Determination of Intrahepatic Exposure to the HIV Protease Inhibitor Ritonavir

Abstract

Accurate knowledge regarding intracellular drug concentrations is crucial for mechanistic understanding of drug efficacy, toxicity and exposure. The aim of this study was to develop an in vitro method to determine the unbound intrahepatic concentration of the model compound ritonavir. First, metabolism and transporter-mediated membrane passage were inhibited by incubating rat hepatocytes with high ritonavir concentrations. Hepatocytes were subsequently allowed to re-equilibrate in fresh albumin-containing medium before determination of extracellular unbound ritonavir concentrations by equilibrium dialysis. The absence of metabolism and carrier-mediated processes allowed deriving free intracellular from free extracellular concentrations. In parallel, total intracellular ritonavir concentrations were determined, enabling calculation of intracellular free fraction (fu,cell). Multiplying the total intracellular ritonavir concentration measured after incubation at a clinically relevant concentration (0.5 µM) with fu,cell resulted in unbound intracellular concentrations. Finally, Kpu,u (intra- to extracellular unbound concentration) was calculated. Fu,cell for ritonavir in rat hepatocytes was 0.0085±0.0023. The Kpu,u in hepatocytes exposed to 0.5 µM ritonavir was 2.83±0.90. This finding is consistent with substantial intrahepatic accumulation of ritonavir, an important property of this widely used pharmacokinetic booster (via CYP3A inhibition). In conclusion we have developed a new in vitro approach to determine the free intracellular concentrations of ritonavir in rat hepatocytes. Experiments are ongoing to determine the free intracellular concentrations of ritonavir in human hepatocytes and of other HIV protease inhibitors.