The effects of pH, temperature and protein concentration on the in vitro binding of flutamide to human serum albumin.

Abstract

Human serum albumin (HSA) is the most abundant protein in plasma with a high ligand-binding capacity. This capacity impacts the pharmacokinetic properties of therapeutic drugs. In the present study, the binding properties of flutamide to HSA at different temperatures, pHs and percentages of HSA were investigated. Thermodynamic parameters were also determined to describe the nature of binding interaction. A modified ultrafiltration method was used for accurate determination of flutamide-HSA parameters. Ultra filtrate samples containing free flutamide were extracted and analyzed by developed HPLC-UV method. Analysis of binding data was performed in terms of Scatchard, Klotz and Hill plots. Kinetic parameters (n, Ka) were found to be affected by temperature, pH and HSA concentration. However, flutamide-HSA-binding level did not show significant differences under different experimental conditions. The negative value of Gibbs free energy (?G) indicated that the binding was spontaneous. Moreover, the negative value for enthalpy and entropy changes suggested that hydrogen bonding and van der Waals forces played major role in the binding of flutamide to HSA. The results suggested a positive cooperation behavior of flutamide-HSA-binding that was affected by pH, temperature and percentage of HSA.