Concentration Dependence of the Unbound Partition Coefficient Kp_uu and Its Application to Correct for...

The unbound partition coefficient (Kp_uu) allows the estimation of intracellular target exposure from free extracellular drug concentrations. Although the active mechanisms controlling Kp_uu are saturable, Kp_uu is commonly determined at a single concentration, which may not be appropriate in cases in which drug concentrations can largely vary, e.g., in plasma in vivo or in vitro IC₅₀ assays. We examined the concentration dependence of Kp_uu in vitro using KAT6A inhibitors with varying potency drop-off in ZR75-1 breast cancer cells to account for exposure-related discrepancies between cellular and biochemical IC₅₀ Considering saturability resulted in a better quantitative bridge between both IC₅₀ values and gave way to a simplified method to determine Kp_uu that is suitable for the prediction of unbound cytosolic drug concentrations without the need to generate f_u,cell estimates from binding studies in cell homogenates. As opposed to the binding method, which destroys cellular integrity, this approach provides an alternative f_u,cell estimate and directly reflects the fraction of unbound drug in the cell cytosol based on Kp saturation (f_u,cyto) of intact cells. In contrast to the binding method, prediction of intracellular KAT6A exposure with this more physiologic approach was able to bridge the average exposure gap between biochemical and cellular IC₅₀ values from 73-fold down to only 5.4-fold. The concept of concentration-dependent Kp_uu provides a solid rationale for early drug discovery to discriminate between pharmacology and target exposure-related IC₅₀ discrepancies. The attractiveness of the approach also lies in the use of the same assay format for cellular IC₅₀, f_u,cyto, and the unbound partition coefficient based on f_u,cyto (Kp_uu,cyto) determination. SIGNIFICANCE STATEMENT: Examination of the yet-unexplored concentration dependence of the unbound partition coefficient led to a new experimental approach that resulted in more reliable predictions of intracellular target exposure and is well suited for routine drug discovery projects.