showed a concentration-dependent aggregation. The higher the human serum albumin concentration, the more the monomer proportion decreases.

Separation of basic proteins by capillary zone electrophoresis with coatings of a copolymer of vinylpyrrolidone and vinylimidazole

Capillary zone electrophoretic (CZE) separation of basic proteins has been achieved with capillary columns modified with copolymers of vinylpyrrolidone (VP) and vinylimidazole (VI). The copolymerization reaction is performed inside the capillary column and involves chemical bonding of the polymer to silica. The electroosmotic flow (EOF) is greatly decreased by this surface modification. The presence of positive charges on the coating surface, due to the cationic property of vinylimidazole at pH below 7, reduces the adsorption of basic proteins onto the silanol groups of the capillary surface. Acidic proteins are irreversibly adsorbed, but rapid separation and good performance reproducibility are obtained with basic proteins. In the case of capillaries modified with VP, the acidic and basic proteins are eluted within 10 min. In this work, we studied the effects of pH and buffer concentration on the magnitude of the EOF, as well as the effect of copolymer composition on the separation efficiency.

Synthesis of New Thiosulfonates and Disulfides from Sulfonyl Chlorides and Thiols

Abstract Aromatic thiols were converted into thiosulfonates or disulfides, under very mild conditions, with fair to excellent yields, by using sulfonyl chloride as oxydant reagent.