Determination of Fraction Unbound and Unbound Partition Coefficient to Estimate Intracellular Free Drug Concentration

Intracellular free drug concentrations are critical to develop pharmacokinetic and pharmacodynamic relationships, estimate therapeutic indices, and predict drug–drug interaction potentials. Free drug concentration at the site of action is most relevant to understand efficacy and toxicity. Because free drug concentrations are difficult to measure directly, indirect methods are often applied. One of the most commonly used indirect methods in drug discovery is to measure total drug concentration and fraction unbound (fu). The free drug concentration is then calculated by multiplying the total drug concentration with fu. Many methods have been developed to measure fu of cells and tissues, such as using cell or tissue homogenate with equilibrium dialysis, or partition coefficient (Kp) of cells at 4 °C (fu = 1/Kp). The method using equilibrium dialysis with tissue or cell homogenate tends to be higher throughput, more reproducible, and cost less. In addition, many tissues are readily available. The method of using a cell Kp at 4 °C is useful in special cases when binding to the specific components in the cell occurs. Determining the unbound partition coefficient (Kpuu) involves measuring the total concentration of both cells/tissues and media/plasma, as well as binding in all the matrices. Intracellular free drug concentration can then be calculated by multiplying Kpuu with free media/plasma concentration. These methodologies are widely applied in drug discovery and development to estimate intracellular free drug concentration and to enable a more accurate prediction of safety and efficacy outcomes in the clinic.