Evolving approaches on measurements and applications of intracellular free drug concentration and Kpuu in drug discovery

Introduction: Intracellular-free drug concentration (C_u,cell) and unbound partition coefficient (Kp_uu) are two important parameters to develop pharmacokinetic and pharmacodynamic relationships, predict drug-drug interaction potentials and estimate therapeutic indices.Area covered: Methods on measurements of C_u,cell, Kp_uu, partition coefficient (Kp) and fraction unbound of cells (f_uc) are discussed. Advantages and limitations of several f_uc methods are reviewed. Applications highlighted here are bridging the potency gaps between biochemical and cell-based assays, in vitro hepatocyte assay to predict in vivo liver-to-plasma Kp_uu, the role of Kp_uu in prediction of hepatic clearance for enzyme- and transporter-mediated mechanisms using extended clearance equation, and structural attributes governing tissue Kp_uu.Expert opinion: C_u,cell and Kp_uu are of growing applications in drug discovery. Methods for measurements of these properties continue to evolve in order to achieve higher precision/accuracy and obtain more detailed information at the subcellular levels. Future directions of the field include the development of in vitro and in silico models to predict tissue Kp_uu, direct measurement of free drug concentration in subcellular organelles, and further investigations into the critical elements governing cell and tissue Kp_uu. Significant innovation is needed to advance this complex, but highly impactful and exciting area of science.