Impact of serum on clearance predictions obtained from suspensions and primary cultures of rat hepatocytes

Abstract

The objective of the present study was to compare two configurations of the hepatocyte model namely suspensions (SH) and conventional primary cultures (CPC) for their ability to predict the hepatic clearance in vivo in the rat and, to investigate the impact of serum on the prediction accuracy.

The metabolic competences of several cytochrome P450 isoenzymes were investigated both in CPC and SH in the presence or absence of serum. Under the same conditions, the in vitro intrinsic clearance of six test compounds metabolised by a variety of phase I and phase II enzymes (antipyrine, RO-X, mibefradil, midazolam, naloxone and oxazepam) were derived from V_max/K_m scaled up to the corresponding in vivo hepatic metabolic clearance.

CYP activities were shown to be stable in both CPC and SH for up to 6 h of incubation, except for the CYP 3A1 activity that decreased in CPC even in the presence of serum. Moreover, the clearances predicted from SH in the presence of serum were closer to the in vivo values than those obtained from CPC.

SH represent a convenient model to assess the hepatic metabolism of xenobiotics, the presence of serum in the incubation medium significantly improved in several instances the quality of the predictions.

Introduction

The prediction of metabolic clearance is essential in both drug discovery and development programs, since it affects parameters such as rate of elimination and oral bioavailability. The determination of enzyme kinetic parameters, K_m, V_max and CL_int is an important aspect in drug discovery and development (Obach, 2001). Especially important are accurate predictions of the hepatic clearance CL_h, since liver is the major organ for metabolism of exogenous compounds. In the context of drug discovery, it is often sufficient to answer the question “is a given compound expected to have a low, intermediate or high hepatic extraction ratio in humans?”. By contrast, a more precise estimate of clearance is requested when drug candidates are selected for clinical development, in order to adequately support the first human studies.

Various in vitro models are available to investigate drug metabolism, such as microsomes, S9 and cellular systems. Considering the broad spectrum of enzyme activities, the physiological cofactor-enzyme levels, the active gene expression and the cell integrity, hepatocytes are considered to be the tool of choice for clearance predictions. Moreover, hepatocytes contain an intact plasma membrane, with all associated functions with the entire complement of transporters, including passive permeation and transporter-assisted uptake and efflux (Houle et al., 2003).

By applying sound pharmacokinetic principles, it has been shown that the human CL_h can be predicted reasonably well from in vitro data (Fabre et al., 1990). Reviews that cover all aspects of the in vitro-to-in vivo scaling have recently been published (Houston, 1994, Houston and Carlile, 1997, Iwatsubo et al., 1997). Although successful predictions of in vivo CL_h have been reported in several cases, a number of issues still need to be clarified to further improve the prediction accuracy. Thus, approaches that are based on physiologically based direct scaling tended to lead to a systematic (about two-fold) underestimate of the in vivo CL_h in humans (Zuegge et al., 2001). Such underestimate might be explained by a number of reasons such as the involvement of active transport processes at the sinusoidal membrane of the hepatocyte and/or inappropriate considerations of both the in vitro and in vivo binding (Ito et al., 1998). Because of the presence of diluted plasma in the incubation medium, the predictions reported with primary cultures of rat and human hepatocytes assumed similar protein binding in both plasma and in vitro (Zuegge et al., 2001). However, the assumption may not be valid for all compounds and needs to be confirmed experimentally.

Recently, a novel in vitro method based on rat and human hepatocytes suspended in serum was developed and showed promising results for predicting the in vivo CL_h in rat and man (Shibata et al., 2000, Shibata et al., 2002). Another issue which requires careful consideration is the instability of the metabolic enzymes during the processing of the liver samples and/or the incubation of hepatocytes. Although a significant degradation of cytochrome P450 (CYP) in hepatocytes upon incubation has been reported in the literature (Maurel, 1996, LeCluyse, 2001) this factor is usually not taken into account and probably impairs the in vitro-to-in vivo scaling (Bousquet-Melou et al., 2002).

The objective of the present work was to compare conventional primary cultures (CPC) to suspensions of rat hepatocytes (SH), in the presence and the absence of serum, for their ability to predict the in vivo CL_h in rat. For this purpose, the stability of various rat liver CYP isoenzymes was determined as a function of time under such conditions. A test set of six compounds covering a broad range of clearance, protein binding and metabolized by a variety of phase I and phase II enzymes was used to evaluate the impact of serum, the hepatocyte configuration and the CYP degradation on clearance predictions.