Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Abstract

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (C max) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC 10) yielded better metrics than higher toxicity thresholds (EC 50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of C max were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC 10 and C max as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

In vitro prediction of organ toxicity: the challenges of scaling and secondary mechanisms of toxicity

“…Nevertheless, the ultimately critical criterion is if the test condition leads to a correct prediction of the clinical situation or not. Also in previous studies on hepatotoxicity (O'Brien et al 2006;Persson et al 2013;Albrecht et al 2019), a similar observation was made that higher concentrations than C max better differentiated between positive and negative control compounds. There are several potential explanations for this.…”

Future perspectives of DILI prediction in vitro

“…Although the study represents an important milestone, these numbers also illustrate a major remaining challenge; even with the very high threshold of 100 a relatively high fraction of in vivo hepatotoxic compounds is predicted as negative by the in vitro test. Several other studies used similar approaches and reported similar limitations (Xu et al 2008;Khetani et al 2013;Albrecht et al 2019;Gu et al 2018;Frey et al 2014;Hewitt et al 2007).…”

Handling deviating control values in concentration-response curves

“…Cytotoxicity with HepG2 cells was analysed using the CellTiter-Blue (CTB) assay as described in Gu et al (2018) according to the SOP in Supplement 3A. HepG2 cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) with 25 mM glucose (Albrecht et al 2019). VPA (CAS number 99-66-1; Sigma Aldrich; product number: PHR1061-1G) was directly dissolved in the culture medium to generate the concentrations indicated in the results section so that no solvent was required.…”