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Charles River scientist using in-vitro methods to accelerate the drug development process. (Image: Charles River) |
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The current pharmaceutical landscape demands safe drugs. To that end, the industry is replete with a seemingly never-ending array of toxicity testing tools and approaches. Cell-based toxicity testing is one of those approaches and is primarily performed at the preclinical stage. Various cell lines are available for toxicity testing, including, most recently, stem cells. Toxicity testing is performed by all of the major types of companies within the industry. As a contract research organization (CRO), Charles River (Wilmington, Mass.) offers a large panel of in vitro toxicity testing. “We think cell-based systems are the way to go,” says Alain Stricker-Krongrad, PhD, chief scientific officer at Charles River.
And the main reason cell-based in vitro toxicity testing is so popular is obvious: It is a cheap way to eliminate toxic compounds earlier. Says Usha Warrior, PhD, technical director, In Vitro Pharmacology at MDS Pharma Services (Bothell, Wash.): “The industry is looking for ways to improve the odds of success for drug discovery while reducing the cost. ‘Fail fast and fail early’ has become the mantra for drug discovery where it is ideal to identify the liabilities early which will eliminate costly drug withdrawals in future.”
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Low power image of iPS cells prior to differentiation. Image taken at 2X magnification. (Images: Cellular Dynamics International) |
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James McKim, PhD, DABT, chief scientific officer, Ceetox Inc., Kalamazoo, Mich. adds, “Late-stage attrition of promising new drug candidates is a major concern for pharmaceutical companies. Although cell-based assays that measure chemical toxicity have been used for many years, it has been the pharmaceutical companies that have improved the technology and applied the data obtained to identify liabilities earlier in the discovery pipeline.” Ceetox specializes in providing in vitro toxicity testing for its pharma customers. The company’s approach is to focus on multiple biochemical endpoints of different essential cellular functions in human cardiomyocytes and rat liver cells such as cell proliferation, mitochondrial function, and cell membrane integrity.
Heart of the matter
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Human induced pluripotent stem cell-derived cardiomycytes, dissociatied and plated as they would be for a cytoxicity screening assay. Image at 10X magnification. |
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As noted above, human cardiomyocytes are a part of a growing list of cell lines currently available for toxicity testing. The reason for this availability is that a major area of cell-based in vitro toxicity testing is cardiotoxicity. Among many examples, one of the cell-based in vitro toxicity testing areas for Charles River is cardiovascular drug testing. For example, the company measures the effect of drug compounds on the activity of the hERG potassium channel by performing high throughput and single-cell patch clamping experiments. Drugs that affect the channel may lead to QT prolongation in humans because of the repolarization of the heart; various drug classes, including antihistamines and some antibiotics, impact the hERG in this way.
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Human induced pluripotent stem cell-derived cardiomycytes in aggregate form. Cardiomyocytes are labeled with red florescent protein (RFP) and each aggregate has approximately 500 to 2000 cardiomyocytes. |
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MDS Pharma Services also uses a number of different cell lines, including cardiomyocytes, in the cell-based in vitro toxicity testing it provides to its pharma customers. They have developed genotoxicity, hepatotoxicity, cardiotoxicity, and immunotoxicity assays. “For the ion channel assays to measure cardiotoxicity, we use transfected cell lines with human hERG, NaV1.5, and CAV1.2 channels,” says Christine O’Day, PhD, senior manager/cell biology group leader at MDS Pharma Services. “For our ion channel assays we use an automated patch clamp assay using PatchXpress (Molecular Devices, Sunnyvale, Calif.).” In addition, MDS Pharma Services is in the process of developing other in vitro toxicity assays such as those for skin using keratinocytes. In terms of readouts, fluorescent markers and antibodies are used for detection of micronuclei and to identify and eliminate false positives in this high content approach for genotoxicity. Fluorescent probes and antibodies are also used for hepatotoxicity testing to monitor cell death, phospholipidosis, and neutral lipid accumulation. The Luminex platform (Luminex Corporation, Austin, Texas) is used for immunotoxicity testing because it provides the ability to multiplex different cytokines or other biomarkers in the same well. Lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay. These assays are semi-automated, using liquid handlers and robotics.
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Close up of a single differentiated human induced pluripotent stem cell-derived cardiomyocyte labeled with sarcomeric Alpha Actinin. |
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Cellular Dynamics International (CDI) has developed human induced pluripotent stem cell (iPSC)-derived human heart cells (cardiomyocytes) for use in cell-based in vitro toxicity assays. CDI has industrialized the production of large scale cultures of iPSCs, which are then differentiated en masse into cardiomyocytes. An antibiotic resistance marker is inserted to allow for genetic selection of purified cardiomyocytes, while eliminating all non-cardiomyocytes. “The cells spontaneously beat in culture like real heart cells, thus exhibiting the electrophysiological behaviors associated with beating as well as standard biochemical and physical characteristics of any human cardiomyocyte,” says Chris Kendrick Parker, BS, chief commercial officer, at CDI. CDI’s pharmaceutical customers perform chemical and electrophysiological assays such as single-patch clamping on the cardiomyocytes in order to determine cardiac safety of their drug candidates.
For the foreseeable future, cell-based toxicity testing will remain a crucial part of preclinical drug testing. Current technologies such as iPS cells and their use in the differentiation and manufacture of various mature, differentiated human cells (e.g. cardiomyocytes) have improved the accuracy of toxicity data, allowing drug developers to more closely predict drug response in humans.
About the Author
James Netterwald is president and CEO of BioPharmaComm LLC, a provider of writing, editing, and consulting services to the life science, pharma-biotech, and public relations industries.
This article was published in Drug Discovery & Development magazine: Vol. 12, No. 10, November/December, 2009, pp. 22-23.
