The in vitro characterization of the drug-protein binding of racemic propafenone, and its active metabolite 5-hydroxypropafenone in human serum, and in solutions of isolated human serum proteins

An accurate plasma concentration-response relationship for propafenone (PF), a potent class 1 antiarrhythmic agent, has not yet been defined. A general pharmacological premise suggests that only the free drug is available to contribute to the observed pharmacological response. It has previously been shown that PF is highly bound to α-l-acid glycoprotein (AAG) which results in a low free PF concentration. The correlation of free PF concentration and response failed to adequately describe the dose response relationship. It has subsequently been shown that upon chronic dosing, two active metabolites, namely 5-hydroxypropafenone (5-OH-PF), and n-depropylpropafenone (n-depropyl-PF) accumulate in humans treated with PF. It is highly likely that the free concentration of PF, in addition to those of 5-OH-PF and n-depropyl-PF, contributes to the observed pharmacological effect following administration of PF at steady-state. To date, no accurate estimation of 5-OH-PF binding in serum has been established. This thesis examines the binding characteristics of PF and 5-OH-PF and their interaction in human serum, and in solutions of AAG, human serum albumin (HSA), high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) using equilibrium dialysis. The binding of PF (2.0 μg/mL) and 5-OH-PF (0.5 μg/mL) was examined in serum when both drug and metabolite were present. The free fraction (FF) of PF and 5-OH-PF in serum was 0.063 ± 0.004 and 0.232 ± 0.020, respectively. Both PF and 5-0H-PF were found to bind to a high affinity, low capacity binding site on AAG, in addition PF showed a second low affinity, high capacity binding site. PF displayed a 10 fold greater affinity for the high affinity binding site on AAG when compared to 5-OH-PF. Both PF and 5-OH-PF showed only one low affinity, high capacity site on HSA of similar affinity. The interaction of PF and 5-OH-PF with HDL, LDL, and VLDL appeared to be due to solubilization, rather than a "true" drug-protein binding interaction, since it correlated well with the concentration of cholesterol within the lipoprotein complex (PF, r²=0.85; 5-OH-PF, r²=0.96). However, PF appeared to show saturable binding to the HDL complex. The uptake of PF and 5-OH-PF was greatest in LDL followed by HDL, and finally VLDL. In serum PF displayed both a high affinity, low capacity, and a low affinity, high capacity binding sites, although a similar observation was expected for 5-OH-PF, only one binding site could be experimentally identified. The uptake of 5-OH-PF by red blood cells (RBC) appeared to be approximately 5 fold greater than that of PF (i.e. The ratio of PF and 5-OH-PF concentration in the red blood cell/plasma was 0.7 ± 0.1 and 3.2 ± 0.5, respectively). When the binding of PF and 5-OH-PF was considered separately, the binding profiles were similar, that is, both drugs showed high affinity binding to AAG, and low affinity binding and/or non-specific binding to other serum proteins such as HSA, HDL, LDL, and VLDL. However, when both drug and metabolite were present, the binding of 5-OH-PF to AAG was found to be reduced. This is thought to occur as a result of the displacement of 5-OH-PF by PF from AAG. Thus, the binding of 5-OH-PF was noted to be more dependent on HSA, and lipoproteins when compared to PF. On the other hand, the binding of PF (2.0 μg/mL), even with the addition of 5-OH-PF, was dependent largely on the concentration of AAG. Although the binding of 5-OH-PF was apparently not altered by the addition of PF in serum, a decrease in the binding of 5-OH-PF by the addition of PF was observed. It is hoped that the understanding gained from this thesis will provide information regarding the relative importance of free PF and 5-OH-PF plasma concentration in future pharmacodynamic studies of PF.Pharmaceutical Sciences, Faculty ofGraduat