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.
Thesaurus Terms: biomedical automation, biomedical equipment development, drug discovery /isolation, drug screening /evaluation, flow cytometry, high throughput technology, image processing, liver cell, mixed tissue /cell culture, tissue engineering, toxicant screening adsorption, clearance rate, cytotoxicity, drug adverse effect, drug metabolism, lead, three dimensional imaging /topography bioimaging /biomedical imaging, biotechnology, fluorescence
