Evaluation of enantioselective binding of fluoxetine to human serum albumin by ultrafiltration and CE – Experimental design and quality considerations

Abstract

Several pharmacokinetic processes are affected by enantioselectivity (ES). At the level of distribution, protein binding (PB) is one of the most important. The enantioselective binding of fluoxetine (FLX) to HSA has been evaluated in this work by ultrafiltration of FLX–HSA mixtures and chiral analysis of unbound fractions by EKC-CD. PB, affinity constants (K) and ES were obtained for both enantiomers of FLX. In order to improve the consistency of the estimations, the evaluation of affinity constants of each enantiomer was performed using two designs, one keeping constant the total concentration of protein and varying the total concentration of the enantiomers, and the other in the opposite way, in both cases via an unusual short-concentration interval strategy to assure model validity. Different mathematical approaches were compared and characterised and some of them, judged as the most consistent under the experimental conditions used, were selected to provide final estimates. Quality considerations include criteria for three critical aspects: (i) detecting/eliminating outliers, (ii) checking the number of binding sites in the protein and (iii) evaluating the robustness of each approach. The differences on estimates from the selected approaches were used as an uncertainty source to delimit the reported values. The ES of HSA for FLX enantiomers was approximate. Estimates include the assumptions of independent and competitive models. In the last case, a SIMPLEX function was designed capable of simultaneously optimizing the non-linear binding models for both enantiomers, thus improving the consistence of results.

Characterizing the interaction between enantiomers of eight psychoactive drugs and highly sulfated‐β‐cyclodextrin by counter‐current capillary electrophoresis

“…, except for terfenadine, baseline resolution was achieved for all the analytes studied and high Rs values were obtained. The Rs values obtained were similar to or higher than those reported in the literature for these compounds with other cyclodextrins, but obtained in most cases using higher CD concentrations than here (Castro‐Puyana et al ., ; Van Eeckhaut et al ., ; Matsunaga et al ., ; Inoue and Chang, ; Rudaz et al ., ; Bonato et al ., ; Perrin et al ., ; Li and Vigh, ; Asensi‐Bernardi et al ., ; Yang et al ., ; Liao et al ., ; Chen et al ., ; Wei et al ., ).…”

“…In order to estimate K ′ and values, effective mobility of the enantiomers was fitted vs [CD] (in molar units on the basis of an average molar mass of 2360 g mol −1 for a substitution degree of 12 sulfates mol −1 ; Chen et al ., ) to equation . Nonlinear estimation was preferred instead of the most popular linear equations, owing to its expected higher robustness (Asensi‐Bernardi et al ., ) and the possible lack of accuracy and precision of linear models owing to errors derived from variable transformation (Foulon et al ., ). The obtained fitted models have good correlation coefficients, R 2 > 0.99 in most of the cases, suggesting that equation appropriately reflects the behavior of the compounds in the HS‐ β ‐CD system.…”

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

“…For the NMF–HSA binding study, routines made/adapted in MATLAB® 4.2 were used for calculations, and the results generated were verified using EXCEL® and STATGRAPHICS®. The mathematical aspects were previously described in detail (Asensi‐Bernardi et al , , and references therein) and they are briefly described here. An accepted mathematical model describing the ligand–HSA interaction, applicable for each enantiomer in the case of a chiral molecule, considering m classes of independent active sites on HSA, each with n i binding sites, is: …”

“…Most of the pharmacokinetic and pharmacological processes exhibit a high degree of enantioselectivity, owing to the characteristic chirality of biomolecules, and this results in differences between the pharmacological activities of drug enantiomers. In fact, very often one of them is most active while the other may produce side‐effects and even toxicity in some cases (Asensi‐Bernardi et al , ). Therefore, investigation of the enantioselectivity of drugs in their pharmacokinetic and pharmacological properties represents a great challenge for clinical pharmacology (Escuder Gilabert et al , ).…”

“…This methodology includes the incubation of drug–protein mixtures at the microliter level, their ultrafiltration through cellulose filters and the chiral analysis of the unbound fraction by EKC‐partial filling technique using heptakis‐2,3,6‐tri‐ O ‐methyl‐ β ‐cyclodextrin (TM‐ β ‐CD) as chiral selector. A recent mathematical approach of our group (Asensi‐Bernardi et al , ) was used to estimate affinity constants ( K 1 ) and verify the stoichiometry ( n 1 ), for both enantiomers of nomifensine, as well as to estimate their enantioselectivity ( ES ). Protein binding in percentage ( PB ) to HSA at physiological concentration levels and total plasma proteins were also estimated and compared with in vivo literature data.…”