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

Abstract

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.

Enantioresolution and Binding Affinity Studies on Human Serum Albumin: Recent Applications and Trends

“…A significant enantioselectivity to HSA was found in these studies; for instance, for zopiclone, the (S)enantiomer displayed more affinity (by a factor of 2) to HSA than the (R)-enantiomer [161]. These studies showed the various advantages of the PFT technique, not only the removal of the UV interference caused by proteins but also allowing a simpler performance with lower reagent consumption [150,[160][161][162].…”

“…As shown in Table 5, phosphate and sodium buffers were the most described. A tris-(hydroxymethyl)aminomethane solution was also reported in the three EKC studies [160][161][162] and, lastly, one study reported a borate buffer [159].…”

“…Furthermore, most of these studies were enantioselective studies where the differences in binding of each enantiomer of the analyzed compounds to HSA were evaluated and compared using, many of them, HSA as a chiral selector [150,152,154,155]. However, a few studies used cyclodextrins for this purpose while HSA was incubated with the samples before the analysis [151,153,161,162].…”

“…Another study showed the first evidence of the enantioselective binding of antihistamines to HSA using an EKC-PFT technique [160]. Furthermore, EKC-PFT was used for chiral separation and HSA binding studies of zopiclone and nomifensine enantiomers [161,162]. A significant enantioselectivity to HSA was found in these studies; for instance, for zopiclone, the (S)enantiomer displayed more affinity (by a factor of 2) to HSA than the (R)-enantiomer [161].…”

Stereoselective binding of chiral drugs to plasma proteins

“…These data suggest that ASA and SA do not affect the binding of PL to AGP because of the different acid-base properties of these drugs. For nomifensine enantiomer E1 (the first elute, protein binding to HSA 40%±5%), other plasma proteins were expected to contribute according to the plasma protein binding (58%±7%), but not for E2 (the second elute, PB 63%±4% and 64%±4% for HSA and plasma, respectively) 32 . Thus, the relative importance of HSA for binding nomifensine enantiomers was confirmed.…”