Hepatocellular Binding of Drugs: Correction for Unbound Fraction in Hepatocyte Incubations Using Microsomal Binding or Drug Lipophilicity Data

Abstract

The need to incorporate the fraction unbound in microsomes (fu mic ) to obtain meaningful drug concentrations for the prediction of intrinsic clearance and cytochrome P450 inhibition potential is widely accepted (Obach, 1996;Ito and Houston, 2005;Rostami-Hodjegan and Tucker, 2007). Recently, two equations based on drug lipophilicity have been developed for prediction of fu mic (Austin et al., 2002;Hallifax and Houston, 2006) that avoid experimental determinations. The limitations of these empirical predictive tools and their applicability for fu mic predictions over a range of lipophilicity and microsomal protein concentrations have been addressed (Gertz et al., 2008).Analogous to applying a correction for microsomal drug binding to in vitro clearance and inhibition parameters, it is important that the fraction unbound in hepatocyte incubations (fu hep ) is also considered for in vitro-in vivo extrapolation (McGinnity et al., 2006;Brown et al., 2007b). However, this need has yet to be broadly applied, perhaps due to the lack of comprehensive demonstration of its value. In a recent study by Austin et al. (2005), the extent of binding between the microsomal and hepatocyte incubations was compared using hepatocyte data (n ϭ 14) and the corresponding fu mic from a previous study (Austin et al., 2002). The authors proposed a linear 1:1 relationship between microsomal and hepatocyte binding for incubations of 1 mg/ml and 10 6 cells/ml, respectively, indicating that the fu hep can be extrapolated from microsomal studies. However, the applicability of this correlation has been questioned because of the small number of compounds investigated (Hallifax and Houston, 2007).To further explore the relationship between binding in microsomal and hepatocyte systems, a detailed analysis was carried out involving 39 drugs. A nonlinear empirical equation is proposed as an alternative to the linear relationship to relate binding between the two systems. In addition, prediction of fu hep directly from the logP/D metric is assessed over a wide range of lipophilicity (Ϫ0.13 to 5.93). The implications of these findings on the estimation of hepatocellular drug concentration for intrinsic clearance and inhibition parameter predictions are discussed. Materials and MethodsA database of 39 drugs and their corresponding fu mic and fu hep values was collated either from in house data or from the literature (Austin et al., 2002(Austin et al., , 2005Brown et al., 2007a;Hallifax and Houston, 2007). In the aforementioned studies, different methods were used to determine the drug binding in hepatocyte incubations, namely oil centrifugation (using live cells), dialysis (using dead cells), and ultrafiltration (using dead cells). Microsomal and hepatocyte binding are defined by eqs. 1 and 2, respectively: ABBREVIATIONS: fu mic , fraction unbound in microsomes; fu hep , fraction unbound in hepatocyte incubations; K a , microsomal protein binding affinity; K p , hepatocyte/medium concentration ratio; V R , ratio of the cell and incubation volume; logP/D...

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

“…Values of fraction unbound (f u ) for each inhibitor in microsomes were determined experimentally (Table 1). These values were used to calculate the intracellular fraction unbound for the hepatocyte studies (Kilford et al, 2008). Both sets of values are listed in Table 1.…”

In Vitro–In Vivo Correlation for Low-Clearance Compounds Using Hepatocyte Relay Method

“…All test compounds had significant turnover under the assay conditions and demonstrated good linearity. Hepatocyte binding was not incorporated for correction of unbound intrinsic clearance, since all compounds have f u,hep (fraction unbound in the hepatocyte incubation) values close to 1 based on the calculated values (Kilford et al, 2008) estimated using log P/D from the ACD software.…”

Evaluation of Normalization Methods To Predict CYP3A4 Induction in Six Fully Characterized Cryopreserved Human Hepatocyte Preparations and HepaRG Cells

“…The plasma concentration, C max, of the perpetrator (inducer compound) in the corresponding clinical trials was either expressed as its unbound concentration, with F up incorporated into the equation or total concentration (without F up in the equation). When unbound C max was used, the in vitro EC 50 was also corrected for the unbound fraction of test compound in the hepatocyte assay (i.e., F uhep ), which was calculated using a quantitative structure-activity relationship (QSAR) model as previously described elsewhere (Kilford et al, 2008). F up and F uhep values for each test compound are reported in Table 4.…”