Does your drug have potential toxicity issues?

Case study data for the validity of 2D and 3D screening assays during early stage drug development.

Drug induced toxicity is responsible for a significant proportion of drug attrition and costly withdrawal during late stage development. In vitro predictive toxicity assays offer a rapid and cost-effective option for identifying potential toxicity concerns at the early lead optimization stage.

Key points to consider when developing and selecting in vitro predictive toxicity assays include the cell type, the readouts employed, throughput, and biological relevance. Potential in vitro models include traditional 2D assays or more complex 3D spheroid assays.

Hepatotoxicity 2D screening as an important tool to assess quickly and early compound innocuity

The 2D hepatoxicity panel conducted in primary rat hepatocytes uses the combination of flow cytometry and microscopy platforms, with a panel of toxicity readouts: cytolysis, mitochondrial membrane potential, cholestasis, lipid accumulation, and reduced glutathion (GSH) depletion.

No alt text provided for this image

 

Figure1: Example of cytolysis data over time for liver toxicity assay (cytolysis probe in green fluorescence).

This panel of assays displays a ~83% correct predictive hit rate in a battery of 30 compounds previously evaluated in acute toxicity assays in rodents. These data demonstrate that rat hepatocytes are a valid and cost-effective alternative for predictive hepatoxicity screening.

No alt text provided for this image

Figure 2: List of compounds tested in vitro and in vivo (TRUE = correct prediction, FALSE = wrong prediction)

These assays can also be performed in primary human hepatocytes in miniaturized format for secondary confirmation screenings or smaller libraries of compounds.

Neurotoxicity 3D hiPS model to screen potential neurotoxicity.

Using complex human 3D spheroid cultures in functional assays can be a very sensitive and powerful tool for early stage predictive toxicity screening. The 3D spheroids microBrain®, generated from human neuron and astrocyte iPSC co-cultures (StemoniX®), exhibit a functional neural network displaying high sensitivity for neurotoxicity assessments (e.g. synchronized calcium oscillation modulation) when compared to standard toxicity readouts in 2D rodent embryonic cultures.

No alt text provided for this image

Figure 3: Example of miniaturized 3D iPS neuronal spheroids StemoniX® microBrain®

The evaluation of calcium flux is a true measure of neuronal spontaneous activity. A set of different known neurotoxic compounds was tested on the microBrain® for 24h followed by calcium staining and oscillation monitoring using a FLIPR Tetra platform.

No alt text provided for this image

Figure 4: Example of screening and data computerized on frequency modulation of different neurotoxins.

Modulation of calcium oscillations was identified for all compounds weither they exhibited a strong inhibition of calcium response (e.g. MPP+, DDT …) or an activation of the oscillation frequency (e.g. 4-AP or benzo(b)fluoranthène). This assay proved to be very sensitive and reliable to assess compounds that can have a modulatory effect of neurons signaling.

This type of approach on primary cells or iPS in 2D or 3D can also be relevant for other predictive toxicity screening in other therapeutic domains, including heart, kidney, skin etc.

 

Does your drug have potential toxicity issues ?

Predictive toxicology provides a rapid and cost-effective way of identifying potential toxicity concerns in early drug development.

The following article provides a valuable overview and describes practical cases using this approach

Please contact us for further information and to learn more about our capabilities.

contact@porsolt.com
www.porsolt.com