Duloxetine (DLX) has been approved for the successful treatment of psychiatric diseases, including major depressive disorder, diabetic neuropathy, fibromyalgia and generalized anxiety disorder. However, since the usage of DLX carries a manufacturer warning of hepatotoxicity given its implication in numerous cases of drug-induced liver injuries (DILI), it is not recommended for patients with chronic liver diseases. In our previous study, we developed an enhanced human-simulated hepatic spheroid (EHS) imaging model system for performing drug hepatotoxicity evaluation using the human hepatoma cell line HepaRG and the support of a pulverized liver biomatrix scaffold, which demonstrated much improved hepatic-specific functions. In the current study, we were able to use this robust model to demonstrate that the DLX-DILI is a human CYP450 specific, metabolism-dependent, oxidative stress triggered complex hepatic injury. High-content imaging analysis (HCA) of organoids exposed to DLX showed that the potential toxicophore, naphthyl ring in DLX initiated oxidative stress which ultimately led to mitochondrial dysfunction in the hepatic organoids, and vice versa. Furthermore, DLX-induced hepatic steatosis and cholestasis was also detected in the exposed EHSs. We also discovered that a novel compound S-071031B, which replaced DLX's naphthyl ring with benzodioxole, showed dramatically lower hepatotoxicities through reducing oxidative stress. Thus, we conclusively present the human-relevant EHS model as an ideal, highly competent system for evaluating DLX induced hepatotoxicity and exploring related mechanisms in vitro. Moreover, HCA use on functional hepatic organoids has promising application prospects for guiding compound structural modifications and optimization in order to improve drug development by reducing hepatotoxicity.
Chemical and Drug Induced Liver Injury , Depressive Disorder, Major , Fatty Liver , Duloxetine Hydrochloride/pharmacology , Humans
Background: Recent breakthroughs in cholangiocarcinoma (CCA) genomics have led to the discovery of many unique identifying mutations, of which HER2 has been found to be overexpressed specifically in cases of extrahepatic CCA. However, whether or not lapatinib (an oral tyrosine kinase inhibitor selective for inhibition of HER2), or a combination of lapatinib and gemcitabine, exerts inhibitory effects on HER2-overexpressed CCA is still unclear. Methods: The effect of lapatinib and a lapatinib-gemcitabine combination treatment on CCA was determined using organoid and cell line models. Cell cycle arrest, apoptosis and proteins involving HER2-dependent downstream signaling pathways were analyzed to assess the effect of lapatinib on HER2+ CCA. The synergistic effect of lapatinib and gemcitabine was interpreted by docking analysis, ABCB1-associated ATPase assay, rhodamine transport assay and LC-MS/MS analyses. Results: dFdCTP, the active metabolite of gemcitabine, is proved to be the substrate of ABCB1 by docking analysis and ATPase assay. The upregulation of ABCB1 after gemcitabine treatment accounts for the resistance of gemcitabine. Lapatinib exerts a dual effect on HER2-overexpressed CCA, suppressing the growth of CCA cells by inhibiting HER2 and HER2-dependent downstream signaling pathways while inhibiting ABCB1 transporter function, allowing for the accumulation of active gemcitabine metabolites within cells. Conclusions: Our data demonstrates that lapatinib can not only inhibit growth of CCA overexpressing HER2, but can also circumvent ABCB1-mediated chemoresistance after gemcitabine treatment. As such, this provides a preclinical rationale basis for further clinical investigation into the effectiveness of a combination treatment of lapatinib with gemcitabine in HER2-overexpressed CCA.
