PREDICTING HUMAN DEVELOPMENTAL TOXICITY OF PHARMACEUTICALS USING HUMAN STEM-LIKE CELLS AND METABOLOMIC RATIOS

• US 20180128814A1
• Filed: 12/20/2017
• Published: 05/10/2018
• Est. Priority Date: 11/02/2012
• Status: Active Grant

• Current Assignee: Stemina Biomarker Discovery Incorporated
• Original Assignee: Stemina Biomarker Discovery Incorporated
• Inventors: Smith, Alan, West, Paul, Palmer, Jessica
• Granted Patent: US 10,209,240 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: G01N 23/2258 Measuring secondary ion emission, e.g. secondary ion mass spectrometry [SIMS] mass-to-charge ratio analysis aspects of SIMS for material analysis G01N27/62
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids
• CPC Class Codes: G01N 33/6815 containing sulfur, e.g. cysteine, cystine, methionine, homocysteine

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.

Claims

1. A method comprising:

culturing undifferentiated human stem cell-like cells (hSLCs) in the presence of a 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; and

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.

2. 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.

3. The method of claim 2, wherein the physical separation method comprises mass spectrometry.

4. The method of claim 3, wherein the mass spectrometry comprises liquid chromatography/electrospray ionization mass spectrometry.

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 measured using a colorimetric or immunological assay.

6. The method of claim 1, wherein the hSLCs comprise human embryonic stem cells (hESCs), human induced pluripotent (iPS) cells, or human embryoid bodies.

7. The method of claim 1, wherein the hSLCs are cultured in a range of concentrations of the test compound.

8. The method of claim 7, wherein the range of concentrations comprises a serial dilution.

9. The method of claim 7, wherein the range of concentrations comprises nine three-fold dilutions.

10. The method of claim 7, wherein the range of concentrations is selected from about 0.04 μ

M to about 300 μ

M, about 4 μ

M to about 30,000 μ

M, and about 0.0001 μ

M to about 10 μ

M.

11. The method of claim 7, wherein the range of concentrations of the test compound comprises the test compound'"'"'s human therapeutic C_max.

12. 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.

13. 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, wherein the one or more additional metabolite comprises arginine, asymmetric dimethylarginine (ADMA), cystathionine, cystine, and/or a fragment, adduct, deduct or loss thereof.

14. The method of claim 13, 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.

15. The method of claim 13, further comprising determining the ratio of the fold change in ADMA, or fragment, adduct, deduct or loss thereof, to the fold change in cystine, or fragment, adduct, deduct or loss thereof.

16. The method of claim 13, further comprising determining the ratio of the fold change in cystathionine, or fragment, adduct, deduct or loss thereof, to the fold change in cystine, or fragment, adduct, deduct or loss thereof.