Enantioselective Binding Of Chiral Drugs, Mexiletine And Ketoprofen, To Plasma Proteins

The binding of drugs to plasma proteins is a major determinant in the disposition of drugs. It is an important factor, which determines the pharmacokinetics and pharmacological effects of drugs. A clear understanding of the plasma protein binding behavior of a therapeutic agent is therefore fundamental to its safe and rational use. Since all proteins are inherently chiral, it should come as no surprise that their interactions with chiral drugs often show stereoselectivity. Therefore, it is important to study the individual enantiomeric differences in the development of an objective picture of drug-protein interaction. In this paper, systematic studies were performed on the enantioselective binding of chiral drugs to plasma proteins using stereoselective HPLC method. The studies are expected to provide a clear understanding of the differences in drug-protein interaction as well as in pharmacokinetic and pharmacodynamics properties between the two enantiomers of chiral drugs in the drug discovery and development process.1. Enantioselective binding of mexiletine to plasma proteinsA stereoselctive reversed-phase HPLC assay to determine S- and R-enantiomers of mexiletine was developed. The conditions for the derivatization, using 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) as a pre-column chiral derivatization reagent, were optimized. The assay was linear from 0.05-40.0 μg·mL^-1 for each enantiomer. The analytical method afforded average extraction recoveries of over 81% for each enantiomer. Intra-day and inter-day variation coefficients were less than 14%. The validated method was applied to quantify the enantiomers of mexiletine for protein binding studies. Ultrafiltration techniques wereused to determine the protein binding of each enantiomer of mexiletine in humanplasma, human serum albumin (HSA) and ai-acid glycoprotein (AGP). The resultsclearly showed that stereoselective binding of two enantiomers of mexiletine withhigher protein binding of the i?-enantiomer than that of the S-enantiomer in humanplasma. The binding of mexiletine enantiomers to plasma from three healthy subjectsshowed the individual differences of binding of mexiletine to plasma protein. Thebinding of the i?-enantiomer was greater than that of the S-enantiomer in all. To assessthe site of the stereoselective binding to plasma protein, the binding of mexiletineenantiomers to HSA and AGP was examined. The binding to AGP was stereoselectivefor i?-enantiomer with the bound fraction of 37.3-24.1% for i?-enantiomer and31.1-21.0% for S-enantiomer, whereas S-enantiomer was bound to a greater extent toHSA with the bound fraction of 37.4-24.3% for ￡-enantiomer and 47.4-40.0% foriS-enantiomer. Although these results demonstrate opposite stereoselective in thebinding of R- and S-enantiomer to AGP and HSA, the stereoselective binding ofi?-enantiomer to AGP predominates in plasma. Both mexiletine enantiomers werebound to one class of binding site on AGP and binding association constants were9.80xl04for i?-enantiomer, 7.56 * lO4 L-mol"1 for ^enantiomer, respectively. Theeffect of pH on the binding of mexiletine enantiomers to HSA was studied. The resultsshowed that HSA possessed the chiral recognition ability to mexiletine enantiomers inthe pH range of 7.4-11.0. The chiral recognition ability to mexiletine enantiomersdisappeared at pH 6.0 and 5.0. AGP was the mixture of three main genetic variants.Binding interaction studies performed with mexiletine enantiomers and the specificliagnds for the AGP variants revealed that mexiletine enantiomers had high bindingaffinity for Fl-S variant of human AGP. The species-dependent bindingstereoselectivity was observed in the mexiletine-protein binding. The S-mexiletinebound to rat and rabbit plasma is more than that of i?-mexiletine, whereas .K-mexiletinebound to human plasma is more than that of S-mexiletine. 5-mexiletine bound to HSAand BSA is more than that of i?-mexiletine, whereas ^-mexiletine bound to AGP andBGP is more than that of S-mexiletine.2. Purification of recombinant human serum albumin domains andtheir bindings with inexiletine and ketoprofenIn an attempt to systematically dissect the chiral drugs binding properties of human serum albumin (HSA) and get an in depth understanding of the binding mechanism, the purification of recombinant HSA domains (HSA DOM I > HSA DOM II-. HSA DOMIII) was established. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding of the drugs to recombinant HSA domains showed that recombinant HSA DOM II and HSA DOMIII possess the chiral recognition ability to mexiletine enantiomers and ketoprofen enantiomers, respectively.