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 - 16 . (canceled) 
     
     
         17 . A method of determining whether a test compound has developmental toxicity potential, the method comprising:
 quantifying an effect of a test compound on levels of a first metabolite in human cells to obtain a first value;   quantifying an effect of the test compound on levels of a second metabolite in the human cells to obtain a second value; and   determining that the test compound has developmental toxicity potential if a ratio of the first value to the second value does not exceed a threshold value.   
     
     
         18 . The method of  claim 17 , wherein the human cells 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. 
     
     
         19 . The method of  claim 17 ,
 wherein the first metabolite is ornithine, or a fragment, adduct, deduct or loss thereof, and   wherein the second metabolite is cystine, or a fragment, adduct, deduct or loss thereof.   
     
     
         20 . The method of  claim 19 ,
 wherein the first value comprises a fold change in levels of the first metabolite in culture media of the human cells cultured in the presence of the test compound in comparison with the human cells cultured in the absence of the test compound, and   wherein the second value comprises a fold change in levels of the second metabolite in culture media of the human cells cultured in the presence of the test compound in comparison with the human cells cultured in the absence of the test compound.   
     
     
         21 . The method of  claim 20 , wherein the threshold value is 0.88. 
     
     
         22 . The method of  claim 20 ,
 wherein the human cells are human embryonic stem cells (hESCs), and   wherein the threshold value is 0.85.   
     
     
         23 . The method of  claim 20 ,
 wherein the threshold value is 0.75, and   wherein the test compound has developmental toxicity potential at a level of exposure that is not cytotoxic.   
     
     
         24 . The method of  claim 17 , wherein at least one of the quantifying steps comprises a physical separation method. 
     
     
         25 . The method of  claim 24 , wherein the physical separation method comprises mass spectrometry. 
     
     
         26 . The method of  claim 25 , wherein the mass spectrometry comprises liquid chromatography/electrospray ionization mass spectrometry. 
     
     
         27 . A method of determining whether a concentration of a test compound is potentially toxic to development, the method comprising:
 quantifying an effect of a concentration of a test compound on levels of a first metabolite in human cells to obtain a first value;   quantifying an effect of the concentration of the test compound on levels of a second metabolite in the human cells to obtain a second value; and   determining that the concentration of the test compound is potentially toxic to development if a ratio of the first value to the second value does not exceed a threshold value.   
     
     
         28 . The method of  claim 27 , wherein the human cells 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. 
     
     
         29 . The method of  claim 27 ,
 wherein the first metabolite is ornithine, or a fragment, adduct, deduct or loss thereof, and   wherein the second metabolite is cystine, or a fragment, adduct, deduct or loss thereof.   
     
     
         30 . The method of  claim 29 ,
 wherein the first value comprises a fold change in levels of the first metabolite in culture media of the human cells cultured in the presence of the test compound in comparison with the human cells cultured in the absence of the test compound, and   wherein the second value comprises a fold change in levels of the second metabolite in culture media of the human cells cultured in the presence of the test compound in comparison with the human cells cultured in the absence of the test compound.   
     
     
         31 . The method of  claim 30 , wherein the threshold value is 0.88. 
     
     
         32 . The method of  claim 30 ,
 wherein the human cells are human embryonic stem cells (hESCs), and   wherein the threshold value is 0.85.   
     
     
         33 . The method of  claim 30 ,
 wherein the threshold value is 0.75, and   wherein the concentration of the test compound is potentially toxic to development but is not cytotoxic.   
     
     
         34 . The method of  claim 27 , wherein at least one of the quantifying steps comprises a physical separation method. 
     
     
         35 . The method of  claim 34 , wherein the physical separation method comprises mass spectrometry. 
     
     
         36 . The method of  claim 35 , wherein the mass spectrometry comprises liquid chromatography/electrospray ionization mass spectrometry. 
     
     
         37 . The method of  claim 27 ,
 wherein the quantifying steps are performed for a plurality of concentrations, and   wherein the determining step is performed for each of the plurality of concentrations.   
     
     
         38 . The method of  claim 37 , wherein one of the plurality of concentrations comprises the test compound's human therapeutic C max .