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An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (C max ) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC 10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.