February 1, 2009 (Vol. 29, No. 3)
Ability to Provide Critical Information Earlier in the Development Process Is Boosting Usage
In vivo molecular imaging can dramatically increase the efficiency of drug development and diagnostics, characterizing target engagement, pharmacokinetics, and pharmacodynamics, according to researchers at Cambridge Healthtech’s recent “In Vivo Molecular Imaging” meeting. Some of the newer in vivo molecular imaging techniques give researchers vital information much earlier in the development process.
At Zygogen, using transparent zebrafish embryos as early test subjects offers the benefits of direct visualization of compound effects and the ability to run assays using 96- or 384-well plates. “These zebrafish embryos also advance the regulatory goal of decreasing the number of mammals used in testing,” said Timothy Baranowski, Ph.D., director of operations.
More importantly, zebrafish let researchers see the effects of a compound on the whole animal early. “At seven days, they are 5.5 to 6 mm long,” Dr. Baranowski noted, allowing the entire embryo to be observed at once with a low-powered microscope. “With fluorescent reporters, you can see quite a bit.”
Zygogen’s Z-Tag technology enhances expression of fluorescent reporter proteins in specific organs and tissues, providing a quantifiable readout for high-throughput imaging, he added. In assays, the same technology highlights the tissue, making it easier to see changes.
“The angiogenesis assay is one of the most validated,” Dr. Baranowski explained. It looks at the network of angiogenic blood vessels in trunks of the embryos. When angiogenesis inhibitors are administered, researchers can quantify the reduction in the number of fluorescently labeled vessels and vessel branches. “The method is quite accurate,” he said. In toxicology and safety pharmacology, zebrafish are moving beyond the traditional role of pathway analysis to helping researchers screen for overt toxicity and a number of organ-specific endpoints such as cardiotoxicity, liver toxicity, or neurotoxicity.
Focusing on innovative imaging agents, the Sidney Kimmel Cancer Center is overcoming the hurdles of crossing the endothelium and epithelium that have limited access for many imaging and therapeutic agents. “We’re mapping the whole vasculature cell surface, including the endothelium, major organs, and certain disease states such as solid tumors,” explained Jan Schnitzer, M.D., scientific director. The center is paying particular attention to the micro-domain of the caveolae. Mapping the proteins in the caveolae is yielding vascular biomarkers that allow penetration into the tissue as well as tight, tissue- and disease-specific immunotargeting.
The approach has implications for tightly targeting gene therapy as well as for delivering other compounds. “This should eliminate systemic side effects and allow usage of dosages that are hundreds, thousands, or even tens of thousands of times lower,” said Dr. Schnitzer. “There’s a difference between specificity and targeting. Specificity is only one element, the in vivo reality is another.
“We find caveolae to be very useful, because their basic mechanisms of action are well-defined”. His team is finding proteins that are well-expressed on the endothelium of one tissue rather than others, and in high concentrations.
“Expression in other tissues—such as deep inside the kidneys—doesn’t really matter if the antibody can’t see the target”, he added. The approach has been successful in delivering agents to the lungs.
The organization is also investigating the value of this approach for tumors and other organs. According to Dr. Schnitzer, clinical trials will commence soon and some toxicological studies have already been completed.
Currently, “nanoSPECT imaging offers best-in-class spatial resolution, but is for small animals only,” noted Jeffry Norenberg, PharmD, associate professor, University of New Mexico Health Sciences Center. The benefits of this approach are threefold. “You can image each animal multiple times so that each animal serves as its own control. This decreases intra-animal variability,” he said. The method also requires fewer animals, as they needn’t be sacrificed to obtain the needed data. And, he continued, “you can use the same techniques to measure tissue response,” eliminating the need to alter imaging techniques as the experiment progresses.
Dr. Norenberg’s lab routinely uses Bioscan’s nanoSPECT to assess drug distribution and action, toxicological studies, and host/pathogen interactions. “It’s one thing to show this in vitro in the cell, and another to show this in vivo in a whole animal,” he said. “We do a lot of translational research, as well as reverse, human to animal studies for further characterization.”
The challenge for the field today is to develop the nanoSPECT technology so that the resolution in man is as good as that in the mouse. It would need as much as 2,000 times better resolution to get the equivalent resolution in man as in the mouse, he explained.
Cell>Point is developing a contrast agent that offers the resolution of PET/CT:F-18DG, but does not require PET to diagnose hyper-metabolic activity in cancers. In oncology, PET imaging shows the primary lesion and the extent of the disease. “Forty percent of the time, physicians change the diagnosis and therapy based on the PET study,” according to David Rollo, M.D., Ph.D., president. Unfortuantely, PET imaging isn’t widely available.
“The issue is that PET requires a cyclotron to produce the imaging agent F-18 FDG (fluorodeoxyglucose). The half-life of FDG is only 110 minutes, so PET centers must be near cyclotrons. Therefore, a large percentage of patients who could benefit from PET scans can’t get them.”
To counteract that, Cell>Point has developed the imaging agent 99mTc-EC-G (ethylene dicysteine glucosamine), which is imaged using a standard SPECT camera that is available in 98% of U.S. hospitals. According to Dr. Rollo, 99mTc-EC-G is also advantageous because it is more tightly targeted for cancer than F-18 FDG.
Basically, Cell>Point’s agent is localized only in rapidly regenerating cells. Therefore, unlike F-18 FDG, it doesn’t localize in infected or inflamed cells. Thus, “while both ECG and FDG localize in cancer cells, ECG is more specific for cancer and has the potential for fewer false positives than does FDG,” Dr. Rollo explained.
“Phase I trials are showing a one-to-one correspondence between results from PET scans and from SPECT scans, without false positives.” Phase II trials began last autumn and are scheduled for completion later this year. ECG is also being evaluated as a cardiac imaging agent.
ECG can also be linked to rhenium187 as a therapy with the potential to affect all cancers, Dr. Rollo noted. “That still has to be proven.” Preclinical work testing this hypothesis for non-Hodgkin’s lymphoma is under way.
Cell>Point plans to develop 99mTc-EC-G as a kit that hospitals or unit-dose pharmacies can keep in stock. It has a six-hour half-life, allowing staged or time-phased imaging.
Promega is developing bioluminescent imaging substrates, focusing on proluciferins for in vivo applications. Currently, according to Dieter Klaubert, Ph.D., director of R&D, “optical imaging has only two options: fluorescence or bioluminescence. We focus on bioluminescence.”
The company’s approach involves making proluciferin. “The proluciferin is converted into a luciferin only when it reacts with a particular enzyme,” explained Kevin Kopish, global product manager. It is protected from an early conversion through a chemical bond that is broken by the enzyme activity.
Promega is also working on another complementary approach in which a luciferase is modified. It, also, only emits light after the transformation process has occurred. Both approaches, therefore, are highly specific, Kopish noted.
The key benefit of this process is its ability to image events within the cell. “A lot of cell models use luciferase, but you can’t see inside the cell. With proluciferin (as an imaging substrate), we can use the same model and know what’s happening inside,” Kopish said.
Promega is also selling a caspase 3 and 7 to detect apoptosis. In that instance, cancer researchers normally would use luciferase to determine tumor volume, but caspase can be used to determine, not only tumor volume, but whether apoptosis is occurring and the extent of apoptosis, often following the introduction of a novel targeted antitumor drug. A beta galactosidase substrate is also in development in another product to image that activity.
One of the goals of this work, Dr. Klaubert said, is to identify agents that appear effective in vitro and use them as a starting point from which to develop agents that are effective in vivo. “The probes to detect enzyme activity for in vitro applications don’t necessarily translate well in vivo,” he explained.
“We’ve developed a new agent, CYP 450. This enzyme detoxifies drugs, so it could have tremendous potential. It’s highly specific and, speaking as a chemist, looks like a perfectly good one for animals.”
The usual benefits of bioluminescence remain intact with these new approaches. They, like the commonly used firefly luciferin, offer 6 logs of dynamic range versus the 4 or 5 log dynamic range common with fluorescence and without the background noise associated with fluorescence.
CRi is combining imaging technology with machine learning. It developed Maestro a few years ago as a small animal imaging box with the ability to multiplex tests and capture reflectant and fluorescent images at multiple wavelengths. Since then, new applications have emerged, according to Richard Levenson, M.D., vp. “The bulk of the work is in preclinical drug development,” he explained, and significant academic work is being undertaken in basic and organism-based biology involving nondestructive testing.
This approach goes beyond simple planar imaging to produce 3-D results. “If you inject a bolus dose of dye into the bloodstream in the tail vein (of a rodent), it has a very predictable path,” Dr. Levenson explained, that allows a 3-D image to be created that includes the major organs. The method is accurate when compared with cryosections of mice.
Maestro lets users separate multiple fluorescent signals in one animal and also eliminates the problem of autoflourescence (in which the entire mouse often glows). The result is that signals are seen clearly at each pixel of the image. When the signals are unmixed, Dr. Levenson said, they are seen clearly against a black background.
The machine-learning strategy uses a learn-by-example method to create a multiclass classifier based upon its automated image-segmentation capability. The beauty of machine learning is that users can train the software to search out certain things by providing examples. “For example, circle the kidneys on an image and it finds the kidneys on other images, too,” Dr. Levenson explained. That capability increases the speed at which quantitative analyses can be performed, he added.
In vivo molecular imaging technology can be helpful in drug development, providing information that eases the decision to kill or advance a compound. But, there are some hurdles. Xavier Tizon, Ph.D., imaging lab manager for Oncodesign, noted that imaging protocols still aren’t standardized. “This is especially true for MRI, which is a complicated modality. Several solutions have been found to diverse technical problems, but those solutions bring variations in the actual imaging data.”
“Another issue, he continued, is the need for radiologists to better understand the image processing software that is used.” The choices made by software programmers are key to the relevance of the measurement. Different choices are made by different vendors, and that makes measured data inconsistent among machines,” Dr. Tizon explained. Further inconsistencies occur through variations in animal-handling procedures such as anesthesia and warming, he added.
Dr. Tizon advocates using a diversity of imaging modalities to obtain the best results. “You need to use them in collaboration,” he insisted, and implement a good quality assurance/quality control plan. “Additionally, choose simple analysis methods and understand when and why a model may fail.”
In vivo molecular imaging can help bridge the gap between early- and late-stage research if—and this is a big if—companies plan ahead. “Researchers may have great inventions that are extremely useful, but they don’t pay attention to details in developing imaging approaches,” James Paskavitz, M.D., medical director at Perceptive Informatics, noted.
“Consequently, they need to be able to think ahead to determine if available ligands or imaging approaches may prove extremely useful in late-stage trials.”
Sometimes, the technology itself becomes a limiting factor. For example, the imaging resolution possible with rats is significantly higher than is possible in humans, leading to smultiple steps to correlate data and the need for many more subjects. The difference between scientific validation and regulatory validation is another potential stumbling block, Dr. Paskavitz said. The bottom line, he added, is to plan several steps ahead.