Applications of high-performance affinity chromatography in the study of drug-protein binding: Examination of interactions between phenytoin and related compounds with human serum albumin
This dissertation involves the detailed examination of phenytoin and its binding properties to human serum albumin (HSA). This was accomplished by implementing various affinity chromatographic techniques including zonal elution and frontal analysis along with columns containing immobilized HSA to provide estimates of equilibrium binding constants. Affinity chromatography with plate height measurements was also used with theory-derived equations to measure the rate constants for the multisite binding of phenytoin to the chromatographic column. Part one discusses the complexity of phenytoin’s multisite binding and work that has been done previously to unravel the binding properties of phenytoin. In addition an expansion of current chromatographic theory describing multisite binding and nonspecific interactions was used to measure rate constants utilizing plate height measurements. Also, frontal analysis was used to measure the overall equilibrium binding constants of this drug-protein binding system. Together these two constants measured at various temperatures were applied to thermodynamic theory to obtain a complete thermodynamic picture for phenytoin binding to HSA. Part two explores the development of a novel near infrared fluorescence and ultrafast immunoextraction/displacement assay (UFIDA) for the analysis of phenytoin’s free drug fraction. The highlights of this work included using microcolumns containing immobilized anti-phenytoin antibodies with sub-millimeter length and capability of extraction of phenytoin containing plasma in the millisecond-timescale range. The use of a phenytoin analog conjugated to a near infrared dye was used to provide the limits of detection required for such an assay. The last part of this dissertation examines a topic sometimes overlooked in protein binding studies, that is the interactions of drug metabolites with their parent compound. It was found that the metabolites of phenytoin do not share every binding site on HSA as their parent drug. However the binding of the metabolites to HSA do exhibit allosteric effects with phenytoin indicating that competition between phenytoin and its metabolites can change phenytoin’s pharmacokinetics.