Rat precision-cut liver slices predict drug-induced cholestatic injury

Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 μM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC

Ex vivo preparations of human tissue for drug metabolism, toxicity and transport

Before new drugs are allowed on the market, their safety and metabolite profile should be extensively tested, as often reactive metabolites are the ultimate toxicant. The exposure of the target cell to the drug and its metabolites is determined by the expression levels of the transporters and the metabolic enzymes in the target cells. Thus to predict drug-induced toxicity not only the intrinsic biological effects of the drug but also the exposure to the ultimate toxicant needs to be predicted. In the past decades, in vitro techniques were increasingly used to predict drug-induced toxicity, not only to reduce the use of experimental animals and to obtain insight in the mechanism, but also to be able to obtain human-specific information by using human cells. Although cellular and subcellular models have been proven to be very useful, they do not fully represent the complex intact tissues, and very often the tissue-specific differentiation is lost in cell lines. In the liver the presence of the Kupffer, stellate, biliary epithelial and endothelial cells are of importance for the toxic effects of drugs. In the intestine the situation is even more complex as the expression levels of the relevant transporters and metabolic enzymes show significant gradients along the length of the intestine. Therefore ex vivo tissue models have proven to be useful to fill the gap between in vitro cell models and the in vivo situation. In this lecture the use of ex vivo models of liver and intestinal tissue are presented. In precision-cut tissue slices, prepared from liver or intestine, all the cell types are present in their original conformation and cell-cell and cell-matrix interactions are intact. Their viability is maintained for up to several days and metabolism and toxic effects can be studied using innovative analytical methods including omics technologies. The Ussing Chamber technique allows to study the vectorial transport of drugs from the lumen to the serosal side and vice versa in addition to their metabolism and toxicity. These techniques can be applied to animal and human tissue and allow to study interspecies differences under identical experimental circumstances. Recent improvements in the technology and applications in prediction of toxicity will be discussed

Prevalidation of liver slices as a model for the early onset of liver fibrosis to test anti-fibrotic drugs

Liver fibrosis is the progressive accumulation of connective tissue that affects the normal function of the liver and will eventually lead to liver cirrhosis. The aim of this study is to prevalidate precision-cut liver slices (PCLS) as an in vitro model to investigate the early onset of liver fibrosis and as a test system for anti-fibrotic drugs. Rat PCLS were incubated up to 48 hours, viability was assessed by ATP content and the gene expression of the fibrotic markers Heat Shock Protein 47 (HSP47), α-Smooth Muscle Actin (αSMA) and Pro-collagen1A1 (PCOL1A1) was determined. Furthermore, during 48 hours the effects of anti-fibrotic drugs inhibiting the PDGF signaling pathway (Gleevec (G), Sorafenib (So) and Sunitinib (Su)) and drugs inhibiting the TGFβ signaling pathway (Valproic acid (Va), Perindopril (Pe)) and drugs that directly affect the collagen expression (Colchicine (Co), Pirfenidone (Pi) and Tetrandrine (Te)), all used at a non-toxic concentration, were determined. In PCLS incubated up to 48 hours an increased gene expression of HSP47, αSMA and PCOL1A1 was found. Addition of G, So or Su resulted in an inhibition of the gene expression of 60, 75 and 80% (G), 35, 60 and 70% (So) and 60, 80 and 90% (Su) of HSP47, αSMA and PCOL1A1 respectively. However, only the highest concentration of Va inhibited the gene expression of the fibrotic markers by 60%. Pe inhibited the gene expression of PCOL1A1 only at the highest concentration by 20%. Co inhibited gene expression of αSMA and PCOL1A1 by 50%, while Pi caused an inhibition of the gene expression of HSP47 (30%), αSMA (60%) and PCOL1A1 (70%) and Te caused only an inhibition of the gene expression of PCOL1A1 by 25%. Rat PCLS incubated for 48 hours show characteristics of early onset of liver fibrosis. This effect is inhibited by anti-fibrotic drugs acting directly on collagen and on the PDGF-pathway, but not on the TGFβ pathway, indicating that PCLS are a model for testing such anti-fibrotic compounds in vitro