Uptake and Intracellular Binding of Lipophilic Amine Drugs by Isolated Rat Hepatocytes and Implications for Prediction of in Vivo Metabolic Clearance

Abstract

ABSTRACT:The hepatic uptake of imipramine and propranolol was investigated after incubation with isolated rat hepatocytes over a wide concentration range (0.04-400 M). The cell-to-medium concentration ratio (K p ) was concentration-dependent and could be described using a two-site binding model incorporating a high affinity/low capacity site and a linear component for a site which was apparently not saturated. Maximum (at 0.04 M) and minimum K p values (at 400 M) were 360 and 280, and 110 and 70 for imipramine and propranolol, respectively. During these incubations, metabolism was inhibited using aminobenzotriazole (an irreversible inhibitor of cytochrome P450). Pretreatment of cells either by freezethawing or with saponin (which permeabilizes the plasma membrane) eliminated the saturable process. The saturable uptake process of imipramine was also inhibited by 18 other lipophilic amine drugs (including propranolol). This uptake component may involve membrane transporter(s), whereas the nonsaturable component probably represents partition into the phospholipid component of membranes. Propranolol was further investigated to determine the impact of high K p values on hepatocellular clearance. The area under the curve for propranolol concentrations in the total incubate (medium including the cells) from the depletiontime profile was substantially greater than the corresponding area under the curve for the drug concentration in the extracellular medium, and this difference approximated the nonsaturable uptake component. It is concluded that the clearance of propranolol in isolated hepatocytes is not rate-limited by hepatic uptake and is directly proportional to unbound drug concentration, being independent of the higher K p value.

Fluorescence Lifetime Imaging of Propranolol Uptake in Living Glial C6 Cells

“…At higher temperatures, the incorporation of propranolol into the membranes of these organelles, due to the lipophilicity of the drug, may lead to leakage into more widespread regions of the cell cytoplasm. The rate of propranolol uptake observed directly in these fluorescence lifetime imaging experiments is in accordance with previous nonimaging measurements using radiochemical analysis [7]. The fluorescence method here, however, not only gives details of uptake but also enables direct imaging of the intracellular location and concentration of the accumulated drug.…”

“…Propranolol is a weakly basic lipophilic compound known to interact and modify the behaviour of bilayer lipid membranes [3][4][5]. Studies have shown propranolol to be taken up into a range of cells including epithelial cells [6] and hepatocytes [7]. In the latter case, it has recently been reported that there are two intracellular sites, one of high affinity and low capacity, and the other a low affinity and nonsaturable site proposed to be the cellular membranes [7].…”

Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

“…Nevertheless these cellular systems represent only one component of the dynamic and interlinked processes occurring in liver tissue. Therefore, it remains a major challenge to predict quantitatively hepatic transport processes based on in vitro data (Bentz et al, 2005;Hallifax and Houston, 2006;Liu and Pang, 2006;Ekins et al, 2007;Webborn et al, 2007). Additional processes, such as nonspecific binding and bidirectional passive diffusion between medium and cells need to be assessed separately from active transport to enable proper mechanistic scaling.…”

Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

“…We recently reported the characteristics of drug uptake into isolated rat hepatocytes of two lipophilic, basic, prototypic drugs: imipramine and propranolol (Hallifax and Houston, 2006b). Hepatocellular uptake studied over a wide concentration range was found to be a combination of high capacity unsaturable intracellular binding and a saturable process that was dependent on cell plasma membrane integrity; the latter process was inhibited by 18 other lipophilic amine drugs.…”

“…Uptake parameters for quinine, fluvoxamine, and fluoxetine are used together with previously described data from our laboratory (Witherow and Houston, 1999;Hallifax and Houston, 2006b) on imipramine, propranolol, and dextromethorphan to form a set of six drugs with a hepatocellular uptake range of 40-fold. Saturable and nonsaturable uptake processes are explored as a function of physiochemical properties.…”