Christelle Medina

Role of thermodynamic and kinetic parameters in gadolinium chelate stability.

In recent years there has been a renewed interest in the physicochemical properties of gadolinium chelates (GC). The aim of this review is to discuss the physicochemical properties of marketed GC with regard to possible biological consequences. GC can be classified according to three key molecular features: 1) the nature of the chelating moiety: either macrocyclic molecules in which Gd3+ is caged in the preorganized cavity of the ligand, or linear, open‐chain molecules; 2) ionicity: the ionicity of the molecule varies from neutral to tri‐anionic agents; and 3) the presence or absence of an aromatic lipophilic moiety, which has a profound impact on the biodistribution of the GC. These parameters can also explain why GC differ considerably with regard to their thermodynamic stability constants and kinetic stability, as demonstrated by numerous studies. The concept of thermodynamic and kinetic stability is critically discussed, as it remains somewhat controversial, especially in predicting the amount of free gadolinium that may result from decomplexation of chelates in physiologic or pathologic situations. This review examines the possibility that the high kinetic stability provided by the macrocyclic structure combined with a high thermodynamic stability (reinforced by ionicity for macrocyclic chelates) can minimize the amount of free Gd3+ released in the body. J. Magn. Reson. Imaging 2009;30:1249–1258.

Magnetic Resonance Molecular Imaging of Thrombosis in an Arachidonic Acid Mouse Model Using an Activated Platelet Targeted Probe

Objective—Atherosclerotic plaque rupture leads to acute thrombus formation and may trigger serious clinical events such as myocardial infarction or stroke. Therefore, it would be valuable to identify atherothrombosis and vulnerable plaques before the onset of such clinical events. We sought to determine whether the noninvasive in vivo visualization of activated platelets was effective when using a target-specific MRI contrast agent to identify thrombi, hallmarks of vulnerable or high-risk atherosclerotic plaques. Methods and Results—Inflammatory thrombi were induced in mice via topical application of arachidonic acid on the carotid. Thrombus formation was imaged with intravital fluorescence microscopy and molecular MRI. To accomplish the latter, a paramagnetic contrast agent (P975) that targets the glycoprotein &agr;IIb&bgr;3, expressed on activated platelets, was investigated. The specificity of P975 for activated platelets was studied in vitro. In vivo, high spatial-resolution MRI was performed at baseline and longitudinally over 2 hours after injecting P975 or a nonspecific agent. The contralateral carotid, a sham surgery group, and a competitive inhibition experiment served as controls. P975 showed a good affinity for activated platelets, with an IC50 (concentration of dose that produces 50% inhibition) value of 2.6 &mgr;mol/L. In thrombosed animals, P975 produced an immediate and sustained increase in MRI signal, whereas none of the control groups revealed any significant increase in MRI signal 2 hours after injection. More important, the competitive inhibition experiment with an &agr;IIb&bgr;3 antagonist suppressed the MRI signal enhancement, which is indicative for the specificity of P975 for the activated platelets. Conclusion—P975 allowed in vivo target-specific noninvasive MRI of activated platelets.

In vivo CEST MR imaging of U87 mice brain tumor angiogenesis using targeted LipoCEST contrast agent at 7 T

LipoCEST are liposome‐encapsulating paramagnetic contrast agents (CA) based on chemical exchange saturation transfer with applications in biomolecular MRI. Their attractive features include biocompatibility, subnanomolar sensitivity, and amenability to functionalization for targeting biomarkers. We demonstrate MR imaging using a targeted lipoCEST, injected intravenously. A lipoCEST carrying Tm(III)‐complexes was conjugated to RGD tripeptide (RGD‐lipoCEST), to target integrin ανβ3 receptors involved in tumor angiogenesis and was compared with an unconjugated lipoCEST. Brain tumors were induced in athymic nude mice by intracerebral injection of U87MG cells and were imaged at 7 T after intravenous injection of either of the two contrast agents (n = 12 for each group). Chemical exchange saturation transfer‐MSME sequence was applied over 2 h with an average acquisition time interval of 13.5 min. The chemical exchange saturation transfer signal was ∼1% in the tumor and controlateral regions, and decreased to ∼0.3% after 2 h; while RGD‐lipoCEST signal was ∼1.4% in the tumor region and persisted for up to 2 h. Immunohistochemical staining revealed a persistent colocalization of RGD‐lipoCEST with ανβ3 receptors in the tumor region. These results constitute an encouraging step toward in vivo MRI imaging of tumor angiogenesis using intravenously injected lipoCEST. Magn Reson Med, 2013.

In vivo imaging of folate receptor positive tumor xenografts using novel 68Ga-NODAGA-folate conjugates.

The overexpression of the folate receptor (FR) in a variety of malignant tumors, along with its limited expression in healthy tissues, makes it an attractive tumor-specific molecular target. Noninvasive imaging of FR using radiolabeled folate derivatives is therefore highly desirable. Given the advantages of positron emission tomography (PET) and the convenience of (68)Ga production, the aim of our study was to develop a new (68)Ga-folate-based radiotracer for clinical application. The chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) was conjugated to folic acid and to 5,8-dideazafolic acid using 1,2-diaminoethane as a spacer, resulting in two novel conjugates, namely, P3246 and P3238, respectively. Both conjugates were labeled with (68/67)Ga. In vitro internalization, efflux, and saturation binding studies were performed using the FR-positive KB cell line. Biodistribution and small-animal PET imaging studies were performed in nude mice bearing subcutaneous KB xenografts. Both conjugates were labeled with (68)Ga at room temperature within 10 min in labeling yields >95% and specific activity ~30 GBq/μmol. The K(d) values of (68/67)Ga-P3246 (5.61 ± 0.96 nM) and (68/67)Ga-P3238 (7.21 ± 2.46 nM) showed high affinity for the FR. (68/67)Ga-P3246 showed higher cell-associated uptake in vitro than (68/67)Ga-P3238 (approximately 72 and 60% at 4 h, respectively, P < 0.01), while both radiotracers exhibited similar cellular retention up to 4 h (approximately 76 and 71%, respectively). Their biodistribution profile is characterized by high tumor uptake, fast blood clearance, low hepatobiliary excretion, and almost negligible background. Tumor uptake was already high at 1 h for both (68)Ga-P3246 and (68)Ga-P3238 (16.56 ± 3.67 and 10.95 ± 2.12% IA/g, respectively, P > 0.05) and remained at about the same level up to 4 h. Radioactivity also accumulated in the FR-positive organs, such as kidneys (91.52 ± 21.05 and 62.26 ± 14.32% IA/g, respectively, 1 h pi) and salivary glands (9.05 ± 2.03 and 10.39 ± 1.19% IA/g, respectively, 1 h pi). The specificity of the radiotracers for the FR was confirmed by blocking experiments where tumor uptake was reduced by more than 85%, while the uptake in the kidneys and the salivary glands was reduced by more than 90%. Reduction of the kidney uptake was achieved by administration of the antifolate pemetrexed 1 h prior to the injection of the radiotracers, which resulted in an improvement of tumor-to-kidney ratios by more than a factor of 3. In line with the biodistribution results, small-animal PET images showed high uptake in the kidneys, clear visualization of the tumor, accumulation of radioactivity in the salivary glands, and no uptake in the gastrointestinal tract. (68)Ga-P3246 and (68)Ga-P3238 showed very high tumor-to-background contrast in PET images; however, the tumor-to-kidney ratio remained low. The new radiotracers, especially (68)Ga-P3246, are promising as PET imaging probes for clinical application due to their facile preparation and improved in vivo profile as compared to the other folate-based PET radiotracers.

Development of new folate-based PET radiotracers: preclinical evaluation of 68Ga-DOTA-folate conjugates

PurposeA number of 111In- and 99mTc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A 68Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of 68Ga from a generator. The aim of the study was to develop a new 68Ga-folate-based PET radiotracer.MethodsTwo new DOTA-folate conjugates, named P3026 and P1254, were synthesized using the 1,2-diaminoethane and 3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propylamine as a spacer, respectively. Both conjugates were labelled with 67/68Ga. Binding affinity, internalization and externalization studies were performed using the FR-positive KB cell line. Biodistribution and PET/CT imaging studies were performed in nude mice, on a folate-deficient diet, bearing KB and HT1080 (FR-negative) tumours, concurrently. The new radiotracers were evaluated comparatively to the reference molecule 111In-DTPA-folate (111In-P3139).ResultsThe Kd values of 67/68Ga-P3026 (4.65 ± 0.82 nM) and 67/68Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order 67/68Ga-P3026 > 67/68Ga-P1254 > 111In-P3139, while almost double cellular retention was found for 67/68Ga-P3026 and 67/68Ga-P1254, compared to 111In-P3139. The biodistribution data of 67/68Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to 111In-P3139. PET/CT images, performed with 68Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours.ConclusionThe DOTA-folate conjugates can be efficiently labelled with 68Ga in labelling yields and specific activities which allow clinical application. The characteristics of the 67/68Ga-DOTA-folates are comparable to 111In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers for FR-positive tumours.

Analytical interference in serum iron determination reveals iron versus gadolinium transmetallation with linear gadolinium-based contrast agents.

ObjectivesThe purposes of this study were to evaluate the risk for analytical interference with gadolinium-based contrast agents (GBCAs) for the colorimetric measurement of serum iron (Fe3+) and to investigate the mechanisms involved. Materials and MethodsRat serum was spiked with several concentrations of all molecular categories of GBCAs, ligands, or “free” soluble gadolinium (Gd3+). Serum iron concentration was determined by 2 different colorimetric methods at pH 4.0 (with a Vitros DT60 analyzer or a Cobas Integra 400 analyzer). Secondly, the cause of interference was investigated by (a) adding free soluble Gd3+ or Mn2+ to serum in the presence of gadobenic acid or gadodiamide and (b) electrospray ionization mass spectrometry. ResultsSpurious decrease in serum Fe3+ concentration was observed with all linear GBCAs (only with the Vitros DT60 technique occurring at pH 4.0) but not with macrocyclic GBCAs or with free soluble Gd3+. Spurious hyposideremia was also observed with the free ligands present in the pharmaceutical solutions of the linear GBCAs gadopentetic acid and gadodiamide (ie, diethylene triamine pentaacetic acid and calcium-diethylene triamine pentaacetic acid bismethylamide, respectively), suggesting the formation of Fe-ligand chelate.Gadobenic acid-induced interference was blocked in a concentration-dependent fashion by adding a free soluble Gd3+ salt. Conversely, Mn2+, which has a lower affinity than Gd3+ and Fe3+ for the ligand of gadobenic acid (ie, benzyloxypropionic diethylenetriamine tetraacetic acid), was less effective (interference was only partially blocked), suggesting an Fe3+ versus Gd3+ transmetallation phenomenon at pH 4.0. Similar results were observed with gadodiamide. Mass spectrometry detected the formation of Fe-ligand with all linear GBCAs tested in the presence of Fe3+ and the disappearance of Fe-ligand after the addition of free soluble Gd3+. No Fe-ligand chelate was found in the case of the macrocyclic GBCA gadoteric acid. ConclusionsMacrocyclic GBCAs induced no interference with colorimetric methods for iron determination, whereas negative interference was observed with linear GBCAs using a Vitros DT60 analyzer. This interference of linear GBCAs seems to be caused by the excess of ligand and/or an Fe3+ versus Gd3+ transmetallation phenomenon.

Entrapment of a neutral Tm(III)‐based complex with two inner‐sphere coordinated water molecules into PEG‐stabilized vesicles: towards an alternative strategy to develop high‐performance LipoCEST contrast agents for MR imaging

Chemical exchange saturation transfer (CEST) probes issued from the encapsulation of a water proton paramagnetic shift reagent into the inner aqueous volume of lipid vesicles provide an emerging class of frequency-selective contrast agents with huge potential in the field of molecular magnetic resonance imaging (MRI). This work deals with the generation of such LipoCEST agents properly designed to optimize, under isotonic conditions, the chemical shift offset of the intra-liposomal water protons as well as the number of exchangeable protons under reasonably low radiofrequency (RF) fields of saturation. The strategy lies in the loading of poly(ethylene glycol)-stabilized nanosized liposomes with uncharged lanthanide chelates, binding more than one water molecule in the first hydration sphere, exemplified here by [Tm(III)-DO3A (H2 O)2 ] complex. The key properties of the probes are demonstrated by complementary NMR investigations. The residence lifetime of the water molecules coordinated to the lanthanide center was outstandingly short (9.5 ± 0.2 ns from (17) O NMR), and indeed relevant for effective LipoCEST responsiveness. The (1) H NMR CEST spectra (7.01 T magnetic field) prove that the theoretically expected optimal sensitivity can be approximated in the nanomolar concentration range, at reasonably low RF presaturation pulses (6.7-12 μT) and saturation frequency offsets of the intra-liposomal water protons beyond 10 ppm, making possible selective irradiation in biological environment. CEST-MRI images (7.01 T magnetic field and 10-12 μT RF pulse) explicitly confirm the interest of these newly conceived LipoCEST agents, indeed among the most efficient ones developed so far under isosmotic conditions.