Nobody likes testing human drugs on animals. While public concerns usually center on humane treatment for the animals, researchers find non-human models of disease expensive and inconvenient. Worse, they are often poor predictors of human results. Besides interspecies differences in physiology, animal studies rely on uniform populations, virtually guaranteeing that they will miss idiosyncratic reactions.
“We look for organ toxicity in drug-naive, young, male animals,” says James Dykens, PhD, associate research fellow at Pfizer in Kent, UK. Dykens adds that actual patients are likely to be older and sicker, often with complex medical histories. As a result, drugs such as troglitazone (Rezulin) and buformin were able to pass animal toxicity tests with flying colors only to later be withdrawn from the market for idiosyncratic toxicity.
In recent years, several trends have made the animal testing problem even more acute. High-throughput screening campaigns continue to push new leads into preclinical studies, but tight company budgets often restrict the availability of expensive laboratory animals. Meanwhile, governments in Europe have squeezed animal testing from both sides; several countries have restricted animal experimentation, while the European Union-wide Registration, Evaluation, and Authorization of Chemicals (REACH) regulation mandates more chemical toxicity testing.
As researchers clamor for better non-animal tests, several companies have stepped into the growing niche of cell-based toxicology assays. Drawing from emerging fields such as nanotechnology and stem cell science, the new assays promise to reduce the need for laboratory animals while simultaneously increasing the success rate of new leads.
Little liver lumps
Many drug side effects involve the liver, so testing leads in hepatocytes is an obvious way to look for toxicity. However, hepatocytes are notoriously finicky. Transformed cell lines do not replicate the behavior of normal liver cells and primary hepatocytes grow unevenly on a petri dish, yielding inconsistent results.
Researchers at Transparent, in Tokyo, Japan, tackled this problem by applying a nanoengineered coating to tissue culture plates. The coating is a sheet of hydrophobic material with regularly spaced, microscopic “holes” of hydrophilic material. “This creates a pattern of adherent and non-adherent areas on the plate. After cell seeding, the hepatocytes migrate to the area where they can adhere, and they settle down there,” says Takeshi Ikeya, PhD, president of Transparent. Ikeya adds that “As a result, the hepatocytes make a spheroid, which reflects the pattern of holes on our plate coating.”
Hepatocytes also form spheroids on ordinary culture plates, but the ball-like structures grow randomly, producing blobs of cells with varying characteristics. Cells in small spheroids act like ordinary hepatocytes in vivo, but large ones often develop necrotic cores that can skew the cells’ responses to drugs. “By providing holes that are 100 mm, our system can make hepatocytes that easily form clots, and don’t build up large aggregations. In this way, our system can maintain a spheroid culture that keeps its aeration and nutrient supply so that the risk of necrosis comes down,” says Ikeya.
Transparent’s coatings allow researchers to grow a grid of uniform hepatocyte spheroids in each well of a 96-well culture plate. The system can accommodate primary hepatocytes isolated by standard protocols, so drug developers can screen their compounds in rodent or human liver cells.
Several companies in Japan and the US are now evaluating the system, and academic researchers hope to push its capabilities even further. “In the academic world in Japan, there has been investigation regarding the use of our product with bone cells and chondrocytes … or stem cells,” says Ikeya.
But like most researchers working on alternatives to animal testing, Ikeya still sees a role for laboratory animals: “It is not possible to recreate all in vivo functions [in cells], thus we don’t intend for our system to directly replace animal systems for hepatic testing. We are able to contribute to a reduction in animal experiments but not replace them.”
The shape they’re in
Besides reducing the need for costly animal tests, some cell-based assays can provide more information than animals. For example, cultured cells often exhibit characteristic shape changes in response to sub-lethal doses of toxins. The changes can take hours or days to occur, though, so researchers turn to automated assays to monitor their cultures.
One such assay is impedance sensing, a technique originally developed in the early 1980s. In its simplest form, an impedance sensing assay involves culturing adherent cells on a conductive surface and pulsing alternating electrical current through them. The cells act as insulators, so if their shapes change to expose more of the underlying surface, the impedance of the system will also change.
As Christian Renken, PhD, applications scientist for Applied Biophysics in Troy, N.Y. explains: “It turns out that very minute motions of the cells as they pull apart from each other … that lets current through actually very quickly, so those very subtle movements [give] quite large signal responses in the impedance measurement.”
Impedance sensing assays have evolved considerably over the years. The latest versions, such as the Electric Cell Substrate Impedance Sensing (ECIS) system from Applied Biophysics, incorporate a complete cell incubator, 96-well plates, and sophisticated data analysis software. Using ECIS, researchers can monitor cells for days or weeks, and parse the raw data to detect even small, brief changes that could indicate low-level toxicity. “If you’re adding something and there’s an initial insult but the cells recover, then you won’t see that in an endpoint assay, but you will see that under our system,” says Renken.
The primary patent on impedance sensing has expired, so Applied Biophysics now competes with other companies making similar systems. For example, Molecular Devices (fomerly MDS Analytical Technologies), in Sunnyvle, Calif. offers a 384-well impedance sensing setup called CellKey. That system’s higher throughput and relatively straightforward operation may appeal to lead developers; Renken says that his company’s ECIS is aimed at earlier-stage researchers who work at medium throughput, and prefer direct access to the raw impedance data.
The expanding market for cellular impedance sensing highlights the drive to reduce animal use in all types of research. “Since a lot of our researchers are true cell biologists … they use this a lot to complement what they’re learning with their mutant mice, [and] guide the experiments they would do in their knockout mice,” says Renken.
The other energy crisis
For some toxicities, though, both animal assays and cellular models have been weak. That’s particularly true for mitochondrial toxicity. Because standard cell culture media have high glucose concentrations, cultured cells can survive even if their mitochondria have been poisoned.
“When the nascent drug would be evaluated in those cell models, the cells wouldn’t … show any toxicity, so the assumption was that the compound was safe, and off it would go into animals and humans, and in both those cases most of these responses were idiosyncratic,” explains Pfizer’s Dykens. Both troglitazone and buformin followed that pattern, with researchers only discovering the drugs’ mitochondrial toxicities after expensive post-marketing failures.
Mitochondrial toxicity assays have been available for years, but they have required special culturing conditions and arcane techniques that don’t fit well in a high-throughput workflow. To get around these problems, Luxcel Biosciences in Cork, Ireland now offers fluorescence-based assays for mitochondrial function. The assays rely on the company’s proprietary oxygen-sensing molecules.
In a typical experiment, a researcher adds the water-soluble Luxcel probes to a cell culture or extracted mitochondria, then puts a layer of mineral oil over the top, preventing additional oxygen from reaching the assay. “As the oxygen is being consumed by the cell there’s less oxygen in the media, so there’s less oxygen to quench the signal of the probe in the media. You get a brighter signal, so as cells respire you get an increase in signal,” says Richard Fernandes, CEO of Luxcel. If a test drug poisons the cells’ mitochondria, the signal is lower or nonexistent.
Because the assays work in standard 96-well or 384-well fluorescent plate readers, drug developers can add them to their standard lead screening process without having to master the intricacies of mitochondrial biology. “We’re hopefully taking what was typically a very specialist area … and converting it into an easy-to-use, real time assay,” says Fernandes.
That idea appears to have caught on with pharmaceutical researchers. Some even hope that mitochondrial assays will eventually become the law of the land. “I will feel that I’ve accomplished my mission if I can get the EMEA [European Medicines Agency] and the FDA [Food and Drug Administration] to mandate mitochondrial assessments for all [new drugs],” says Dykens, adding that “we’re still trying to work out the structure-activity relationships, and it’s very complicated, so we have very low predictivity. Under those circumstances you just have to empirically determine whether the compound has a mitochondrial liability.”
Other companies are also working on making traditional cellular assays more user-friendly. At HemoGenix in Colorado Springs, Colo., researchers developed an updated version of the decades-old colony-forming cell assay for hemotoxicity. The assay, called HALO, measures the proliferation of blood stem cells quantitatively with a luminescent output. Colony-forming cell assays that would have entailed hours of laborious, subjective colony counting can be done in minutes on the HALO platform, with full automation and up to 384-well capacity.
Since introducing it in 2002, the company has made several updates to the system. Drug developers can now determine whether a lead is likely to alter blood cell counts by itself, or in combination with other drugs the target patients are likely to take. “Now companies are seeing that they can save quite a bit of time and quite a bit of money by performing these assays up front before they even start doing animal testing. Because the assays can be very easily performed using human material, they get a lot of very predictive information out of the results that they get,” says Ivan Rich, PhD, CEO of HemoGenix.
The company is now working on a system that will mimic in vivo drug metabolism, allowing drug developers to test precursor molecules as well as active compounds and breakdown products. “When a drug is metabolized it may produce very active or even inactive metabolites and then the question is, well is it those metabolites that are causing problems, and what those problems might be,” says Rich.
Like other assay developers, Rich believes that in vitro tests will continue to reduce or replace animal assays: “There’s certainly been a very big push towards alternative methods, and … in order to be able to predict what is going to happen in the human patient, it’s important to be able to use human cells.”
About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and government for more than a decade.
This article was published in Drug Discovery & Development magazine: Vol. 13, No. 4, May 2010, pp. 10-13.
Filed Under: Drug Discovery