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

ABSTRACT

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.

KEYWORDS:

• Assay disconnect
• clearance prediction
• fraction unbound
• intracellular free drug concentration
• hepatocytes
• partition coefficient
• unbound partition coefficient
• tissue distribution

Article highlights

• C_u,cell and Kp_uu are typically measured using indirect methods by determining total concentration and fraction unbound of cells and media.

• Several methods are available to measure f_uc. Tissue or cell homogenate with equilibrium dialysis is the most commonly used method.

• Hepatocytes in the presence of 4% bovine serum albumin (BSA) can be used to predict in vivo liver-to-plasma Kp_uu with good accuracy.

• Incorporation of Kp_uu in clearance prediction using the extended clearance equation improves prediction accuracy for hepatic clearance involving both enzyme- and transporter-mediated mechanisms.

• C_u,cell and Kp_uu help to bridge potency disconnect between biochemical and cell-based assays.

• Structure attributes governing tissue Kp_uu are valuable information to enable design of compounds with optimal tissue exposure.

Abbreviations

ADH, alcohol dehydrogenase; ADME, absorption, distribution, metabolism and excretion; AFE, average fold error; ALDH, aldehyde dehydrogenase; AO, aldehyde oxidase; AUC, area under the curve; BCRP, breast cancer resistance protein; BSA, bovine serum albumin; CES, carboxylesterase; CL_int, intrinsic clearance; CL_int,efflux, intrinsic efflux clearance; CL_int,met, intrinsic metabolic clearance; CL_int,pass, passive intrinsic clearance; CL_int,uptake, intrinsic uptake clearance; C_t,cell, total cellular drug concentration; C_t,media, total media drug concentration; C_t,plasma, total plasma drug concentration; C_t,tissue, total tissue drug concentration; C_u,cell, intracellular free drug concentration; C_u,liver, free liver drug concentration; C_u,media, free drug concentration in the media; C_u,plasma, free plasma drug concentration; C_u,tissue, free tissue drug concentration; DDI, drug-drug interaction; EC₅₀, half maximal effective concentration; FBS, fetal bovine serum; F_ic, intracellular bioavailability; f_uc, fraction unbound of cells; f_u,media, fraction unbound of media; f_up, plasma fraction unbound; f_ut, tissue fraction unbound; HBD, hydrogen bond donor; HHEP, human hepatocytes; HLM, human liver microsomes; IC₅₀, half maximal inhibitory concentration; IC₅₀’, apparent half maximal inhibitory concentration; IC_50,u, free half maximal inhibitory concentration; IVIVE, in vitro-in vivo extrapolation; KATs, acetylation of histones via lysine acetyltransferases; K_i, inhibition constant; K_I, inactivation constant; k_inact, maximum rate of inactivation; Kp, partition coefficient; Kp_uu, unbound partition coefficient; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LogD, Log₁₀ of distribution coefficient between octanol and buffer; MAPK14, mitogen-activated protein kinase 14; MDCK-LE, low efflux Madin-Darby canine kidney cells; M-PER, mammalian protein extraction reagent; MRP1, multidrug resistance protein 1; MRPs, multidrug resistance proteins; NaCT, sodium citrate transporter; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; NASH, nonalcoholic steatohepatitis; OAT2, organic anion transporter 2; OATP, organic-anion-transporting polypeptide; PBMC, peripheral blood mononuclear cell; PD, pharmacodynamics; PET, positron emission tomography; P-gp, P-glycoprotein; PK/PD, pharmacokinetics / pharmacodynamics; SAR, structure-activity relationship; SULT, sulfotransferase; TI, therapeutic index; TPSA, topological polar surface area; UGTs, uridine 5’-diphospho-glucuronosyltransferases

Acknowledgments

The authors greatly appreciate the help of Sophia M. Shi in editing the manuscript.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.