While the zebrafish has become a popular model organism for developmental biologists and geneticists, at least one company is using this hardy translucent fish as a drug screening tool.
For the past five years or so, a contract research organization called Phylonix, located in Cambridge, Mass., has been attempting to exploit the model organism’s well-known advantages — translucency for easy imaging, rapid developmental cycles, short generation time, and the ability to produce viable organisms in spite of a wide variety of mutations — for high-content drug screening.
Just over a month ago, Phylonix received a vote of confidence from the US National Cancer Institute in the form of a $993,463 Phase II SBIR Grant to further develop live zebrafish apoptosis assays for drug screening. The Phase II grant — typically awarded to a company that has proved the commercial viability of its technology following a Phase I grant — is the third such grant awarded to Phylonix in the last three years for live zebrafish-based assays. In 2001, the company was awarded one such grant to develop organ toxicity screening assays, and in 2002, it received one for angiogenesis.
Organ toxicity assays are crucial in drug screening, as they can predict where a drug might fail later on in clinical trials before a company has invested prodigious sums to take it that far. Meanwhile, angiogenesis and apoptosis assays are more important for drug specificity in certain disease states. Both are heavily studied in cancer, and apoptosis assays are also crucial for screening drugs in heart disease, stroke, AIDS, autoimmunity, and degenerative disease.
The most recent grant money, Phylonix said, will be used to further develop the zebrafish apoptosis assays using acridine orange dye staining. In addition, the company is planning to develop a quantitative, vital dye assay using automated liquid handling and a microplate reader to increase throughput.
Somewhere in between cellular assays and small animal testing, the assays open up a new avenue in high-content screening, said Chaoyong Ma, Phylonix’ business development manager.
“While the concept of high-content screening was first proposed by a company doing cell-based assays,” Ma said, “I think the concept is more applicable to in vivo assays, because you get more information. Multiple parameters are much more predictive in a whole organism.”
The assays are performed in standard microwell plates, as the fish embryos can be maintained in as little as 100 microliters during the first six days of their development. Chemical compounds can then be added directly to the wells, and the results of the assay examined using with different basic microscopes or plate readers depending on the type of assay being performed.
Several companies have similar assays on the market that utilize very small organisms such as C. elegans and Drosophila, but Ma said that zebrafish are still more relevant to human health because they are vertebrates. But the assays still beg the question: How relevant to human disease are they as compared to mouse or rat testing, or even to cell-based assays using mammalian cells?
Ma said this question is one of those that potential users most frequently ask company representatives.
“I think the underlying validation of using species that are farther away from humans than mammals is that the underlying genetic pathways and physiology are still highly conserved,” he said. “If you look at the genetic sequences, there is a very high conservation between the homologs of the human and zebrafish genes — especially in the functional domain. Often they are almost identical.
“Because a lot of drugs act on the functional domains,” he added, “there is a high degree of similarity in drug response.” Ma also said that there is a significant body of research published — most of it by Phylonix — that shows a great deal of similarity between drug responses in zebrafish and humans. He cited, in particular, the response to anti-angiogenesis drugs.
Phylonix claims that even with the high level of relevance, the assays feature many of the positive aspects of cell-based assays, even trumping a few. Specifically, the company cites short assay time, single dosing, low drug usage, easy embryo maintenance, and low cost. Each assay is estimated to cost less than $100, which is a bit pricier than most cell-based assays, but cheaper than small-animal studies.
Russ Lebovitz, president of Houston-based drug development company C Sixty, told Inside Bioassays that the company uses Phylonix’ assays primarily for lead optimization in evaluating its drug candidates based on a class of small molecules known as fullerenes. The company recently signed an agreement with Merck to license these compounds for animal studies and further development.
“We’re a very early-stage drug-discovery company, and so we’re trying to rapidly find the best in class for each of our compounds,” Lebovitz said. “We’re interested in things from distribution to efficacy to toxicity. We use the zebrafish to … rapidly discern which of the structural features [of our lead compounds] might be related to toxicity or efficacy in one cell type versus another.”
Lebovitz said that the company finds the zebrafish assays to be “much more effective than cultured cells, particularly for things like toxicity, where even in the very early zebrafish, we’re looking at a number of functional organ systems that aren’t that different in a lot of their basic biochemistry than what’s going on in mammalian systems.”
Lebovitz characterized the assays as being like “multidimensional, primary screens,” adding that it’s almost as simple to perform as it is with cultured cells. And although the cost is more than that with typical primary screens, he said, “the cost of making a mistake early on and going too far down the drug-development road is much higher, and this is the benefit of the zebrafish assays.”
Ma said that Phylonix has a number of other biotech and pharmaceutical customers, but that he could not disclose them due to confidentiality agreements.
As it stands right now, Phylonix remains a contract research organization, performing the assays for its customers at its Cambridge laboratories. However, Ma said, this does not rule out the possibility that the company will eventually license the assays to pharmaceutical customers for in-house use, as it has intellectual property rights to most of the methods.
In addition, although Phylonix currently uses basic microscopy platforms provided mostly by Zeiss, it is in discussion with several other companies about designing “a more complicated and detail-specific reader to provide more content-rich data,” Ma said.