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Lighting up in vitro and in vivo work
November 2008
by Jeffrey Bouley  |  Email the author

As scientists and clinicians in the drug discovery and development realm become increasingly keen to understand the complex, underlying biology of disease processes—and as good cell studies and animal studies become increasingly critical for weeding out bad drug candidates early—optical imaging technologies, also known as molecular imaging, are stepping up to the plate to help accelerate the process.

"If drug discovery and drug development are about cycles of optimization as medicinal chemists put compounds through analysis to improve outcomes, then the more times you can go through that cycle, particularly before you get to the clinical stage, the better your outcomes will be," notes Stephen Oldfield, the director of imaging marketing at Caliper Life Sciences. "Most importantly, you want to get animal studies into the cycle as soon as possible both to accelerate the process and help build confidence that a drug is worthy of human clinical trials, and optical imaging technologies, particularly bioluminescence and fluorescence, are being used more often, and earlier in the process, to do just that."

One sign of the growing importance and acceptance of optical imaging technology in discovery and development can be found in the movement of nuclear medicine professionals into pharmaceutical company roles, notes Dr. Robert Atcher, president of the Society of Nuclear Medicine. He says more and more of his colleagues in recent years have been lured from jobs in diagnostic nuclear medicine to work at pharmas to lend their talents in molecular imaging and radiolabeled imaging to drug development.

"Drug discovery is a very dynamic environment and I believe, as many other people do, that we should move more into the examination of living organisms because to understand cell biology, we need to understand function and relation of the proteins in a complex environment of living cells or living organisms and first of all in humans," says Dr. Georgyi Los, senior scientist and imaging group leader for Promega Corp. "Imaging technologies today give us a unique opportunity to look into the cells and into the organisms, and fluorescent tagging in particular gives us an opportunity to follow the proteins in this living environment without sacrificing animals, which is important to really understanding cell biology."

Bioluminescence vs. fluorescence

As just noted by Los, fluorescence offers some advantages over its major competitor, bioluminescence. But both types of luminescent technologies have their place.
Bioluminescence has an advantage over fluorescence in that it is extremely sensitive, with an excellent operation range and good range of linearity in measurements, Los explains.

Dr. Keith V. Wood, head of research at Promega, agrees, and adds that the technology also tends to be faster—an important consideration as real-time imaging becomes ever more critical—and says bioluminescence is also more robust.

"However, luminescence has some limitations in terms of spatial resolutions, and some spectral limitations because there are three useful colors of light generated by different luciferases, whereas we have an entire rainbow of naturally fluorescent proteins or fluorescent dyes that have a potential to label multifunctional reporters," Los notes.

He predicts that in the near future, bioluminescence will go toward miniaturization in ultra-high-throughput screening because of the signal-to-noise ratios, and he says that even today, numerous pharmaceutical companies, one of the more notable being Merck, are using 1,536 well plates, and even 3,456 well plates, for such applications.

"This can only be done with a robust luminescence signal," Los asserts. "I also believe that luminescence could be used in development of sensors that will measure second messengers, enzyme activity, or protein-protein interaction. Most importantly, this can be done in living cells in the real time."

For its part, fluorescence offers a broader spectral range, says Wood. Also, because you need a light source to create an excited state, researchers have more options for exploiting those excited states. Using polarized light to do fluorescence polarization or time-gating for time-resolved fluorescence are two such examples.

Another drawback of bioluminescence is that you must initiate a chemical reaction to create the excited state. Because researchers are limited in how fast they can drive a chemical reaction, this means the brightness of luminescence is often more limited than fluorescence. So, for times when researchers really need brightness, such as in cellular imaging or flow cytometry, fluorescence has an edge in generating a clear signal and the advantages of better signal-to-noise ratio offered by bioluminescence are less critical.

Another consideration is the fact that fluorescence can be used in the clinics, and fluorescent dyes are gaining increasing favor with FDA, whereas luciferase is not approved for use in humans.

"Bioluminescence doesn't translate well to the clinics," Oldfield notes. "I cannot really imagine a scenario in which people would want to be treated with cells that express luciferase. But fluorescence allows you to apply compounds to people that will bind to what you're looking at and allow you to follow biological processes through the body, and for that reason, fluorescence is generating a lot of excitement."

Natural fluorescent proteins have been in the conversation for probably the past 20 years, Los says, but it is only recently that they are becoming "a workhorse for cell biologists to look at things like translocation, protein interactions, signaling pathways and other areas that it was not possible to get a good read on even a few years ago."

Also, fluorescent imaging is a technique that can generate a great deal of good information at relatively low cost, Oldfield says, which is why he would like to see it more widely adopted. Ironically, this may also limit its rapid acceptance by some researchers.

"I've seen many people try to downplay the results of fluorescent imaging because it's so easy to get the data out. Scientists often don't need a dedicated person to operate the technology for them as they do imaging studies on animals. I've seen people lined up down the halls to use the equipment—it looks like LaGuardia some days with the lines—with people waiting to put animals through the systems. People are doing 50 to 100 animals in some industrial settings, and that is certainly something I would consider 'high-throughput' where animals are concerned at least."

Mixing it up

Where both bioluminescence and fluorescence could truly shine is if researchers more aggressively combined them with other imaging technologies, like using molecular imaging along with CT, MRI and PET scans.

"We see PET being used more and more not just in clinical diagnostics but in clinical work, preclinical work and even surrogate marker studies," says Dave Pendleton, senior director of PET perfusion imaging at Lantheus Medical Imaging, a company focused on diagnostic imaging applications. "I think PET has huge potential in drug discovery work, and I think that using it in conjunction with something like fluorescence could be very valuable."

Oldfield agrees that combining techniques is critical to better drug discovery and development, and he advocates both the combination of bioluminescence and fluorescence in research, as well as the use of both or either with other high-tech imaging modalities like CT and PET scanners. And he says he would love to see development of fluorescent markers that could also serve as MRI contrast agents.

"I'm seeing more data using a combination of optical imaging with luciferase or fluorescent dyes and then CT scans, which can give you an exquisitely sensitive and quantitative picture of exactly where and what a molecule's effects are in animal models," Oldfield says.

"I believe it is important to use multiple imaging tools, and I would like to see more drug developers and discovery biologists use a combination of different imaging modalities, like luciferase and natural fluorescent proteins with PET, for example, to get real-time readings from three different perspectives," adds Los.

Cost and transparency

One of the major challenges in wider use of imaging technologies is that as the tools become more complex, they may require special skills, Los notes, "and so we need proper training and education of scientists and drug screeners."

Another challenge, he says, is the cost of the technology. A contemporary confocal microscope is about $300,000 and costs more if you go after additional features beyond the basics, Los notes. High-content screening instruments run $400,000 to $800,000. Super-resolution microscopy equipment is priced in the range of $1.3 million.

"So this kind of instrumentation is difficult for regular scientists to afford," he says. "What we need are more core imaging facilities at various universities or pharmaceutical companies where researchers can go and get time on instruments instead of having to get them into their own labs."

Also needed is more transparency, Los says, and not in the sense of transparent business practices or transparent research methods.

"One of the areas that needs more development is near-infrared fluorescent proteins and ligands and luciferase that can illuminate in the near-red part of the spectrum," he says. "Tissues are more transparent the closer you get to the infrared end of the spectrum, and that means higher sensitivity and better resolution."

Pushing the development and use of more molecular imaging biomarkers will also be critical to expanding the use of imaging technologies, notes the Society of Nuclear Medicine's Atcher.

"We and other organizations have talked with FDA about the need to be more flexible on the use of experimental biomarkers in assessing the effectiveness of drugs in addition to—or instead of—classical criteria," he says. "The FDA has actually been very accepting of this concept and is definitely making that part of their Critical Path Initiative, which is critical for researchers to make better decisions early on."

Success stories

Often, it's the researchers and not the companies that come up with the breakthroughs on the technology side, and that's no different in optical imaging.

Last year, for example, molecular virology and biomedical engineering professor Peixuan Guo at Purdue University created a single-molecule imaging system to view DNA, RNA and other tiny biological molecules smaller than the 200-nanometer diffraction limit of standard microscopy equipment. In doing so, he helped settle a seven-year debate over the shape and structure of a biological "motor" of interest in nanomedicine for diagnosing and treating diseases like cancer, AIDS and influenza.

The system, called a single-molecule dual viewing total internal reflection fluorescence imaging system (SMDV-TIRF) is an improvement of total internal reflection microscopy, with a laser combiner to control the release of two or more lasers as the light source.

At Sandia National Laboratories this summer, researchers used a custom confocal microscope for optical imagining of naturally occurring nanotubes that may serve as tunnels to protect retroviruses and bacteria in transit from diseased to healthy cells—a fact that may explain why vaccines fare poorly against some microorganisms.

The specialized instrument used at Sandia allowed researcher Carl Hayden and others to achieve pixel-by-pixel examination protein interactions with the membranes of the nanotubes by detecting the spectrum and lifetimes of fluorescent labels on the proteins. DDN



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