Determination of the free fraction and relative free fraction of drugs strongly bound to plasma proteins.

This report describes a new method for the determination of protein binding and relative protein binding (ratio of f(u) for different species) for compounds strongly bound to proteins. The method used is based on the distribution of the drug in plasma water, plasma proteins, and blood cells. Incubations were performed in diluted plasma. In diluted plasma, the erythrocyte/plasma distribution was determined with greater precision than in undiluted plasma. Formulae were derived for calculating f(u) in undiluted plasma based on the f(u) values determined in diluted plasma. These formulae are also valid in the event of more than one independent binding site in plasma. All incubations with plasma of different species were performed using rat erythrocyte suspensions, thereby making it possible for relative f(u) values in different species to be calculated without knowing the absolute free fractions. This method avoids the determination of the erythrocyte/buffer distribution in cases where it is sufficient to know relative f(u) values (e.g., exposure comparisons). Relative protein binding can also be quantified for compounds that tend to adsorb to surfaces of vials or test tubes, thus avoiding errors caused by adsorption when quantifying the drug in a protein-free aqueous solution. This method was validated by making comparisons of free fraction values obtained by the method herein described with those obtained by either ultrafiltration or equilibrium dialysis for two compounds that bind predominantly to albumin and another compound that binds to alpha(1)-acid glycoprotein. The results confirm our method produces identical free fractions in comparison with other established techniques. In addition, the range of applications of our method is much wider. Copyright 2000 Wiley-Liss, Inc.