High Content Toxicology: CellCiphr® Premier
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In March 2012, Cyprotex presented a poster on their CellCiphr® research. The study focused on using a PBPK (physiologically based pharmacokinetic) modeling approach to estimate exposure, combined with CellCiphr® high content screening data for a panel of mechanistic toxicity endpoints, to greatly increase the predictive power for the determination of safety risk. |
- Hepatotoxicity is one of the main reasons for drug withdrawals, accounting for 37% of all therapeutics taken off the market between 1994 and 2006.1
- CellCiphr® Premier is a cell-based assay that combines an extended panel of toxicologically-relevant endpoints with Cyprotex’s CellCiphr® classifier to provide one of the most reliable clinical hepatotoxicity predictors available (sensitivity = 70% and specificity = 100%).
- Both HepG2 (replicating cells) and primary rat hepatocytes (metabolically competent cells) are investigated at multiple time points (extending from 4 hrs to 5 days to identify early and late stage toxic effects).
- The data can be integrated with actual or predicted Cmax to correct for exposure-related effects.
- The extended panel of end points allows CellCiphr® Premier to offer enhanced predictivity for hepatotoxicity risk over the standard CellCiphr® panels.
The rapid expansion of HCS technology throughout the pharmaceutical industry and academic research centers validates the usefulness of the information-rich screening approach.
2Zanella F, Lorens JB and Link W (2010) Trends Biotechnology 28(5); 237-245
Protocol
CellCiphr® Premier profiling protocol
| Instruments | Cellomics ArrayScan® VTI (Thermo Scientific) |
|---|---|
| Analysis Method | High Content Screening with CellCiphr® Classifier System |
| Cell Types | HepG2 (replicating) and primary rat hepatocytes (metabolically competent) |
| Toxicity Markers | Extended panel of toxicity markers including: Apoptosis Cell cycle arrest Cell loss Cytoskeletal disruption DNA fragmentation and damage response Glutathione depletion Mitochondrial function Mitosis marker Nuclear size Oxidative stress Phospholipidosis Reactive oxygen species Steatosis Stress kinase activation |
| Test Article Concentration | 10 point dose response curve in duplicate |
| Data Delivery | CellCiphr® toxicity report including: AC50 Safety ranking Safety alert Heat maps (normalized to Cmax if available) |
Data
Data from CellCiphr® Premier
Figure 1a
Reactive Oxygen Species (ROS) Assay. Rat hepatocytes were stained with Hoechst and CM-H2DCFDA after 4 hour incubation with vehicle (DMSO) or Compound 1, which induces ROS. CM-H2DCFDA enters the cell and reacts with ROS to create a fluorescent signal. ROS levels were determined using the ArrayScan® VTI.
Figure 1b
Glutathione (GSH) Assay. Rat hepatocytes were stained with Hoechst and monochlorobimane after 18 hour incubation with vehicle (DMSO) or Compound 2, which depletes GSH. Monochlorobimane reacts with cellular GSH. GSH levels were determined in the cytoplasm of the cells using the ArrayScan® VTI.
Figure 1c
Representative heat maps for CellCiphr® Premier. The heatmaps demonstrate the pattern of endpoint response for the six compounds in a set. The AC50 of the endpoint response is shown using a color scale from red (highly toxic) to green (safe) with the bar at the right showing the full range of the AC50 scale. The heatmaps demonstrate various biomarkers at different time points giving rise to a ‘toxicity risk fingerprint’ for each compound. These data can be used to determine the mechanistic pathways causing the toxic response and the time course over which it occurs. The AC50 data from the different endpoints, for example GSH and ROS activity, are represented by specific boxes on the heatmaps, as demonstrated by the grey connectors that link to the detailed images shown in Figures 1a and 1b.
Reactive Oxygen Species (ROS) Assay. Rat hepatocytes were stained with Hoechst and CM-H2DCFDA after 4 hour incubation with vehicle (DMSO) or Compound 1, which induces ROS. CM-H2DCFDA enters the cell and reacts with ROS to create a fluorescent signal. ROS levels were determined using the ArrayScan® VTI.
Figure 1b
Glutathione (GSH) Assay. Rat hepatocytes were stained with Hoechst and monochlorobimane after 18 hour incubation with vehicle (DMSO) or Compound 2, which depletes GSH. Monochlorobimane reacts with cellular GSH. GSH levels were determined in the cytoplasm of the cells using the ArrayScan® VTI.
Figure 1c
Representative heat maps for CellCiphr® Premier. The heatmaps demonstrate the pattern of endpoint response for the six compounds in a set. The AC50 of the endpoint response is shown using a color scale from red (highly toxic) to green (safe) with the bar at the right showing the full range of the AC50 scale. The heatmaps demonstrate various biomarkers at different time points giving rise to a ‘toxicity risk fingerprint’ for each compound. These data can be used to determine the mechanistic pathways causing the toxic response and the time course over which it occurs. The AC50 data from the different endpoints, for example GSH and ROS activity, are represented by specific boxes on the heatmaps, as demonstrated by the grey connectors that link to the detailed images shown in Figures 1a and 1b.
| CellCiphr® Toxicity Profiling Report | Sensitivity | Specificity |
|---|---|---|
| HIAT Method3 | 35% | 100% |
| CellCiphr® Premier | 70% | 100% |
Table 1
Sensitivity and Specificity of CellCiphr® Premier to Predict Human Hepatotoxicity.
A blind set of 47 compounds were screened in CellCiphr® Premier to establish the sensitivity and specificity of the model. Data generated were compared with existing data from the HIAT method. Mechanistic endpoints were normalized for Cmax to predict toxicity relative to exposure. The compounds chosen were known to be poorly sensitive (i.e.: they were mainly false negatives) in the HIAT method. In summary, the CellCiphr® Premier method shows greatly increased sensitivity over the HIAT method for this set of compounds.
Sensitivity and Specificity of CellCiphr® Premier to Predict Human Hepatotoxicity.
A blind set of 47 compounds were screened in CellCiphr® Premier to establish the sensitivity and specificity of the model. Data generated were compared with existing data from the HIAT method. Mechanistic endpoints were normalized for Cmax to predict toxicity relative to exposure. The compounds chosen were known to be poorly sensitive (i.e.: they were mainly false negatives) in the HIAT method. In summary, the CellCiphr® Premier method shows greatly increased sensitivity over the HIAT method for this set of compounds.
References
1 Dykens JA and Will Y (2007) Drug Discovery Today 12; 777-785
2 Zanella F et al., (2010) Trends Biotechnol 28(5); 237-245
3 Xu JJ et al., (2008) Toxicol Sci 105(1); 97-105
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