A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds

Abstract

Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73-82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy-three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost-effective stand-alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively.

In Vitro Micropatterned Human Pluripotent Stem Cell Test (µP-hPST) for Morphometric-Based Teratogen Screening

“…We have established the first in vitro hPSC-based and fully quantitative morphometric assay which captured both cell differentiation and cell migration processes, and successfully eliminated inter-species variations 14 . Refined and validated using 30 compounds, our µP-hPST exhibited the highest accuracy compared with other existing in vitro teratogen screening models using animal embryos and cells 8 , 10 , 22 , 24 , 27 , 28 , or hPSCs 6 , 7 .…”

“…For in vitro teratogen screenings, some studies have examined the morphological changes due to teratogenic disruptions in both cell differentiation and migration using animal embryos or cells, such as the rat whole embryo culture (rWEC) assay 22 , the zebrafish embryo culture (ZEC) assay 23 , 24 , and a newly developed assay using mouse embryoid body (mEB) by Marikawa’s group 25 , 26 . However, these assays are under performing (~70–80% accuracy for the rWEC and ZEC assays) 27 . We have established the first in vitro hPSC-based and fully quantitative morphometric assay which captured both cell differentiation and cell migration processes, and successfully eliminated inter-species variations 14 .…”

Goldilocks’ Determination of What New In Vivo Data are “Just Right” for Different Common Drug Development Scenarios, Part 1

“…Lastly, some understanding of cross‐species binding, functional activity, and potency are an important component to providing ADME confidence using animal models. In vitro EFD data. For this article, we assume that the in vitro EFD assays (whole embryo culture, zebrafish, embryonic stem cells, other similar tests alone, or in combination) correctly predict animal EFD toxicity 80 to 85% of the time, as this seems to be the emerging window of performance (Augustine‐Rauch, Zhang, & Panzica‐Kelly, ; Maglich et al., ; Sogorb et al., ). Currently, these are most useful for small molecules that are not limited by species specificity.…”

Using Zebrafish for Investigating the Molecular Mechanisms of Drug-Induced Cardiotoxicity

“…Cardiotoxicity might occur as a side effect of chemotherapeutical drugs or might develop due to exposure to certain chemicals. Lately, zebrafish embryos were shown to be very useful in toxicology studies, particularly to screen for developmental toxicants [ 8 , 9 ] and environmental pollutants such as pesticides and heavy metals as well as nanoparticles [ 10 , 11 ]. Small molecules can be added directly into the fish water within multiwell plates and absorbed via diffusion by the fish.…”