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Protein binding displacement interactions between propafenone, 5-hydroxypropafenone and other antiarrhythmic drugs, HPLC analysis of indocyanine green Wang, Jing

Abstract

Propafenone (PF) is a class I antiarrhythmic agent used to treat ventricular and superventricular tachyarrhythmias. After oral administration, PF undergoes extensive metabolism. 5-hydroxypropafenone is one of the major active metabolites of PF. In this thesis, the displacement of PF and 5-hydroxypropafenone in purified human α₁-acid glycoprotein (AAG), albumin, serum and whole blood was examined using equilibrium dialysis. Free and bound PF and 5-hydroxypropafenone concentrations were analyzed by gas-chromatography with electron-capture detection (GC-ECD). Several antiarrhythmic agents (e.g. propranolol, verapamil, lidocaine, phenytoin and quinidine) and acetyl salicylic acid (ASA) were used as displacing agents. These displacing agents were chosen as specific probe for binding to recognized sites of the plasma protein molecules. The binding of 5-hydroxypropafenone to isolated human AAG phosphate buffer solution was studied over the concentration range of 50-20,000 ng/ml. One class of binding site was found. Saturation of binding sites occurred at concentrations of 5-hydroxypropafenone greater than 2,000 ng/ml. In purified solutions of human AAG or albumin, propranolol, verapamil, ASA and quinidine did not displace PF from its binding sites to a statistically significant degree. Phenytoin displaced PF to a statistically significant degree from its binding sites in albumin but not in AAG solution. Lidocaine caused a statistically significant increase in PF free fraction in solution of either AAG or albumin. 5-Hydroxypropafenone free fraction was not affected by verapamil ASA and propranolol, but increased with the addition of lidocaine in purified human AAG solution. In purified human albumin solutions, ASA caused a significant increase in 5-hydroxypropafenone, while no displacing effect was observed with either lidocaine, propranolol or verapamil. In human serum, quinidine, phenytoin and lidocaine caused a statistically significant increased PF free fraction. Neither ASA, quinidine nor phenytoin caused a statistically significant displacement of 5-hydroxypropafenone from its binding site in serum. Quinidine also displaced PF from its binding sites in whole blood while no displacing effect was observed with phenytoin. Quinidine and lidocaine were the most potent displacing agents for PF protein binding. They exhibited the displacement through competitive inhibition in purified human AAG. None of the displacing agents used seemed to be a potent inhibitor of 5-hydroxypropafenone protein binding in serum. Since PF is a drug known to exhibit very significant first-pass metabolism, we wished to prepare to study certain pharmacological interventions which might alter hepatic blood flow, and, hence PF clearance. Before such a study of PF could be conducted, we required a reliable measure of hepatic blood flow. The dye indocyanine green (ICG) affords such an estimate. We, therefore, set out to duplicate and refine on existing HPLC ICG assay. In early attempts, the detection limit of our HPLC analysis of ICG in human serum was 0.80 μg/ml. The analysis method of ICG in rat plasma gave an apparently good on-column detection limit (3.8 ng) using current detection technology available in our laboratory. However, we still could not detect the low concentration ICG samples as reported. This is, in part, due to the low injection volume that can be accommodated by our microbore HPLC system and more importantly the limitation of maximum detection wavelength available (600 nm). Fluorometric of ICG is not feasible and the chemical nature of this compound limit the use of solvent-solvent extraction method for sample preparation. With the manipulation of the sample extraction procedure, such as the use of solid phase extraction, or modification of our current liquid sample introduction configuration devices, it might be possible to develop a better sample preparation procedure in order to detect the low concentration which most likely will be encountered during the course of animal and/or human experimentation with ICG.

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