Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs

Abstract

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

Keywords: DILI; drug safety; drug-induced liver injury; microphysiological system; spheroid cultures.

Fig. 1.

Description of the 152 FDA-approved drug set. Schematic representations of the drugs in relation to the DILI concern class (DILI concern, left panel), Hepatotoxicity-related warning (Drug label, center panel), DILI description on the drug label (DILI description, right panel).

Fig. 2.

Plasma concentrations and cytotoxicity of drugs tested in hLiMTs. a) Total plasma Cmax values, b) IC50_ATP values, and c) IC50_ATP-to-total plasma Cmax ratio (C2C) of 152 FDA-approved drugs are plotted against their DILI concern class. Statistical differences between classes using Dunn’s multiple comparisons tests are shown, together with the P-value.

Fig. 3.

C2C ratio in relationship with drug label information. a) C2C ratio of 152 FDA-approved drugs plotted against their hepatotoxicity safety warning and b) C2C ratio of 98 FDA-approved drugs (“^vNo-DILI-concern” and “^vMost-DILI-concern”) plotted against their DILI description. Statistical differences between groups using Dunn’s multiple comparisons tests are denoted, along with their P-values. A dotted line is drawn at C2C = 176.

Fig. 4.

C2C scores in relationship to DILI concern class and anatomical or pharmacological group. a) “Nervous system” anatomical group. b) “Cardiovascular system” anatomical group. c) “Alimentary tract and metabolism” anatomical group. d) “Anti-infectives for systemic use” pharmacological group. Statistical differences between groups using Dunn’s multiple comparisons tests are denoted, along with their P-values. A dotted line is drawn at C2C = 176.

Fig. 5.

Reproducibility across experimental run and hepatocyte lot. C2C ratios of 20 FDA-approved drugs measured twice in hLiMTs made with 3 different 10-donor hepatocyte lots: Lot IPHH_32 (open circles), lot IPHH_24 (black diamond), and lot IPHH_18 (gray triangle). The initial measurements made in lot IPHH_32 are depicted as full black circles. A dotted line is drawn at C2C = 176.