Predicting human developmental toxicity of pharmaceuticals using human stem-like cells and metabolomics
Inventors
West, Paul R. • WEIR-HAUPTMAN, April M. • Smith, Alan M. • Donley, Elizabeth L. R. • Cezar, Gabriela G.
Assignees
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Publication Number
US-8703424-B2
Publication Date
2014-04-22
Expiration Date
Abstract
The invention provides biomarker profiles of metabolites and methods for screening chemical compounds including pharmaceutical agents, lead and candidate drug compounds and other chemicals using human stem-like cells (hSLCs) or lineage-specific cells produced therefrom. The inventive methods are useful for testing toxicity, particularly developmental toxicity and detecting teratogenic effects of such chemical compounds. Specifically, a more predictive developmental toxicity model, based on an in vitro method that utilizes both hSLCs and metabolomics to discover biomarkers of developmental toxicity is disclosed.
Core Innovation
The invention relates to an all-human in vitro method of screening the teratogenicity of a test compound using human stem cell-like cells (hSLCs). The approach culturing hSLCs in the presence and in the absence of the test compound provides comparative measurements of metabolite-related changes associated with developmental toxicity and teratogenicity.
The method determines fold changes in arginine and fold changes in asymmetric dimethyl arginine (ADMA) associated with hSLCs cultured in the presence of the test compound compared with hSLCs cultured in the absence of the test compound. The teratogenicity decision is based on determining the ratio of the fold change in arginine to the fold change in ADMA, using defined ratio bounds to indicate teratogenicity or non-teratogenicity.
The disclosed framework further supports metabolomics-based assessment beyond arginine and ADMA by correlating changes in small-molecule metabolite profiles measured in the system. Mechanistic biomarker candidates and pathways highlighted include arginine/ADMA/urea cycle, glutamate/GABA/neuronal development, and citric acid cycle intermediates such as succinic acid, together with additional enriched metabolic networks including nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, and glutathione metabolism.
Claims Coverage
The independent claim describes one core inventive concept: classifying teratogenicity by measuring fold changes in arginine and asymmetric dimethyl arginine (ADMA) in human stem cell-like cells (hSLCs) cultured with versus without a test compound, and interpreting the resulting ratio using defined threshold bounds. Dependent claims refine the claim coverage by specifying embodiments of hSLCs, narrowing metabolite identification via physical separation and mass spectrometry modalities, and expanding the set of metabolite fold-change determinations using specified metabolites or tables.
Screening teratogenicity using hSLCs with arginine and ADMA fold-change ratio
A method of screening the teratogenicity of a test compound by culturing human stem cell-like cells (hSLCs) in the presence and in the absence of the test compound, determining fold change in arginine and fold change in asymmetric dimethyl arginine (ADMA), and determining the ratio of the fold change in arginine to the fold change in ADMA, wherein a ratio of less than 0.9 or greater than 1.1 is indicative of the teratogenicity of the test compound and a ratio of greater than 0.9 and less than 1.1 is indicative of the non-teratogenicity of the test compound.
hSLC embodiment selection
The method further defines that the hSLCs include human embryonic stem cells (hESCs), human induced pluripotent (iPS) cells, or human embryoid bodies.
Metabolite identification using physical separation
The method further includes identifying metabolites using a physical separation method.
LC-electrospray mass spectrometry for metabolite analysis
The method further specifies that the mass spectrometry is performed as liquid chromatography/electrospray ionization mass spectrometry.
Fold-change determination for specified small-molecule set
The method further includes determining a fold change in at least five of the small molecules listed in Table 8.
Fold-change determination for specified metabolites
The method further determines a fold change for one or more specified metabolites including succinic acid, GABA, isoleucine, aspartic acid, malic acid, glutamic acid, and histidine.
Overall claim coverage is centered on classifying teratogenicity from a specific arginine-to-ADMA fold-change ratio measured in hSLCs cultured with versus without the test compound. Dependent coverage further narrows or expands the implementation by specifying hSLC types, adding metabolite identification by physical separation and LC/electrospray mass spectrometry, and adding additional metabolite fold-change determinations including metabolites from specified lists or tables.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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