Electrokinetic chromatographic estimation of the enantioselective binding of nomifensine to human serum albumin and total plasma proteins

This report is the first evidence of enantioselective binding of nomifensine to human serum albumin (HSA) and plasma proteins. The overall process with HSA included: (i) consistent experimental design along two independent sessions; (ii) incubation of nomifensine–HSA designed mixtures; (iii) ultrafiltration for separating the unbound enantiomers fraction; (iv) electrokinetic chromatography (EKC) using heptakis‐2,3,6‐tri‐O‐methyl‐β‐cyclodextrin as chiral selector to provide experimental data for enantiomers (first, E1, and second, E2, eluted ones); and (v) a recent direct equation allowing univariate tests and robust statistics to provide consistent parameters and uncertainty. A significant enantioselectivity to HSA (2.7 ± 0.1) was encountered, related to a 1:1 stoichiometry and log affinity constants of 3.24 ± 0.10 and 3.67 ± 0.08 for E1 and E2, respectively. The protein binding (PB) estimated at physiological concentration levels was 40 ± 5 and 63 ± 4% for E1 and E2, respectively. The use of synthetic human sera allowed in vitro estimation of the total plasma PB for the racemate (61 ± 5%; coincident with in vivo values), and its enantiomers (58 ± 7 and 64 ± 4% for E1 and E2, respectively). Comparison allowed the relative importance of HSA respect to other plasma proteins for binding nomifensine to be established. Copyright © 2012 John Wiley & Sons, Ltd.     

Retention of structurally diverse drugs in human serum albumin chromatography and its potential to simulate plasma protein binding

The retention behavior of 39 structurally diverse neutral, basic and acidic drugs was investigated on an HSA stationary phase using PBS buffer (pH 7.0) and acetonitrile or 2-propanol as organic modifiers. Extrapolated or directly measured log k_w values as well as isocratic retention factors were correlated with plasma protein binding data taken from the literature. Retention factors determined in the presence of 10% acetonitrile led to high quality 1:1 correlation with apparent log K_HSA values. The derived reference equation was successfully validated using a secondary set of 24 drugs. Further analysis of HSA retention into more fundamental properties revealed the involvement of anionic species in solute-stationary phase interactions, expressed by the negatively charged fraction, besides the partitioning mechanism which was reflected by lipophilicity. Protonation of basic drugs, although less important, may also influence retention, leading to reduced partitioning into the HSA surface as a net effect, while it seems to have no effect on HSA binding. The above results were further confirmed by linear solvation energy relationships (LSER).     

Determination of L-tryptophan-serum albumin binding by high-performance liquid chromatography

Summary Two high-performance liquid chromatography (HPLC) techniques were developed for the determination of binding constants in the interaction of serum albumin with L-tryptophan: internal calibration and external calibration. The results obtained were compared with those obtained by the classical method of equilibrium dialysis and by gel filtration. While all the methods are equally reliable, the internal and external calibration techniques seem to be superior in their simplicity, speed and convenience.     

Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA–HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β‐cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K₁), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K₁ values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S‐omeprazole, S‐OME) and its R‐enantiomer (R‐omeprazole, R‐OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10³ M⁻¹ and 5.36 × 10³ M⁻¹, respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs.     

Studies on the binding of vinpocetine to human serum albumin by molecular spectroscopy and modeling

The interaction between vinpocetine(VPC) and human serum albumin(HSA) in physiological buffer(pH 7.40) was investigated by fluorescence,FT-IR,UV-vis absorption and molecular modeling.VPC effectively quenched the intrinsic fluorescence of HSA via static quenching.The binding site number n and apparent binding constant K_a,corresponding thermodynamic parametersΔG,ΔH andΔS at different temperatures were calculated.The synchronous fluorescence and FT-IR spectra were used to investigate the structural change of HSA molecules with addition of VPC.Molecular modeling indicated that VPC could bind to the site I of HSA and hydrophobic interaction was the major acting force,which was in agreement with the binding mode study.     

Multivariate optimization approach for chiral resolution of drugs using human serum albumin in affinity electrokinetic chromatography-partial filling technique

The enantiomeric resolution of chiral compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer, protein and compound solutions) and protein solution plug length. In this paper multivariate optimization approaches for chiral separation of four basic drugs (alprenolol, oxprenolol, promethazine and propranolol) using HSA as chiral selector in AEKC-partial filling technique are studied. The experimental conditions to achieve maximum resolution are optimized using the Box-Behnken experimental design. Partial least squares and pareto charts are used to analyse the main effects on the resolution. The experimental resolutions observed for all compounds studied in optimum conditions agree with the estimated values based on response surface models. The results obtained show that the range of experimental conditions that provided enantioresolution narrows as hydrophobicity of analytes decreases. This fact can be explained by assuming that hydrophobicity controls the interaction of basic compounds with HSA.     

Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for enantioresolution of each compound were: brompheniramine, pH 8.5, [HSA] 180 μM, SPL 180 s; chlorcyclizine, pH 6.5, [HSA] 180 μM, SPL 150 s; chlorpheniramine, pH 8.25, [HSA] 160 μM, SPL 150 s; hydroxyzine, pH 7.0, [HSA] 180 μM, SPL 150 s; and orphenadrine, pH 7.8, [HSA] 160 μM, SPL 150 s. pH and the quadratic term of pH seem to be the most critical factors that determine enantioresolution of antihistamines. The validity of the developed methodologies to enantiomeric quality control of antihistamines in pharmaceutical formulations is demonstrated analyzing the content of brompheniramine, chlorpheniramine and hyroxyzine enantiomers in commercially available pharmaceutical formulations containing racemic mixtures of compounds. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make them suitable for quality control of the enantiomeric composition of antihistamines in pharmaceutical preparations.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Spectroscopic studies on binding of Puerarin to human serum albumin

The interaction between Puerarin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Puerarin can strongly quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the binding of Puerarin to HSA changed slightly molecular conformation of HSA. Furthermore, the thermodynamic functions ΔH⁰ and ΔS⁰ for the reaction were calculated to be −9.067 kJ mol⁻¹ and 54.315 J mol⁻¹ K⁻¹ according to van’t Hoff equation. These data suggested that both hydrogen bond and hydrophobic interaction play a major role in the binding of Puerarin to HSA, which is in good agreement with the result of molecular modeling study.     

Prediction of human serum albumin-drug binding affinity without albumin

A new liquid chromatographic system was developed to measure protein-drug binding affinity indirectly without albumin and was evaluated using log nK values of drugs measured by a modified Hummel-Dreyer method using purified human serum albumin. The retention factors of acidic and basic drugs were measured by reversed-phase and ion-exchange liquid chromatography in sodium phosphate buffer, pH 7.40, containing 50 vol.% methanol at 37 °C. The bonded phases were pentyl, guanidino and carboxyl phases. The combined retention factors were correlated with the log nK values measured by a modified Hummel-Dreyer method because glycosylation of human serum albumin did not significantly affect log nK value. The correlation coefficients were 0.949 (n=7) for acidic drugs and 0.978 (n=5) for basic drugs. The log nK values of 26 acidic and 18 basic drugs were predicted from their retention factors measured by reversed-phase and ion-exchange liquid chromatography.     

Studies on the binding of nevadensin to human serum albumin by molecular spectroscopy and modeling

The binding of nevadensin to human serum albumin (HSA) in aqueous solution was investigated for the first time by molecular spectroscopy and modeling at pH 7.4. Spectrophotometric observations are rationalized in terms of a static quenching process and binding constant (K_a, K_b) and the number of binding sites (n ≈ 1) were evaluated by fluorescence quenching methods. Thermodynamic data showed that nevadensin was included in the hydrophobic cavity of HSA mainly via hydrophobic interactions. The value of 3.09 nm for the distance r between the donor (HSA) and acceptor (nevadensin) was derived from the fluorescence resonance energy transfer. Spectrophotometric techniques were also applied to investigate the structural information of HSA molecules on the binding of nevadensin and the results showed that the binding of nevadensin to HSA did not change significantly molecular conformation of HSA in our experimental conditions. Furthermore, the study of molecular modeling also indicated that nevadensin could strongly bind to the site I (subdomain IIA) of HSA mainly by a hydrophobic interaction and there are hydrogen bond interactions between nevadensin and the residues Arg-218, Arg-222, Lys-195, and Asp-451. As compared to the other flavonoids, the flavonoids containing methoxy groups which are in aromatic rings can bind to HSA with higher affinity.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (R _o ) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).     

Fluorescence study on the interaction of human serum albumin with bromsulphalein

The binding of bromsulphalein (BSP) with human serum albumin was investigated at different temperatures, 298 and 308 K, by the fluorescence spectroscopy at pH 7.24. The binding constant was determined by Stern-Volmer equation based on the quenching of the fluorescence HSA in the presence of bromsulphalein. The effect of various metal ions on the binding constants of BSP with HSA was investigated. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: DeltaH and DeltaS possess small negative (9.3 kJ mol(-1)) and positive values (22.3 J K(-l)mol(-l)), respectively. The experimental results revealed that BSP has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The binding constants between BSP to HSA were remarkable and independent on temperature. The binding constants between HSA and BSP decreased in the presence of various ions, commonly decreased by 30-55%. The hydrophobic force played a major role in the interaction of BSP with HSA. All these experimental results and theoretical data clarified that BSP could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design.     

Evaluation of alternatives to warfarin as probes for Sudlow site I of human serum albumin: characterization by high-performance affinity chromatography

Warfarin is often used as a site-specific probe for examining the binding of drugs and other solutes to Sudlow site I of human serum albumin (HSA). However, warfarin has strong binding to HSA and the two chiral forms of warfarin have slightly different binding affinities for this protein. Warfarin also undergoes a slow change in structure when present in common buffers used for binding studies. This report examined the use of four related, achiral compounds (i.e., coumarin, 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, and 4-hydroxycoumarin) as possible alternative probes for Sudlow site I in drug binding studies. High-performance affinity chromatography and immobilized HSA columns were used to compare and evaluate the binding properties of these probe candidates. Binding for each of the tested probe candidates to HSA was found to give a good fit to a two-site model. The first group of sites had moderate-to-high affinities for the probe candidates with association equilibrium constants that ranged from 6.4 x 10(3)M(-1) (coumarin) to 5.5 x 10(4)M(-1) (4-hydroxycoumarin) at pH 7.4 and 37 degrees C. The second group of weaker, and probably non-specific, binding regions, had association equilibrium constants that ranged from 3.8 x 10(1)M(-1) (7-hydroxy-4-methylcoumarin) to 7.3 x 10(2)M(-1) (coumarin). Competition experiments based on zonal elution indicated that all of these probe candidates competed with warfarin at their high affinity regions. Warfarin also showed competition with coumarin, 7-hydroxycoumarin and 7-hydroxy-4-methycoumarin for their weak affinity sites but appeared to not bind and/or compete for all of the weak sites of 4-hydroxycoumarin. It was found from this group that 4-hydroxycoumarin was the best alternative to warfarin for examining the interactions of drugs at Sudlow site I on HSA. These results also provided information on how the major structural components of warfarin contribute to the binding of this drug at Sudlow site I.     

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C₄₄), 83% carbamylated (C₈₃) and 85% acetylated (A₈₅) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C₈₃ and A₈₅ HSAs while C₄₄ HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (K_SV) and bimolecular quenching rate constant (k_q) suggested, NA-HSA complex formation, binding constant (K_a) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the K_a value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.     

Enantioseparation of phenotiazines by affinity electrokinetic chromatography using human serum albumin as chiral selector: application to enantiomeric quality control in pharmaceutical formulations

Nowadays, there is a special interest within the pharmaceutical laboratories to develop single enantiomer formulations and consequently a need for analytical methods to determine the enantiomeric purity of drugs. The present paper deals with the enantiomeric separation of promethazine and trimeprazine enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length, is carried out to obtain enantioresolution of promethazine and trimeprazine. The estimated maximum and optimum resolution of trimeprazine and prometazine enantiomers (Rs = 1.74 and 2.01, respectively) corresponded to the following experimental conditions: pH 7.5; [HSA] 170 μM and plug length 190 s and pH 7.6; [HSA] 170 μM and plug length 170 s, for trimeprazine and prometazine, respectively. The developed methodologies were applied for the enantiomeric quality control of promethazine and trimeprazine enantiomers in commercially available pharmaceutical formulations. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make it suitable for quality control of the enantiomeric composition of promethazine and trimeprazine in pharmaceutical preparations.