Evaluation of a Novel Renewable Hepatic Cell Model for Prediction of Clinical CYP3A4 Induction Using a Correlation-Based Relative Induction Score Approach.

Metabolism enzyme induction-mediated drug-drug interactions need to be carefully characterized in vitro for drug candidates to predict in vivo safety risk and therapeutic efficiency. Currently, both the Food and Drug Administration and European Medicines Agency recommend using primary human hepatocytes as the gold standard in vitro test system for studying the induction potential of candidate drugs on cytochrome P450 (CYP), CYP3A4, CYP1A2, and CYP2B6. However, primary human hepatocytes are known to bear inherent limitations such as limited supply and large lot-to-lot variations, which result in an experimental burden to qualify new lots. To overcome these shortcomings, a renewable source of human hepatocytes (i.e., Corning HepatoCells) was developed from primary human hepatocytes and was evaluated for in vitro CYP3A4 induction using methods well established by the pharmaceutical industry. HepatoCells have shown mature hepatocyte-like morphology and demonstrated primary hepatocyte-like response to prototypical inducers of all three CYP enzymes with excellent consistency. Importantly, HepatoCells retain a phenobarbital-responsive nuclear translocation of human constitutive androstane receptor from the cytoplasm, characteristic to primary hepatocytes. To validate HepatoCells as a useful tool to predict potential clinical relevant CYP3A4 induction, we tested three different lots of HepatoCells with a group of clinical strong, moderate/weak CYP3A4 inducers, and noninducers. A relative induction score calibration curve-based approach was used for prediction. HepatoCells showed accurate prediction comparable to primary human hepatocytes. Together, these results demonstrate that Corning HepatoCells is a reliable in vitro model for drug-drug interaction studies during the early phase of drug testing.

Three-Dimensional Spheroids With Primary Human Liver Cells and Differential Roles of Kupffer Cells in Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) remains a challenge and a leading risk for drug discovery. Three-dimensional liver spheroids made from primary human hepatocytes (PHHs) with, or without, other liver cell types can provide more physiological relevance. In comparison to conventional 2-dimensional monolayer culture, our tests with 100 drugs of known DILI status indicate that PHH spheroids are significantly more sensitive in detecting drug-induced hepatotoxicity. To evaluate the role of Kupffer cells (KCs) in drug-induced liver toxicity, we have established conditions for generating co-culture spheroids with PHH and KCs. Inflammatory responses as shown by interleukin 6 secretion can be recapitulated in co-culture spheroids when treated with endotoxin lipopolysaccharides. KCs potentiated the cytotoxicity induced by trovafloxacin in co-culture spheroids at 48 h, but the differences between PHH spheroids and co-culture spheroids became less obvious after a 5-day treatment. Interestingly, a protective role of KCs was shown in co-culture spheroids treated with both acetaminophen and lipopolysaccharides. Additional tests with 14 DILI compounds comparing PHH spheroids and co-culture spheroids showed differential roles of KCs that were compound dependent. In summary, these 3-dimensional liver spheroid models are useful tools to understand the complex mechanisms underlying DILI.

Comparative membrane channel size and activity of botulinum neurotoxins A and E.

In an effort to compare the molecular basis of differential toxic activity of botulinum neurotoxin A (BoNT/A) and BoNT/E, we have analyzed their membrane channel activity by measuring calcein release from liposomes. Both BoNT/A and /E showed a same level of membrane channel activity that was specifically blocked by IgG specific to the neurotoxins. With the use of fluorescein-labeled dextran, we determined that the size of the channel is at least 24.2 A which is appropriate for the translocation of a protein of 50 kDa (the light chain of BoNT). These findings would suggest that the difference in the toxicity level of the two BoNT serotypes might reflect differences in either endopeptidase activity or their binding to receptor(s).