Molecular imaging is broadly defined as the visualization of molecular and cellular processes on either a macro- or microscopic level. Because of its high spatial resolution and ability to noninvasively visualize internal organs, magnetic resonance (MR) imaging is widely believed to be an ideal platform for in vivo molecular imaging.^[1] For this reason, MR contrast agents that can detect molecular events are an active field of research.^[2] One group of compounds that has shown particular promise is enzyme-activated MR contrast agents.

Enzyme-activated MR contrast agents are compounds that cause a detectable change in image intensity when in the presence of the active form of a certain enzyme. This makes them useful for in vivo assays of enzyme activity. They are distinguished from current, clinical MR contrast agents that give only anatomical information,^[3] such as aqueous gadolinium compounds, by their ability to make molecular processes visible. Enzyme-activated contrast agents are powerful tools for molecular imaging. To date, β-galactosidase-activated contrast agents have attracted the most attention in the literature, although there no theoretical reason that other enzymes could not be used to activate contrast agents. Also, mechanisms other than enzyme activation, such as Ca²⁺-dependent activation, can theoretically be used.^[2]

In general, enzyme-activated agents contain a paramagnetic metal ion which can affect the T₁ or T₂ relaxation times for nearby water molecules. However, the metal ions are unable to interact with the water until an enzyme-catalyzed reaction takes place. Steric hindrance or coordination with other ions prevents water from accessing the paramagnetic center prior to the enzymatic reaction.^[4]

Two distinct β-galactosidase-activated contrast agents have been reported. Both consist of a Gd(III) ion complexed with a tetraazamacrocycle. At the N-10 position, a two-carbon chain links the gadolinium-tertaazamacrocycle complex to a molecule of galactose. The galactose is linked to the complex by a β-glycosidic bond at its C-1 position.^[4]

The two forms of the contrast agent differ only in the location of a single methyl group. The first class, known as the α-series, has a methyl group bound to the carbon that is α to the tetraazamacrocycle. The other class, called the β-series, has a methyl attached to the β carbon relative to the tetraazamacrocycle. The position of this methyl group is significant for the structure of the agent, and thus determines the mechanism by which the non-active compound shields the Gd(III) ion from interacting with water. The α-series adopts a conformation in which the sugar lies directly over the paramagnetic center, thus sterically prohibitting water from accessing the gadolinium. The β-series, on the other hand, blocks water from the gadolinium by coordinating with a carbonate ion. There is no evidence that the stereochemical orientation of the methyl-bearing carbon affects the either the enzyme-catalyzed cleavage or the ability of the sugar to exclude water from the gadolinium ion.^[4]

Studies have shown that the α-series is far more effective at blocking water from the paramagnetic center prior to cleavage.^[4] The need to coordinate with a carbonate ion and the lower level of signal suppression inherent to the β-series make the α-series a better candidate for use in research and clinical medicine.

In a tissue where active β-galactosidase is present, the sugar will be cleaved from the rest of the compound. This permits water to access the paramagnetic center, and causes the magnetic relaxation properties of the surrounding water molecules to change. This change in relaxation times will, in turn, visibly alter the signal intensity of images of the tissue obtained from MR scans.

The mechanism proceeds in the same manner as all other β-galactosidase-catalyzed cleavages. The carboxyl group on a glutamic acid side chain within the enzyme acts as an acid catalyst, hastening the cleavage of the glycosidic bond at the C-1 position in the sugar. This cleavage gives water access to the paramagnetic center. The result of the enzyme-catalyzed reaction is a free galactose molecule and an activated contrast agent.

There are obvious potential uses to this technology, both in research and clinical medicine.

Once the contrast agent has been activated by cleavage of the sugar group, the signal enhancing effects will only diminish if the gadolinium is washed out of the compartment containing it, or if water’s access to the metal group is again inhibited. So, to prevent permanent enhancement of the MR signal, cells must either have a way to export the gadolinium group to the bloodstream, or they must be able to replace the cleaved sugar group. There is no data in the literature indicating that either approach is feasible in vivo, suggesting that these methods may result in permanent signal amplification.

Another challenge is the delivery of the contrast agents to target cells. In the sole paper describing in vivo use of enzyme-activated MR contrast agents, the agent was delivered to embryonic cells via a micropipette. However, the authors of the paper acknowledge that this is not a feasible approach for many research projects,^[5] and it presents a clear impediment to clinical use. There is active research in using the cell’s native import machinery to load contrast agents.^[7]