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The vertebrate nervous system is characterized by myelination, a fundamental biological process that protects the axons and facilitates electric pulse transduction. Damage to myelin is considered a major effect of autoimmune diseases such as multiple sclerosis (MS). Currently, therapeutic interventions are focused on protecting myelin integrity and promoting myelin repair. These efforts need to be accompanied by an effective imaging tool that correlates the disease progression with the extent of myelination. To date, magnetic resonance imaging (MRI) is the primary imaging technique to detect brain lesions in MS. However, conventional MRI cannot differentiate demyelinated lesions from other inflammatory lesions, and therefore cannot predict disease progression in MS. To address this problem, we have prepared a Gd-based contrast agent, termed MIC (Myelin Imaging Compound), which binds to myelin with high specificity. In this work, we demonstrate that MIC exhibits a high kinetic stability towards transmetallation with promising relaxometric properties. MIC was used for in vivo imaging of myelination following intracerebroventricular infusion in the rat brain. MIC was found to distribute preferentially in highly myelinated regions and was able to detect regions of focally induced demyelination.

The advent of powerful new imaging tools such as confocal and two-photon microscopy, MRI, PET, SPECT, and ultrasound have provided scientists and clinicians with the ability to acquire in vivo images of anatomy and physiology in whole animals and humans [1–3]. Each of these in vivo imaging techniques possesses unique strengths and weaknesses with varying spatial and temporal resolution limits [4]. Further, the development of sophisticated reporter probes for cell tracking, gene expression, and gene monitoring therapeutic paradigms is rapidly changing the way these techniques are being applied. For example, advances in optical microscopy have revolutionized the way living cells and intact tissues are studied [5]. As a result, cellular events, biochemical processes, and tissue morphogenesis can be followed in developing embryos at submicron resolution. MRI employs nonionizing radiation and has become an integral tool in clinical radiology, and more recently in experimental settings [6–8]. The intensity of the signal in each voxel is dependent on several intrinsic parameters, such as the proton density, longitudinal (R1) and transverse (R2) NMR relaxation rates, and extrinsic parameters, such as magnetic field strength, pulse sequence, and the presence of contrast agents (CAs). CAs for a variety of imaging methods have been in use for decades in diagnostic radiology and developmental biology [9–13]. The application of these CAs is well documented and the fundamental chemical principles for the synthesis and characterization of these complexes are known [14–16]. A rapidly growing body of literature documents the clinical effectiveness of paramagnetic CAs [17–19]. Currently, eight such CAs are in clinical use or clinical trials and more than 30% of all clinical MRI examinations employ CAs [20]. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation-enhancement agents and decrease the T1 and T2 relaxation times of nearby spins [21]. Some paramagnetic ions decrease T1 without causing substantial line broadening [T1 agents; e.g., Gd(III)], while others induce drastic line broadening (T2 agents; e.g., superparamagnetic iron oxide) [22]. The lanthanide ion Gd(III) has generally been chosen as the metal ion for CAs because it has a high magnetic moment (l = 63 lB ) and a symmetric electronic ground state (S) [23]. Transition metal ions such as high-spin Mn(II) and Fe(III) are candidates owing to their high magnetic moments. The mechanism of T1 relaxation is generally a through-space dipole–dipole interaction between the unpaired electrons of the paramagnetic metal ion and the protons of coordinated or diffusing water molecules. The longitudinal and transverse relaxation rates (Ri) of a CA solution are proportional to the CA’s relaxivity rip (Eq. 1) [20, 23]

A multinuclear NMR study of the interaction between phenylboronic acid (PBA) and sialic acid (Neu5 Ac) has been performed. The latter compound is known to be overexpressed on the cell surface of tumor cells. The results of this investigation suggest that the binding of PBA to sialic acid is pH dependent. 17O NMR experiments with glycolic acid as the model compound prove that an interaction at the alpha-hydroxycarboxylate occurs at pH < 9, while a study with threonic and erythronic acids shows that the PBA group interacts selectively with the vicinal diol functions at higher pH. Similarly, Neu5 Ac binds PBA through its alpha-hydroxycarboxylate at low pH (< 9) and through its glycerol side chain at higher pH values. The conditional stability constant of the phenylboronate ester at pH 7.4 is 11.4. On cell surfaces, sialic acid is connected to the neighboring sugar unit through the 2-hydroxy group. To mimic this the 2-alpha-O-methyl derivative of Neu5 Ac was included in this study. The erythro configuration of the hydroxy substituents prevents stable-complex formation at positions C7 and C8 and, consequently, the strongest interaction is observed at positions C8 and C9, leading to a five-membered 2-boron-1,3-dioxalate. In addition, a relatively small amount of the C7-C9 six-membered complex was observed. Molecular modeling studies confirm that the C8-C9 boronate complex has the lowest energy.

Four new ligands for lanthanide ions based on the H3do3a (=1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) structure and bearing one N-sulfonylacetamide arm were synthesized, i.e., H4dota-NHSO2R=10-{2-[(R)sulfonylamino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acids 1a–e. A 15N-NMR study of the 15N-labelled Eu3+ complex of one such ligands, 1d, showed that the coordination of the N-sulfonylacetamide arm involves the carbonyl O-atom rather than the N-atom. The relaxometric properties of the corresponding Gd3+ complexes were investigated as a function of pH and temperature. These complexes have relaxivities in the range 4.5–5.3 mM−1 s−1, at 20 MHz and 25°, and are characterized by a single H2O molecule in their inner coordination sphere. The mean residence lifetime of this molecule is relatively long (500–700 ns) compared to other anionic complexes. The slow rate of H2O exchange can be justified by the extensive delocalization of the negative charge on the N-sulfonylacetamide arm. The long residence time of the coordinated H2O allowed the observation of the effect of the prototropic exchange on the relaxivity. The study of the interaction between the complex [Gd(1e)]- and HSA revealed a weak affinity constant highlighting the importance of a localized negative charge on the complex to promote a strong interaction with the protein.

Myelination is one of the most fundamental biological processes in the development of vertebrate nervous systems. Abnormal or disrupted myelination occurs in many acquired or inherited neurodegenerative diseases, including multiple sclerosis (MS) and various leukodystrophies. To date, magnetic resonance imaging (MRI) has been the primary tool for diagnosing and monitoring the progression of MS and related diseases; however, any change in signal intensity of conventional MRI reflects a change only in tissue water content, which is a nonspecific measure of the overall changes in macroscopic tissue injury. Thus, the use of MRI as a primary measure of disease activity was shown to be disassociated from the clinical outcome due to the lack of specificity for myelination. In order to increase the MRI specificity for myelin pathologies, we designed and synthesized the first Gd-based T(1) MR contrast agent (MIC) that binds to myelin with high specificity. In this Communication, we demonstrate that MIC localizes in brain regions in proportion to the extent of myelination. In addition, MIC possesses promising MR contrast properties, which allow for direct detection of myelin distribution through T(1) mapping in the mouse brain.

New cationic gadolinium complexes have been synthesized and their 1H and 17O relaxometric properties investigated in view of their possible use as MRI contrast agents. The reaction of the bicyclic anhydride of ethylenedioxydiethylenedinitrilotetraacetic acid (H4egta) with NH3, isobutylamine and phenylpropylamine gave the octadentate bis-amide ligands 1–3 in high yields. The gadolinium(III) complexes were prepared from GdCl3 and the acid form of the ligands. The stability constants of the complexes were obtained from potentiometric data and are about five orders of magnitude lower than for the parent [Gd(egta)]− complex. The magnetic field dependence of the proton relaxivity at 6, 25 and 39 °C was quantitatively analysed in order to obtain the relaxation parameters and indicated that one water molecule occupies a site of the co-ordination sphere of the complexes. The temperature dependence of the relaxivity, measured at 20 MHz, suggested that the water exchange rate, kex = 1/τM, is sensibly reduced compared to that of the parent compound. 17O NMR data were acquired at 2.1 T and allowed an accurate estimation of τM which, at 298 K, resulted to be 230, 133 and 211 ns for Gd·1, Gd·2 and Gd·3 respectively. The two complexes bearing hydrophobic residues on the amide nitrogens form non-covalent adducts with serum albumin whose relaxivity is limited by the water exchange rate.

The formation of a macromolecular adduct between a suitably functionalized GdIII complex and an exogeneous substance such as a poly-β-cyclodextrin (see picture) forms the basis of the novel route to contrast agents for magnetic resonance imaging endowed with high relaxivity discussed herein.

We have prepared the first MRI prodrug-procontrast complex by conjugating doxorubicin to a Gd(III) chelate using an acid-labile linker. The relaxometric properties of the adduct and pH activation are reported.

Myelin is a membrane system that fosters nervous impulse conduction in the vertebrate nervous system. Myelin sheath disruption is a common characteristic of several neurodegenerative diseases such as multiple sclerosis (MS) and various leukodystrophies. To date, the diagnosis of MS is obtained using a set of criteria in which MRI observations play a central role. However, because of the lack of specificity for myelin integrity, the use of MRI as the primary diagnostic tool has not yet been accepted. In order to improve MR specificity, we began developing MR probes targeted towards myelin. In this work we describe a new myelin-targeted MR contrast agent, Gd-DODAS, based on a stilbene binding moiety and demonstrate its ability to specifically bind to myelin in vitro and in vivo. We also present evidence that Gd-DODAS generates MR contrast in vivo in T1-weighed images and in T1 maps that correlates to the myelin content.

The development of gadolinium chelators that can be easily and readily linked to various substrates is of primary importance for the development high relaxation efficiency and/or targeted magnetic resonance imaging (MRI) contrast agents. Over the last 25 years a large number of bifunctional chelators have been prepared. For the most part, these compounds are based on ligands that are already used in clinically approved contrast agents. More recently, new bifunctional chelators have been reported based on complexes that show a more potent relaxation effect, faster complexation kinetics and in some cases simpler synthetic procedures. This review provides an overview of the synthetic strategies used for the preparation of bifunctional chelators for MRI applications.

The scope of Magnetic Resonance Imaging (MRI) is moving beyond anatomical and functional imaging to also convey information at the molecular level. Molecular MRI is enabled by the introduction of protein-targeted contrast agents[1] as well as “smart” or activatable contrast agents.[2] MR contrast agents induce relaxation of tissue water, and the extent of this relaxation enhancement, termed relaxivity (r1), depends on a number of molecular factors including the hydration state of the contrast agent and its rotational diffusion rate. In a seminal paper, Meade and colleagues demonstrated that the relaxivity of a specifically designed contrast agent could be changed in the presence of the enzyme beta-galactosidase, thereby creating an imaging agent whose signal was activated by the presence of the enzyme.[3, 4] Numerous publications have followed in the last decade that describe “smart” agents responsive to other enzymes, to pH, to specific metal ion concentrations, to partial oxygen pressure and to temperature.[5, 6] The impressive gains in smart agent development have been slow to make their way into in vivo imaging studies, however. This can be appreciated from equation 1 which relates the water relaxation rate (1/T1) to r1. MR signal is a function T1 (Eq. 1, 1/T10 = relaxation rate in absence of agent), which depends on both r1 and contrast agent concentration ([Gd]). In vitro, the gadolinium concentration is known and fixed; any signal change is due to relaxivity change. In vivo, the agent concentration is unknown, will change with time, and may vary in diseased versus normal tissue. 1T1=r1⋅[Gd]+1T10 (1) A smart agent to noninvasively map pH with both high temporal and spatial resolution would have broad utility. Decreased extracellular pH is associated with cancer and ischemic diseases such as stroke, ischemic heart disease, and kidney disease.[7] pH could be a very useful biomarker to identify disease and monitor response to therapy, but it remains a challenge to routinely assess pH in vivo. Implanting a pH electrode is invasive and offers little spatial information. 31P NMR can measure pH via the pH-dependent chemical shift of inorganic phosphate.[8] Other papers have described using exogenous agents with pH-sensitive chemical shifts.[7, 9] Yet, these NMR spectroscopic techniques are limited by low sensitivity resulting in trade offs in imaging time (longer, more averages required) and resolution (lower, bigger volume elements required). Chemical exchange saturation transfer (CEST) agents have also been described for pH imaging, but these also require millimolar concentrations for detection.[10, 11] Recently hyperpolarized 13C-carbonate MR was used to image pH.[12] There are several Gd-based smart agents whose relaxivity is pH dependent due to changes in complex hydration with pH.[6, 7] To address the problem of complex concentration, Aime et al. proposed a R2/R1 ratiometric method[13] but given the relatively large R2 present in living tissues compared to R1, the in vivo accuracy of such an approach has yet to be proven. Combining fluorine MRI for quantification with a pH sensitive Gd-based agent has also been suggested,[14] although the sensitivity of F-19 imaging is in the millimolar range. An early pH sensitive agent was GdDOTA-4AMP.[15] This agent was used to map pH in vivo in renal acidosis[16] and brain tumor[17] models. To estimate the in vivo concentration of agent, these investigators first injected GdDOTP, which has pH-independent relaxivity, and imaged. They assumed that the pharmacokinetics of GdDOTP was the same as for GdDOTA-4AMP and that differences in the signal vs time curves for GdDOTP and GdDOTA-4AMP were due to differences in relaxivity. These studies demonstrated the potential for in vivo pH mapping and showed that MRI with GdDOTA-4AMP was sensitive enough to detect pH differences. The limitations of this approach were the need for two sequential injections and the assumption that both contrast agents have identical pharmacokinetics. Positron emission tomography (PET) offers exquisite sensitivity and the ability to perform absolute quantification. Quantitative PET imaging is routinely used in human and animal studies, for example to measure neuroreceptor occupancy levels[18] or to measure tissue perfusion.[19] The recent application of MR-compatible avalanche photodiode detector technology has now made it possible to have a functioning PET detector inside the MR magnet.[20, 21] This allows for the simultaneous acquisition of PET and MR data, and ability to obtain both temporally and spatially registered imaging data sets. We hypothesized that simultaneous MR-PET imaging with a bimodal MR-PET smart agent would result in quantification of both concentration and relaxivity. This dual label approach could enable a range of quantitative smart probes for in vivo applications. Here, a bimodal MR-PET agent designed for quantitative pH imaging at concentrations commonly used for in vivo MRI (0.1–1 mM) is described. The established pH sensitive MR agent GdDOTA-4AMP was modified to incorporate a fluorine atom (either 18F or 19F). The highly charged, hydrophilic GdDOTA-4AMP necessitated a strategy to introduce the 18F atom under aqueous conditions, and we chose the versatile Cu(I)-catalyzed Huisgen cycloaddition (“click reaction”) for this purpose.[22] GdDOTA-4AMP-F was prepared in six steps (Scheme 1) with a 25% overall yield starting from an established bifunctional chelator, tBu protected DOTAGA, 1.[23] 1 was activated and coupled with propargylamine, and subsequently deprotected in neat TFA to give 3. The introduction of the phosphonate groups was accomplished by coupling with aminomethyl-phosphonic acid diethyl ester, followed by mild deprotection of the phosphonate groups with trimethylsilyl bromide in DMF. The formation of the Gd(III) complex from the chloride salt, followed by reaction of fluoroethylazide and the alkyne intermediate 6, was performed in one pot. [F-18]fluoroethylazide was prepared in two steps from 2-azidoethanol, while the [F-19] version was prepared in two steps from 2-fluoroethanol (see Supp. Info).[22] Scheme 1 Synthesis of Gd-DOTA-4AMP-F and structure of Gd-DOTA-4AMP. The introduction of the fluorine-containing moiety into GdDOTA-4AMP-F did not modify the pH dependence of the longitudinal relaxivity with respect to the parent compound GdDOTA4-AMP.[24] GdDOTA-4AMP-F retains a monotonic decrease in relaxivity between pH 6.0 and 8.5 (Figure 1). In this pH range the relaxivity varied between 7.4 and 3.9 mM−1s−1 (60 MHz, 37 °C) when measured in an isotonic salt mixture. When the relaxivity was measured in rabbit plasma, the profile was found to be very similar to the profile measured in the salt solution. This indicates little if any protein binding and suggests that the pH-relaxivity relationship will be valid in vivo. Figure 1 Relaxivity of GdDOTA-4AMP-F as a function of pH (37 °C, 1.4 T) in presence of 135 mM NaCl, 5 mM KCl, and 2.5 mM CaCl2 (filled diamonds), and in rabbit plasma (open circles). The chemical concentration required for MR contrast is orders of magnitude higher than for PET imaging. For this reason, F-19 and F-18 versions of the probe were prepared separately, and subsequently mixed to produce a low specific activity MR-PET agent. Simultaneous MR-PET imaging was performed on a series of samples with varying pH using a clinical 3T MRI with a MR-compatible human PET scanner insert.[21] Figure 2 shows simultaneous MR-PET images acquired on phantoms where the T1 varied (MR, 2A) but the probe concentration was constant (PET, 2B); or where T1 was constant (2C) but the probe concentration varied (2D). Figure 2 eloquently displays the limitations of using an MR responsive agent without independent knowledge of the agent concentration. Note that in both sets of phantoms, the pH is varied. The only way to obtain pH values from the images is to combine both the PET and MR datasets. Figure 2 T1-weighted MR images (A and C, T1 values (ms) listed) and PET images (B and D, PET intensities (a.u.) listed) of phantoms at pH 6.5 (tube 1), 6.8 (2), 7.1 (3), 7.4 (4), and 7.8 (5). Phantoms in images A and B have the same concentration (0.45 mM); phantoms ... Since the PET signal is linear with radiochemical concentration, the unknown agent concentration can be determined by comparing the PET images with a series of standards. For the MR data, the relationship between r1 and pH can be measured (see Fig 1) and this was repeated at 3T where a similar linear relationship between r1 and pH between pH 6 to 8.5 was obtained. From these two standard curves, the PET and MR imaging data can be analyzed to estimate the pH of the samples. Figure 3 shows the good correspondence between pH measured by electrode and pH calculated from the MR and PET images. Figure 3 pH obtained from PET-MR image analysis versus pH measured by a glass electrode. The solid line is a linear fit of the data, while the dotted line represents a 1:1 correspondence. In conclusion, this communication describes a smart MR-PET agent that can quantitatively and non-invasively report on pH. Imaging data were obtained on a commercial clinical MRI with a prototype human PET camera at agent concentrations routinely encountered in clinical MRI (0.1 – 1 mM). This augurs well for the application of GdDOTA-4AMP-F to image pH changes in vivo. The combination of PET for quantifying concentration and MR for quantifying T1 allows for the simultaneous determination of relaxivity. For smart MR probes where relaxivity is proportional to an environmental stimulus, this bimodal imaging approach enables direct quantification of the stimulus, pH in this case. We note that this bimodal MR-PET strategy is generally applicable to other smart MR probes.

Simulations were performed to understand the relative contributions of molecular parameters to longitudinal (r(1)) and transverse (r(2)) relaxivity as a function of applied field, and to obtain theoretical relaxivity maxima over a range of fields to appreciate what relaxivities can be achieved experimentally. The field-dependent relaxivities of a panel of gadolinium and manganese complexes with different molecular parameters, water exchange rates, rotational correlation times, hydration state, etc. were measured to confirm that measured relaxivities were consistent with theory. The design tenets previously stressed for optimizing r(1) at low fields (very slow rotational motion; chelate immobilized by protein binding; optimized water exchange rate) do not apply at higher fields. At 1.5 T and higher fields, an intermediate rotational correlation time is desired (0.5-4 ns), while water exchange rate is not as critical to achieving a high r(1). For targeted applications it is recommended to tether a multimer of metal chelates to a protein-targeting group via a long flexible linker to decouple the slow motion of the protein from the water(s) bound to the metal ions. Per ion relaxivities of 80, 45, and 18 mM(-1) s(-1) at 1.5, 3 and 9.4 T, respectively, are feasible for Gd(3+) and Mn(2+) complexes.

The detection of sialic acid in living systems is of importance for the diagnosis of several types of malignancy. We have designed and synthesized two new lanthanide ion ligands (L1 and L2) that are capable of molecular recognition of sialic acid residues. The basic structure of these ligands consists of a DTPA-bisamide (DTPA, diethylenetriamine pentaacetic acid) whose amide moieties each bear both a boronic function for interaction with the diol groups in the side chain of sialic acid, and a functional group that is positively charged at physiologic pH values and is designed to interact with the carboxylate anion of sialic acid. The relaxometric properties of the Gd3+ complexes of these two ligands were evaluated. The relaxivity of the GdL1 complex has a significant second-sphere contribution at pH values above the pKa of its phenylboronic acid moiety. The interaction of the Gd3+ complexes of L1 and L2 with each of several saccharides was investigated by means of a competitive fluorescent assay. The results show that both complexes recognize sialic acid with good selectivity in the presence of other sugars. The adduct formed by GdL2 with sialic acid has the higher conditional formation constant (50.43+/-4.61 M(-1) at pH 7.4). The ability of such complexes to recognize sialic acid was confirmed by the results of a study on the interaction of corresponding radiolabeled complexes (153SmL1 and 153SmL2) with C6 glioma rat cells. 153SmL2 in particular is retained on the cell surface in significant amounts.

Contrast agents for magnetic resonance imaging are mainly Gd3+ complexes of octadentate polyaminopolycarboxylates and one water molecule in the first coordination sphere of the metal ion. Both the Ln3+ ion and the nitrogen atoms in these complexes may be chiral. Therefore, various isomers may occur for these complexes, which interconvert in aqueous solution. Here studies on the configurations and conformations of Ln3+ complexes of acyclic and cyclic ligands are reviewed. Furthermore, the structures of new generations of contrast agents that are able to target organs or to sense their biochemical environment are discussed. Molecular recognition is an important issue for these compounds.

A novel ligand based on a pyridine-containing macrocycle bearing two acetic and one methylenephosphonic arms (PCP2A) has been synthesized. An efficient synthesis of PCP2A is based on the macrocyclization reaction between 2,6-bis(chloromethyl)pyridine and a 1,4, 7-triazaheptane derivative bearing a methylenephosphonate group on N-4. The Gd(III) complex of PCP2A displays characteristic properties which make it a very promising contrast agent for improved applications in magnetic resonance imaging. In fact it shows (i) a very high stability constant (log K(GdPCP2A) = 23.4) which should guarantee against the in vivo release of toxic free Gd(III) ions and free ligand molecules and (ii) a relaxivity that is about 2 times higher than the values reported for contrast agents currently used in the clinical practice. Its high relaxivity is the result of the presence of two water molecules in the inner coordination sphere and a significant contribution from water molecule(s) hydrogen bonded to the phosphonate group. Moreover, the inner sphere water molecules are involved in an exchange with the bulk water which is relatively fast. This property is important for the attainment of an even higher relaxivity once the molecular reorientation rate of the [GdPCP2A(H(2)O)(2)](-) moiety is lengthened by means of conjugation to a macromolecular substrate.