Indirectly, tissue is the issue

Chemistry
By using a paramagnetic CR—that is, the magnetism of its unpaired electrons influences ¹H₂O—the CR is detected indirectly through its ability to affect signal strength. The chemical mechanism of this effect is implied in Figure 1a, which shows the diethylenetriaminepentaacetate (DTPA^5–) chelate of gadolinium(III) [GdDTPA^2–], also known as Magnevist. The CR-bound water molecule directly coordinates with the Gd(III) ion, bringing its hydrogen protons close to the seven unpaired (4f) electrons of the Gd(III), which greatly affect the former’s magnetism.

After perturbation by a resonant radio-frequency pulse, the tissue ¹H₂O signal returns to equilibrium, or relaxes. The paramagnetism of the Gd(III) ion catalyzes this relaxation. The equilibration of the component of the ¹H₂O vector along, for example, the direction of the instrument’s magnetic field is referred to as longitudinal relaxation, and it is characterized by the longitudinal relaxation time (T₁). The CR causes the value of T₁ to decrease and, with the appropriate (T₁-weighted) pulse sequence, increases the portion of ¹H₂O signal detected. Thus, tissue regions entered by the CR appear brighter in the post-CR image.

Figure 1a illustrates equilibrium water exchange, which is extremely rapid. The average lifetime of a water molecule bound to GdDTPA^2– is 130 ns at 37 °C. This allows all water molecules that can chemically access the chelate to be influenced by the paramagnetism of the CR during a very brief period of time; it is important because it facilitates the detection of reagent when there are as few as two CR molecules for every million water molecules. Of course, a “prototropic” proton-exchange mechanism—whether hydrolytic (cleaving the bound water) or protolytic (exchanging some other CR proton with a free water proton)—could accomplish the same thing. Now, a thorough appreciation of this “no-reaction” exchange reaction (K = 1) is yielding a wealth of principles that could very well lead to a golden age for MRI CRs. GdDTPA^2– and similar compounds now approved for use in humans are very popular in clinical MRI. By 1999, an estimated 2–4 million doses were being administered per year.

Physiology
The CR is usually injected intravenously and therefore arrives at the region of interest dissolved in the blood plasma (and thus the enhancement rate can be used to measure perfusion) and is designated CR_p. It is generally agreed that the GdDTPA^2–-style CRs do not cross viable cytolemmae (cell membranes), including those of blood cells. When CRs leave a blood vessel, they are thought to do so through the junctions between endothelial cells lining the vessel wall,which are almost totally closed in most regions of the healthy brain—the well-known blood–brain barrier. Thus, CRs serve as excellent reporters for even a slightly compromised blood–brain barrier, like that found in multiple sclerosis lesions and in certain tumors as a consequence of angiogenesis.

Once CRs are in the extravascular volume, they occupy the extracellular space and are therefore designated CR_o (outside), where they can directly bind with extracellular water, ¹H₂O_o. Because T₁ is an intensive property of ¹H₂O, its CR-induced change depends directly on the molar ratio of CR_o to H₂O_o. This means that T₁ determination allows direct measurement of the thermodynamic concentration, [CR_o], in the space in which CR is actually distributed. Furthermore, because most water is intracellular (¹H₂O_i), the change in the tissue ¹H₂O T₁ from the entire voxel (image volume element) by a CR allows evaluation of the kinetics with which water moves across the cell membrane. This is characterized by the average lifetime of a water molecule inside a cell, t_i, and is inversely proportional to the cytolemmal water permeability, which is affected by aquaporin regulation.

Taken together, these parameters also allow measurement of the intra- and extracellular volume fractions. These are intravoxel quantities, and thus determining them for each pixel in the field of view will allow the production of intra- and extracellular volume maps, as well as a cytolemmal water permeability map. The [CR] threshold for detection is sufficiently reduced at higher magnetic fields that the equilibrium transcytolemmal water-exchange kinetics (though unaltered) can appear to be faster in research instruments than in clinical scanners.

CR development
Figure 1b depicts a modification of GdDTPA^2– entailing the attachment of a diphenylcyclohexylphosphodiester residue, a ligand for serum albumin binding sites. This modified CR, designated MS-325 (AngioMARK), is thus targeted directly for the blood. Another feature of MS-325 exploits the relaxivity, r₁, the coefficient that relates the ¹H₂O T₁ to [CR] in vitro. The r₁ of free MS-325 is 6.6 (mM)^–1s^–1. However, when the CR binds to albumin, its r₁ increases to 30–50 (mM)^–1s^–1. This means that the CR is reversibly activated on binding to human serum albumin and is much more effective at reducing ¹H₂O T₁. Thus, it can be detected at significantly lower levels. This well-known effect (termed proton relaxation enhancement in the older literature) is caused by the reduced rotation rate of a CR molecule bound to a macromolecule and will be immensely important in future MRI CR development. Randall Lauffer and colleagues at EPIX Medical (Cambridge, MA) have renamed this phenomenon the receptor-induced magnetization enhancement (RIME) mechanism, and they suggest that the molecular rotation rate constant may be reduced by 2 orders of magnitude in this case.

Another important feature of MS-325 is that its strong binding to albumin takes place in rapid equilibrium. Therefore, free CR can be eliminated from the body by renal excretion, just like GdDTPA^2– and other analogues. In contrast, most other putative blood pool agents require a hepatic route for elimination. Because the equilibrium concentration of free MS-325 is low and essentially undetected, however, its elimination through the kidney is much slower than, for example, that of GdDTPA^2– (t_1/2 1.5 h).

Figure 1c depicts a modification, Egad Me, of another related CR, the 1,4,7,10-tetraazacyclododecane-N,N´,N´´,N´´´´-tetraacetate chelate of Gd(III) (GdDOTA^–), which takes direct aim at the exchange reaction. Here, a galactopyranose ring is appended to the GdDOTA^– structure in such a way that it blocks the Gd water-binding site and thus severely inhibits the water-interchange reaction. Although this makes for a poor CR, the researchers chose the galactopyranosyl moiety precisely because it can act as a cleavable ligand specifically for the common marker enzyme β-galactosidase (β-gal). β-Gal can excise the blocking group and expose the Gd site to water, creating a product molecule that is a considerably better CR, and thus an irreversible activation.

Figure 2. EgadMe in action. At the two-cell stage, each cell of the two tadpoles was microinjected with the less active form of EgadMe. However, for the embryo of the tadpole on the right, one cell (only) was also microinjected with the mRNA that codes for β-galactosidase. (A) Unenhanced MRI image. (B) Pseudocolor MRI image. The unilateral activation of EgadME is indicated by the blue glow. (Reprinted with permission from Nat. Biotechnol. 2000, 18, 321–325.)

They are now considering other approaches that do not require microinjection for loading CR into cells and that do not require such high CR levels. Using the same exchange-blocking principle, the investigators have developed a dimeric Gd(III) CR that is reversibly activated by elevated Ca²⁺ levels, via a Ca²⁺-induced conformational change.

Summary
Because of the water exchange chemical reaction underlying their detection, MRI CRs directly report their own local concentration within an image voxel and can be made activatable, either reversibly or irreversibly. Researchers are taking advantage of these properties in order to expand the diagnostic armamentarium of CR-enhanced MRI.

Further reading

• Caravan, P.; et al. Chem. Rev. 1999, 99, 2293–2352.
• Landis, C. S.; et al. Magn. Reson. Med. 2000, 44, 563–574.
• Li, W.-H.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc. 1999, 121, 1413–1414.
• Louie, A. Y.; et al. Nat. Biotechnol. 2000, 18, 321–325.
• Shellock, F. G.; Kanal, E. J. Magn. Reson. Imag. 1999, 10, 477–484.
• Springer, C. S. Physicochemical principles influencing magnetopharmaceuticals. In NMR in Physiology and Biophysics; Gillies, R. J., Ed.; Academic Press: San Diego, 1994; pp 75–99.
• Springer, C. S.; Rooney, W. D. Proc. Int. Soc. Magn. Reson. Med. 2001, 9, 2241.
• Yankeelov, T. E.; Rooney, W. D.; Springer, C. S. Proc. Int. Soc. Magn. Reson. Med. 2001, 9, 2251.
• Zhang, S.; Wu, K.; Sherry, A. D. Angew. Chem. 1999, 38, 3192–3194.