Cell-based assays are powerful tools for monitoring response to external activation signals in living cells. Recognition of this fact has been responsible for roughly 40% growth in the industry in 2008, according to SMI Conferences, which recently sponsored a symposium on the topic. The meeting showcased new developments, including many different automated platforms, new assays for hepatotoxicity, and assays based on the application of transient gene expression, transmembrane receptors, and pluripotent stem cells.
“Of the many reasons for escalating clinical costs, failure due to toxicity holds the promise of being successfully addressed with new, discovery-stage tools,” said Katya Tsaioun, Ph.D., president of Apredica. Dr. Tsaioun argued that while technologies such as toxicogenomics and high-content imaging have the potential to hold down costs in the long run, predictive assays could speed clinical trials and lower their costs in a much shorter time frame.
According to Dr. Tsaioun, toxicity is a chronic and growing problem in drug development. She cited 12 drug withdrawals from the market between 1999 and 2001, three of which were due to toxicity that was missed during the development stage. A particularly egregious example was the antibiotic Trovafloxacin, which was withdrawn from the market due to its association with hepatotoxicity.
Studies have found animal testing to be only 50% accurate in predicting hepatotoxicity, with a large number of false positives and false negatives. “Improving hepatotoxic predictivity with an assay of 60% sensitivity and 90% specificity would save at least $150 million per year,” Dr. Tsaioun stated.
Hepatoxicity is a poorly understood phenomenon; in some cases, patients may worsen even after treatment with a suspected agent is terminated. Tests that look for reactive intermediates generated by drug metabolism have proven inadequate for the task since they look at only a single mechanism. Dr. Tsaioun has developed a multiparameter assay that evaluates a number of markers and is predictive of hepatoxicity even in the absence of cellular death. This approach encompasses a number of features, including cell loss, nuclear morphology, DNA content, cell membrane permeability, mitochondrial membrane potential changes, and cytochrome C release from the mitochondria.
Dr. Tsaioun and her colleagues use quantitative algorithms that score responses from 0 to 100%. The methodology was validated using several agents known to be or not to be hepatoxic, which provided a well-founded set of values on the basis of which each agent can be ranked.
“We believe that in vitro approaches with human cells are powerful tools for evaluation of the hepatotoxicity risk of early leads,” Dr. Tsaioun stated. “No one readout or assay is sufficient to definitively predict hepatotoxicity, but a multiparameter cell-based approach can yield insights when single mechanism readouts fail. Certainly, they are more cost-effective at this stage of development than animal studies and potentially more accurate.”
James Brady, Ph.D., director of technical applications at MaxCyte, believes that transient transfection systems offer a host of advantages that have largely been ignored. The company’s MaxCyte STX® system is used for developing cell-based assays. “We prefer transient transfection over stable transfection for a variety of reasons,” he said. “These include decreased time and cost of production, better control of the expression process, and the ability of the system to handle expression of toxic gene products.”
The MaxCyte system uses an electroporation technology designed to move small molecules, antigens, and nucleic acids into all varieties of cells. Using the green fluorescent protein gene as a convenient marker, Dr. Brady demonstrated that a number of well-known cell lines such as VERO, NIH 3T3, and CHO could be transfected with greater than 90% efficiency.
Following transfection, cells can be stored and recovered through cryopreservation with minimal loss of activity. Dr. Brady described a number of case studies in which different targets were successfully transfected such as the β2 adrenergic receptor into CHO cells and the M1 muscarinic receptor into HEK 293 cells. Transient and stably transfected cell lines show comparable levels of performance, he added.
Ion-channel expressing stable cell lines are especially problematic, thus representing an apt target for the MaxCyte technology. Their construction and validation is time consuming and expensive, and because expression of multiple subunits may involve a number of different genes (up to four in some cases), multiple selection with a number of antibiotics could be required. The presence of several antibiotics can lower cell viability and performance.
Using data shared by MaxCyte STX customers, Dr. Brady illustrated how multisubunit calcium channels can be expressed efficiently in transiently transfected HEK 293 cells. He also showed how the transfected cells can be used to screen ion-channel inhibitors using an automated calcium flux assay that is widely used for high-throughput screening.