November 2004

Targeted contrast media play to strengths of MRI

Novel imaging agents boost modality's resolution and expand its clinical utility

By: James Brice

Although many applications are still in preclinical trials, researchers are moving along a broad front to exploit MR's potential as a molecular imaging tool. Contrast agent development efforts are creating precise targeting mechanisms and vastly more amplification of MR signal.

While MR spectroscopy is recognized as an inherent metabolic modality, MR's clinical repertoire is expanding to include detection of early angiogenesis, inflammation, extracellular glucose, pH, hypoxia, and temperature.

TARGETED PROBES

Targeted MR probes are equipped with protein fragments, or integrins, which latch on to receptors that are particularly abundant on the surface of specific types of cells.

Two major areas of application are envisioned for such agents. Targeted agents that carry a large gadolinium payload are well suited for intravascular applications such as detecting atherosclerotic plaques. Receptor-targeted probes that penetrate vessel walls easily are good candidates for solid-tumor diagnosis.

Dr. Samuel A. Wickline, codirector of the cardiovascular division at Washington University in St. Louis, is developing a liquid perfluorocarbon nanoparticle encapsulated in a lipid coating capable of carrying various payloads. Wickline's group and their collaborators at Kereos, a St. Louis-based biotechnology company, have incorporated up to 90,000 gadolinium particles on the surface of the probe.

The payload generates extremely high relaxation rates in low concentrations. Its R1 relaxivity is about 20, more than four times the rate of conventional MR media, according to Dr. Gregory M. Lanza, an assistant professor of medicine at Washington University. Because the particle is targeted, the per-particle effect of the gadolinium load boosts the R1 relaxivity to about two million.

At the Society for Molecular Imaging meeting in September, Patrick Winter, Ph.D., an assistant professor of radiology at Washington University, discussed results from two studies that used two different formulations of the nanoparticle. One measured its ability to detect early angiogenic processes associated with atherosclerosis. An avb3 integrin, a biomarker for angiogenesis expressed on neovascular endothelium, served as the targeting mechanism. The other study looked at the success of a drug transported by the particle to the angiogenic sites.

The agent was studded with gadolinium, avb3, and fumagillin, a lipophilic agent that inhibits cell proliferation and migration. It has been studied for antiangiogenic therapy in both cancer and atherosclerosis, Winter said. The two studies examined the aortas of rabbits with induced atherosclerosis.

In the first study, black blood images revealed the vessel wall surrounding the lumen and provided colorized MR enhancement that mapped a localized, patchy distribution of angiogenesis. The targeted agent generated higher localized enhancement than nanoparticles without avb3 two hours after injection. The targeted agent did not enhance the aortas of rabbits that did not have induced atherosclerosis.

Looking at the MR signal over the entire imaged aorta, Winter found that a high enhancement rate first observed two hours after treatment persisted one week later. Among rabbits treated with the targeted drug, high enhancement was seen soon after injection, indicating successful drug delivery. A week later, the enhancement level fell significantly, suggesting that the desired antiangiogenic effects of the fumagillin had taken place.

Dr. Dmitri Artemov, an assistant professor of radiology at Johns Hopkins School of Medicine, has had success homing in on breast tumors in animal models with a two-stage labeling protocol targeting HER-2/neu receptors. During the first step, Artemov introduced nonspecific biotinylated antibodies that selectively identified the targeted tumors. After the antibody cleared, avid gd-DTPA was injected and imaging was performed. In most cases, the sequence produced a strong, specific signal corresponding to the number of Her-2/neu receptors on the tumor cells' surfaces. The highest expression level was three million receptors per cell, enabling Artemov to induce a strong signal with only four or five gadolinium molecules per particle.

TARGETING uMUC-1 ANTIGEN

Also working with mice in an in vivo preclinical study, Anna Moore, Ph.D., a cellular biologist at Massachusetts General Hospital's Center for Molecular Imaging Research, demonstrated that underglycosylated MUC-1 tumor antigen could be another good cancer biomarker for targeted MR contrast. Her experimental protocol could help diagnose and characterize many types of cancers. Moore said at the 2004 International Society for Magnetic Resonance in Medicine meeting that uMUC-1 is a common feature of epithelial cell adenocarcinomas, including 90% of breast cancers and nearly all permutations of pancreatic, colorectal, lung, prostate, and gastric cancers. Surrounding normal cells are devoid of the marker, and it is destroyed or downregulated during successful therapy.

The probe used to target the antigen was designed for either MR or optical imaging. It consists of a superparamagnetic, aminated dextran-coated nanoparticle and Cy5.5 dye, a near-infrared optical agent. For targeting, the probes were conjugated with EPPT1, a peptide with a known affinity for uMUC-1.

MR and near-infrared imaging procedures were performed before and 24 hours after the probe was injected in mice implanted with uMUC-1-positive and -negative tumors. Transverse and coronal T2 imaging revealed a significant loss of signal in the uMUC-positive tumors but no change in those that were uMUC-negative. The finding corresponded to bright signal emitted from the uMUC-positive cancer during in vivo optical imaging and follow-up in vitro imaging of histological samples.

VERSATILE SPIOS

Iron oxide nanoparticles have gained prominence for their potential ability to identify vulnerable plaques. Macrophages gravitate to inflammation, thought to be a contributor to plaque instability. Such macrophages may themselves destabilize plaques by eroding the fibrous cap that retains their thrombus-triggering lipid cores. Iron oxides are rapidly taken up by macrophages, thereby creating a source of MR T2 relaxation that can identify the sites of problematic atherosclerotic disease.

Superparamagnetic iron oxides such as Feridex, a dextran-coated SPIO, or Sinerem, an ultrasmall iron oxide, have established themselves as good liver-enhancing agents and leading candidates as enhancers of early angiogenesis and vulnerable plaque as well.

SPIOs are also frequently the agent of choice for cell trafficking, Artemov said in a lecture at the ISMRM meeting. Signal amplification is the key role of contrast for this set of rapidly evolving applications. Cells can accumulate a huge number of contrast particles, and the test's sensitivity rises as more contrast accumulates.

Two approaches enable SPIOs to cross cellular membranes, Artemov said. Transferin receptors can play a role: The complexes can be conjugated with transfection agents such as transactivator proteins (Tat) or amphiphilic peptides that help them move across the membrane. The agents can also be mixed with poly-L-lysine peptide or other transfection agents. This technique has been shown in swine studies to be useful for labeling mesenchymal stem cells that are tracked with MR imaging after implantation in injured myocardium.

Dr. Moritz F. Kircher's research at the Center for Molecular Imaging Research at MGH illustrates another application of intracellular contrast agents for cell trafficking. His group developed CLIO-HD, a highly derivatized cross-linked iron oxide nanoparticle, for efficient intracellular labeling of various types of cells and in vivo MR tracking at near-single-cell resolution. The agent was used for the first time to label OVA-specific CD8 T cells to track the T-cell recruitment to intact tumors in vivo. Measurable amounts of the agent were detected 36 hours after injection. The method's sensitivity was as low as three cells per voxel.

At GE's global research center in Schenectady, NY, chemist Edward Urgaris, Ph.D., has developed a long cylindrical polymer strand strung with gadolinium particles that has shown promise in preclinical tests as a new way to measure the permeability of angiogenic vessels. The framework can hold a number of gadolinium particles, and its shape and slithering behavior appear to contribute to easy penetration of neovascular walls, said Michael C. Mantalto, Ph.D., project leader in GE's molecular imaging technology program.

PROMISE OF CEST

Researchers are examining the potential of proton chemical exchange-dependent saturation transfer (CEST) for a future generation of highly amplified targeted agents. First described by Robert Balaban, Ph.D., chief of the Laboratory of Cardiac Energetics at the National Institutes of Health, CEST is based on the understanding that most molecules in the body exchange protons with bulk water, Balaban said at the 2004 Society for Cardiovascular Magnetic Resonance meeting. The MR saturation transfer technique allows the user to irradiate a millimolar pool of metabolites. The energy is transferred to a nearby pool of water where the chemical exchange can produce a several-thousand-fold increase in enhancement

"This is a tremendous amplification caused by direct chemical exchange," Balaban said.

Hydroxyls or ammonia can trigger CEST and produce about a 7000-fold enhancement. Because the chemical exchange is naturally pH-sensitive, the probes are highly pH-sensitive, creating opportunities to measure pH with CEST.

However, CEST also has inherent concentration and sensitivity problems. These have been addressed with paraCEST, the invention of A. Dean Sherry, Ph.D., a professor of radiology at the University of Texas Southwestern in Dallas, and Silvo Aime, Ph.D., a professor of chemistry at the University of Torino in Italy.

The development of paraCEST is based on the fact that it is hard to saturate protons located next to bulk water during CEST imaging without saturating the water itself. Aime and Sherry discovered paramagnetic agents that allow the targeted protons to be shifted slightly farther from bulk water.

"If you make the exchange rate large enough, the chemical shift that defines all this can be much faster, and you still see an effect that translates into an amplified signal," Sherry said.

CEST and paraCEST create opportunities to use MR imaging to recognize specific metabolites because chemical exchange rates depend on how fast the proton spins move from one molecule to another, he said. A specific molecule, for example, can be found to bind to extracellular glucose to slow down the exchange rate of hydrogen through water. The exchange rate of water containing glucose can be compared with the rate of water without glucose to measure sugar concentration. Using this principle, paraCEST could potentially map the extracellular distribution of glucose in any organ.

"If you have an imaging agent that will allow you to look at the distribution of metabolites in the brain, liver, or kidney, it could be quite important in helping diagnose diabetes and other disease," Sherry said.

ParaCEST can also be used to measure pH, temperature, lactate, and the concentration of various metabolites.

Such metabolic strategies represent the brightest vision of MRI's future, according to Thomas J. Meade, Ph.D., a professor of biochemistry at Northwestern University. In the mid-1990s, Meade designed the first activatable gadolinium MR contrast agents. EgadMe, for example, consisted of caged gadolinium that was switched off because it was isolated from water. Specific enzymes could open the cage and switch the gadolinium on to alert the physician to their presence.

New agents developed by Meade's group reversibly bind to calcium and to six different classes of enzymes including caspases, matrix metalloproteases, glucuronidases, and kinases.

"Now you have a means to track gene expression, because what makes the enzymes? Genes do," Meade said. "Instead of looking at MRI for anatomy, you are going to get physiological information."