Prediction of Hepatic Clearance and Availability by Cryopreserved Human Hepatocytes: An Application of Serum Incubation Method

Abstract

ABSTRACT:A novel and convenient method was established for the prediction of in vivo metabolic clearance in human liver. The present method applied the in vitro-in vivo extrapolation paradigm previously established in rats to the in vitro data obtained from cryopreserved human hepatocytes. Predicted hepatic availability and clearance were compared with the reported oral bioavailability and plasma clearance in humans for 14 clinically used drugs (naloxone, buspirone, verapamil, lidocaine, imipramine, metoprolol, timolol, antipyrine, diazepam, quinidine, caffeine, propranolol, diclofenac, and phenacetin). A large interindividual variation was observed in the intrinsic metabolic clearance among separate cryopreserved preparations from different subjects. The prediction generally resulted in a marked underestimation when the biologically based scaling (cells/kg) from pooled preparation of two selected lots, which were 3-to 4-fold larger than the biologically based scaling factor. These data suggested that the calibration of inherent interindividual variation of metabolic activities among different cryopreserved preparations of human hepatocytes to obtain the empirical scaling factor, which is applicable only to the preparation used, was an essential step for more reliable and quantitative prediction of in vivo metabolic activity in humans.Hepatic clearance for the metabolism of compounds kinetically consists of two major determinants: intrinsic (metabolic) clearance of the unbound compound and unbound fraction of compound in the blood (or plasma when corrected by the blood-to-plasma partition). Generally, the intrinsic clearance for the unbound compound is measured in vitro by the incubation of isolated hepatocytes or subcellular fractions such as S-9 and microsomes in the protein-free medium. The in vitro metabolic parameters thus obtained are extrapolated by using anatomical parameters such as cell numbers and protein content in the intact liver for the prediction of in vivo metabolic activity (Houston and Carlile, 1997;Iwatsubo et al., 1997;Obach, 1999). Separate experiments necessarily are further carried out to measure the unbound fraction in the plasma. Many technical problems including adsorption of compounds to the apparatus during the equilibrium dialysis and ultra-filtration often hamper the accuracy of the evaluated values (Bertilsson et al., 1979;Desoye, 1988). To improve the accuracy and avoid complexity for predicting in vivo metabolic clearance from in vitro experiments, we have recently developed a novel and convenient in vitro method for predicting in vivo metabolic clearance by using freshly isolated rat hepatocytes suspended in rat serum (Shibata et al., 2000). Oral bioavailability and hepatic clearance for 16 widely used compounds were well predicted directly from the in vitro metabolic clearance values obtained from a single incubation without separate evaluation of unbound fraction in the plasma. The purposes of the present study were to 1) determine whether the same methodology was applicable ...

A Unified Model for Predicting Human Hepatic, Metabolic Clearance From in Vitro Intrinsic Clearance Data in Hepatocytes and Microsomes

“…CL int, in vivo data were provided in several reports (Shibata et al, 2002;Naritomi et al, 2003). Relationships obtained using the parallel tube model to compute CL int, in vivo were very similar to those reported (data not shown).…”

“…CL int, in vitro data derived from hepatocyte (Lave et al, 1997a,b;Lau et al, 2002;Shibata et al, 2002;Naritomi et al, 2003) or microsomal incubations (Carlile et al, 1999;Obach, 1999;Naritomi et al, 2001;Andersson et al, 2004) were generated in the authors' laboratory and collated from several published studies (Tables 1-3). Incubation conditions for data generated in the authors' laboratory have been detailed previously (Austin et al, 2002;McGinnity et al, 2004).…”

“…CL int, ub, in vivo was calculated from values of CL h , the fu p , and blood-toplasma concentration ratio (R B; fraction unbound in blood, fu b ϭ fu p /R B ) reported for each drug according to the well stirred or dispersion liver model (Shibata et al, 2002). Where values of R B were not provided or readily available, this parameter was assumed to be unity for neutral and basic compounds and 0.55 for acids.…”

“…Additional protein was either autologous (human) serum (Shibata et al, 2002) or 10% fetal calf serum (FCS) for incubations conducted with cultured cells for up to 72 h (Lave et al, 1997a,b;Schneider et al, 1999). CL int, in vitro was estimated using only an unbound fraction in plasma (fu p ) correction for incubations that included serum, since binding to plasma proteins was deemed to be greater than any hepatocyte binding (Shibata et al, 2002;Austin et al, 2005).…”

Mass Transfer Modeling of Hepatic Drug Elimination Using Local Volume Averaging Approach

“…Gauss-Seidel iterative method with a convergence criterion of 10 -6 was used for solving the system of algebraic equations simultaneously in MATLAB. Simulation was performed for seven drugs, lidocaine, metoprolol, verapamil, caffeine, timolol, diazepam, and phenacetin (Shibata et al, 2002), at a hepatic perfusion rate of 1500 mlmin -1 and a sinusoidal porosity of 0.12 (Bonfiglio et al, 2010) for a time-course of 200 s following a 5 mg IV dose of each drug. The tortuosity of the liver tissue was calculated according to the tortuosity of porous media consisting of layer by layer parallel rods as (Perry and Green, 2008): Table 1 shows the reported pharmacokinetic properties of each drug used for the simulation.…”