The Amphioxus ACTIVTox System

1. The Amphioxus ACTIVTox� System A human tumor-derived, aneuploid cell line proposed as a in vitro high throughput screen for predicting human hepatotoxicity. The system was introduced to the Hepatotoxicity Steering Committee at the last meeting in October. Derived from HepG2 cells. Cytotoxicity is the primary endpoint. Given the inherent limitations of any immortalized cell line for predicting complex human organ toxicity, additional supporting information is required.

2. The Amphioxus ACTIVTox� System The system appears to have low cytochrome P-450 activity compared to freshly isolated hepatocytes. Although hepatic CYP1A plays a minor role in the metabolism of most drugs, CYP1A is relatively high (characteristic of such systems) and the rest of the activities are low, leaving the cell metabolically unbalanced. While the induction of CYP3 can be achieved, the mechanistic inhibition of CYP3A4 is of greater clinical concern, and CYP induction per se is not directly relevant to predictive toxicity screening. Comparison of data to that in the literature for HepG2 reveals many similarities. Thus, clarification of potential advantages would be desirable.

3. The Amphioxus ACTIVTox� System More comparative data with human primary hepatocytes, including a wider range of compounds, would be desirable (i.e. head to head comparison with HepG2). Phase 2 metabolic capability (e.g. glucuronidation), an important detoxification pathway in vivo, has not been demonstrated. Would this give inaccurate cytotoxicity rankings for agents whose toxicity is influence by the status of these pathways (e.g. acetaminophen and Trovan)? With cytotoxicity as the end point, how are the data interpreted in regard to relevant human exposure levels and the important influences of absorption, distribution, meatabolism and excretion which govern the in vivo response?

4. The Amphioxus ACTIVTox� System Trovan appears toxic at concentrations that are not consistent with clinical data. In the second cytotoxicity study conducted with Trovan a decrease in absorbance occurred at a much lower drug concentration that in the first study. Was a control included to ensure that the drug does not quench absorbance. It is not clear if protein binding is accounted for in the assay. We would be interested in learning more about assay conditions. We suggest a meeting of the Preclinical Subcommittee with Amphioxus to gain a better understanding of the potential utility of this cell line as presented.