For Early Toxicity Testing
This very affordable screen requires only 6 mg of compound and provides data in less than three weeks.
The development of a new drug from concept to market requires 12 to 15 years of research and development and nearly $1 billion. It is estimated that 40% to 60% of new drugs that enter preclinical animal testing are dropped because of unexpected toxicity.
Late stage failure of promising new compounds can be reduced by building more robust data sets that are focused on toxicity. Well designed cell-based in vitro screening systems that have in vivo relevance can be used to significantly improve the drug selection process.
Compounds should be selected to move into preclinical animal studies based on standard drug attributes and safety. CeeTox offers a cell-based in vitro toxicity screen that can be used in early discovery to help select the candidates that will have the highest probability of success in animal studies.
This evaluation system is based on a systems-biology approach that incorporates the effects of the test compound on multiple biochemical processes, and over a wide range of exposure concentrations. By building a temporal component into the analysis, it is possible to identify the most sensitive subcellular target of toxicity. An internal database of several hundred compounds can be used to compare toxicity profiles of NCEs to known drugs and chemicals. This system was developed over a period of 7+ years and validated with in vivo results. A proprietary algorithm used by CeeTox was developed and fine-tuned based on in vivo experiments. Using this multi-parametric approach combined with dose-response and our algorithm, a predictive value (Ctox) can be determined. The Ctox is an estimate of the sustained blood concentration where toxicity would first be expected to occur in a rat 14-day repeat dose study.
This panel consists of 9 different standard assays to measure biochemical processes that are important for cell health. Each assay is performed using a range of exposure concentrations (1-300 µM).
- Membrane integrity (1)
- Mitochondrial function (2)
- Cell proliferation (1)
- Apoptosis (1)
- Oxidative stress (2)
- Interaction with Pgp (1)
- Solubility (1)
- Add-on assays include:
- Metabolic stability
- Metabolic activation
- BrdU incorporation for S-phase
- Production of reactive-oxygen species
- Proprietary algorithm processes all data and provides an estimated blood concentration where toxicity would be expected to occur (ctox value)
- Rapid assessment of large chemical libraries
- Predictive of and correlated to in vivo blood levels
- Low incidence of false positives (<1%) and false negatives (<5%)
- Robotics platform
- Rapid turnaround time
- Minimal compound requirement, typically 6 mg or less to perform all assays
- Subcellular target information
- Detailed interpretive report
- Database for comparative analysis of toxicity
- Rank-ordering of potential lead candidates based on efficacy and toxicity
- Monitoring of changes in toxicity with changes in chemistry
Example Data Sets
Rotenone is a potent inhibitor of liver mitochondrial respiration. Following a 24-hour exposure, a strong concentration-dependent reduction in markers of mitochondrial function and cell proliferation was measured. At exposures of 1 and 10 µM, cell death (open squares) was also evident (graph below). When dose-response profiles overlap at 24 hours, it may not be possible to ascertain which endpoint is most sensitive. Cell replication requires a large amount of ATP. The purple line in the graph at 6 hours, shows a significant reduction in ATP levels that cannot be accounted for by reduced cell proliferation (blue). Another marker of mitochondrial function is [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a tetrazolium dye (MTT), which is reduced by mitochondrial dehydrogenases (red). MTT reduction coincides with a loss in cell number, indicating that ATP depletion is the primary event leading to mitochondrial damage, and eventually to cell death.
Camptothecin is a well-known anti-tumor class drug with significant toxicity associated with the molecule. After 24 hours of exposure, mitofunction markers fall below 50% response at 1µM. In comparison, at 6 hours mitofunction markers are stable, with only cell number being reduced.
Camptothecin is a potent inducer of apoptosis and this is supported in the graphs below, depicting changes in caspase-3 activation with time and dose. Note the effect of time on the response profiles. Clearly for this compound, disruption of mitochondrial function, leading to loss of cytochrome c and a subsequent activation of caspase-3, is clearly the underlying mechanism of this response.