Automated Microscopy of 3-D Liver Tissue for ADME/Tox

描述（由申请人提供）：ADME/Tox评估的传统方法，包括HepG 2细胞、肝切片、微粒体、肝细胞（单层、悬浮液和冷冻保存）和动物，不能完全预测人体功能，并且具有以下一个或多个缺点：表现出短期功能，不完美、可变、昂贵、不可扩展和/或供应有限（参考Segenix，Inc.）拥有核心专利技术，可实现原代人肝基质细胞和肝组织实质细胞的三维共培养体外生长。大量研究表明，该组织在长期体外培养（48天大鼠，80天人类）和体内植入（大鼠和犬）后保留了生理和生化功能，并提供了对模型毒素的生理反应。缺乏体外系统预测人类ADME/Tox功能是困扰制药行业的关键问题。2000年，制药行业估计花费了30亿美元用于药物ADME/Tox研究。不良ADME（41%）和肝毒性（22%）是导致候选药物未能成为新药的主要原因。ADME/Tox的先导化合物失败可能会对公司业绩产生破坏性影响，因为临床试验中的候选药物丢失，延迟新药上市，黑盒警告标签对FDA批准的药物施加了重大限制，以及从市场上撤回批准的药物。由于药物引起的肝损伤，至少有五种已批准的药物已被撤出市场，每种药物的平均成本为5亿美元。工程化的人肝组织是可扩展的、可再现的和特异性的，并且被设计为提供正常的、患病的和多态的组织。这种方法使ADME/Tox研究能够在药物发现过程的早期进行，成本较低。在药物发现过程的早期成功整合这些工程化的人体组织将需要小型化、自动化和高通量，并在细胞和分子水平上保留关于组织功能的详细信息。Q3 DM高性能扫描细胞仪是满足这些要求的理想技术。该系统利用自动聚焦、图像分割和灯稳定方面的进步来提供第一个完全自动化的荧光图像细胞仪，其提供流式细胞术细胞度量的超集，具有相等或更好的自动化和直接访问原位细胞。基于细胞的测定能够鉴定以特定方式与所选分子靶标相互作用的化合物。在整个细胞以及复杂的细胞相互作用的背景下发生的高通量测定可以利用多重报告分子准确地可视化和定量。这些基于细胞的系统近似生理条件，并产生高度复杂的信息，需要复杂的图像分析工具和简化的数据处理。然而，有许多应用，特别是ADME/Tox，细胞反应往往是不够的。对于ADME/Tox，由组织提供的细胞-细胞通讯和结构组织对于评估外源性物质存在下的组织特异性反应和功能是必要的。为了满足这一需求，本提案中概述的工作旨在利用为单层细胞成像开发的Q3 DM高性能显微镜系统，对Segenix工程化三维工程化肝组织中的细胞和细胞过程（包括蛋白质表达和酶功能）进行可视化。将在ADME/Tox评估应用中测试所得的高通量三维组织图像分析平台，以评估在三维体外肝共培养物中提供肝细胞组织特异性功能维持的主要候选物和机制。如果成功，将开发系统组件，使全球以及特定应用的组织成像用于药物发现和开发。

DESCRIPTION (provided by applicant): Traditional methods of ADME/Tox assessment, including HepG2 cells, liver slices, microsomes, hepatocytes (monolayer, suspension and cryopreserved), and animals are not fully predictive of human function and have one or more of the following drawbacks: exhibit short-term function, are imperfect, variable, expensive, not scaleable and/or limited in supply ref. Segenix, Inc. possesses patented core technologies enabling the in vitro growth of three-dimensional co-cultures of primary human liver stroma and parenchyma cells that function as liver tissue. Numerous studies have demonstrated this tissue retains both physiological and biochemical functions in long-term in vitro culture (48 day rat, 80 day human) and following in vivo implantation (rats and dogs), as well as provides a physiologic response to model toxins ref. The absence of in vitro systems predictive of human ADME/Tox function is a critical problem plaguing the pharmaceutical industry. An estimated $3.0 billion was spent on drug ADME/Tox studies by the pharmaceutical industry in 2000 ref. Poor ADME (41%) and hepatotoxicity (22%) are the principle reasons for failure of lead candidates to become new drugs ref. Failure of lead compounds for ADME/Tox can have devastating effects on company performance through loss of lead candidates in clinical trials, delays in bringing new drugs to market, black box warning labels imposing significant limitations on FDA approved drugs, and withdrawal of approved drugs from the market. At least five approved drugs have been pulled off the market due to drug-induced liver injury at an average cost of $500 million per drug 'ef. The engineered human liver tissues are scalable, reproducible and specific and designed to provide normal, diseased and polymorphic tissues. This approach enables ADME/Tox studies to be performed earlier in the drug discovery process when costs are lower. Successful incorporation of these engineered human tissues earlier in the drug discovery process will require miniaturization, automation, and high throughput with retention of detailed information on tissue function at the cellular and molecular levels. The Q3DM high performance scanning cytometry instrumentation is an ideal technology to meet these requirements. The system utilizes advances in autofocus, image segmentation and lamp stabilization to provide the first fully automated fluorescence image cytometer that provides a superset of the flow cytometry cell metrics with equal or better automation and direct access to in situ cells. The cell-based assays enable identification of compounds that interact with a selected molecular target in a specific manner. High throughput assays that occur in the context of the whole cell as well as complex cellular interactions can be visualized and quantified accurately utilizing mutiplex reporter molecules. These cell-based systems approximate physiological conditions and yield highly complex information which requires sophisticated image analysis tools and streamlined data handling. However, there are many applications, specifically, ADME/Tox, for which cellular response, is often insufficient. For ADME/Tox, the cell-cell communication and structural organization afforded by a tissue is necessary to assess tissue-specific response and function in the presence of xenobiotics. To meet this need, the work outlined in this proposal aims to leverage the Q3DM high performance microscopy system developed for monolayer cell-based imaging into visualization of cells and cellular processes (including protein expression and enzyme function) in Segenix's engineered, three-dimensional engineered liver tissue. The resultant high throughput three-dimensional tissue image analysis platform will be tested in the applications of ADME/Tox assessment of lead candidates and mechanisms affording maintenance of tissue specific function to hepatocytes in three-dimensional in vitro liver co-cultures. If successful, system components will be developed that enable global as well as application-specific use of tissue imaging for drug discovery and development.