HepG2 (C3A) spheroids show higher sensitivity compared to HepaRG spheroids for drug-induced liver injury (DILI).

High-throughput, automation-friendly and therapeutically-predictive assays are needed in early drug discovery in order to prioritise compounds and reduce the risk of new drugs causing Drug-Induced Liver Injury (DILI). We evaluated the suitability of high-throughput 3D liver spheroid models of HepG2 (C3A clone) and HepaRG cell lines to predict DILI in early drug development. Spheroids were formed in 384-well ultra-low-attachment plates and dosed via direct acoustic droplet ejection at nine half-log spaced concentrations per compound. Spheroid viability was quantified with an ATP endpoint after a 4-day incubation with 150 drugs with known DILI liability. We derived a margin of safety for each cell line defined as the ratio between the IC50 values generated for each compound to their maximum plasma concentration Cmax which resulted in optimal classification accuracy. The margin of safety can be used to estimate a maximum safe Cmax for compounds in early drug discovery for which Cmax is not yet known. Both cell lines had similar level of accuracy in predicting DILI, with HepG2 spheroids being more sensitive. HepG2 spheroids had a sensitivity of 58% and a specificity of 83%, while HepaRG spheroids had a sensitivity of 47% and specificity of 86%. Ninety-nine of the 150 compounds were used to compare the relative sensitivities of HepG2 and HepaRG spheroids. HepaRG spheroids were more sensitive to 7 compounds and HepG2 spheroids were more sensitive to 34 compounds. In conclusion, across a diverse group of drugs HepG2 spheroids were more predictive of DILI compared to HepaRG spheroids.

A multi-center preclinical study of gadoxetate DCE-MRI in rats as a biomarker of drug induced inhibition of liver transporter function.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Idiosyncratic toxicity: the role of toxicophores and bioactivation.

Drugs and chemicals can undergo enzyme-catalyzed bioactivation reactions within cellular systems, with the formation of reactive chemical species. These reactive metabolites can lead to thiol depletion, reversible protein modification (glutathionylation and nitration), further irreversible protein adduct formation and subsequent irreversible protein damage. The incorporation of potentially reactive chemical moieties - toxicophores - within new therapeutic agents should be limited. However, this cannot always be prevented, particularly when the structural feature responsible for toxicity is also responsible for the pharmacological efficacy. The identification and further knowledge of critical levels of thiol depletion and/or covalent modification of protein will aid in the development of new drugs. Importantly, the identification of drug-thiol conjugation should provide a warning of potential problems, yet not hinder the development of a potentially therapeutically useful drug.