Lenka Michalcová

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†

The binding ability of a drug to plasma proteins influences the pharmacokinetics of a drug. As a result, it is a very important issue in new drug development. In this study, affinity capillary electrophoresis, capillary electrophoresis with frontal analysis, and Hummel Dreyer methods with internal and external calibration were used to study the affinity between bovine serum albumin and salicylic acid. The binding constant was measured by all these approaches including the equilibrium dialysis, which is considered to be a reference method. The comparison of results and other considerations showed the best electrophoretic approach to be capillary electrophoresis-frontal analysis, which is characterized by the high sample throughput with the possibility of automation, very small quantities of biomacromolecules, simplicity, and a short analysis time. The mechanism of complex formation was then examined by capillary electrophoresis with frontal analysis. The binding parameters were determined and the corresponding thermodynamic parameters such as Gibbs free energy ΔG(0), enthalpy ΔH(0), and entropy changes ΔS(0) at various temperatures were calculated. The results showed that the binding of bovine serum albumin and salicylic acid was spontaneous, and that hydrogen bonding and van der Waals forces played a major role in the formation of the complex.

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis-frontal analysis.

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the bodys systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis-frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis-frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.

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

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.

Implantation of posterior chamber phakic intraocular lens for myopia and hyperopia - long-term clinical outcomes

PURPOSE To evaluate long-term refractive outcomes of implantable collamer lens (ICL) implantation and late postoperative complications. METHODS We assessed outcomes of patients who underwent ICL implantation (type ICM V4 for myopia, ICH V3 for hyperopia, TICM V4 for astigmatism) in our department between 1998 and 2013. It comprised 62 eyes (40 myopic and 22 hyperopic). The average follow-up period was 10.5 years. We evaluated: uncorrected and best-corrected visual acuity (UCVA and BCVA), spherical equivalent (SE), ICL vault, endothelial cell density and late postoperative complications. RESULTS In myopes, the average UCVA was 1.0±0.37 and BCVA 1.18±0.38, in hyperopes 0.78±0.19 and 1.14±0.18, respectively. The average SE in myopes, whose target refraction was emmetropia, was -0.6±0.83 Dsf, in hyperopes +0.73±0.93. Central ICL vault was 206.16μm±105.94, (range 10-427) in myopes, 195.5μm±109.09, (range 20-404) in hyperopes. The most common late postoperative complication was cataract formation. Three myopic eyes (7.5%) developed symptomatic anterior subcapsular opacities with loss of at least two lines of BCVA. Cataract significantly affecting visual acuity occurred in 5 myopic eyes (12.5%) and 2 hyperopic eyes (9.09%). In these eyes, ICL removal and cataract surgery was performed. CONCLUSIONS In our experience, ICL implantation in moderate and high ametropia was effective and relatively safe. The most common late complication was cataract formation. This complication can be managed effectively surgically with good refractive outcomes without loss of BCVA.

MicroScale thermophoretic investigation of deferiprone interaction with selected biometals

MicroScale Thermophoresis (MST) method has been developed and applied for investigating the interaction of deferiprone (iron-chelator) with different biometals.The experiments were performed on Monolith NT. LabelFree MicroScale Thermophoresis system. Pretest scanning indicated good fluorescence intensity of deferiprone proper for label free experiments. Different concentrations of the intended metals in the range of 0.048 - 500.0 µM were titrated against 100 µM fixed concentration of deferiprone dissolved in 0.1 M Tris buffer at pH 7.4. The results indicated significant interactions of deferiprone with Cu2+, Zn2+, Ni2+, Co2+ and Fe3+. The data fitted to Hill equation indicates more than 1:1 stoichiometry. No binding of deferiprone with Mg2+, Ca2+ and Mn2+ were observed. The technique shows a fast and simple approach to study the binding of deferiprone to different biometals.