Characterization of drug interactions with alpha(1)-acid glycoprotein using high performance affinity chromatography

This dissertation focuses on characterization of drug interactions with α 1-acid glycoprotein (AGP) using high performance affinity chromatography (HPAC). The first part examines the interactions between propranolol and AGP using hydrazide-linked AGP supports in HPAC columns. The binding of R- and S-propranolol to a hydrazide-linked AGP column was evaluated by zonal elution and frontal analysis. The results indicated that propranolol had a single binding site on AGP with association constants of 0.8 (±0.2) × 106 M-1 for R-propranolol and 1.0 (±0.1) × 106 M -1 for S-propranolol at pH 7.4 and 37°C. The results confirm that a hydrazide-linked AGP column can be used as a model for soluble AGP in this system. This work also examined the ability of drug screening on the AGP column and a good correlation (correlation coefficient of 0.954) between the retentions in the HPLC system and the association constants from the literatures for 11 drugs was observed. Part two examines the applications of hydrazide-linked AGP supports in chiral separations by using R- and S-propranolol as model solutes. The effects of various chromatographic conditions were examined in this work including temperature, mobile buffer pH, ionic strength and organic modifier. Finally, the chromatographic conditions used to separate R- and S-propranolol on the hydrazide-linked AGP column was optimized. Under the final optimized conditions identified in this study it was possible to separate R- and S-propranolol with a resolution of 1.38 in less than 5 min. Part three examined the interactions between carbamazepine and AGP using hydrazide-linked AGP columns. Equations for a two-site binding system were derived for both frontal analysis and zonal elution. In general, two classes of binding sites for carbamazepine were found on the AGP column with association equilibrium constants that ranged from 0.7 to 3.1 × 106 M-1 between 5 and 45°C for high affinity site and 1.9 to 7.8 × 104 M-1 for the low affinity sites. It was also found that the higher affinity site made up an average of 4% (range, 3-6%) of the total carbamazepine binding regions in the AGP column. Possible sources of this binding site heterogeneity were considered. It was found that these two ligand populations were most likely a property of the original AGP preparation rather than a result of the immobilization process. The last part focused on the development and characterization of a new immobilization method for HPAC. The amount of protein that can be entrapped in a silica chromatographic support was examined. The oxidation degree of glycogen, the amount of oxidized glycogen and type of silica were optimized. The entrapped protein was found to have better specific activities than that obtained in covalent immobilization methods