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. 2018 Jun 1;163(2):655-665.
doi: 10.1093/toxsci/kfy058.

Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions

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Free PMC article

Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions

Sabine U Vorrink et al. Toxicol Sci. .
Free PMC article

Abstract

High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.

Figures

Figure 1.
Figure 1.
Scheme of the experimental outline. The experimental schedule for all experiments included in this study is shown for both human and animal cells.
Figure 2.
Figure 2.
Overview of the compound panel tested in this study. A, Pie chart showing the classification of 123 compounds into 70 that have been associated with DILI events in the clinic (shades of red) and 53 that have not been reported to cause clinically apparent liver injury (green). B, Venn-diagram visualizing the reported DILI patterns for the 70 DILI positive compounds. C, Pie chart depicting the distribution of the 123 tested compounds across therapeutic areas. D, Therapeutic cmax serum concentrations of DILI positive and DILI negative medications are overall similar. E, Most drugs tested in this study are extensively metabolized by the liver. Overall, the DILI negative compounds show a slight tendency for less hepatic metabolism compared with DILI positive drugs.
Figure 3.
Figure 3.
Compounds implicated in clinical DILI events exhibit substantial hepatotoxicity in the CD-spheroid system. Overview of the experimentally determined viabilities for DILI negative (green) and DILI positive (red) compounds at 1×, 5×, and 20× of the therapeutic serum concentration (cmax) after 2 weeks of exposure. Viability as determined by ATP quantifications relative to untreated controls is shown. The dashed line indicates viability of the respective control spheroids (100%). The gray shaded box highlights the viability interval in which compounds are classified as hepatotoxic (<80%). Note that viability decreases dose-dependently when hepatocytes are exposed to DILI positive but not to DILI negative compounds. Error bars indicate SD **, ***, and **** indicate p < .01, p < .001, and p < .0001 in a 2-tailed heteroscedastic t-test, respectively.
Figure 4.
Figure 4.
The CD-spheroid model faithfully flags compounds with hepatotoxic liabilities. Results of experimental hepatotoxicity assessments are shown for compounds causing severe DILI (n = 36; A) for drugs with DILI concern (n = 34; B) and for DILI negative compounds (n = 53; C). Red boxes indicate that the average hepatocyte viability was decreased to <80% of the respective controls with statistical significance (p < .05, mostly n = 6–8 biological replicates per condition). If these conditions are not met, the evaluation is classified negative (green). Note that drugs for which toxicity was indicated at low concentrations followed by nontoxic evaluations at higher concentrations were predicted to be DILI negative (iproniazid and propranolol). D, Overall, the spheroid model flagged 69% of DILI positive compounds as hepatotoxic without any FP results (specificity 100%). * Note that the highest exposure concentration for gabapentin was 17.3× cmax.
Figure 5.
Figure 5.
The CD-spheroid model can distinguish between hepatotoxic and nonhepatotoxic structural analogues. Hepatotoxicity of the structurally related antimalarials primaquine (DILI negative) and amodiaquine (DILI positive; A), the antidiabetic PPAR agonists rosiglitazone (DILI negative) and troglitazone (DILI positive; B), the antidepressant amoxapine (DILI negative) and the antipsychotic clozapine (DILI positive; C), the anxiolytics buspirone (DILI negative) and nefazodone (DILI positive; D), as well as the endothelin receptor antagonists ambrisentan (DILI negative) and bosentan (DILI positive; E) were compared in 3D PHH spheroids. Note that all 5 DILI positive compounds were clearly identified as hepatotoxic, whereas their DILI negative analogues revealed no toxic liability. The dashed line indicates viability of respective control spheroids (100%). The gray shaded boxes highlight the viability interval in which compounds are classified as hepatotoxic (<80%). *, **, and *** indicate p < .05, p < .01, and p < .001 in a 2-tailed heteroscedastic t-test, respectively.
Figure 6.
Figure 6.
Cross-comparison of spheroids generated from primary hepatocytes of important preclinical animal model species. A, Viability quantifications of spheroids established from primary hepatocytes from BL6 and CD1 mice, as well as from Wistar rat, minipig, rhesus monkey, and human after 2 weeks of exposure are shown (n = 6–8 biological replicates per condition). Different shades of green and red indicate exposure to 1×, 5×, and 20× the therapeutic exposure levels (cmax) in humans for DILI negative and DILI positive compounds, respectively. The dashed line indicates viability of the respective species controls (100%). The grey shaded boxes highlight the viability interval in which compounds are classified as hepatotoxic (<80%). Error bars indicate SD. B, Based on data shown in A, predictions about the toxic liability of compounds across species are shown. Red and green boxes indicate hepatotoxic and nonhepatotoxic predictions, respectively. C, Scatter plots depicting the concordance between human (abscissa) and animal (ordinate) viability quantifications for each of the 11 tested compounds. Note that while the concordance between toxicity evaluations across species are overall similar at the highest (20×) exposure concentration, toxicity predictions at low (1×), and intermediate (5×) concentrations are highly discrepant, suggesting substantial variability in thresholds at which hepatotoxicity manifests across species.

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