Interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin

Abstract

A B S T R A C T The interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin was studied by equilibrium dialysis. A 20-fold variation of buffer ionic strength (0.017-0.340) caused no significant change in the warfarin association constant. But the binding strength rose significantly as the pH was increased from 6.0 to 9.0 and then declined at pH 10.0. The 6-, 7-, and 8-hydroxywarfarin metabolites showed a 7-to 23-fold reduction in binding strength at pH 10.0. These data indicate that the molecular basis of the interaction is nonelectrostatic and that the introduction of polar hydroxyl groups on the coumarin nucleus by metabolism reduces its hydrophobic binding surface. The interaction was markedly exothermic and showed a positive entropy (increased molecular disorder), which suggests cooperative hydrogen and hydrophobic bonding as the molecular basis for the binding of warfarin to albumin.The marked albumin binding and nonpolar character of warfarin explains the respective absence and presence of the unchanged drug in urine and plasma of warfarintreated patients, while the more polar character and lesser albumin binding of the metabolites probably determines their absence in plasma and presence in urine. The relatively marked binding to albumin of the 4'-hy- INTRODUCTIONIn previous studies of the interaction of the coumarin anticoagulant warfarin sodium and human plasma albumin, the drug was strongly bound to plasma albumin when analyzed by equilibrium dialysis (1). While the strength of the interaction was studied as a function of temperature, no experiments were performed at different levels of buffer pH and ionic strength. Such experiments were performed in the present study which together with the thermodynamic data allowed us to analyze the molecular basis of the warfarin-albumin interaction.Countercurrent distribution analysis of plasma of patients receiving clinical doses of warfarin shows essentially only the unchanged drug and similar analysis of urine reveals only hydroxylated metabolites of warfarin (2, 3). To determine if the absence of the metabolites in plasma and the absence of unchanged drug in the urine could be correlated with albumin binding, we also studied the four known metabolites by equilibrium dialysis. Based on these data, a hypothesis is presented for the pharmacodynamics of warfarin in man. METHODS Warfarin sodiumMaterial. The equilibrium dialysis technique, the crystalline human albumin and warfarin sodium, the preparation of the cellophane bags, the experimental techniques employed, the measurement of free and bound drug, and the determination of the standard free energy change were exactly as described previously (1). The low concentrations of warfarin sodium were varied over a 16-fold range (6-100 ,moles/ liter), which evaluates mainly the association constant of the first warfarin anion bound (k1). The concentration of The Journal of Clinical Investigation Volume 48 1969 193 albumin used in all experiments was 0.4% (57.9 /hM/liter). The e...

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

“…Compared with our f u measurements for 9S-and 9R-warfarin (0.4%), we found that most warfarin metabolites were less tightly bound in human plasma (2.1%-3.4% for 49-OH, 6-OH, 7-OH, and 8-OH warfarin), in agreement with previous reports. 39 The f u values we measured in human plasma for 5-OH and 10-OH warfarin and the warfarin alcohols were all ,1.0%. In contrast, the f u in DMEM/10% FBS for all warfarin-related compounds tested was significantly higher than in human plasma.…”

Interpretation of Protein-Drug Interaction through Fraction Bound and Relative Contribution of Secondary Sites

“…13) is the comparison of this warfarin binding study with the study of O'Reilly (17). The data for warfarin in Table I …”

Interaction Between 5-Dimethylaminonaphthalene-1-Sulfonamide and Human Serum Albumin

“…water, peaks at a longer wavelength (580 nm) than that in a relatively nonpolar solvent, such as dioxane (500 nm) [3,4], Thus, the increased fluorescence at 485 nm of DNSA in the presence of HSA suggests that these compounds interact at a nonpolar site(s) and, therefore, bond hydrophobically. Furthermore, other compounds, such as warfarin and phenylbutazone, which are known to form hydrophobic bonds, displace DNSA from HSA [5,6,11].…”

“…Other drug classes, which are hydrophobically bound to albumin, also displace DNSA from HSA (table II) [5,6,11]. In this regard, warfarin and phenylbutazone are among the most potent.…”