The interplay of multiple ADME mechanisms, prediction of hepatic drug-drug interactions from in vitro

This thesis was previously held under moratorium in Chemistry Department (GSK) from 23rd September 2014 until 14th November 2016.Rosuvastatin has poor passive membrane permeability and its uptake into the liver is mediated predominately by the transporter, Organic Anion Transporting Polypeptide 1B1 (OATP1B1). Cyclosporin A (CsA) is a potent inhibitor of a range of transporters, including OATP1B1 and clinical drug-drug interactions (DDI) have been reported with rosuvastatin. The aim of this study was to determine the uptake kinetics of rosuvastatin in human hepatocytes using a mechanistic model and to determine the inhibitory effect of CsA upon those kinetics. These data may then allow the extrapolation of in vitro to in vivo kinetics and provide an understanding of the interplay between different disposition mechanisms, with particular regard to the potential to predict DDI.This study was divided into method development and experimental phases. In the development phase, paediatric hepatocytes from a single donor were used to develop the methods. The uptake parameters (Vmax, Km,u, Pdiff,u, Fucell and CLuptake) using estradiol-17β-D-glucuronide (EG) and rosuvastatin were determined using a mechanistic two-compartment model developed by Menochet et al (2012a). The uptake of rosuvastatin was also determined using sodium free media, which prevents the efficient functioning of the uptake transporter Sodium Taurocholate Dependent Transporter (NTCP), another transporter thought to contribute to the uptake of rosuvastatin. Inhibition parameters (IC50) of the uptake of EG and rosuvastatin by CsA and rosuvastatin and rifampicin were determined.The uptake kinetic parameters of EG and rosuvastatin in the paediatric human hepatocytes were in agreement with the quoted literature values for adult human hepatocytes. The hepatocytes were robust enough to be used for method development and to plan for the future studies. The IC50 values for EG and rosuvastatin as the probe substrates using CsA and rifampicin as inhibitors were in agreement with quoted literature values and suggested a predominate role for OATP1B1 in EG and rosuvastatin uptake. In these paediatric human hepatocytes NTCP did not appear to play a role in the uptake of rosuvastatin.The paediatric human hepatocyte data were used to help define and refine the studies conducted in the experimental phase of the study. The same uptake parameters for rosuvastatin were determined in human hepatocytes from three adult donors using the mechanistic two-compartment model. The time of the incubation was extended to 60 minutes to ensure that steady state kinetics were reached. Inhibition of the uptake of rosuvastatin was determined with co- and pre-incubation of CsA and its main metabolite, AM1. The time-dependent nature of the inhibitors have been studied by ourselves, but not in human hepatocytes [Gertz et al 2012]. There did not appear to be an effect of co- versus pre-incubation of the inhibitors.The mechanistic two-compartment model was also used to determine the uptake parameters for a GSK compound, namely GSK2879552, using the same adult human hepatocytes from three adult donors. However, the hepatic uptake clearance (CLuptake) values suggested that active saturable uptake of GSK2879552 was not evident. This may explain the high variability observed between the three donors for the uptake parameters (Vmax, Km,u, Pdiff,u and Fucell) and the high coefficient of variation observed about each parameter. These data provided a useful learning with regards to understanding the limitations of the model.Rosuvastatin has poor passive membrane permeability and its uptake into the liver is mediated predominately by the transporter, Organic Anion Transporting Polypeptide 1B1 (OATP1B1). Cyclosporin A (CsA) is a potent inhibitor of a range of transporters, including OATP1B1 and clinical drug-drug interactions (DDI) have been reported with rosuvastatin. The aim of this study was to determine the uptake kinetics of rosuvastatin in human hepatocytes using a mechanistic model and to determine the inhibitory effect of CsA upon those kinetics. These data may then allow the extrapolation of in vitro to in vivo kinetics and provide an understanding of the interplay between different disposition mechanisms, with particular regard to the potential to predict DDI.This study was divided into method development and experimental phases. In the development phase, paediatric hepatocytes from a single donor were used to develop the methods. The uptake parameters (Vmax, Km,u, Pdiff,u, Fucell and CLuptake) using estradiol-17β-D-glucuronide (EG) and rosuvastatin were determined using a mechanistic two-compartment model developed by Menochet et al (2012a). The uptake of rosuvastatin was also determined using sodium free media, which prevents the efficient functioning of the uptake transporter Sodium Taurocholate Dependent Transporter (NTCP), another transporter thought to contribute to the uptake of rosuvastatin. Inhibition parameters (IC50) of the uptake of EG and rosuvastatin by CsA and rosuvastatin and rifampicin were determined.The uptake kinetic parameters of EG and rosuvastatin in the paediatric human hepatocytes were in agreement with the quoted literature values for adult human hepatocytes. The hepatocytes were robust enough to be used for method development and to plan for the future studies. The IC50 values for EG and rosuvastatin as the probe substrates using CsA and rifampicin as inhibitors were in agreement with quoted literature values and suggested a predominate role for OATP1B1 in EG and rosuvastatin uptake. In these paediatric human hepatocytes NTCP did not appear to play a role in the uptake of rosuvastatin.The paediatric human hepatocyte data were used to help define and refine the studies conducted in the experimental phase of the study. The same uptake parameters for rosuvastatin were determined in human hepatocytes from three adult donors using the mechanistic two-compartment model. The time of the incubation was extended to 60 minutes to ensure that steady state kinetics were reached. Inhibition of the uptake of rosuvastatin was determined with co- and pre-incubation of CsA and its main metabolite, AM1. The time-dependent nature of the inhibitors have been studied by ourselves, but not in human hepatocytes [Gertz et al 2012]. There did not appear to be an effect of co- versus pre-incubation of the inhibitors.The mechanistic two-compartment model was also used to determine the uptake parameters for a GSK compound, namely GSK2879552, using the same adult human hepatocytes from three adult donors. However, the hepatic uptake clearance (CLuptake) values suggested that active saturable uptake of GSK2879552 was not evident. This may explain the high variability observed between the three donors for the uptake parameters (Vmax, Km,u, Pdiff,u and Fucell) and the high coefficient of variation observed about each parameter. These data provided a useful learning with regards to understanding the limitations of the model