Sensitivity enhancement of capillary electrophoresis-frontal analysis-based method for characterization of drug-protein interactions using on-line sample preconcentration

Capillary electrophoresis-frontal analysis is one of the most frequently used approaches for the study of plasma protein-drug interactions as a substantial part of new drug development. However, the capillary electrophoresis-frontal analysis typically combined with ultraviolet-visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on-line sample preconcentration. According to the knowledge of the authors this combination has never been used to characterize plasma protein-drug binding. It resulted in a fully automated and versatile methodology for the characterization of binding interactions. Further, the validated method minimalizes the experimental errors due to a reduction in the manipulation of samples. Moreover, employing an on-line preconcentration strategy with capillary electrophoresis-frontal analysis using human serum albumin-salicylic acid as a model system improves the drug concentration sensitivity 17-fold compared to the conventional method. The value of binding constant (1.51 ± 0.63) · 10⁴ L/mol obtained by this new capillary electrophoresis-frontal analysis modification is in agreement with the value (1.13 ± 0.28) ·10⁴ L/mol estimated by a conventional variant of capillary electrophoresis-frontal analysis without the preconcentration step, as well as with literature data obtained using different techniques.     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.     

Interaction of glycyrrhetinic acid, furosemide and hydrochlorothiazide with bovine serum albumin and their displacement interactions: capillary electrophoresis and fluorescence quenching study

Licorice is the most widely used crude drug in traditional Chinese medicine. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizic acid, which is the main bioactive ingredient of licorice. In this work, capillary electrophoresis-frontal analysis (CE-FA) was applied to study the binding of bovine serum albumin with GA and two diuretics: furosemide (FU) and hydrochlorothiazide (HZ). The binding parameters of GA were determined by Scatchard analysis, which showed that there are two kinds of binding sites in bovine serum albumin for GA. However, the results showed that the CE-FA method was not suitable for the interaction study of FU and HZ. Therefore, utracentrifugation-CE was used to probe the binding characteristic of these two drugs and the results showed only one kind of binding site for them under the studied conditions. Displacement interactions between these drugs were also investigated by utracentrifugation-CE method and the results showed that GA hardly displaces HZ while it can slightly displace FU and FU can slightly displace HZ. For comparison, the binding of these drugs was also studied by the fluorescence quenching method and the data were processed by the Stern-Volmer quenching equation. Results showed that the binding constants were basically consistent for two methods for all drugs studied. The number of binding sites on one protein molecule was well consistent for FU and HZ while it was quite different for GA.     

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.     

Analysis of drug interactions with serum proteins and related binding agents by affinity capillary electrophoresis: A review

Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug–protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.     

High-throughput capillary electrophoresis frontal analysis method for the study of drug interactions with human serum albumin at near-physiological conditions

The application of the short-end capillary injection to capillary electrophoresis frontal analysis (CE-FA) to study the interaction between basic, neutral and acid drugs towards human serum albumin (HSA) at near-physiological conditions is presented. The compounds selected display a wide range of binding affinities and the results obtained were in good agreement with those reported in the literature. An equation for the estimation of the number of primary binding sites and their corresponding affinity constants is developed isolating the experimentally measured variables in just one axis. The proposed CE-FA method to screen drug interactions with HSA under physiological conditions is simple, rapid and cost-effective what may facilitate its implementation in the drug discovery process.     

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.     

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.     

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs

In this study, two capillary electrophoresis–based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l -tryptophan (l -TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l -TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.     

Complexation between low-molecular-weight cationic ligands and negatively charged polymers as studied by capillary electrophoresis frontal analysis

The potential of using capillary electrophoresis frontal analysis for the study of low-molecular-weight ligand-polyelectrolyte interactions was assessed. The interaction of the ligands 1-propylpyridinium bromide, 2-propylisochinolinium bromide, and paraquat with the polymer dextran sulfate was investigated as a function of polymer concentration and ionic strength of the buffer solution. Linear binding isotherms were obtained and association constants were determined. The complex formation was independent of the dextran sulfate concentration at low ionic strength. Ligand-polyelectrolyte interactions were strongly dependent on the ionic strength. The interaction of the divalent cation paraquat with the dextran sulfate was much stronger than the interactions of the monovalent cationic ligands with the polyelectrolyte. The binding data obtained were in accord with results obtained by equilibrium dialysis. Capillary electrophoresis frontal analysis has the potential to become a valuable tool for characterization of ligand-polyelectrolyte interactions in drug design as well as in other areas.     

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling

Stereoselective recognition of chiral compounds can be used for mapping of surface interaction sites on proteins. Iron-free human serum transferrin is a suitable chiral selector in capillary electrophoresis used in native form in solution. Separation of optical isomers of tryptophan-methylester, tryptophan-ethylester and tryptophan-butylester and various drugs were studied in capillary zone electrophoresis applying a distinct transferrin zone prior to sample injection. Changes in the electrophoretic patterns (i.e., in the migration properties) of the molecules reflected the possible interactions with the protein. The tryptophan derivatives and eight drugs possessed stereoselective interactions, seven drugs showed interactions without appreciable chiral separation, and the others did not present any direct complexation with the protein molecules. Molecular modelling was performed to characterize the binding areas at the iron binding site of iron-free transferrin. The docking of tryptophan derivatives on transferrin showed that the R-enantiomers possess a stronger complexation with transferrin, whereas the S-enantiomers are bound by weaker interactions, which is in excellent agreement with the capillary electrophoresis results, where the R-enantiomers were always retarded stronger by transferrin. A ranking of drugs by the lipo score parameter of the docking shows an accordance with the stereoselective interactions by the protein.     

Chromatographic and electrophoretic studies of protein binding to chiral solutes

Protein interactions are important in determining the transport, metabolism and/or activity of many chiral compounds within the body. This review examines data that have been obtained on these interactions by various chromatographic and electrophoretic methods, especially those based on either high-performance liquid chromatography or capillary electrophoresis. Zonal elution, frontal analysis and vacancy methods are each considered, as are approaches that employ either soluble or immobilized proteins. There are a variety of different items that can be learned about a solute-protein system through these techniques. This includes information on the binding constants and number of binding sites for a solute-protein system, as well as the thermodynamic parameters, rate constants, interaction forces and binding site structure for the protein and solute. Numerous examples are provided throughout this review, as taken from the literature and from work performed within the author's laboratory.     

Drug-plasma protein binding assay by electrokinetic chromatography-frontal analysis

We developed a rapid, microscale and reliable analytical method for binding of drugs to plasma proteins using capillary electrophoresis (CE) with ionic cyclodextrins (CD) combined with frontal analysis. These CDs were used as pseudostationary phases of electrokinetic chromatography (EKC). The CD-modified EKC (CDEKC) approach allowed us to separate anionic drugs from plasma proteins, whereas CZE could not separate these drugs from plasma proteins because they had a similar mobility like plasma proteins. CDs uniquely interact with these drugs but not with plasma proteins. Therefore, CDEKC could be coupled with frontal analysis to measure the binding of anionic drugs to plasma proteins. The binding values obtained by CDEKC were highly consistent with those determined by the ultrafiltration method. Our CDEKC approach should expand the applicability of CE to protein binding analysis.     

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 body's 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.     

Characterization of antihistamine-human serum protein interactions by capillary electrophoresis

An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this paper the characterization of the interaction between antihistamines (cationic drugs) towards human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. Antihistamines present a wide-ranging behaviour with respect to their affinities towards plasma proteins. Orphenadrine, phenindamine, tripelenamine and tripolidine principally bind to HSA; carbinoxamine, dimetindene and etintidine principally bind to AGP; brompheniramine, chlorpheniramine and ranitidine present an important binding to lipoproteins and/or globulins and finally, chlorcyclizine, cinarizine, cyclizine, doxylamine, hydroxyzine, perphenazine and terfenadine do not bind to lipoproteins and/or globulins but bind to HSA and AGP in different extension. The interaction of antihistamines with HSA is determined by the hydrophobicity (direct relationship) and the polar surface area (indirect relationship) of the compounds. The steric parameters and hydrogen bonding character of compounds seems to be related with the binding of antihistamines to AGP. The antihistamine-HSA affinity constants were evaluated and the K(1) values ranged from 7 x 10(2)M(-1) (for doxylamine) to 4 x 10(4)M(-1) (for phenindamine).     

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.     

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 ΔG⁰, enthalpy ΔH⁰, and entropy changes ΔS⁰ 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.     

Affinity capillary electrophoresis is a powerful tool to identify transthyretin binding drugs for potential therapeutic use in amyloidosis

In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.     

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.