Integration of an in vitro renal cellular set of tools with a systems biology modelling to predict the renal contribution to the pharmacokinetics

A suite of hepatic metabolic in vitro tools, such as recombinant enzymes and hepatocytes, have been integrated with systems biology modelling (SBM) to predict drug pharmacokinetics (PK) in animals. This approach has become a stalwart in the discovery of new drugs over the last 5 years and has led to a significant reduction in the use of animals as well as a better mechanistic understanding of the processes that drive the PK of a drug. However, limited research has been carried out on in vitro renal cellular tools integrated with SBM to predict the renal contribution to the PK of a drug. Currently, renal drug clearance (CLr) is predicted in human by carrying out invasive PK studies in animals and investigating whether an empirical relationship (ER) exists between the different species. In many cases an ER is not established due to species differences in membrane transporter activity and the prediction of the renal contribution to the PK of a drug for a target species becomes uncertain. In addition to the prediction of CLr, an established renal in vitro cellular set of tools in conjunction with SBM would be able to predict renal drug-drug interactions (DDIs). Vertex Pharmaceuticals are currently setting up both primary proximal (PTC) and primary distal tubular cell (DTC) monolayers in a transwell format using established methods from rat, dog and human kidney tissues. The proposed project involves (a) knowledge transfer of the transwell cultures from Vertex to the University of Nottingham and measuring expression levels of the main nephron membrane drug transporters (year 1). Then (b) the apparent permeabilities for a set of drugs that have in vivo CLr data in all 3 species will be measured using the cell monolayers (year 2). A physiological based PK (PBPK) mathematical model will be developed for the kidney that includes in vitro inputs for permeability at PTC and DTC sites in the nephron as well as for pH, GFR and kidney blood flow (years 2-3). The predicted CLr from this mathematical model will be compared with the clinically measured values from the literature and conclusions drawn. Further studies will be carried out that investigate the effect of established renal drug transport inhibitors and/or gene silencing methods on the apparent permeability of the drugs investigated in the cell monolayers described above and the PBPK model will be used to predict the impact of transporter inhibition on the PK of the victim drug and then compared with known clinical renal DDIs (year 3-4). Techniques used will include cell culturing in transwell plates and gene silencing techniques. Protein (transporter) expression will include immunofluorescence, dot blot and western blot. Drug permeability measurements will involve bidirectional transepithelial flux measurements of substrates across monolayers of pure PTC and DTC and will be measured at steady state using LC/MS/MS. PBPK models will be built using commercial PK modelling software. The industrial partners are very interested in understanding both the prediction of CLr and renal drug-drug interactions using an in vitro based approach integrated with SBM. Thus, the project proposed crosses two key BBSRC priorities of (1) a systems biology approach in which experimental biology is closely-integrated with mathematical or computational modelling in a synergistic way to answer biological questions that would be not be possible by empirical approaches alone and (2) the 3 Rs in research using animals.