Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomic ratios
Inventors
Smith, Alan • West, Paul • Palmer, Jessica
Assignees
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Publication Number
US-10209240-B2
Publication Date
2019-02-19
Expiration Date
Abstract
This present invention provides rapid, reproducible, biomarker-based screening methods for the developmental toxicity testing of compounds. The methods are designed to identify the exposure level at which a test compound perturbs metabolism in a manner predictive of developmental toxicity. In particular, the perturbation of two metabolites, ornithine and cystine, is measured, wherein a ratio of the fold change in ornithine to the fold change in cystine of less than or equal to about 0.88 is indicative of the teratogenicity of a test compound.
Core Innovation
The invention relates to methods for screening the teratogenicity of a test compound using undifferentiated human stem cell-like cells (hSLCs). hSLCs are selected from human embryonic stem cells (hESCs), human induced pluripotent (iPS) cells, human embryoid bodies, and hSLC-derived lineage-specific cells, and are cultured in the presence of the test compound and in the absence of the test compound.
Fold change determination is performed for ornithine and for cystine in the culture media. The method determines the fold change of ornithine, or a fragment, adduct, deduct or loss thereof, and the fold change of cystine, or a fragment, adduct, deduct or loss thereof, in the culture media in the presence of the test compound compared with the absence condition.
Teratogenicity classification is then determined from a ratio of the fold changes. A ratio of the fold change in ornithine, or fragment/adduct/deduct/loss, to the fold change in cystine, or fragment/adduct/deduct/loss, is used for classification, where a ratio of less than or equal to 0.88 is indicative of teratogenicity and a ratio greater than 0.88 is indicative of non-teratogenicity.
Claims Coverage
The claim coverage identifies one independent claim and centers on four inventive features: culturing undifferentiated hSLCs with and without a test compound, measuring fold changes for ornithine and cystine in culture media, and classifying teratogenicity from an ornithine-to-cystine fold-change ratio using the stated threshold.
Undifferentiated hSLC-based teratogenicity screening with comparison conditions
A method comprising culturing undifferentiated human stem cell-like cells (hSLCs) in the presence of the test compound and in the absence of the test compound, wherein hSLCs are selected from the group consisting of human embryonic stem cells (hESCs), human induced pluripotent (iPS) cells, human embryoid bodies, and hSLC-derived lineage-specific cells.
Ornithine fold-change determination in culture media
Determining the fold change in ornithine, or fragment, adduct, deduct or loss thereof, in the culture media of undifferentiated hSLCs cultured in the presence of the test compound in comparison with hSLCs cultured in the absence of the test compound.
Cystine fold-change determination in culture media
Determining the fold change in cystine, or fragment, adduct, deduct or loss thereof, in the culture media of undifferentiated hSLCs cultured in the presence of the test compound in comparison with hSLCs cultured in the absence of the test compound.
Ornithine-to-cystine fold-change ratio classification with threshold
Determining the ratio of the fold change in ornithine, or fragment, adduct, deduct or loss thereof, to the fold change in cystine, or fragment, adduct, deduct or loss thereof, wherein a ratio of less than or equal to 0.88 is indicative of the teratogenicity of the compound, and a ratio of greater than 0.88 is indicative of the non-teratogenicity of the compound.
The claims are directed to a biomarker-ratio method that compares undifferentiated hSLCs cultured with versus without a test compound, measures ornithine and cystine fold changes in culture media, and classifies teratogenicity based on the stated ornithine-to-cystine ratio threshold.
Stated Advantages
Assay and throughput advantages are described, including a short LC gradient and reduced instrument time.
Reported performance metrics include training set and test set accuracies, sensitivity/specificity.
Documented Applications
The method is applied for developmental toxicity screening and prediction, including application to compounds with unknown human teratogenicity.
The method is used as a teratogenicity classifier based on the ornithine/cystine fold-change ratio.
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