Received for publication February 19, 2008.
Revised May 8, 2008.
Accepted for publication May 9, 2008.

The Functional Consequences of Active Hepatic Uptake on CYP Inhibition in Rat and Human Hepatocytes

Abstract

A series of CYP inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin and pitavastatin) using hepatocytes and recombinant CYPs. The uptake was shown to be temperature-dependent and inhibited by estrone sulphate, signifying an active component. At the lowest concentrations tested, the inhibitors concentrated up to 1000-fold in rat hepatocytes, but demonstrated only 5-fold greater CYP inhibition relative to recombinant rat CYPs, indicating high intracellular binding. Inhibitor accumulation was considerably lower in human hepatocytes and an increase in inhibitory potency relative to recombinant human CYPs was not obvious. This study highlights several technical and conceptual issues when studying CYP inhibition mediated by compounds actively transported across the basolateral hepatocyte membrane. Primarily, the incubation medium concentration once the inhibitor has fully accumulated into the hepatocytes rather than the starting medium concentration, along with the extent of intracellular binding, must be considered as a foundation for in vitro-in vivo extrapolations. Additionally, it is suggested that if the K_m value for the active uptake process is close to the CYP inhibition K_i, hepatocytes may be used only to establish the free drug accumulation ratio at a clinically relevant drug concentration and this information, along with the (recombinant CYP) K_i value, utilised to simulate the likely impact of active hepatic uptake on CYP inhibition in vivo.

Key words: active transport, CYP inhibition, cytochrome P450, drug transport, drug-drug interactions, hepatic transport, hepatic uptake, hepatocytes, in vitro-in vivo prediction