The Caco-2 cell line in studies of drug metabolism and efflux

Preclinical prediction of intestinal drug absorption is a continuous challenge in drug development. The absorption of a drug from the gastrointestinal tract is a complicated process, involving passive membrane permeability parameters as well as many active transport and metabolism components. Therefore, well characterised and reliable in vitro methods for studying drug absorption are constantly devised and under refinement. In this work, the Caco-2 cell line, a widely used model for intestinal drug absorption, was assessed as a platform to study the interplay of phase II metabolism and MRP (multidrug resistance associated protein) -mediated efflux. Expression and function of several metabolic enzymes and efflux proteins have been observed in the intestine and many drug conjugates produced by UDP-glucunorosyltransferases (UGTs) and sulfotransferases (SULTs) are substrates for the apical MRP2 and/or the basolateral MRPs. The kinetics of these interactions is complex, but the human origin and intestinal-like differentiation under appropriate culture conditions appoint Caco-2 cells as a potential platform for these studies. The Caco-2 cell line studied was thoroughly characterised with regards to different efflux proteins and UGT enzymes. The expression and functionality of MDR1 (multidrug resistance protein 1, P-glycoprotein) and several MRP proteins as well as UGT enzymes were observed in the studied cells, while the expression of SULTs and GSTs (glutathione-S-transferases) have been previously reported by other groups working with Caco-2. In fully differentiated Caco-2 monolayers the expression of most MRPs and UGTs was significantly higher compared to less differentiated cells grown for shorter periods or in flasks, an important observation with direct implications for the sensitivity and specificity of higher throughput cell-based screening assays. Other factors such as the passage number of the cells and the use of inducers also affected the mRNA expression levels. Based on the observed efflux and phase II metabolism activities, a Caco-2 based screening method was developed for compounds interacting with MRP2, either directly or via their phase II metabolites. The kinetics of these interactions were investigated more closely in permeability experiments, where conjugation of model compounds and the efflux of their metabolites (indomethacin glucuronide, paracetamol sulfate and naphthol glucuronide) were detected. Substrate or product inhibition of the UGT enzyme(s) was evident at higher 1naphthol concentrations, whereas the complementary role of basolateral efflux proteins was observed at the highest indomethacin concentration as the apical efflux was saturated. Pharmacokinetic modelling could be utilized as a tool for further interpretation of the results. The combined results of these studies go a long way in improving our understanding of the Caco-2 cell line and its suitability as a model system for drug absorption and metabolism in the intestine.