Capillary electrophoresis/frontal analysis versus equilibrium dialysis in dexamethasone sodium phosphate‐serum albumin binding studies

Abstract

Plasma protein binding of drugs may have significant effect on its pharmacodynamic, toxicological and pharmacokinetic properties, since only the free drug can pass across biological membrane and get to its specific site of action. Many drugs show a high affinity to albumin which is the most abundant plasma protein. In the present study capillary electrophoresis in the frontal analysis mode (CE/FA), as promising technique for assessment of drug-protein interaction was used. The free drug concentration was measured from height of the frontal peak and calculated based on the external drug standard in absence of protein. With a known concentration of total drug, the percentage of protein bound drug was determined. The binding parameters were also estimated based on the equilibrium dialysis experiment which is considered to be a reference method. This study was designed to examine the interaction of dexamethasone sodium phosphate (DXM) with BSA and HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate buffer, I = 0.17). Using fixed, at physiological level, HSA and BSA concentrations and increasing DXM concentrations, the number of binding sites (n) and binding constant (K(a) ) was calculated from both nonlinear regression fitting and Scatchard Plot. Despite some differences, it can be concluded that the CE/FA is comparable with equilibrium dialysis, but since the first one offers advantages such as low sample consumption, short analysis time, and high separation efficiency, it can be used in high-throughput screening of drug protein binding at the early stage of drug discovery. Interspecies differences in binding of a drug to albumins have been observed and it should be taken into account in interpretation of the results.

Comparison of various capillary electrophoretic approaches for the study of drug–protein interaction with emphasis on minimal consumption of protein sample and possibility of automation†

“…Small differences although still insignificant according the accomplished t ‐test could be given by volume shift associated with the presence of proteins, nonspecific adsorption of compounds on the cell wall and dialysis membrane, and the Donnan effect . Similar results especially in the case of number of binding sites were obtained by Gonciarz et al in their studies of dexamethasone‐serum albumin's interactions performed both by CE–FA and ED .…”

Optimising the controlled release of dexamethasone from a new generation of PLGA-based microspheres intended for intravitreal administration

“…Interaction between drugs and plasma proteins is a rapid process (Schmidt 2010). Gonciarz A et al (Gonciarz 2012) studied the binding between Dxm and HSA in PBS buffer solution (pH 7.4) using capillary electrophoresis/ frontal analysis. In the light of their results, they concluded that there is a strong interaction between both molecules (88% Dxm union) with an intermediate degree of affinity (Scatchard equilibrium association constant Ka (M -1 ) 1.08 x 10 4 ).…”

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis

“…As can be seen from Table , the CE‑FA based method is the best candidate especially in early screening stage of drug development, having advantages such as low sample consumption, automation without necessity of special equipment and the possibility of studying both low‐ and high‐affinity interactions. However protein adsorption onto the capillary wall may occur during CE‐FA analyses, and so a thorough rinsing procedure must be applied between the analyses for optimal robustness, especially if a bare capillary is used . Thermodynamics parameters can be acquired by the ITC‐based method, on the other hand large sample consumption, a requirement for purified samples and problem with estimating very high and very low affinity processes are its limitations .…”

“…ED is considered a reference method for such studies, but it suffers from drawbacks, such as long equilibrium time, volume shifts associated with oncotic pressure and nonspecific adsorption. Also the Donnan effect may occur for charged proteins and skew the measurements . ITC, CD and ED instrumentations can be supplemented with the special autosamplers for automation but their using is usually associated with the substantial sample consumption increasing.…”