Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomic ratios

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.

1 . A method of classifying a test compound as a teratogen or a non-teratogen, the 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;   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;   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;   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 about 0.88 is indicative of the teratogenicity of the test compound; and   a ratio of greater than about 0.88 is indicative of the non-teratogenicity of the test compound.   
     
     
         2 . A method of predicting teratogenicity of a test compound, the 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;   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;   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;   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 about 0.88 is indicative of the teratogenicity of the test compound; and   a ratio of greater than about 0.88 is indicative of the non-teratogenicity of the test compound.   
     
     
         3 . A method for validating a test compound as a teratogen, the 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;   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;   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;   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 about 0.88 is indicative of the teratogenicity of the test compound; and   a ratio of greater than about 0.88 is indicative of the non-teratogenicity of the test compound.   
     
     
         4 . A method for determining the exposure concentration at which a test compound is teratogenic, the method comprising:
 culturing undifferentiated human stem cell-like cells (hSLCs) in a range of concentrations of the test compound and in the absence of the test compound;   determining the fold change in ornithine, or fragment, adduct, deduct or loss thereof, in the culture media of undifferentiated hSLCs cultured in each concentration of the test compound in comparison with hSLCs cultured in the absence of the test compound;   determining the fold change in cystine, or fragment, adduct, deduct or loss thereof, in the culture media of undifferentiated hSLCs cultured in each concentration of the test compound in comparison with hSLCs cultured in the absence of the test compound;   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, for each concentration of test compound, wherein:   a ratio of less than or equal to about 0.88 at a given concentration of the test compound is indicative of the teratogenicity of the test compound at that given concentration; and   a ratio of greater than about 0.88 at a given concentration of the test compound is indicative of the non-teratogenicity of the test compound at that given concentration.   
     
     
         5 . The method of  claim 1 , wherein the cystine, or fragment, adduct, deduct or loss thereof, and/or ornithine, or fragment, adduct, deduct or loss thereof, are identified using a physical separation method. 
     
     
         6 . The method of  claim 5 , wherein the physical separation method comprises mass spectrometry. 
     
     
         7 . The method of  claim 6 , wherein the mass spectrometry comprises liquid chromatography/electrospray ionization mass spectrometry. 
     
     
         8 . The method of  claim 1 , wherein the cystine, or fragment, adduct, deduct or loss thereof, and/or ornithine, or fragment, adduct, deduct or loss thereof, are measured using a colorimetric or immunological assay. 
     
     
         9 . The method of  claim 1 , wherein the hSLCs comprise human embryonic stem cells (hESCs), human induced pluripotent (iPS) cells, or human embryoid bodies. 
     
     
         10 . The method of  claim 1 , wherein the hSLCs are cultured in a range of concentrations of the test compound. 
     
     
         11 . The method of  claim 10 , wherein the range of concentrations comprises a serial dilution. 
     
     
         12 . The method of  claim 10 , wherein the range of concentrations comprises nine three-fold dilutions. 
     
     
         13 . The method of  claim 10 , wherein the range of concentrations is selected from about 0.04 μM to about 300 μM, about 4 μM to about 30,000 μM, and about 0.0001 μM to about 10 μM. 
     
     
         14 . The method of  claim 10 , wherein the range of concentrations of the test compound comprises the test compound's human therapeutic C max . 
     
     
         15 . The method of  claim 1 , wherein the hSLCs are cultured at a concentration of the test compound comprising the test compound's human therapeutic C max . 
     
     
         16 . The method of  claim 1 , further comprising detecting one or more additional metabolites associated with hSLCs cultured in the presence of the test compound in comparison with hSLCs cultured in the absence of the test compound. 
     
     
         17 . The method of  claim 16 , wherein one or more additional metabolite comprises arginine, ADMA, cystathionine, and/or a fragment, adduct, deduct or loss thereof. 
     
     
         18 . The method of  claim 1 , further comprising determining the ratio of the fold change in arginine, or fragment, adduct, deduct or loss thereof, to the fold change in ADMA, or fragment, adduct, deduct or loss thereof, wherein:
 a ratio of less than at least about 0.9 or greater than at least about 1.1 is indicative of the teratogenicity of the test compound; and   a ratio of greater than at least about 0.9 and less than at least about 1.1 is indicative of the non-teratogenicity of the test compound.