High Content Toxicology: CellCiphr™ Cytotoxicity Profiling in HepG2 Cells
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Drug toxicity is typically a combination of multiple mechanisms. A single experimental approach is unlikely to be predictive of the complexity involved in cellular toxicity.
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High Content Screening uses fluorescence imaging to simultaneously analyse multi-parametric indicators of cellular toxicity. It can detect cell death as well as mechanisms of cell death and can cover a wide spectrum of cytopathological changes.1,2
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The CellCiphr™ cytotoxicity profiling assay assesses a panel of 10 key toxicity markers in HepG2:
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Cell Loss
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Cell cycle arrest
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Nuclear size
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Oxidative stress
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Stress kinase activation
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DNA damage response
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Mitochondrial function I
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Mitochondrial function II
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Mitosis marker
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Cytoskeletal disruption
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Using Cyprotex’s proprietary database of in vitro profiling and in vivo toxicity data, the CellCiphr™ Classifier system can rank and classify unknown compounds against known toxic effects improving the accuracy of toxicity prediction.
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For drug-discovery programs using CellCiphr™ toxicity profiling to filter out toxic compounds at the start of the hit to lead stage, Cyprotex projects a saving in direct costs of over $91 million. For programs using CellCiphr™ to selectively advance compounds with reduced risk of attrition due to toxicity, Cyprotex projects a $35 million increase in value for the typical clinical pipeline.
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‘The CellCiphr™ Cytotoxicity Profiling system offers a rapid, inexpensive, cell-based method capable of accurate and sensitive identification of compounds associated with severe in vivo toxicity with the additional benefit of no occurrence of false positives.’ |
4Vernetti L, Irwin W, Giuliano KA, Gough A, Johnson K and Taylor DL (2009)
In Drug Efficacy, Safety and Biologics Discovery: Emerging Technologies and Tools (Ed. Ekins S and Xu JJ) John Wiley & Sons, New Jersey; 53-74 |
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CellCiphr™ cytotoxicity profiling in HepG2 cells protocol
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Instruments |
Cellomics ArrayScan® VTI (Thermo Scientific) |
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Analysis Method |
High Content Screening with CellCiphr™ Classifier System |
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Cell Type |
HepG2 |
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Toxicity Markers |
10 key toxicity markers (cell loss, cell cycle arrest, nuclear size, oxidative stress, stress kinase activation, DNA damage response, mitochondrial function I, mitochondrial function II, mitosis marker and cytoskeletal disruption) |
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Test Compound Concentration |
10 point dose response curve in duplicate at 1 hr, 24 hr and 72 hr exposure |
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Data Delivery |
CellCiphr™ toxicity report (see table 1) |
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CellCiphr™ Toxicity Profiles are analysed with proprietary visual and quantitative data mining tools including CellCiphr™ Classifiers, correlation analysis, and cluster analysis.
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Data from CellCiphr™ cytotoxicity profiling in HepG2 cells
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Figure 1
Representative high content screening images for untreated (image on left) and treated (image on right) HepG2 cells illustrating key toxicity markers.
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CellCiphr™ Toxicity Profiling Report |
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CellCiphr™ Safety Risk Index |
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Maximum Tolerated Dose |
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Earliest Toxic Indicator |
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Most Sensitive Toxic Indicator |
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General Indicators of Toxicity |
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Mechanistic Indicators of Toxicity |
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CellCiphr™ correlation analysis, including comparison with other compounds in the project, compounds in the reference database, and CellCiphr™ ToxProfile Similarity plots |
Table 1
Data deliverables within the CellCiphr™ Cytotoxicity Profiling Report.
Figure 2
Example of a CellCiphr™ Toxicity Profiling Report
The CellCiphr™ Classifier profiles unknown compounds against an extensive set of reference compounds for which safety data are available. The profiles for the reference compounds are used to create a proprietary classification algorithm that provides a rank order of risk of failure in safety studies (Safety Risk Index).
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References
1 Abraham VC et al., (2008) J Biomol Screen 13(6); 527-537
2 Xu JJ et al., (2008) Toxicol Sci 105(1); 97-105
3 Vernetti L et al., (2009) In Drug Efficacy, Safety and Biologics Discovery: Emerging Technologies and Tools Ed. Ekins S and Xu JJ; 53-74
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