In Vitro Gene Expression Model Predicting Drug Induced Liver Disease

预测药物性肝病的体外基因表达模型

• 批准号: 7746775
• 负责人: BRUCE E. SELIGMANN
• 金额: $ 22.38万
• 依托单位: HTG MOLECULAR DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-21 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7746775
• 关键词: Address; Animal Model; Bile Acids; Biological Assay; Biological Markers; Biological Models; Blood Cells; Cell Line; Cells; Chemicals; Data; Disease; Dose; Drug Transport; Early identification; Electron Microscopy; Exhibits; Gene Expression; Gene Expression Profile; Genes; Genome; Goals; Hepatocyte; Human; In Vitro; Industry; Investigation; Link; Lipids; Literature; Liver; Liver diseases; Measurement; Measures; Metabolic Diseases; Metabolic Pathway; Metabolism; Methods; MicroRNAs; Modeling; Molecular; Molecular Profiling; Nuclease Protection Assays; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Process; Property; Publishing; Qualifying; Quantitative Structure-Activity Relationship; Rattus; Relative (related person); Reporting; Series; Services; Small Business Innovation Research Grant; Staining method; Stains; Structure; Structure-Activity Relationship; System; Testing; Time; Tissues; analog; base; cell type; commercialization; density; design; drug discovery; drug induced liver disease; drug metabolism; improved; in vitro Assay; in vitro Model; in vitro testing; in vivo; lipid metabolism; nile red; novel; programs; public health relevance; response; species difference

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.

描述（由申请方提供）：磷脂病（PLDosis）或脂质代谢紊乱是许多药物诱导性肝病的一个因素。药物诱导的、剂量依赖性的基因组表达改变，无论是直接原因还是影响，都可以提供功能表型效应的分子特征（例如不良代谢）。PLDosis的连续鉴定是基于电子显微镜，但是已经发表了基于基因表达和荧光脂质染色的评估PLDosis的高通量方法。大多数系统使用细胞系，尽管有些使用次优培养的原代肝细胞，但没有一个系统提供用于QSAR的精确剂量响应数据。已知细胞系缺乏在原代肝细胞和肝脏中发现的代谢途径。在这个I期SBIR计划中，我们将联合收割机用于测量基因表达的新方法-qNPA-与最佳培养的（体外培养的）人原代肝细胞相结合，将其与Hep-G2系统进行比较，并在来自先前报告的大量（~63）参考化合物中分析与PLDosis相关的所有（~86）基因。我们将在先前用于基因表达（24小时）、尼罗红（48小时）和电子显微镜（72小时）的时间点评价基因表达，以便可以直接比较结果。我们将对所有化合物的所有基因进行剂量响应测量，验证该测定可以提供对QSAR有用的EC 50数据。最后，将选择一组集中的基因作为PLDosis特征，并将评价原代肝细胞相对于HEp-G2和基于qNPA的基因表达相对于尼罗红的益处。如果成功，我们将提交第二阶段申请，以扩大模型，包括miRNA和其他可能更具选择性的基因，并包括其他肝脏疾病的基因特征识别。公共卫生相关性：磷脂病（PLDosis）或脂质代谢紊乱是许多药物性肝病的一个因素。在这个I期SBIR项目中，我们建议将联合收割机基因表达方法，定量核酸酶保护试验（qNPA）与体外培养的原代肝细胞相结合，以提供一种敏感的体外模型，用于在药物发现过程的早期识别PLDosis。qNPA提供了优于PCR的上级精确度。原代肝细胞具有完整的肝细胞代谢和药物转运的所有机制，而Hep-G2等细胞系则没有;因此，我们的模型应该是一个大大改进的和更相关的细胞系统。我们将在涵盖其他评估PLDosis方法所用时间点的时间点验证其他基因，以证明提供精确剂量反应数据和EC 50值的能力，这些数据和EC 50值可供药物化学家用于“设计”化学系列的PLDosis责任。我们的目标是生成所需的数据，以确定使用qNPA基因表达/原代肝细胞模型与使用其他方法相比的优势。在第二阶段，我们将改进该模型，并将其扩展到其他肝脏疾病，大鼠原代肝细胞系统，以及使用循环血细胞的体内生物标志物系统。