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Feature Articles : Feb 1, 2008 ( )
Driving Discovery from Inside Out
In Vivo Imaging Revitalizes Research Methodologies
In vivo imaging is rapidly gaining popularity as a faster and less expensive method for enhancing drug discovery and development. Its keen ability to noninvasively gauge drug efficacy and trail compounds inside living organisms places it at the forefront of new technologies. It could eventually replace traditional survival-based endpoints, especially since the FDA now accepts surrogate endpoints to prove drug efficacy.
Scientists at Cambridge Healthtech’s “In Vivo Molecular Imaging” meeting held late last year highlighted new research and insights into this burgeoning field. These include development of novel targeted tracers, new methods for cost-effective optical imaging, and functional imaging driven by positron emission tomography (PET) and its close cousin SPECT (single photon emission computed tomography).
Angiogenesis takes place in many pathological processes that range from cancer to myocardial infarctions. Vascular endothelial growth factor (VEGF) and its receptors play a critical role in angiogenesis. Consequently, this signaling pathway is the therapeutic target for several approved and a multitude of experimental antiangiogenic drugs.
Joseph Backer, Ph.D., CEO at SibTech (www.sibtech.com), is pursuing molecular imaging of VEGF receptors with targeted tracers. “VEGF receptors are major drug targets, so clinical imaging of these receptors will be valuable for diagnostics, patient segmentation, monitoring treatments, and developing drug regimens.”
Dr. Backer said that SibTech is converting growth factors such as VEGF into targeting vectors for contrast agent delivery. “The use of tracers based on growth factors is important because they can be delivered into targeted cells only by active receptors. Therefore, imaging with such tracers would provide information regarding receptor functionality.”
One challenge to this strategy has been the small size of growth factors. “With something that small, where evolution stringently selected each amino acid, it is a problem to find safe sites for attaching contrast agents. To solve this problem, we have developed a short fusion tag containing cysteine (Cys-tag) for site-specific derivatization.”
As it turns out, different proteins can be expressed with this tag and then safely and site specifically derivatized with a variety of imaging and therapeutic payloads. “In collaboration with Stanford University, and with support from NIH/NCI SBIR grants, we use Cys-tagged single-chain VEGF as a standardized platform for developing tracers for PET, SPECT, and near-infrared fluorescent imaging modalities,” noted Dr. Backer.
Currently, there are no contrast agents for imaging VEGF receptors. “We expect molecular imaging of VEGF receptors with our scVEGF-based strategies will greatly facilitate progress in development of anti-angiogenic therapies. We plan to take our lead SPECT tracer, scVEGF/99mTc, into clinical trials soon.”
Nanoparticles that can be detected by optical fluorescence and radioscintigraphy are being used as important tools for diagnostic imaging, according to Rao V. L. Papineni, Ph.D., research scientist R&D, Carestream Molecular Imaging (www.carestreamhealth.com). Kodak X-Sight Nanospheres are approximately 16 nanometers in diameter and are composed of an organic polymer that has more than 200 active surface amines that are amenable for surface nanoengineering, Dr. Papineni reported.
“We derivatized the nanoparticles with various small compounds, peptides, or proteins through simple chemistries. This allowed us to create nanoparticles with both radioactive isotopes and a targeting peptide for the noninvasive imaging of tissue.” These multimodal nanoparticles provide a platform for dual optical and nuclear imaging applications for tumor detection and molecular imaging of specific tissues or organs in vivo.
Dr. Papineni reported that the embedded fluorochromes in Kodak X-Sight Nanospheres make them brighter reporters in both in vivo and in vitro applications.
“The nanoparticle is sufficiently small to enable FRET between two spectral overlapping donor and acceptor nanoparticles. We found that the Kodak X-Sight Nanospheres remain in blood circulation for a prolonged time during biodistribution studies, which potentially allows the sequestration and accumulation of these nanoparticles in tumors due to enhanced permeability and retention effect.”
Carestream’s research program includes both preclinical and clinical platforms with a long-term goal of bringing optical imaging to the many facets of clinical imaging such as endoscopy, laparoscopy, and physician’s front-office diagnostics.
Noninvasive Visible Light
Optical imaging measures light produced by biological or chemical moieties and provides the opportunity to noninvasively visualize processes in a live animal. Caliper Life Sciences (www.caliperls.com) uses luciferase-based bioluminescence reporters and fluorescent tags (proteins and small molecules) to monitor migration and behavior of various types of cells.
“Every imaging modality has its place in research as well as drug discovery, development, clinical trials, and routine patient treatment,” according to Peter Lassota, Ph.D., divisional vp, imaging biology and oncology at Caliper. “PET and SPECT are rather costly at nearly $1,200 per patient or mouse model. Caliper has developed technology that uses light-producing reporters in dark mice and light-producing animal models to provide researchers with information about drug effects on particular pathways.”
This helps verify the mechanism of action of a drug in an animal using the same method that is used for cell-based assays in vitro. Additionally, compound testing in these models provides immediate information about bioavailability of the compound in the target tissue, he explained. So, light-producing animal models expand applications for in vivo real-time imaging for drug discovery as well as toxicology.
According to Dr. Lassota, visible light imaging has advantages over other types of technologies. “In oncology, for example, the classical way of looking for drug effect on a mouse tumor is to monitor tumor size. We, on the other hand, label tumor cells with luciferase and then treat the mice with the drug under evaluation. Using a special CCD camera, we can sensitively detect decreases in light that correlate with decreases in cell number. You can see changes much more quickly this way as opposed to waiting for the tumor size to shrink. In some cases we are able to obtain data within one to two days.”
Noninvasive in vivo imaging can provide information that would be impossible by any other technique, reported Antony Gee, Ph.D., director, PET and radiotracer development, GlaxoSmithKline Clinical Imaging Centre, Imperial College London (cic.gsk.co.uk).
“PET follows the fate of molecules labeled with short-lived positron-emitting radionucleotides in living subjects in a dynamic and quantitative manner. The technology uses the basic building blocks of life: radioactive carbon-11, nitrogen-13, and oxygen-15, which have short radioactive half-lives (10, 20, and 2 minutes respectively). This limits radiation dose to the subject to a level similar to what would be obtained on a transatlantic flight.”
Dr. Gee suggested that to understand why PET is such a valuable technology for drug discovery, traditional drug discovery must be considered first. After a molecule is identified, medicinal chemists collaborate to improve and then test in cloned receptors, in vitro tissue homogenates or slices, and in vivo preclinical models. There are several iterations of these steps before you can get a suitable candidate to test in greater detail in animals (e.g., toxicology and kinetic properties).
“Pending the results of those studies the compound properties are often further optimized by further iterations of chemical manipulation and in vitro testing until a few compounds look sufficiently promising to administer to humans.”
One of the key advantages of PET is that it can be used in humans early in the above process, Dr. Gee reported. “It provides a noninvasive way to immediately monitor in humans using low doses (typically less than 10 micrograms) of the therapeutic, which is usually below the pharmacological or toxicological threshold for the drug.”
Additionally, such testing provides critical information as to how much the drug occupies receptors, if it gets to the organ intended for treatment, and its appropriateness for treating specific tumors. It is also useful for stratifying heterogeneous patient populations and characterizing disease mechanisms and abnormal biochemistry, he noted.
Another use of PET is for functional genomics. “The Genome Project, for example, has uncovered hundreds of previously uncharacterized enzymes and receptors,” Dr. Gee said. “Using PET, we can capitalize on and better understand the role of these targets in human disease and normophysiology, which will be a driver for a new generation of therapeutics for these targets.”
Monitoring of HER2
Among the most studied cancer markers today is human epidermal growth factor receptor-2 (HER2/neu/c-erbB-2), which plays a key role in many cellular processes, including cell growth, differentiation, cell survival, cell adhesion, and migration.
“We are monitoring HER2 expression in vivo. Normal epithelial cells express little or no HER2 protein, yet many tumors such as cancers of the breast, lung, ovaries, and B-cells overexpress it. Also, HER2-positive status is associated with resistance of cancers to therapy,” reported Jacek Capala, Ph.D., D.Sc., principal investigator, radiation oncology branch, NIH (cis.nci.nih.gov).
Dr. Capala explained that HER2 expression may be different on tumors that metastasize. “Different metastases show expression levels that often are distinct from the primary tumor. Thus, what really is needed to adequately diagnose and select appropriate therapies is assessment of the global expression of these receptors within the body.”
To address this problem, Dr. Capala’s team created a molecular in vivo probe using HER2-specific molecules developed by Affibody (www.affibody.com). “These are not antibodies, but rather stable and highly soluble alpha-helical proteins. They present several advantages over antibodies.”
They are relatively small (6–7 kDa, or 20–25X less than antibodies), bind to HER with high affinity, and can be made by chemical synthesis or by bacterial expression along with a unique cysteine to be used for thiol-based conjugation. “We have applied maleimide chemistry to attach the positron-emitting radionuclide 18F to this Affibody molecule.”
Dr. Capala’s group next correlated the signal observed by PET for receptor expression by administering the new tracer to athymic nude mice bearing subcutaneous tumors with three different levels of HER2 expression, very high, high, or low. “Our results suggest that the radio tracer can be used to assess HER2 expression in vivo and to monitor possible changes of receptor expression in response to therapeutic interventions.”
Academia and Industry
Scientists in academia seek to understand innate and complex biological processes, while scientists in industry seek to use such knowledge to develop therapeutics. Finding ways to amalgamate both disciplines could reap huge benefits for each side.
J. James Frost, M.D., Ph.D., professor of diagnostic radiology, Yale University, explained that about five years ago Yale and Pfizer (www.pfizer.com) entered into a 10-year cooperative research agreement for drug discovery and molecular imaging related to Pfizer’s development of a drug molecule aimed at serotonin receptors that might be used in patients with depression. Researchers wanted to know if the new drug candidate got into the brain and targeted the 5HT-1B receptors.
“The Yale PET Center team synthesized a related 5HT-1B compound with an added radioisotope and performed PET scanning studies in healthy humans. The drug lit up the relevant serotonin-receptor areas, including those associated with depression,” explained Dr. Frost.
The key to such partnering is that the agreements are mutually beneficial. “We are seeing this around the country for many applications such as cardiovascular, oncology, diabetes, and pain.”
The emerging technology of in vivo imaging holds great promise for cutting costs and speeding clinical trials. Most agree, though, that challenges remain before the technology and instrumentation can completely replace traditional methods.
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