Functional Consequences of Active Hepatic Uptake on Cytochrome P450 Inhibition in Rat and Human Hepatocytes

A series of cytochrome P450 (P450) inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin, and pitavastatin) using hepatocytes and recombinant P450s. The uptake was shown to be temperature-dependent and was inhibited by estrone sulfate, 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 P450 inhibition relative to recombinant rat P450s, indicating high intracellular binding. Inhibitor accumulation was considerably lower in human hepatocytes and an increase in inhibitory potency relative to recombinant human P450s was not obvious. This study highlights several technical and conceptual issues in the study of P450 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 P450 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 P450) K i value, may be used to simulate the likely impact of active hepatic uptake on P450 inhibition in vivo.