Research Project
7746775
DESCRIPTION (provided by applicant): Phospholipidosis (PLDosis) or lipid metabolism disorders are a factor in many drug induced liver diseases. Drug induced, dose-dependent, alterations in the expression of sets of genes, whether direct cause or effect, can provide a molecular signature of the functional phenotypic effect(s) (e.g. adverse metabolism). Definitive identification of PLDosis is based on electron microscopy, but high throughput methods to assess PLDosis have been published based on gene expression and fluorescent lipids staining. Most systems use cell lines, though some have used sub-optimally cultured primary hepatocytes, but none provides precise dose response data used for QSAR. It is known that cell lines lack metabolic pathways found in primary hepatocytes and the liver. In this Phase I SBIR program, we combine a novel method for measuring gene expression-qNPA--with optimally cultured (sandwich-cultured) human primary hepatocytes, compare this to a Hep-G2 system, and profile all the (~86) genes that have been linked to PLDosis across a large set (~63) of reference compounds taken from previous reports. We will evaluate gene expression across the time points used previously for gene expression (24hr) Nile Red (48 hr) and electron microscopy (72 hr) so that a direct comparison of results can be made. We will perform dose response measurements for all the genes for all the compounds, validating that the assay can provide EC50 data useful for QSAR. Finally, a focused set of genes will be selected as a PLDosis signature, and the benefit of primary hepatocytes vs HEp-G2 and of qNPA-based gene expression versus Nile Red will be evaluated. If successful, we will submit a Phase II application to expand the model to include miRNA and additional, potentially more selective genes, and to include identification of gene signatures for other liver diseases. PUBLIC HEALTH RELEVANCE: Phospholipidosis (PLDosis) or lipid metabolism disorders are a factor in many drug induced liver diseases. In this Phase I SBIR program, we propose to combine a gene expression method, the quantitative Nuclease Protection Assay (qNPA) with sandwich-cultured primary hepatocytes to provide a sensitive in vitro model for identifying PLDosis early in the drug discovery process. qNPA provides a level of precision superior to that of PCR. Primary hepatocytes have all the mechanisms of liver cell metabolism and drug transport intact, while cell lines such as Hep-G2 do not;therefore, our model should be a greatly improved and more relevant cell system. We will validate additional genes across time points that cover the time points used by other methods of assessing PLDosis to demonstrate the ability to provide precise dose response data and EC50 values that can be used by medicinal chemists to "design out" the PLDosis liability of chemical series. Our goal is to generate the data required to determine what the advantages of using the qNPA gene expression/primary hepatocyte model are compared to using other methods. In Phase II we will improve the model and extend it to other liver diseases, to a rat primary hepatocyte system, and to an in vivo biomarker system using circulating blood cells.
