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OBJECTIVES: Ultrasmall particles of iron oxide (USPIO) possess superparamagnetic properties and are used as negative contrast agent in magnetic resonance imaging (MRI) because of their strong T(2) and T(2)* effects. Besides this method, electron paramagnetic resonance (EPR) offers the unique capability to quantify these particles. The objective of this study was to evaluate a molecular marker for non invasive diagnosis and monitoring of inflammation. During inflammation cell adhesion molecules such as E-selectin are expressed on the endothelial cell surface. An E-selectin ligand was coupled to pegylated USPIO particles. MATERIALS AND METHODS: Inflammation was induced by intramuscular injection of Freund's Complete Adjuvant in male NMRI mice. After intravenous injection of grafted or ungrafted USPIO particles, iron concentration in inflamed muscles was quantified ex vivo by X-band EPR. Particle accumulation was also assessed in vivo by L-Band EPR, as well as by T(2)-weighted MRI. RESULTS: We determined the mean iron oxide concentration in inflamed muscles after injection of grafted or ungrafted UPSIO particles, which was 0.8% and 0.4% of the initially injected dose, respectively. By L-band EPR, we observed that the concentration of the grafted USPIO particles in inflamed muscles was twice higher than for the ungrafted particles. Using MRI experiments, a higher signal loss was clearly observed in the inflamed muscle when grafted particles were injected in comparison with the ungrafted particles. CONCLUSION: Even taking into account a non specific accumulation of iron oxides, the targeting of USPIO particles with E-selectin ligands significantly improved the sensitivity of detection of inflamed tissues.

The elimination of hepatocyte-directed particulate contrast agents has not been studied in the same detail as particles eliminated mainly by the mononuclear phagocyte system. The aim of the present study was to elucidate the fate of these particles by a multidisciplinary approach.After intravenous injection of AMI-HS particles directed to the hepatocytes, rats were killed and cytological studies, by both electron microscopy and histochemistry, and spectroscopic studies of the bile were performed. The data were compared with a dynamic magnetic resonance study of the heart and liver.The particles were rapidly cleared from the blood by Kupffer cells and hepatocytes and then found first in the vascular and later in the biliary pole of the hepatocytes. After 24 hours, a relaxometric characterization of the bile showed the presence of unchanged particles in the bile.These results show the capacity of the liver to excrete unchanged AMI-HS particles directly into the bile.

Rationale and objectives Liposomal gadolinium (Gd)-HP-DO3A has been evaluated as a contrast agent for liver magnetic resonance imaging. The influence of various liposomal physicochemical properties on the liver uptake and contrast efficacy was investigated in various ex vivo and in vivo liver models. Methods Liposomes of different size and membrane properties were prepared. The liposome size ranged from 74 to 304 nm. Two types of phospholipid compositions were studied; a mixture of hydrogenated phosphatidylcholine (HPC) and hydrogenated phosphatidylserine (HPS) with a phase transition temperature (Tm) of 51 degrees C and, a blend composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) displaying a Tm of 41 degrees C. Ex vivo tissue relaxometry and in vivo liver imaging were used to study the influence of liposome composition on the liver uptake and contrast efficacy of intravenously injected liposomes. The influence of liposome size and composition on the kinetics of liver uptake and imaging effect was assessed ex vivo in the perfused rat liver. Results The HPC/HPS preparations showed generally a higher and faster liver uptake than the DPPC/DPPG preparations due to a higher stability in blood/perfusate (high Tm) and to the HPS component. The liposome size modulated the extent and kinetics of liver uptake; the larger the size, the faster and more extensive was the liver uptake. Both types of liposome preparations were shown to be efficient liver susceptibility agents both ex vivo and in vivo due to their uptake by the Kupffer cells of liver. The lack of full correlation between the extent of liver uptake and degree of contrast enhancement might be attributed to different regimes of susceptibility-based relaxation. Conclusions The present study has demonstrated the influence of key liposomal physicochemical properties on the liver uptake and contrast efficacy of liposome-encapsulated Gd chelates, exemplified by Gd-HP-DO3A.

RATIONALE AND OBJECTIVES Proper fitting of the nuclear magnetic resonance dispersion (NMRD) profiles to the numerous factors governing nuclear relaxation in paramagnetic systems requires knowledge of some parameters usually obtained by other techniques. The rotational correlation time (tau R) for example can be measured by carbon-13, hydrogen-2, or oxygen-17 NMR. Discrepancies between values reported in the literature might be attributed to the different concentration ranges used so far in these modalities. In the present work focussing on commercial nonspecific contrast agents, the influence of the solution composition (type and concentration of the complexes and of the counterions) has been examined with regard to the water proton relaxation enhancement and molecular dynamics. METHODS The proton relaxation rate enhancement of Magnevist, Dotarem, Omniscan, and ProHance was measured in aqueous solution up to a concentration of 0.5 M. In the same concentration window, the rotational correlation times were obtained from the study of deuterium relaxation rates of the diamagnetic deuterated analogs (lanthanum complexes) of the gadolinium chelates. RESULTS Above 50 mM, the relaxation rate enhancement versus concentrations strongly deviates from linearity. Magnevist, a clinical formulation containing two meglumine counterions per molecule of paramagnetic complex, exhibits the largest concentration effect. A slowing down of the molecular dynamics accounts for this behavior as confirmed by the analysis of the rotational correlation times obtained by deuterium relaxometry. At low concentrations (< or = 50 mM), tau R values obtained by proton NMRD analysis and by deuterium relaxation are in very good agreement. CONCLUSIONS This study shows that NMR analyses of small molecular weight complexes should be carried out on solutions containing no more than 50 mM to avoid the biaising effects of concentration. On the other hand, the benefitting relaxivity enhancement induced by highly concentrated solutions has to be taken into account in the context of bolus injection or vesicular entrapment.

RATIONALE AND OBJECTIVES This study illustrates the synergistic effects of relaxation- and susceptibility-based contrast enhancement. Using a combination of gadolinium (Gd) and dysprosium (Dy) complexes and a sequence capable of taking advantage of the particular relaxation behavior of this combination, the difference between compartmentalized and noncompartmentalized regions was significantly enhanced. METHODS Magnetic resonance imaging of the rat kidney was performed before and immediately after the administration of a combination of Gd and Dy chelates (Gd-DTPA-BMA and Dy-DTPA-BMA). RESULTS The signal intensity (SI) of the renal parenchyma was reduced by 85%, whereas the collecting tubes had a 100% increase of their SI as demonstrated by a short repetition time (600 msec), long echo time (50 msec), and spin-echo sequence. CONCLUSIONS The high R2* effect, specific to the compartmentalized tissues, associated with the moderately high R1 and R2 developed in the remaining areas, results in an important improvement in tissue differentiation, which potentially is useful for the evaluation of pathological changes as in tubular necrosis.

RATIONALE AND OBJECTIVES Several works have shown that the hepatobiliary magnetic resonance imaging contrast agent manganese dipyridoxyl diphosphate (MnDPDP) partly releases its metallic ion and exhibits cardiovascular effects that are supposed to arise from the free manganese ions (Mn++). In the current study, the cellular internalization of Mn by the isolated rat heart is monitored through the mechanical function of the organ and the relative broadening of the P-31 nuclear magnetic resonances. METHODS Rat hearts were perfused with manganese chloride (MnCl2; 15 and 25 microM) or MnDPDP (25 microM). Variations of the linewidths, heights, and surfaces of phosphocreatine and adenosine triphosphate peaks were monitored. Cardiac function was monitored simultaneously through heart rate, left ventricular pressure, and coronary flow. RESULTS Influx of Mn++ induces a significant broadening of the P-31 resonances of adenosine triphosphate and phosphocreatine because of a strong scalar paramagnetic interaction between the nuclei and the ion. Compared with MnDPDP administered at the same concentration, MnCl2 induced a more pronounced and dose-dependent line broadening as well as a coronary vasodilation. Calcium channel blockers (nifedipine and verapamil) and EDTA inhibit MnCl2 influx. Similarly, verapamil, EDTA, and DPDP reduce the alterations provoked by MnDPDP. CONCLUSIONS The effects of MnDPDP are smaller but of the same type than those induced by MnCl2. Their inhibition by calcium channel blockers (verapamil and nifedipine) and by an excess of strong chelators such as DPDP or EDTA confirms that they originate from a partial release of Mn++ by the contrast agent.

RATIONALE AND OBJECTIVES Bolus injection of iodinated contrast media has been observed to alter myocardial mechanical function, but the consequences on cellular metabolism are poorly documented. Modifications of metabolic parameters (intracellular pH as well as adenosine triphosphate [ATP], phosphocreatine, and inorganic phosphate contents) and of mechanical function (coronary flow, heart rate, and left ventricular developed pressure) were simultaneously recorded on isolated rat hearts perfused over 2 minutes with a high osmolality contrast medium (HOCM) and two low osmolality contrast media (LOCM). In addition, the effects of test solutions mimicking the ionicity and the osmolality of LOCM were evaluated. METHODS Isovolumic rat hearts were submitted to a 2-minute perfusion with oxygenated Radioselectan, Hexabrix, and Omnipaque (320 mgI/mL) at 37 degrees C. Metabolic parameters were obtained by P-31 nuclear magnetic resonance spectroscopy at 4.7 Tesla. Noniodinated ionic and nonionic solutions also were tested for comparison. RESULTS HOCM irreversibly impairs the metabolic and mechanical functions, whereas ionic and nonionic LOCM and test solutions induce transient cardiac failure but no permanent alteration of the metabolic or mechanical parameters. CONCLUSION In this protocol, HOCM causes irreversible degradation of the biochemical status and definitive heart failure, whereas ionic and nonionic LOCM only induce transient changes of myocardial function. Treatments with the LOCM do not induce any modification of the ATP and PCr content, and, at the end of the reperfusion period, the mechanical function is equivalent to that of control hearts. Depending on the ionic content of the solutions (iodinated or not), the evolution of the ventricular developed pressure after injection differs from one group of hearts to another. From these experiments, it is concluded that ionic imbalance and viscosity of the solutions, rather than iodine content or hyperosmolality, should be considered the causes of heart failure.

The MR relaxation properties of ferrioxamine-B, a chelate of iron, were investigated in vitro and in vivo to establish the potential use of the compound as a paramagnetic contrast agent. Whereas the paramagnetic relaxivity of ferrioxamine-B is such that, compared to gadolinium-DTPA (Gd-DTPA), two to three times higher concentrations are necessary to produce the same relaxation effects, the toxicity of the iron ion should be much lower because of the availability of physiological metabolic pathways. Preliminary experiments in three dogs under invasive cardiovascular monitoring demonstrated that high-dose bolus application (0.1-0.3 mmol/kg body weight) of ferrioxamine-B leads to a precipitous blood pressure drop to almost zero, lasting for several minutes. This reaction seems most likely the result of a negative inotropic effect of ferrioxamine-B. In order to reduce these side effects ferrioxamine was modified to a nonionic derivative, PEG-ferrioxamine-B. In vivo experiments with this compound did not demonstrate any substantial change in blood pressure. Dynamic MR imaging of the kidneys and the liver was performed after bolus injection of the compound in six dogs. The results indicate that PEG-ferrioxamine-B produces effects very similar to Gd-DTPA, resulting in T1-mediated signal intensity increases in the liver and in the early stages of passage through the kidneys. During the phase of medullary concentration, T2 effects seem to dominate visualization of the renal medulla. The nonionic PEG-ferrioxamine-B derivative appears to offer an alternative to gadolinium-containing chelates as an MR contrast agent.

Rationale and objectives A possible approach for the extension of the vascular residence time of contrast agents relies on the renal reabsorption mechanisms of some molecules such as glucose. In this study, various small-molecular-weight glucosyl derivatives of gadolinium diethylenetriaminepentaacetic (Gd-DTPA) were synthesized and their vascular half-life was studied. Methods Several Gd-DTPA-bisamides carrying glucosyl groups bound by different linkers were prepared. The pharmacokinetics and biodistribution of these compounds were determined on Wistar rats. Results The sugar moieties linked to Gd-DTPA efficiently reduce the renal excretion of some derivatives. The interaction with renal carrier has not been clearly demonstrated, nor was any interaction observed with blood components. Conclusions Two of the new glucosylated derivatives of Gd-DTPA (Gd-DTPA-BC2-β-cellobionA 2 and Gd-DTPA-BC4-β-glucosylA 7) can be proposed as blood-pool MR contrast agents, considering their vascular remanence.

Colet J-M, Haverbeke YV, Muller RN. Evidence for attachment of magnetic starch microspheres to Kupffer cells receptors in excised and perfused rat liver. Invest Radiol 1994;29: S223–S22S.

The suitability of paramagnetic complexes as magnetic resonance contrast agents depends on various factors such as their relaxivity, stability, selectivity, and the inertness toward transmetallation by endogenous ions. The transmetallation of a series of 18 gadolinium complexes by the Zn2+ ion was studied in vitro by proton relaxometry.Transmetallation was analyzed through the evolution of the paramagnetic longitudinal relaxation rate of water protons at 37 degrees C in pH = 7 phosphate buffer solutions containing 2.5 mmol/L of the gadolinium complexes and 2.5 mmol/L zinc chloride. The measurements were performed at 0.47 T over a period of at least 3 days.The results confirm the high stability of macrocyclic systems and a high sensitivity of Gd-diethylenetriamine-pentaacetic acid (DTPA) derivatives to transmetallation by Zn2+ ions. The decreasing order of stability with respect to metal exchange is as follows: Gd-macrocyclicsGd-C-functionalized DTPAGd-DTPAprimary and secondary Gd-DTPA bisamides. The ternary bisamide analyzed in this study [Gd-DTPA-1,11-bisbismethylamino-1,11-dioxo-3,6,9-triaza-3,6,9-tris(carboxymethyl)undecane] is more stable than the parent compound Gd-DTPA.A simple relaxometric protocol has been successfully developed to study the in vitro transmetallation process of gadolinium complexes. The importance of the functionalization and substitution of the DTPA-like complexes is clearly shown.

Immunocontrast agents used for magnetic resonance imaging require antibodies that preserve the immunoreactivity while containing a high number of chelated paramagnetic ions.Anti-CEA F(ab')2 fragments were coupled to polylysine-Gd-DOTA and polylysine-Gd-DTPA. A paramagnetic load as high as n = 24 to 28 metal ions per antibody was reached.The immunoreactivity of the gadolinium (Gd)-labeled anti-CEA F(ab')2 immunoconjugates was 80% to 85%. Compared with that of commercial chelates, the relaxivity (R1) increase is as follows: Gd-DTPAGd-DOTAGd-H2OPL-Gd-DTPA24-28PL-Gd-DTPA24-28 F(ab')2PL-Gd-DOTA24-28PL-Gd-DOTA24-28 F(ab')2. 1H nuclear magnetic relaxation dispersion data of immunoconjugates showed that the high relaxivity enhancement was the result of a reduction of the molecular tumbling rate. Twenty-four hours after intravenous injection of 50 micrograms (1 mumol Gd/kg) of Gd-labeled immunoconjugates to nude mice grafted with human colorectal carcinoma LS 174T, the tumor uptake was 10% to 15%, resulting in an increase of R1 of up to 15% to 20% versus noninjected mice. No difference was found between PL-Gd-DTPA24-28 F(ab')2 and PL-Gd-DOTA24-28 F(ab')2 immunoconjugates for tumor, liver, and kidney uptake. A high signal intensity of tumor was observed in 50% of the tested mice.

Observation of the relaxivity of MRI contrast media over a wide range of magnetic fields is not only necessary for predicting their efficiency at any field but also compulsory for understanding and improving their mechanisms of action. The best experimental approach to this problem is the field cycling method, which allows the exploration of nuclear relaxation over a broad interval of magnetic field intensity but requires a specially dedicated instrument called a relaxometer. Particularly relevant are the relaxivity profiles of the two chelates Gd-DOTA and Gd-DTPA. Both show an important decrease from low to high fields within the current imaging range (0.02 T to 1.5 T). Although high field relaxivities of these chelates are similar, Gd-DTPA becomes less efficient in facilitating water protons relaxation at fields lower than 0.15 T. This behavior has to be related to different electronic relaxation times due to a different chelate symmetry.