Abstract
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 (Cmax) 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 EC10 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.
Highlights
Prediction of drug-induced liver injury (DILI) remains a major challenge [1,2,3]
An advantage of the published procedure is that it allows for the optimization of an in vitro test system by calculating two novel metrics - the toxicity esti mation index (TEI) and toxicity separation index (TSI) - where the former determines how well hepatotoxic blood concentrations in vivo can be estimated for hepatotoxic compounds, and the latter determines the degree of separation of hepatotoxic from non-hepatotoxic test sub stances
We established a technique that allows for the determina tion of hepatotoxicity in relation to oral doses and blood concentrations [4] based solely on CTB test results generated in cultured human he patocytes as the single in vitro readout
Summary
Prediction of drug-induced liver injury (DILI) remains a major challenge [1,2,3]. Recently, an in vitro/in silico method was established that determines the risk of human DILI in relation to oral doses and blood concentrations of a test compound [4]. An advantage of the published procedure is that it allows for the optimization of an in vitro test system by calculating two novel metrics - the toxicity esti mation index (TEI) and toxicity separation index (TSI) - where the former determines how well hepatotoxic blood concentrations in vivo can be estimated for hepatotoxic compounds, and the latter determines the degree of separation of hepatotoxic from non-hepatotoxic test sub stances These metrics are calculated for a specified set of test com pounds based on i) the concentration where cytotoxicity in cultured human hepatocytes first becomes evident, and ii) the maximal blood concentration (Cmax) of a dose of interest obtained by pharmacokinetic modeling. We previously demonstrated that considering the expression of a set of toxicity indicator genes in addition to cyto toxicity to determine the lowest in vitro alert concentration resulted in a better TEI than cytotoxicity alone [4]
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