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.