High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells.

Drug-induced liver injury (DILI) results in the termination of drug development or withdrawal of a drug from the market. The establishment of a predictive, high-throughput preclinical test system to evaluate potential clinical DILI is therefore required. Here, we established a high content analysis (HCA) assay in human hepatocyte cell lines such as the HepaRG with normal expression levels of CYP enzymes and HepG2 with extremely low expression levels of CYP enzymes. Clinical DILI or non-DILI compounds were evaluated for reactive oxygen species (ROS) production, glutathione (GSH) consumption, and mitochondrial membrane potential (MMP) attenuation. A proportion of DILI compounds induced ROS generation, GSH depletion, and MMP dysfunction, which was consistent with reported mechanisms of DILI of these compounds. In particular, DILI compounds that deplete GSH via reactive metabolites exhibited a more marked decrease in intracellular GSH or increase in ROS production in HepaRG cells than in HepG2 cells. Comparison of the two cell lines with different levels of CYP expression might help clarify the contribution of metabolism to hepatocyte toxicity. These results suggest that the HCA assay in HepaRG and HepG2 cells might help improve the accuracy of evaluating clinical DILI potential during drug screening.

Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614.

We evaluated antidiabetic effects of 3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3 H-benzimidazole-5-carboxamide (FK614), a benzimidazole derivative without a thiazolidinedione structure, which was obtained using C57BL/KsJ-db/db mice (db/db mice). In db/db mice, the potency of FK614 for hypoglycemic effect was comparable to that of rosiglitazone and approximately 15-fold greater than that of pioglitazone. FK614 also showed a potent attenuating effect on hypertriglyceridemia in db/db mice, as well as rosiglitazone and pioglitazone. In C57BL/6J-ob/ob mice (ob/ob mice), ED(50) values of FK614 and pioglitazone for hypoinsulinemic effect were 1.3 and 11.8 mg/kg, respectively. FK614 also improved the impaired glucose tolerance in ob/ob mice. In normal rats, FK614 did not influence plasma glucose and insulin levels but significantly decreased both plasma triglyceride and nonesterified fatty acid levels. FK614 was found to activate peroxisome proliferator-activated receptor (PPAR)gamma-mediated transcriptional activity in the reporter gene assay as well as thiazolidinedione derivatives, although its maximum effect was less than that of thiazolidinedione derivatives. In rat toxicity studies, hemodilution effects for FK614 were less than that for rosiglitazone. Overall, these studies suggest that FK614 improves insulin resistance in such animal models through activation of PPARgamma-mediated transcriptional activity and that it would be a new therapeutic candidate with potential for the treatment of type 2 diabetic patients.