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.