Determination of Unbound Partition Coefficient and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake

Abstract

Unbound partition coefficient (K puu ) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. K puu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transportertransfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher K puu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high K puu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different K puu values in human hepatocytes (K puu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. K puu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (K m ) of SLC13A5 was estimated to be 24 mM for PF-06649298 in human hepatocytes. In vitro K puu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo K puu of liver-to-plasma, illustrating the potential of this approach to predict in vivo K puu from in vitro systems.

A Novel Unified Approach to Predict Human Hepatic Clearance for Both Enzyme- and Transporter-Mediated Mechanisms Using Suspended Human Hepatocytes

“…We have developed a novel experimental approach to predict human clearance for enzyme-and transporter-mediated mechanisms. This method was developed on the basis of liver-to-plasma (Kp uu ) work previously published (Riccardi et al, 2016(Riccardi et al, , 2017. With these hypotheses as a basis: 1) The metabolic, passive, and uptake intrinsic clearance rates in the in vitro hepatocyte assay, in the presence of 4% BSA, are equivalent to in vivo rates, and 2) the metabolic intrinsic clearance rates in the in vitro hepatocyte assay, in the absence of 4% BSA, are equivalent to in vivo rates.…”

“…Presaturation method was also used for highly bound compounds (Riccardi et al, 2015). Calculation of fraction unbound has been discussed previously (Riccardi et al, 2015(Riccardi et al, , 2016(Riccardi et al, , 2017.…”

Quantitative Prediction of CYP3A4 Induction: Impact of Measured, Free, and Intracellular Perpetrator Concentrations from Human Hepatocyte Induction Studies on Drug-Drug Interaction Predictions

“…The f u,cell value was determined by correcting the fraction unbound in homogenate (f u,hom ) by a dilution factor (D) as previously reported (Mateus et al, 2013;Riccardi et al, 2016). Based on a hepatocyte volume of 4 ml for 1 Â 10 6 cells (Reinoso et al, 2001), D was determined to be 25.…”

“…time point. K puu,in vitro values can be concentration-dependent (Riccardi et al, 2016), so a follow-up study was performed in which the cell and medium concentrations were measured following incubation with the full range of inducer concentrations for 24 hours. The K puu,in vitro values decreased as the nominal concentrations of bosentan and rifampicin increased ( Fig.…”

“…To date, numerous methods have been proposed for determining intracellular unbound concentrations of drugs; however, as highlighted in a recent white paper, no systematic evaluation has been conducted to determine which method is the most reliable (Ulvestad et al, 2011;Zhu et al, 2014). Estimation of the f u,cell using cell homogenate has been widely used in recent years (Mateus et al, 2013;Chien et al, 2016;Riccardi et al, 2016). Furthermore, the K puu,in vitro values estimated using this method were reported to be in agreement with the fold difference in IC 50 estimated in human liver microsomes versus cell lines, providing confidence in the estimated f u,cell (Riccardi et al, 2016).…”