Impact of hepatic transporter multiplicity and loss-of-function on hepatocellular distribution and excretion of drugs

The objective of this research project was to develop preclinical and clinical tools to assess hepatocellular exposure and multiplicity of transport. A variety of model systems were employed, including membrane vesicles, sandwich-cultured hepatocytes (SCH), rat isolated perfused livers (IPLs), and in vivo human studies; a truly translational approach. This research focused primarily on three drugs to evaluate transporter function: 99mTechnetium-mebrofenin (MEB), ritonavir (RTV), and rosuvastatin (RSV). A semi-physiologically-based pharmacokinetic (semi-PBPK) modeling and in vitro systems translational approach suggested a transporter-mediated drug-drug interaction (DDI) between MEB and RTV at both hepatic and extrahepatic sites of MEB distribution, observed using a novel clinical protocol. Rather than a specific multidrug resistance-associated protein (MRP) 2 probe, this clinical study demonstrated the utility of MEB as a model organic anion to evaluate DDIs in overall (uptake and efflux) transporter-mediated hepatobiliary clearance. A novel application of classic approaches to determine whole tissue distribution (differential centrifugation, equilibrium dialysis) was investigated with a set of probe drugs in the SCH model system, highlighting the finding that total tissue accumulation and intracellular unbound concentrations can be predicted using SCH, including the relative contribution of active transport vs. binding/sequestration as potential mechanisms of hepatocellular accumulation. A novel uptake and efflux protocol in SCH, coupled with pharmacokinetic modeling, was developed in order to elucidate the relative contribution of basolateral and biliary efflux to the hepatocellular elimination of RSV. This approach, along with IPL data, revealed that basolateral and biliary efflux represent alternative pathways with a quantitatively similar contribution to total hepatocellular excretion of RSV; the role of basolateral efflux in the hepatocellular disposition of RSV has not been investigated previously. RSV transport was evaluated in human SCH and membrane vesicles prepared from MRP3- and MRP4-overexpressing cells, demonstrating that RSV is an efficient MRP4 substrate. This research resulted in a number of important and novel contributions to the preclinical and clinical "toolkit" available to study hepatic drug disposition. These developments will significantly enhance the ability to assess and predict altered hepatobiliary drug disposition due to DDIs, genetic variation and/or disease states, and will ultimately lead to improved safety and efficacy of medications.