Enantiomeric separation of basic drugs with partially filled serum albumin as chiral selector in capillary electrophoresis.

A reliable method is presented for the chiral separation of three basic drugs (mexiletine, chlorpheniramine and propranolol) with serum albumins (human and porcine, HSA and PSA) as chiral selectors by capillary electrophoresis in combination with the partial filling technique. Based on the systematic optimization of operation variables, the chiral separation of mexiletine, chlorpheniramine and propranolol was achieved in the pH 7.4 phosphate buffer by using HSA, PSA and PSA as selectors, respectively. The chiral recognition ability of HSA and PSA was compared. HSA and PSA show a different chiral recognition ability for each of the three drugs. In addition, the association constants between enantiomeric drugs and proteins were determined to be 2.00 and 3.80 x 10(2) M(-1) for mexiletine and HSA, 0.59 and 1.12 x 10(3) M(-1) for chlorpheniramine and PSA, and 0.87 and 1.42 x 10(3) M(-1) for propranolol and PSA. The method for the chiral separation and determination of association constants possesses the advantages of simple performance, effective avoiding of the interference of the UV detection from protein, and lowering of the reagent consumption.     

Evaluation of enantioselective binding of antihistamines to human serum albumin by ACE

The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with alpha(1)-acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein.     

Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector

The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.     

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.     

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.     

Potential of human serum albumin as chiral selector of basic drugs in affinity electrokinetic chromatography-partial filling technique

The enantiomeric resolution of 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 (BGE), protein, and compound solutions), and plug length. In this paper, the possibility of using HSA as chiral selector for enantioseparation of 28 basic drugs using this methodology is studied. The effect of the physicochemical parameters, the structural properties of compounds, and compound-HSA protein binding percentages over their chiral resolution with HSA is outlined. Based on the results obtained, a decision tree is proposed for the "a priori" prediction of the capability of HSA for enantioseparation of basic drugs in AEKC. The results obtained indicated that enantioresolution of basic compounds with HSA depends on the hydrophobicity, polarity, and molar volume of compounds.     

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.     

Analysis of conformational change of human serum albumin using chiral capillary electrophoresis

The conformational change of human serum albumin (HSA) was studied through its binding with basic drug-mexiletine by chiral capillary electrophoresis. The effects of the conformational change of HSA resulted from pH, thermal, acute vibration, and alcohol on its chiral selectivity to mexiletine were investigated in detail. This study offers a simple and complementary method to investigate the binding of proteins with drugs and the characteristic of conformational change of protein. The method is easy to perform, high speed, low reagent consumption, and no modification is required to the commercially available CE instrument.     

人血清白蛋白柱上药物的手性拆分

考察了４种酸性药物和１种中性药物对映体在人血清白蛋白手性固定相上的保留行为。这５种药物与人血清白蛋白结合的亲和力高，难于实现快速分离，作者提出在流动相中加入短链脂肪酸－正己酸，可快速手性拆分非诺洛芬、萘普生和布洛芬。酮基布洛芬对映体分离选择性随乙腈浓度升高而增大，流动相中加入适量异丙醇可使对映体选择性大大增加（α～１．２３），华法令同样可取得很好分离。     

Development of a disulfiram-modified human serum albumin-based HPLC column

Summary A human serum albumin (HSA)-based HPLC column has been modified in situ by disulfiram, an alcohol-deterrent drug reported to bind cys₃₄, the only free cysteine in HSA, under physiological conditions. The reversible and covalent binding of disulfiram was found to change the binding properties of the protein, giving rise to a new selector which performed differently from the native albumin-based stationary phase. When low concentrations of disulfiram were used as mobile phase modifier, reversible binding resulted in a cooperative allosteric effect with improved selector performance. Covalent modification resulted in markedly reduced affinity for binding of the drugs to sites I or II, while still maintaining enantioselectivity. This study has enabled the monitoring of interactions of disulfiram with potentially coadministered drugs, and the preparation of a chiral selector with different drug affinity and enantioselectivity.     

On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach

In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-β-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.     

Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF₆)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF₆ which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2 × 10⁵ ± 0.3 L mol⁻¹ and 0.8 × 10⁴ ± 0.4 L mol⁻¹ for l- and d-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers–HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that l-fenoprofen replaced d-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.     

Evaluation of enantioselective binding of basic drugs to plasma by ACE

The present paper deals with the evaluation of the stereoselective binding of antihistamines (brompheniramine, chlorpheniramine, hydroxyzine, orphenadrine and phenindamine), phenothiazines (promethazine and trimeprazine) and a local anesthetic (bupivacaine) to human plasma proteins. Since all of them are drugs highly bound to proteins, a methodology to determine the bound fraction of each drug enantiomer was proposed. This methodology includes the incubation of samples containing plasma and racemic drug, ultrafiltration of the mixture and the chiral separation of enantiomers in the bound drug fraction using affinity EKC (AEKC)-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of some antihistamines such as brompheniramine, hydroxyzine, orphenadrine and phenindamine and the phenothiazines, promethazine and trimeprazine, to human plasma proteins. The binding of phenindamine to plasma presented the highest enantioselectivity (ES) (ES = 2.5) followed by trimeprazine (ES = 1.5) and promethazine (ES = 1.4).     

Characterization of interaction kinetics between chiral solutes and human serum albumin by using high-performance affinity chromatography and peak profiling

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.     

Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives

A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes.     

Stereochemical resolution of enantiomeric 2-aryl propionic acid non-steroidal anti-inflammatory drugs on a human serum albumin based high-performance liquid chromatographic chiral stationary phase

Summary The native enantioselectivity in binding of human serum albumin (HSA) towards 2-aryl propionic acid non-steroidal anti-inflammatory drugs (2-APA-NSAIDs, the profens) was found to be preserved when the protein was immobilized within a commercially available diol high-performance liquid chromatographic column. High capacity factors were obtained, reflecting the previously observed extensive binding of the 2-APA-NSAIDs to free HSA. The capacity factors were modified by the addition of octanoic acid to the mobile phase. Chiral resolution of the enantiomers of all nine 2-APA-NSAIDs studied was achieved. Preliminary studies show that in addition to being a useful chiral analytical tool for this therapeutically important series of compounds, the HSA chiral stationary phase may provide useful information on the affinity and binding mechanism of small molecules to HSA.     

Chiral separation of bupivacaine enantiomers by capillary electrophoresis partial-filling technique with human serum albumin as chiral selector

Capillary electrophoresis (CE) is a powerful technique for enantiomer separations due to its intrinsic high separation efficiencies, speed of analysis, low reagent consumption and small sample requirements. However, some chiral selectors present strong background UV absorption providing high detection limits. The present paper deals with the application of the partial-filling technique to the separation of bupivacaine enantiomers by capillary electrophoresis using human serum albumin (HSA) as chiral selector. In this procedure the cationic surfactant cetyltrimethylammonium bromide (CTAB) was used as a dinamic capillary coating in order to reduce the electro-osmotic flow and detect both bupivacaine enantiomers out of the chiral selector plug. Several experimental conditions such as CTAB concentration, pH, HSA concentration and plug length, background electrolyte concentration, temperature and voltage were studied. Under the selected conditions it is possible to detect the separated enantiomers out of the HSA plug in less than 4 min using 50 mM Tris pH 8 as background electrolyte with 50 microM CTAB, at 30 degrees C and using a separation voltage of 25 kV. The proposed methodology was then validated for analytical purposes and applied to the analysis of pharmaceutical preparations commercially available. The results obtained with the proposed methodology were in good agreement with those declared by the manufacturers. The simplicity, sample throughput, accuracy, reproducibility and low cost of the proposed method make it suitable for the control of the enantiomeric composition of bupivacaine in pharmaceuticals.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

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.     

Betulinic acid binding to human serum albumin: a study of protein conformation and binding affinity

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be K(BA)=1.685+/-0.01 x 10(6) M(-1), indicating a strong binding affinity. The secondary structure of the HSA-BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA+drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.