TeamChip for High-Throughput, Predictive Human Metabolism and Toxicology: Phase I

用于高通量、预测性人体代谢和毒理学的 TeamChip：第一阶段

• 批准号: 8251456
• 负责人: MOO-YEAL LEE
• 金额: $ 50万
• 依托单位: SOLIDUS BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251456
• 关键词: Acetaminophen; Adenoviruses; Affect; Animals; Biological Assay; CYP2C9 gene; CYP3A4 gene; Cell Culture Techniques; Cells; ChIP-on-chip; Chemicals; Clinical Trials; Cryopreservation; Development; Encapsulated; Environmental Pollutants; Enzymes; Evaluation; Exposure to; Freezing; Generations; Genes; Hepatocyte; Hepatotoxicity; Human; Human body; In Vitro; Individual; Infection; Institutes; Investments; Lead; Libraries; Liver; Measurement; Measures; Metabolic; Metabolism; Methods; Modeling; Organ; Parents; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plastics; Population; Predisposition; Process; Protein Isoforms; Proteins; Protocols documentation; Public Health; Recombinants; Reporter; Research; Research Personnel; Safety; Screening procedure; Simulate; Small Business Technology Transfer Research; Speed; Staging; Subgroup; System; Technology; Testing; Therapeutic; Time; Toxic effect; Toxicology; Transfection; Treatment Protocols; Validation; Viral; Western Blotting; Xenobiotic Metabolism; Xenobiotics; base; cDNA Library; cell type; commercialization; design; drug candidate; drug development; drug discovery; environmental chemical; flexibility; high risk; high throughput analysis; in vivo; monolayer; programs; response; tool; toxicant; troglitazone

DESCRIPTION (provided by applicant): Solidus Biosciences, Inc. in partnership with Rensselaer Polytechnic Institute, will focus on further development, validation, and commercialization of its proprietary "Transfected Enzyme and Metabolism Chip" (or TeamChip) for high-throughput analysis of systematic drug candidate and chemical metabolism and toxicology. The TeamChip is being developed to mimic first-pass metabolism of the human liver and to predict enzyme-specific hepatotoxicity. A library of human cells expressing different combinations of metabolic enzymes on the TeamChip will be prepared by transfecting metabolic genes using a viral delivery system into human cells encapsulated in three-dimensional (3D) matrices (as small as 60 nL) arrayed on a plastic chip. Thus, the reactivity of target compounds with individual human metabolic enzymes or combinations of enzymes in the human liver or other organ types can be assessed and quantified at speeds commensurate with predictive human toxicity assessment of early stage drug candidates and environmental chemicals. The specific aims/milestones of this Phase II STTR proposal are to: 1. Construct recombinant adenoviruses that carry genes for metabolic enzymes from a human liver cDNA library (e.g., representative CYP450 isoforms and conjugative metabolic enzymes), demonstrate gene transfection on monolayers of Hep3B cells using fluorogenic/luminescent substrates, and measure different levels of adenoviral enzyme expression by Western blot assays. 2. Prepare the TeamChip containing Hep3B cells expressing various combinations of metabolic enzymes and identify metabolic genes whose differential expression affects the cellular response to model compounds. 3. Optimize on-chip cryopreservation protocols for recombinant adenoviruses and transfected Hep3B cells. Demonstrate metabolism-induced toxicity with cryopreserved TeamChips and compare the results with non- frozen counterparts. In vitro technologies that can be used to quickly assess large numbers of compounds for toxicity remain limited. A critical component of safety evaluation is metabolism and toxicology of chemicals (e.g., drug candidates and environmental chemical toxicants), which reflects the susceptibility of chemicals to be metabolized by human metabolic enzymes and the toxicity of parent compounds and their metabolites. Current approaches to chemical safety assessment are costly, time consuming, and use large amounts of compound and large numbers of animals. Thus, there is great potential and opportunity to apply the TeamChip as a safety assessment tool that can be used to evaluate whether and how specific metabolic enzymes contribute to the toxicity of drug candidates and chemical toxicants. This capability may also be used to predict differences among individuals in drug and chemical metabolism and toxicity. PUBLIC HEALTH RELEVANCE: The drug discovery process is an investment-intensive, high-risk endeavor that results in low yields of effective and safe drugs; a problem that is confounded by the significant lack of information that exists in predicting the metabolic fate of drug candidates, in general, and in predicting the reactivity of drug candidates in the human body. The proposed Phase II STTR project for the development of Solidus Bioscience's TeamChip technology has significant relevance to public health by providing pharmaceutical researchers with in vitro information needed to predict the in vivo metabolism of drug candidates, and thus help to decide which compounds are brought forward for lead optimization and the ultimate development of better and safer drugs. Furthermore, this research is relevant to the prioritization of industrial and environmental chemicals in terms of their safety and use.

描述（由申请人提供）：Solidus Biosciences, Inc. 与伦斯勒理工学院合作，将重点关注其专有的“转染酶和代谢芯片”（或 TeamChip）的进一步开发、验证和商业化，用于系统候选药物以及化学代谢和毒理学的高通量分析。 TeamChip 的开发目的是模拟人类肝脏的首过代谢并预测酶特异性肝毒性。 TeamChip 上表达不同代谢酶组合的人类细胞文库将通过使用病毒递送系统将代谢基因转染到封装在排列在塑料芯片上的三维 (3D) 基质（小至 60 nL）中的人类细胞中来制备。因此，可以以与早期候选药物和环境化学品的预测人类毒性评估相当的速度评估和量化目标化合物与人类肝脏或其他器官类型中的单个人类代谢酶或酶组合的反应性。 第二阶段 STTR 提案的具体目标/里程碑是： 1. 构建携带来自人肝脏 cDNA 文库的代谢酶基因（例如代表性 CYP450 亚型和接合代谢酶）的重组腺病毒，证明使用荧光/发光底物对单层 Hep3B 细胞进行基因转染，并测量不同水平的 通过蛋白质印迹测定检测腺病毒酶的表达。 2. 准备含有表达各种代谢酶组合的 Hep3B 细胞的 TeamChip，并鉴定其差异表达影响细胞对模型化合物的反应的代谢基因。 3. 优化重组腺病毒和转染Hep3B细胞的片上冻存方案。使用冷冻保存的 TeamChips 展示代谢诱导的毒性，并将结果与​​非冷冻对应物进行比较。 可用于快速评估大量化合物毒性的体外技术仍然有限。安全性评价的一个重要组成部分是化学品（例如候选药物和环境化学毒物）的代谢和毒理学，它反映了化学品被人体代谢酶代谢的敏感性以及母体化合物及其代谢物的毒性。目前的化学品安全评估方法成本高昂、耗时，并且使用大量化合物和大量动物。因此，将 TeamChip 作为安全评估工具应用具有巨大的潜力和机会，可用于评估特定代谢酶是否以及如何影响候选药物和化学毒物的毒性。这种能力还可用于预测个体之间在药物和化学代谢以及毒性方面的差异。 公共卫生相关性：药物发现过程是一项投资密集型、高风险的工作，导致有效且安全的药物产量低；总体而言，在预测候选药物的代谢命运以及预测候选药物在人体内的反应性方面，存在严重缺乏信息而造成的问题。拟议的用于开发 Solidus Bioscience 的 TeamChip 技术的 II 期 STTR 项目与公共健康具有重大意义，它为药物研究人员提供了预测候选药物体内代谢所需的体外信息，从而有助于决定提出哪些化合物用于先导化合物优化以及最终开发更好、更安全的药物。此外，这项研究与工业和环境化学品在安全和使用方面的优先顺序相关。