Unbound Ritonavir Concentrations in Rat and Human Hepatocytes

Knowledge regarding intracellular drug exposure is crucial to gain mechanistic understanding of hepatic disposition. This study aims to develop an approach to determine unbound intracellular concentrations (Cu,cell) of ritonavir. Ritonavir was selected as a model drug as incubations with high ritonavir concentrations inhibited all saturable processes involved in ritonavir disposition including metabolism and transporter-mediated membrane passage. Following this incubation, hepatocytes were re-equilibrated in fresh protein-containing medium before determination of extracellular unbound ritonavir concentrations. In the absence of metabolism and transport, unbound intracellular and unbound extracellular concentrations were identical. In parallel, total intracellular ritonavir concentrations (Ccell) were determined, enabling the calculation of intracellular free fractions (fu,cell). Additionally, Ccell was determined after exposing hepatocytes to a therapeutically relevant concentration (0.5 μM). Multiplication of this concentration with fu,cell resulted in Cu,cell. Finally, Kpu,u (intracellular unbound drug accumulation ratio) was calculated. Exposure of rat and human hepatocytes to 0.5 ^M ritonavir resulted in Cu,cell of 12 ± 1 and 8 ± 1 nM. Corresponding Kpu,u values were 2.7 ± 0.5 and 1.4 ± 0.2. We present an in vitro method to determine Cu,cell of ritonavir in intact hepatocytes. Cu,cell obtained at clinically relevant extracellular concentrations are in accordance with concentrations known to inhibit cytochrome P450 and are achieved because of ritonavir accumulation in hepatocytes. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2378-2387, 2015