Investigations of the Effects of Ethanol on Warfarin Binding to Human Serum Albumin

Abstract

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37°C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 ± 0.2 μM, 0.1% ethanol 5.86 ± 0.1 μM, 0.3% ethanol 5.83 ± 0.2 μM, 0.5% ethanol 6.76 ± 0.1 μM, 1% ethanol 7.01 ± 0.1 μM, 3% ethanol 9.9 ± 0.7 μM, 5% ethanol 13.01 ± 0.1 μM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6.62 ± 1.6 μM, 0.1% ethanol 6.81 ± 1.1 μM, 0.3% ethanol 8.26 ± 2.5 μM, 0.5% ethanol 8.86 ± 1.9 μM, 1% ethanol 11.01 ± 4.2 μM, 3% ethanol 20.75 ± 2.4 μM, 5% ethanol 21.67 ± 2.2 μM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.

Warfarin: an environment‐dependent switchable molecular probe

“…The traditional difficulties associated with correlating warfarin concentration with biological activity are reflected in the results of studies examining its binding to proteins involve in the bioavailabilty and even the direct action of warfarin. Human serum albumin (HSA) (Ha et al, 2000;Il'ichev et al, 2002;Petersen et al, 2002) and cytochrome P2C9 (CYP2C9) (He et al, 1999) and even binding site models such as the cyclodextrins (Ishiwata and Kamiya, 1997) have been examined. In these cases, the binding sites demonstrate preferences for different isomers, Table 1.…”

Effect of Ethanol on the Binding of Warfarin Enantiomers to Human Serum Albumin

“…It was reported that ethanol (1700 or 4250 mM; 10 or 25% (v/v)) changes the a-helical structure in HSA 12) and structural changes in Site I due to ethanol (above 17 mM; 0.1% (v/v)) were suggested. 7) Hence, these previous data suggested that the conformation of the warfarin binding pocket was altered upon the binding of ethanol. Fitos et al suggested that differences in the microenvironments of the binding site selectively increased the binding of S-warfarin to HSA by the presence of S-lorazepam acetate.…”

“…7) In addition, it was recently reported that an excipient changes the unbound fraction of naproxen, warfarin and digitoxin to commercial albumin preparations.…”

The Effect of Ethanol on the Hydrolysis of Ester-Type Drugs by Human Serum Albumin

“…27) On the other hand, ethanol have a significant effect on the fluorescence intensity of HSAbound warfarin indicating structural changes in the binding site. 15) It is considered that the enhanced hydrolysis of aspirin by HSA containing low levels of fatty acids was inhibited by the displacement of fatty acid and changes of surrounding environment on site I in the presence of ethanol.…”

“…Various drugs can pharmacokinetically or pharmacodynamically interact with alcohol. 14) Ha et al 15) reported that more than 2 vol% ethanol change the conformation of HSA and the binding of warfarin to HSA. Michnik and Drzazga 16) studied that the thermal stability of HSA was changed by more than 2 vol% ethanol.…”