Study of the enantioselective binding between BOF-4272 and serum albumins by means of high-performance frontal analysis

Abstract

High-performance forntal analysis (HPFA) was incorporated in an on-line HPLC system for the study of the enantioselective binding of BOF-4272, a new xanthine oxidase inhibitor, with human, bovine and rat serum albumins. This HPLC system consists of a HPFA column (diol-silica column), an extraction column (C 4 column) and a chiral separation column (β-cyclodextrin immobilized silica column), which were connected in series via two column switching valves. After the direct injection of a solution of 0.5–400 μM racemic BOF-4272 and 550 μM serum albumin onto the HPFA column, BOF-4272 was eluted, under a mild mobile phase condition (phosphate buffer, pH 7.4, ionic strngth 0.17), as a zonal peak containing a plateau region. The drug concentration in the plateau region is the same as that for the unbound drug concentration in the sample solution. A given volume of this plateau region was transferred into the extraction column, and subsequently the extracted BOF-4272 was transferred into the chiral separation column to determined the unbound concentration of each enantiomer. The binding between BOF-4272 and the serum albumins was enantioselective and species dependent. The unbound concentration of the (+)-isomer in rat serum albumin solution was 1.04–1.14 times larger than that of the antipode, while the unbound concentration of the (−)-isomer in bovine serum albumin solution was 1.04–1.16 times larger than that of the antipode. The enantioselectivity of the binding between BOF-4272 and human serum albumin was concentration dependent. When the total concentration of racemic BOF-4272 was low (0.5 μM or 5 μM), the unbound concentration of the (+)-isomer was 1.15 or 1.06 times larger than that of the (−)-isomer. On the contrary, the unbound concentration of the (−)-isomer was 1.05 or 1.34 times larger than the (+)-isomer in case of the higher total drug concentration (50 μM or 400 μM). Based on the Scatchard analysis of the binding between human serum albumin and BOF-4272 enantiomers, it was found that this change is due to the enantiomeric difference in the binding constant ( K) and the number of binding site per protein moleculer ( n); K = 1.22·10 5 M −1 and n = 2.30 for the (+)-isomer, and K = 2.32·10 5 M −1 and n = 1.3- for the (−)-isomer.