Characterization of the protein binding of chiral drugs by high-performance affinity chromatography. Interactions of R- and S-ibuprofen with human serum albumin.

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3 x 10(5) M-1 at pH 6.9 and 25 degrees C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1 x 10(5) M-1 and 1.2 x 10(5) M-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. The chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.

In vitro metabolism studies on oxamniquine and related compounds by chiral liquid chromatography.

A previously developed method based on alpha 1-acid glycoprotein for the resolution of the enantiomers of the Pfizer antischistosomal drug oxamniquine was used to examine possible enantioselectivity in the in vitro microsomal hydroxylation of a metabolic precursor, UK-3883, but was found to be limited by the poor operational stability of the analytical column ("EnantioPac") employed. As an alternative approach, a "Pirkle" covalently-bonded dinitrobenzoyl leucine column was used, with simple precolumn solute derivatization to the carbamate to improve chromatographic performance. The method allowed preliminary examination of the stereochemistry of the in vitro biotransformation, hydroxylation of UK-3883 to oxaminquine, which yielded evidence for substrate enantioselectivity in favour of the dextrorotatory enantiomer of UK-3883.

The in situ acetylation of an immobilized human serum albumin chiral stationary phase for high-performance liquid chromatography in the examination of drug-protein binding phenomena.

The in situ modification of an immobilized human serum albumin (HSA) high-performance liquid chromatographic chiral stationary phase by p-nitrophenyl acetate is reported. This procedure, which is thought to affect primarily a single reactive tyrosine residue within the protein structure, influenced the chromatographic retention and enantioselectivity factors of a wide range of solutes. For certain solutes, increases in both capacity factor and chiral resolution were observed. Ultrafiltration studies on representative test solutes using free HSA, treated in a similar manner to the immobilized protein, gave similar results as the chromatographic observations, indicating that the latter effects are not artifactual results of immobilization. The effect of the modification of HSA on the binding behavior of drugs reportedly sharing the site predominantly affected by the derivatization, namely, the indole-benzodiazepine binding site, varied greatly. This observation suggests that the affected binding area is not a single, tightly structurally defined site.

Stereochemical aspects of benzodiazepine binding to human serum albumin. II. Quantitative relationships between structure and enantioselective retention in high performance liquid affinity chromatography.

Previously determined retention data for a series of benzodiazepine (BDZ) derivatives, comprising nine achiral compounds, four single enantiomers, and 18 individual isomers of nine racemates, on a chiral stationary phase based on immobilized human serum albumin (HSA) were analyzed to define quantitative relationships between structure and enantiospecific retention. Structural parametrization of the agents was done by means of hydrophobic fragmental constants and electronic and steric parameters obtained by computational chemistry methods. A structural descriptor was identified, a submolecular measure of polarity about the stereogenic center, that accounted for the stronger electrostatic interactions of the second-eluting enantiomer with the HSA chiral stationary phase. Quantitative structure-enantiospecific retention relationships were derived for both enantiomeric series and for achiral compounds, and structural requirements for binding to HSA were determined. Two types of binding sites were postulated. For BDZs in the P-conformation, binding to HSA involved a hydrophobic region with steric restrictions. For BDZs in the M-conformation, a hydrophobic region was also involved, as well as a cationic region that interacted electrostatically with carbon C(3) of the diazepine system and substituents at that carbon. These differences lead to different binding patterns for BDZ enantiomers and provide a rationalization for the diversified behavior of individual BDZs that was observed in previous displacement studies.

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.

High-performance liquid chromatographic resolution of oxamniquine enantiomers: application to in vitro metabolism studies.

A method is described for the HPLC analysis of oxamniquine enantiomers in liver fraction incubates, using a second-generation alpha 1-acid glycoprotein-based column (Chiral-AGP). Oxamniquine is extracted from the incubation media by liquid-liquid extraction, using diethyl ether. The dried residue is redissolved in eluent, filtered, then injected directly onto the analytical column. The extraction method affords recoveries of oxamniquine of approximately 93%, at concentrations up to 525 micrograms/ml, with an average relative standard deviation of 5.9%. The limit of detection of the method (to give an SNR = 2 at 246 nm) is 0.3 ng on-column for the first eluting, laevorotatory enantiomer and 2.3 ng for the dextrorotatory isomer. The method allowed study of the depletion of oxamniquine enantiomers in liver postmicrosomal incubates. In the rat, a turnover of 21.9% was observed, with no apparent enantioselectivity. Similar observations were made for a mouse liver subcellular fraction incubation. The absence of enantioselectivity in this biotransformation may be attributable to the low substrate specificity of the oxidase or dehydrogenase enzymes involved.

Computer-aided optimisation of drug enantiomer separation in chiral high-performance liquid chromatography.

The advent of several new column materials for the resolution of chiral compounds in high-performance liquid chromatography has opened up new possibilities for the analysis of drug enantiomers both in the dosage form and in bioanalytical studies. The utility of simplex optimisation, modified simplex and response surface mapping are considered with reference to the antischistosomal drug, oxamniquine, separated on an alpha 1-acid glycoprotein column. The resolution of the enantiomers of three closely related benzodiazepines, temazepam, oxazepam and lorazepam, is attempted on three new column systems: cellulose triacetate, beta-cyclodextrin and the reversed-phase column porous graphitic carbon with beta-cyclodextrin as a mobile phase additive.

Stereochemical aspects of benzodiazepine binding to human serum albumin. I. Enantioselective high performance liquid affinity chromatographic examination of chiral and achiral binding interactions between 1,4-benzodiazepines and human serum albumin.

The displacement of a series of 1,4-benzodiazepine (BDZ) drugs from a chiral stationary phase, based upon human serum albumin, for high performance liquid chromatography was investigated. The different displacement patterns obtained using various mobile phase additives could not be interpreted in terms of binding of the solutes to a single site. The observations were better described by considering the attachment of the BDZs to several loci on the protein. Two main mechanisms of binding were discerned, a nonstereoselective mode, which affected all solutes and seemed to occur at a large number of locations on the protein, and a highly stereoselective mode, which involved only one enantiomer of chiral BDZs and presumably one conformation of certain achiral solutes. The stereoselective binding mode encompassed at least four different sites, each of which displayed slightly different structural requirements. It is suggested that the nomenclature currently used to describe drug binding to human serum albumin may be misleading. Rather than the use of site I or site II, it may be preferable to adopt the terms type I and type II binding, according to the displacement patterns of the compound concerned. This approach would retain the conceptual simplicity of the current notation, while avoiding misleading implications of the exact molecular locus of binding.