Part one in a three-part series on advances in ADME-Tox
Smaller organisms like zebrafish allow researchers to perform inexpensive high-throughput screens and detect toxicity early in discovery
The use of living organisms to test the safety and efficacy of potential drug compounds has long been an industry mainstay. But despite their importance, only a few useful
models have been developed. Over the last several years, researchers have worked to create new tools using transgenic animals, juvenile animals, and novel small-animal models. Still, advances in in vivo technologies lag behind areas like in vitro and in silico modeling, but they will clearly continue to play a role.
“The contribution of in silico and in vitro studies is in fact increasing, but on the other hand, in vivo studies and studies with radiolabeled compounds are still needed and will be needed in the coming years. I think many people have hope that at a certain time in vivo animal studies will be no longer be needed, but right now, that doesn’t seem realistic,” says Willem Meuldermans, PhD, senior director, global preclinical pharmacokinetics, Johnson & Johnson Pharmaceutical Research and Development (J&JPRD), Beerse, Belgium.
Their mechanistic toxicology group uses molecular biology methods to help predict and anticipate toxicity issues with compounds as they are selected for development, and to better define toxic mechanisms of action for compounds after toxicities are identified. The compound of interest is administered to animals, short-term transcriptomic responses are evaluated by microarray, and the microarray analysis suggests potential mechanisms of toxicity. The hypotheses generated from this and other analyses like pathology or clinical chemistry are tested in either animals or cultured cells.
Another area gaining increasing attention, and being investigated at JJPRD, is the use of juvenile animals for pediatric programs, says Geert Mannens, PharmD, a research fellow in Beerse. J&J was one of the first companies to have a special department for pediatric development, he adds, and more recently, regulatory agencies have sought comments on draft guidances for the study of toxins in juvenile animals. “Until recently,” Meuldermans says, “there was the idea that it was very difficult to predict data in your pediatric formulations from juvenile animal studies, but because of the increasing knowledge about the ontogeny of metabolizing enzymes and so on, it seems to be becoming a request from most of the regulatory authorities.”
Meuldermans adds that the increasing sensitivity of many analytical and bioanalytical instruments are enabling researchers to enhance in vivo models. For example, lower sample aliquots are required and more samples can be taken from one rodent. “Until now, you could only collect a limited number of samples from one rat or one mouse, but because only tens of microliters are needed now, you can sample more very quickly.” And while it is not new, quantitative whole body auto radiography with phosphor imaging, in which radioactive compounds are added to rats or other rodents, is evolving. “This is really replacing studies that were previously done with dissection of a whole number of different tissues and organs.”
Meuldermans and Mannens say tools such as these are used at most major pharmaceutical companies, but researchers are working on newer tools that have yet to be embraced, or in some cases, even noticed by industry. One promising area is emerging model organisms which enable animal studies at the earliest stages of development. They have potential uses in library design and characterization, hit prioritization, and preclinical efficacy and toxicity. Mammalian models, on the other hand, are largely restricted to later-stage development.
Randall Peterson, PhD, acknowledges the utility of in vivo tools like mice, rats, dogs, and guinea pigs. “One trend is to improve those sustaining technologies, make them more human-like using transgenic lines, getting to more and more complex mammalian models. That of course, is very important, and that will continue,” says Peterson, of the cardiovascular research center at Massachusetts General Hospital, Boston. But he is more interested in emerging in vivo tools such as roundworms, fruit flies, and zebrafish. “These technologies are less sophisticated but they’re also much easier and much cheaper, and so they enable entirely new applications that you wouldn’t have dreamed of doing before . . . They’re allowing people to look at toxicity much earlier in the process.”
The zebrafish model emerged over the last few years. Its use has grown dramatically in the basic sciences, especially in academia, but it is just beginning to be used by the
click the image to enlarge
Zebrafish embryos in the well of a 384-well assay plate. (Source: Randall Peterson, PhD)
pharmaceutical industry. Unlike the less complex invertebrates C. elegans roundworm and fruit fly, the zebrafish genome is not fully sequenced. In addition, zebrafish embryos hatch in four days and can eat and swim. Their intestinal epithelial cells are polarized and express digestive enzymes, their hepatocytes secrete bile, and their pancreatic islets and acini produce insulin and carboxypeptidase. The embryos are transparent, so every event in early development can be observed, and quantitative end point assays can be performed using microplate readers.
“If you have a high-throughput, fast, cheap, easy way to assess toxicity you could imagine prioritizing hundreds of compounds that come out of an early screen. You could even take a whole library of 100,000 compounds and prefilter it, weeding out problematic ones up front. So you’re finding the problem compounds at a much earlier stage and you’re saving a lot of money.”
QT prolongation is one area that Peterson’s lab has studied intensively using zebrafish. QT prolongation, which is associated with increased risk of lethal ventricular arrhythmias, is a side effect that can occur in a number of drugs. Peterson’s lab developed a system that lets them put a fish in each well of a 384-well plate. The system then goes to each well and records a heart rate for each individual fish. “We’ve used that system to test 100 FDA approved drugs, of which we know 23 caused QT prolongation in humans. What we found was that this system could detect 22 out of 23 of them causing a slowing of the heart rate in the zebrafish.”
The zebrafish model is also able to identify drug-drug interactions known to occur in humans, including the severe QT prolongation that can occur when Propulsid (cisapride) is combined with erythromycin. “These instances demonstrate that this system combines the advantages of high-throughput in vitro techniques with the physiological relevance of the whole animal. In an in vitro assay, you could never identify these complex drug-drug interactions that you need to have a whole organism to detect.”
Phylonix Inc., Cambridge, Mass., is a contract research organization that provides zebrafish assays for drug discovery and screening. Founded in 1999, the company has customers who run the gamut from academic and government labs, small biotechnology companies, and small and large pharmaceutical companies, says Patricia McGrath, president and chief executive officer. One company they work with is C Sixty Inc., Houston, a small company that is seeking to develop drugs based on carbon-60 fullerenes, a molecule of 60 carbon atoms that forms a hollow sphere 1 nm in diameter. The molecule has numerous points of attachment that allow for grafting of chemical groups three-dimensionally, a key to rational drug design. “They like the speed with which they can get data from the zebrafish, and they’ve been modifying their compounds based on the results they get in the fish. They’ll change the structure of the fullerenes depending on whether they get a toxicity readout.”
Filed Under: Drug Discovery