Pharmacokinetic and in vivo evaluation of a self‐assembled gadolinium(III)‐iron(II) contrast agent with high relaxivity

A high‐molecular weight tetrametallic supramolecular complex [(Ln‐DTPA‐phen)₃Fe]^? (Ln = Gd, Eu, La) has been obtained upon self‐assembly around one iron(II) ion of three 1,10‐phenantroline‐based molecules substituted in 5′‐position with the polyaminocarboxylate diethylenetriamine‐N,N,N′,N′,N′‐pentaacetate, DTPA‐phen^4?. The ICP‐MS measurements indicated that the lanthanide:iron ratio is 3:1. Photoluminescence spectra of [Eu‐DTPA‐phen]^? and of [(Eu‐DTPA‐phen)₃Fe]^? are nearly identical, implying that the first coordination sphere of the lanthanide(III) ion has not been changed upon coordination of phenantroline unit to iron(II) ion. NMRD measurements revealed that at 20 MHz and 310 K the relaxivity of the [(Gd‐DTPA‐phen)₃Fe]^? is equal to 9.5 ± 0.3 s^?1 mM^?1 of Gd (28.5 s^?1 per millimole per liter of complex) which is significantly higher than that for Gd‐DTPA (3.9 s^?1 mM^?1). The pharmacokinetic parameters of [(Gd‐DTPA‐phen)₃Fe]^? in rats indicate that the elimination of [(Gd‐DTPA‐phen)₃Fe]^? is significantly slower than that of Gd‐DTPA and is correlated with a reduced volume of distribution. The low volume of distribution and the longer elimination time (T_e1/2) suggest that the agent is confined to the blood compartment, so it could have an important potential as a blood pool contrast agent. The biodistribution profile of [(Gd‐DTPA‐phen)₃Fe]^? 2 h after injection indicates significantly higher concentrations of [(Gd‐DTPA‐phen)₃Fe]^? as compared with Gd‐DTPA in kidney, liver, lungs, heart and spleen. The images obtained on rats by MR angiography show the enhancement of the abdominal blood vessels. The signal intensity reaches a maximum of 55% at 7 min post‐contrast and remains around 25% after 90 min. MRI‐histomorphological correlation studies of [Gd‐DTPA‐phen]^? and [(Gd‐DTPA‐phen)₃Fe]^? showed that both agents displayed potent contrast enhancement in organs including the liver. The necrosis avidity tests indicated that, in contrast to the [Gd‐DTPA‐phen]^? precursor complex, the supramolecular complex [(Gd‐DTPA‐phen)₃Fe]^? exhibits necrosis avidity. Copyright © 2006 John Wiley & Sons, Ltd.     

A neutral polydisulfide containing Gd(III) DOTA monoamide as a redox‐sensitive biodegradable macromolecular MRI contrast agent

This work aims to develop safe and effective gadolinium (III)‐based biodegradable macromolecular MRI contrast agents for blood pool and cancer imaging. A neutral polydisulfide containing macrocyclic Gd‐DOTA monoamide (GOLS) was synthesized and characterized. In addition to studying the in vitro degradation of GOLS, its kinetic stability was also investigated in an in vivo model. The efficacy of GOLS for contrast‐enhanced MRI was examined with female BALB/c mice bearing 4T1 breast cancer xenografts. The pharmacokinetics, biodistribution, and metabolism of GOLS were also determined in mice. GOLS has an apparent molecular weight of 23.0 kDa with T₁ relaxivities of 7.20 mM^?1 s^?1 per Gd at 1.5 T, and 6.62 mM^?1 s^?1 at 7.0 T. GOLS had high kinetic inertness against transmetallation with Zn²⁺ ions, and its polymer backbone was readily cleaved by L‐cysteine. The agent showed improved efficacy for blood pool and tumor MR imaging. The structural effect on biodistribution and in vivo chelation stability was assessed by comparing GOLS with Gd(HP‐DO3A), a negatively charged polydisulfide containing Gd‐DOTA monoamide GODC, and a polydisulfide containing Gd‐DTPA‐bisamide (GDCC). GOLS showed high in vivo chelation stability and minimal tissue deposition of gadolinium. The biodegradable macromolecular contrast agent GOLS is a promising polymeric contrast agent for clinical MR cardiovascular imaging and cancer imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessment of tumor angiogenesis: dynamic contrast‐enhanced MRI with paramagnetic nanoparticles compared with Gd‐DTPA in a rabbit Vx‐2 tumor model

The purpose of this study was to evaluate the suitability of a macromolecular MRI contrast agent (paramagnetic nanoparticles, PNs) for the characterization of tumor angiogenesis. Our aim was to estimate the permeability of PNs in developing tumor vasculature and compare it with that of a low molecular weight contrast agent (Gd‐DTPA) using dynamic contrast‐enhanced MRI (DCE). Male New Zealand white rabbits (n = 5) underwent DCE MRI 12–14 days after Vx‐2 tumor fragments were implanted into the left hind limb. Each contrast agent (PNs followed by Gd‐DTPA) was evaluated using a DCE protocol and transendothelial transfer coefficient (K_i) maps were calculated using a two‐compartment model. Two regions of interest (ROIs) were located within the tumor core and hindlimb muscle and five ROIs were placed within the tumor rim. Comparisons were performed using repeated measures analysis of variance (ANOVA). The K_i values estimated using PNs were significantly lower than those obtained for Gd‐DTPA (p = 0.018). When PNs and Gd‐DTPA data were analyzed separately, significant differences were identified among tumor rim ROIs for PNs (p < 0.0001), but not for Gd‐DTPA data (p = 0.34). The mean K_i for the tumor rim was significantly greater than that of either the core or the hindlimb muscle for both contrast agents (p < 0.05 for each comparison). In summary, the extravasation of Gd‐DTPA was far greater than that of PNs, suggesting that PNs can reveal regional differences in tumor vascular permeability that are not otherwise apparent with clinical contrast agents such as Gd‐DTPA. These results suggest that PNs show potential for the noninvasive delineation of tumor angiogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

The effects of intramolecular H‐bond formation on the stability constant and water exchange rate of the Gd(III)‐diethylenetriamine‐N′‐(3‐amino‐1,1‐propylenephosphonic)‐N,N,N″,N″‐tetraacetate complex

The binding interaction of metal chelates to biological macromolecules, though driven by properly devoted recognition synthons, may cause dramatic changes in some property associated with the coordination cage such as the thermodynamic stability or the exchange rate of the metal coordinated water. Such changes are due to electrostatic and H‐bonding interactions involving atoms of the coordination cage and atoms of the biological molecule at the binding site. To mimic this type of H‐bonding interactions, lanthanide(III) complexes with a DTPA–monophosphonate ligand bearing a propylamino moiety (H₆NP–DTPA) were synthesized. Their thermodynamic stabilities and the exchange lifetime of the coordinated water molecule (for the Gd‐complex) were compared with those of the analog complexes with DTPA and the parent DTPA–monophosphonate derivative (H₆P–DTPA). It was found that the intramolecular H‐bond between the ε‐amino group and the phosphonate moiety in NP–DTPA complexes causes displacements of electric charges in their coordination cage that are markedly pH dependent. In turn, this affects the characteristic properties of the coordination cage. In particular it results in a marked elongation of the exchange lifetime of the coordinated water molecule. Copyright © 2007 John Wiley & Sons, Ltd.     

Enhanced MRI relaxivity of Gd3+‐based contrast agents geometrically confined within porous nanoconstructs

Gadolinium chelates, which are currently approved for clinical MRI use, provide relaxivities well below their theoretical limit, and they also lack tissue specificity. Recently, the geometrical confinement of Gd³⁺‐based contrast agents (CAs) within porous structures has been proposed as a novel, alternative strategy to improve relaxivity without chemical modification of the CA. Here, we have characterized and optimized the performance of MRI nanoconstructs obtained by loading [Gd(DTPA)(H₂O)]^2? (Magnevist®) into the pores of injectable mesoporous silicon particles. Nanoconstructs with three different pore sizes were studied, and at 60 MHz, they exhibited longitudinal relaxivities of ~24 m m ^?1 s^?1 for 5–10 nm pores and ~10 m m ^?1 s^?1 for 30 – 40 nm pores. No enhancement in relaxivity was observed for larger pores sizes. Using an outer‐sphere compound, [GdTTHA]^3?, and mathematical modeling, it was demonstrated that the relaxivity enhancement is due to the increase in rotational correlation times (CA adsorbed on the pore walls) and diffusion correlation times (reduced mobility of the water molecules), as the pore sizes decreases. It was also observed that extensive CA adsorption on the outer surface of the silicon particles negates the advantages offered by nanoscale confinement. Upon incubation with HeLa cells, the nanoconstructs did not demonstrate significant cytotoxicity for up to 3 days post incubation, at different particle/cell ratios. In addition, the nanoconstructs showed complete degradation after 24 h of continuous agitation in phosphate‐buffered saline. These data support and confirm the hypothesis that the geometrical confinement of Gd³⁺‐chelate compounds into porous structures offers MRI nanoconstructs with enhanced relaxivity (up to 6 times for [Gd(DTPA)(H₂O)]^2?, and 4 times for [GdTTHA]^3?) and, potentially, improved stability, reduced toxicity and tissue specificity. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacokinetics and magnetic resonance imaging of biodegradable macromolecular blood‐pool contrast agent PG–Gd in non‐human primates: a pilot study

The purpose of this study was to evaluate poly(L ‐glutamic acid)‐benzyl‐DTPA–Gd (PG–Gd), a new biodegradable macromolecular magnetic resonance imaging contrast agent, for its pharmacokinetics and MRI enhancement in nonhuman primates. Studies were performed in rhesus monkeys at intravenous doses of 0.01, 0.02 and 0.08 mmol Gd/kg. T₁‐weighted MR images were acquired at 1.5 T using fast spoiled gradient recalled echo and fast spin echo imaging protocols. The small‐molecule contrast agent Magnevist was used as a control. PG–Gd in the monkey showed a bi‐exponential disposition. The initial blood concentrations within 2 h of PG–Gd administration were much higher than those for Magnevist. The high blood concentration of PG–Gd was consistent with the MR imaging data, which showed prolonged circulation of PG–Gd in the blood pool. Enhancement of blood vessels and organs with a high blood perfusion (heart, liver, and kidney) was clearly visualized at 2 h after contrast injection at the three doses used. A greater than proportional increase of the area under the blood concentration–time curve was observed when the administered single dose was increased from 0.01 to 0.08 mmol/kg. By 2 days after PG–Gd injection, the contrast agent was mostly cleared from all major organs, including kidney. The mean residence time was 15 h at the 0.08 mmol/kg dose. A similar pharmacokinetic profile was observed in mice, with a mean residence time of 5.4 h and a volume of distribution at steady‐state of 85.5 ml/kg, indicating that the drug was mainly distributed in the blood compartment. Based on this pilot study, further investigations on the potential systemic toxicity of PG–Gd in both rodents and large animals are warranted before testing this agent in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

Molecular recognition of sugars by lanthanide (III) complexes of a conjugate of N,N‐bis[2‐[bis[2‐(1, 1‐dimethylethoxy)‐2‐oxoethyl]amino]ethyl]glycine and phenylboronic acid

A novel conjugate of phenylboronic acid and an Ln(DTPA) derivative, in which the central acetate pendant arm was replaced by the methylamide of L ‐lysine, was synthesized and characterized. The results of a fit of variable ¹⁷O NMR data and a ¹H NMRD profile show that the water residence lifetime of the Gd(III) complex (150 ns) is shorter than that of the parent compound Gd(DTPA)^2? (303 ns). Furthermore, the data suggest that several water molecules in the second coordination sphere of Gd(III) contribute to the relaxivity of the conjugate. The Ln(III) complexes of this conjugate are highly suitable for molecular recognition of sugars. The interaction with various sugars was investigated by ¹¹B NMR spectroscopy. Thanks to the thiourea function that links the phenylboronic acid targeting vector with the DTPA derivative, the interactions are stronger than that of phenylboronic acid itself. In particular, the interaction with N‐propylfructosamine, a model for the glucose residue in glycated human serum albumin (HSA), is very strong. Unfortunately, the complex also shows a rather strong interaction with hexose‐free HSA (K_A = 705 ± 300). Copyright © 2007 John Wiley & Sons, Ltd.     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of rHA labeled with Gd–DTPA for blood pool imaging and targeted contrast delivery

A new contrast agent was developed by linking Gd–DTPA chelate to recombinant human albumin in the laboratory. The molar relaxivity of the new agent was tested in aqueous solution at B₀ 1.5 T and temperature 20°C. The soluble compound had a higher molar longitudinal relaxivity and molar transverse relaxivity in water (r₁ = 7.2 s^?1 mM ^?1, r₂ = 18.4 s^?1 mM ^?1) than those measured for Gd–DTPA solution (r₁ = 3.5 s^?1 mM ^?1, r₂ = 5.5 s^?1 mM ^?1). The performance of the compound as a blood pool agent was investigated with soluble and microparticulate forms of the compound and comparisons were made with Gd–DTPA and the polymeric blood‐pool agent, Gadomer. T₁‐weighted imaging experiments show that the soluble compound acts as a highly effective blood pool agent with hyperintensity in the vasculature persisting beyond 2 h post administration, compared with free Gd–DTPA, which was cleared from the blood pool after approximately 10 min. The clearance kinetics of the new agents were examined, due to the incomplete elimination within 14 days post injection; both rHA labeled compounds are probably not suitable for development as routine blood pool contrast media. However, with free sites on the Gd‐loaded rHA molecule, there are possibilities for binding the agent to antibodies in the laboratory, which was demonstrated, and thus there exist potential applications for in vivo molecular imaging with this agent. Copyright © 2010 John Wiley & Sons, Ltd.     

Cellular labeling with Gd(III) chelates: only high thermodynamic stabilities prevent the cells acting as ‘sponges’ of Gd3+ ions

MR‐labeling of cells may be carried out by adding a Gd‐based contrast agent to the incubation media. The amount of gadolinium internalized in HTC and C6 cells upon incubation with Gd–DTPA–BMA is circa one order of magnitude higher than those found with Gd–DTPA, Gd–DOTA and Gd–HPDO3A, respectively. The comparison of relaxometric and mass spectrometry determinations allows us to establish that only a minor fraction of intact Gd–DTPA–BMA is internalized into the cells. Moreover the binding/uptake behavior shown by Gd–DTPA–BMA resembles that found when GdCl₃ is added to the incubation medium. We suggest that the lower stability of Gd–DTPA–BMA is responsible for a shift in the dissociation equilibrium that results in the net transfer of Gd³⁺ ions on the cell membrane followed by a slower internalization process. The transmetallation process is mediated by components of the incubation media, among which a dominant role is represented by phosphate anions. The uptake of Gd³⁺ ions is clearly reflected in the drastic decrease of cell viability observed for cells labeled with Gd–DTPA–BMA. Copyright © 2006 John Wiley & Sons, Ltd.     

Pharmacokinetics of Gd(DO3A‐Lys) and MR imaging studies in an orthotopic U87MG glioma tumor model

Pharmacokinetics of Gd(DO3A‐Lys), a macrocyclic gadolinium‐based magnetic resonance imaging (MRI) contrast agent functionalized with a lysine derivative, was studied in Wistar rats. Kinetic data were fitted using a two‐compartment model and revealed Gd(DO3A‐Lys) to have a distribution half‐life, t_1/2(α), of 1.3 min, an elimination half‐life, t_1/2(β), of 24.9 min and a large volume of distribution, V_D, of 0.49 L/kg indicative of the agent being able to rapidly distribute into tissues and organs. Contrast‐enhanced magnetic resonance angiography (CE‐MRA) in an orthotopic U87MG glioma mouse model demonstrated considerable enhancement of both the tumor and surrounding vasculature after intravenous administration of Gd(DO3A‐Lys). Applying dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) in the glioma of different sizes further showed distinct uptake characteristics and patterns of enhancement, which suggests the potential for differentiating changes at different stages of tumor growth. Our results indicate that Gd(DO3A‐Lys) could be a promising candidate for glioma MR imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

A dual CT‐MR dendrimer contrast agent as a surrogate marker for convection‐enhanced delivery of intracerebral macromolecular therapeutic agents

The feasibility of using Gd dendrimer‐based macromolecules (Gd‐G8 dendrimer) as a dual CT and MR contrast agent for monitoring convection‐enhanced delivery of therapy in the brain is evaluated both in vitro and in vivo with optimal dosing established. In vitro CT attenuation values of the Gd‐based agents (～6.0 HU mM ^?1) were ～1.6 times greater than iodine‐based agents and the attenuation of the Gd‐DTPA was comparable to Gd‐G8 dendrimer. Visible enhancement was observed on both CT and MR using Gd‐G8 dendrimer over a range of 23–78 mM ; however, a concentration of at least 47 mM in Gd was required for adequate delineation of the injection site on both CT and MR. MR offers greater sensitivity than CT in estimating the volume of distribution (V_d) and effectively quantified the agent's concentration and diffusion using T₁ mapping at much lower concentrations of Gd (<10 mM in [Gd]). Copyright © 2008 John Wiley & Sons, Ltd.     

Connexin 43‐targeted T1 contrast agent for MRI diagnosis of glioma

Glioblastoma multiforme is the most aggressive form of brain tumor. Early and accurate diagnosis of glioma and its borders is an important step for its successful treatment. One of the promising targets for selective visualization of glioma and its margins is connexin 43 (Cx43), which is highly expressed in reactive astrocytes and migrating glioma cells. The purpose of this study was to synthesize a Gd‐based contrast agent conjugated with specific antibodies to Cx43 for efficient visualization of glioma C6 in vivo. We have prepared stable nontoxic conjugates of monoclonal antibody to Cx43 and polylysine–DTPA ligands complexed with Gd(III), which are characterized by higher T₁ relaxivity (6.5 mM^?1 s^?1 at 7 T) than the commercial agent Magnevist® (3.4 mM^?1 s^?1). Cellular uptake of Cx43‐specific T₁ contrast agent in glioma C6 cells was more than four times higher than the nonspecific IgG‐contrast agent, as detected by flow cytometry and confocal analysis. MRI experiments showed that the obtained agents could markedly enhance visualization of glioma C6 in vivo after their intravenous administration. Significant accumulation of Cx43‐targeted contrast agents in glioma and the peritumoral zone led not only to enhanced contrast but also to improved detection of the tumor periphery. Fluorescence imaging confirmed notable accumulation of Cx43‐specific conjugates in the peritumoral zone compared with nonspecific IgG conjugates at 24 h after intravenous injection. All these features of Cx43‐targeted contrast agents might be useful for more precise diagnosis of glioma and its borders by MRI. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative imaging of the tissue contrast agent [Gd(DTPA)]2− in articular cartilage by laser ablation inductively coupled plasma mass spectrometry

Laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS) is an emerging analytical technique in the generation of quantitative images of MR contrast agent distribution in thin tissue sections of articular cartilage. An analytical protocol is described that includes sample preparation by cryo‐cutting of tissue sections, mass spectrometric measurements by LA‐ICP‐MS and quantification of gadolinium images by one‐point calibration, standard addition method (employing matrix‐matched laboratory standards) and isotope dilution analysis using highly enriched stable Gd‐155 isotope (abundance 92 vs 14.8% in the [Gd(DTPA)]^2? contrast agent). The tissue contrast agent concentrations of [Gd(DTPA)]^2? in cartilage measured in this work are in agreement with findings obtained by magnetic resonance imaging and other analytical methodologies. The LA‐ICP‐MS imaging data also confirm the observation that the spatial distribution of [Gd(DTPA)]^2? in the near‐equilibrium state is highly inhomogeneous across cartilage thickness with the highest concentration measured in superficial cartilage and a strong decrease toward the subchondral bone. In the present work, it is shown for the first time that LA‐ICP‐MS can be applied to validate the results from quantitative gadolinium‐enhanced MRI technique of articular cartilage. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo MRI assessment of a novel GdIII‐based contrast agent designed for high magnetic field applications

Gd₃L is a trinuclear Gd³⁺ complex of intermediate size, designed for contrast agent applications in high field magnetic resonance imaging (H₁₂L is based on a trimethylbenzene core bearing three methylene‐diethylenetriamine‐ N,N,N″,N″‐tetraacetate moieties). Thanks to its appropriate size, the presence of two inner sphere water molecules and a fast water exchange, Gd₃L has remarkable proton relaxivities at high magnetic field (r₁ = 10.2 vs 3.0 mM ^?1 s^?1 for GdDOTA at 9.4 T, 37°C, in H₂O). Here we report an in vivo MRI feasibility study, complemented with dynamic γ scintigraphic imaging and biodistribution experiments using the ¹⁵³Sm‐enriched analog. MRI experiments were performed at 9.4 T in mice with Gd₃L and the commercial contrast agent gadolinium(III)‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate (GdDOTA). Gd₃L was well tolerated by the animals at the dose of 8 µmol Gd kg^?1 body weight. Dynamic contrast enhanced (DCE) images showed considerably higher signal enhancement in the kidney medulla and cortex after Gd₃L injection than after GdDOTA injection at an identical dose. The relaxation rates, ΔR₁, were calculated from the IR TrueFISP data. During the excretory phase, the ΔR₁ for various tissues was similar for Gd₃L and GdDOTA, when the latter was injected at a three‐fold higher dose (24 vs 8 µmol Gd kg^?1 body weight). These results point to an approximately three times higher in vivo relaxivity (per Gd) for Gd₃L relative to GdDOTA, thus the ratio of the relaxivities of the two compounds determined in vitro is retained under in vivo conditions. They also indicate that the two inner sphere water molecules per Gd in Gd₃L are not substantially replaced by endogenous anions or other donor groups under physiological conditions. Gd₃L has a pharmacokinetics typical of small, hydrophilic complexes, involving fast renal clearance and no retention in the blood pool. The dynamic γ scintigraphic studies and the biodistribution experiments performed in Wistar rats with ¹⁵³Sm‐enriched ^*Sm₃L are also indicative of a fast elimination via the kidneys. Copyright © 2008 John Wiley & Sons, Ltd.     

Contrast enhancement by lipid‐based MRI contrast agents in mouse atherosclerotic plaques; a longitudinal study

The use of contrast‐enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium‐based contrast agents to enhance atherosclerotic plaques in ApoE^?/? mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer‐term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T₁‐weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR₆₆₄ incorporated in the contrast agents and quantification of tissue and blood Gd–DTPA. Both micelles and liposomes enhanced contrast in T₁‐weighted MR images of plaques in the aortic arch. The average contrast‐to‐noise ratio increased after liposome or micelle injection to 260 or 280% respectively, at 24 h after injection, compared with a pre‐scan. A second wave of maximum contrast enhancement was observed around 60–72 h after injection, which only slowly decreased towards the 1 week end‐point. Confocal fluorescence microscopy and whole body fluorescence imaging confirmed MRI‐findings of accumulation of micelles and liposomes. Plaque permeation of contrast agents was not strongly dependent on the contrast agent size in this mouse model. Our results show that intraplaque accumulation over time of both contrast agents leads to good plaque visualization for a long period. This inherent intraplaque accumulation might make it difficult to discriminate passive from targeted accumulation. This implies that, in the development of targeted contrast agents on a lipid‐based backbone, extensive timing studies are required. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorometry,a fast screening technique for non‐covalent binding of contrast agents to human serum albumin?

The non‐covalent interactions of gadolinium‐based MRI contrast agents with macromolecules, such as human serum albumin (HSA), increase their efficacy. The identification of contrast agents that interact with HSA is a crucial first step in the complex, lengthy and expensive developmental process of a new potential HSA‐targeting contrast agent. Fluorometry has been used as a possibly simpler and more effective tool of screening. In this study, the affinity of four compounds (Gd–DTPA, Gd–BOPTA, Gd–EOB–DTPA and MS‐325) for HSA was investigated. The results show that the fluorescence method is a convenient tool that can easily detect this kind of non‐covalent interaction owing to the small amount of required compound, the simplicity of the procedure and the popularity of the instrument, compared with the other approaches reported in the literature. However, fluorescence screening tests should be interpreted with caution since false‐negative results will occur when the binding site of a gadolinium‐based agent is far away from the location of the sole Trp residue of HSA or when an unsuitable site‐marker is selected. Copyright © 2013 John Wiley & Sons, Ltd.     

Novel Gd(III)‐based probes for MR molecular imaging of matrix metalloproteinases

Two novel Gd‐based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd–DOTA chelate was conjugated to the N‐terminal position through an amide bond, either directly to proline (compd Gd–K11) or through a hydrophilic spacer (compd Gd–K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C‐terminus of the peptide sequence. Gd–K11 and Gd–K11N have a good affinity for β‐cyclodextrins (K_D 310 and 670 µ m respectively) and for serum albumin (K_D 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP‐2 and MMP‐12. Upon MMP‐dependent cleavage, the CAs lose the C‐terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic‐to‐hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd–K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre‐treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd‐contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd‐contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and evaluation of a polydisulfide with Gd–DOTA monoamide side chains as a biodegradable macromolecular contrast agent for MR blood pool imaging

Macromolecular Gd(III)‐based contrast agents are effective for contrast‐enhanced blood pool and cancer MRI in preclinical studies. However, their clinical applications are impeded by potential safety concerns associated with slow excretion and prolonged retention of these agents in the body. To minimize the safety concerns of macromolecular Gd contrast agents, we have developed biodegradable macromolecular Gd contrast agents based on polydisulfide Gd(III) complexes. In this study, we designed and synthesized a new generation of the polydisulfide Gd(III) complexes containing a macrocyclic Gd(III) chelate, Gd–DOTA monoamide, to improve the in vivo kinetic inertness of the Gd(III) chelates. (N₆‐Lysyl)lysine‐(Gd–DOTA) monoamide and 3‐(2‐carboxyethyldisulfanyl)propanoic acid copolymers (GODC) were synthesized by copolymerization of (N₆‐lysyl)lysine DOTA monoamide and dithiobis(succinimidylpropionate), followed by complexation with Gd(OAc)₃. The GODC had an apparent molecular weight of 26.4 kDa and T₁ relaxivity of 8.25 m m ^?1 s^?1 per Gd at 1.5 T. The polymer chains of GODC were readily cleaved by l ‐cysteine and the chelates had high kinetic stability against transmetallation in the presence of an endogenous metal ion Zn²⁺. In vivo MRI study showed that GODC produced strong and prolonged contrast enhancement in the vasculature and tumor periphery of mice with breast tumor xenografts. GODC is a promising biodegradable macromolecular MRI contrast agent with high kinetic stability for MR blood pool imaging. Copyright © 2013 John Wiley & Sons, Ltd.     

First in vivo MRI assessment of a self‐assembled metallostar compound endowed with a remarkable high field relaxivity

{Fe[Gd₂bpy(DTTA)₂(H₂O)₄]₃}^4? is a self‐assembled, metallostar‐structured potential MRI contrast agent, with six efficiently relaxing Gd³⁺ centres confined into a small molecular space. Its proton relaxivity is particularly remarkable at very high magnetic fields (r₁ = 15.8 mM ^?1 s^?1 at 200 MHz, 37°C, in H₂O). Here we report the first in vivo MRI feasibility study, complemented with dynamic γ scintigraphic imaging and biodistribution experiments using the ¹⁵³Sm‐enriched compound. Comparative MRI studies have been performed at 4.7 T in mice with the metallostar and the small molecular weight contrast agent gadolinium(III)‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate ([Gd(DOTA)(H₂O)]^? = GdDOTA). The metallostar was well tolerated by the animals at the concentrations of 0.0500 (high dose) and 0.0125 (low dose) mmol Gd kg^?1 body weight; (BW). The signal enhancement in the inversion recovery fast low angle shot (IR FLASH) images after the high‐dose metallostar injection was considerably higher than after GdDOTA injection (0.1 mmol Gd kg^?1 BW), despite the higher dose of the latter. The high‐dose metallostar injection resulted in a greater drop in the spin‐lattice relaxation time (T₁), as calculated from the inversion recovery true fast imaging with steady‐state precession (IR TrueFISP) data for various tissues, than the GdDOTA or the low dose metallostar injection. In summary, these studies have confirmed that the approximately four times higher relaxivity measured in vitro for the metallostar is retained under in vivo conditions. The pharmacokinetics of the metallostar was found to be similar to that of GdDOTA, involving fast renal clearance, a leakage to the extracellular space in the muscle tissue and no leakage to the brain. As expected on the basis of its moderate molecular weight, the metallostar does not function as a blood pool agent. The dynamic γ scintigraphic studies performed in Wistar rats with the metallostar compound having ¹⁵³Sm enrichment also proved the renal elimination pathway. The biodistribution experiments are in full accordance with the MR and scintigraphic imaging. At 15 min post‐injection the activity is primarily localized in the urine, while at 24 h post‐injection almost all radioactivity is cleared from tissues and organs. Copyright © 2006 John Wiley & Sons, Ltd.