In Vivo MR Imaging of Fibrin in a Neuroblastoma Tumor Model by Means of a Targeting Gd-Containing Peptide.

PURPOSE: A magnetic resonance imaging contrast agent based on a tetrameric Gd-DTPA-like system linked to a fibrin-targeting peptide (Gd-F) has been designed for in vivo tumor characterization. PROCEDURES: Gd-F was synthesized following Fmoc-SPPS strategy. Binding was measured using soluble fibrin DD(E) fragment and a dried fibrin assay. Contrast efficiency was tested on human and mouse clots and in vivo on Neuro2A tumor model. An anti-thrombotic drug was used to evaluate Gd-F sensitivity for changes in fibrin availability at the tumor site. RESULTS: The high relaxivity of Gd-F (42 mM(-1) s(-1), per molecule) yielded a strong signal enhancement in human and murine clots. High contrast was also measured in vivo in Neuro2A tumors, with a persistent enhancement in tumor rim and stroma. Upon treatment with an anti-thrombotic drug, the contrast uptake was significantly reduced in the tumor area confirming the specificity of the probe. CONCLUSIONS: Gd-F resulted to be an efficient probe for tumor delineation and for monitoring fibrin deposits during tumor progression and anti-thrombotic therapy.

B25716/1: a novel albumin-binding Gd-AAZTA MRI contrast agent with improved properties in tumor imaging.

The aim of this study is to describe the synthesis of, relaxometric characterization of, pharmacokinetic properties of, and animal imaging experiments with a new, low molecular weight gadolinium complex with high binding affinity toward serum albumin. The gadolinium(III) chelate (B25716/1) is based on the structure of the heptadentate ligand 1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxycarbonylmethyl)]amino-6 methylperhydro-1,4-diazepine (AAZTA) covalently conjugated to an analogue of deoxycholic acid. The study was conducted as a comparison with that of an analogous complex based on the octadentate diethylenetriaminepentaacetic acid ligand B22956/1 (whose albumin binding properties were previously assessed). The structural modification with respect to B22956/1 leads to a system that can host two coordinated water molecules in fast exchange with bulk water with potential higher efficiency as an MRI contrast agent. On interaction with human serum albumin the expected-field-independent-relaxation enhancement is not observed, possibly as a consequence of the displacement of one of the two inner-sphere water molecules of the gadolinium complex. At clinically relevant magnetic fields, however, the plasma relaxivity of B25716/1 is markedly higher than that shown by B22956/1, owing to concomitant synergistic contributions from the electronic correlation time and water molecules in the second coordination sphere. The capability of B25716/1 to enhance tumor regions in magnetic resonance images was assessed in vivo at 3 T on a xenograft tumor mouse model prepared with PC-3 cells. B25716/1 displays signal enhancements approximately double those observed for B22956/1, in agreement with the findings of the in vitro relaxivity investigations.

In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-(13)C]glutamine.

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized (13)C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-(13)C]glutamine, which provides a highly sensitive (13)C MR marker. With this preparation of hyperpolarized [5-(13)C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-(13)C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

Evaluation of a soft atherosclerotic lesion in the rabbit aorta by an invasive IVUS method versus a non-invasive MRI technology.

The intravascular ultrasound (IVUS) modality has rapidly gained acceptance for the measurement of arterial plaque thickness and for anatomical characterization. In view, however, of the growing interest in the direct assessment of plaque size after therapeutic modalities directly reducing plaque burden, a non-invasive method such as magnetic resonance imaging (MRI) may be of help for repeated evaluations. The two methods were compared directly on a focal plaque developed at the abdominal aortic level by a combination of local electric lesion followed by a hypercholesterolemic diet. The plaque was fully characterized histopathologically at intervals up to 120 days from lesion induction, and maximal plaque formation was detected at 90 days from electrical injury. Plaques could be well assessed by IVUS at each time point analyzed and data correlated very well to histopathologic findings (r = 0.969, P = 0.0014). The MRI technology provided reliable determinations only at 90 days after lesion induction, i.e. at maximal plaque formation, with excellent correspondence to IVUS determinations (r = 0.989, P = 0.0111). Altogether these findings indicate that the non-invasive MRI technology, when applied to the analysis of arterial plaques of adequate size, can be used successfully for plaque determination, with results comparable to the invasive IVUS technique.

Comparative study between gadobenate dimeglumine and gadobutrol in rats with brain ischemia: evaluation of somatosensory evoked potentials.

RATIONALE AND OBJECTIVES: The present study had two main objectives: to validate a rat model of brain ischemia in terms of somatosensory evoked potentials (SEPs) and to compare, using the validated model, the potential activity on the somatosensory function of the new, approved contrast agent gadobenate dimeglumine with that of gadobutrol, a specific contrast agent, for magnetic resonance imaging of the brain. METHODS: Rats were prepared for SEP recording at least 5 days before ischemia induction. Ischemia was induced by 30-minute right middle cerebral artery occlusion and 3-day reperfusion. The SEP responses, evoked contralaterally to the stimulated upper limb, were recorded from the primary somatosensory cortical area. Model validation, on day 3 after occlusion, was performed using iopromide, an iodinated contrast agent poorly neurotolerated in rats, intravenously injected at 7 g I/kg. The comparative study between gadobenate dimeglumine and gadobutrol was performed at an intravenously injected dose of 2 mmol/kg. Somatosensory evoked potential responses were measured as peak latencies (P2 and N2) and peak-to-peak amplitude (P2N2). The brain concentration of iopromide was measured by high-performance liquid chromatography, whereas that of the gadolinium contrast agent was measured by inductively coupled plasma-atomic emission spectrometry analysis; given the absence of metabolism for both contrast agents, the gadolinium content values can be interpreted as representing unmetabolized contrast agent. RESULTS: In the ischemic rats, the SEP responses of the lesioned hemisphere showed significant increases in P2, N2, and interpeak N2-P2 latencies and a significant reduction in peak-to-peak (P2N2) amplitude. In the validation experiments, iopromide dramatically increased the P2N2 amplitude of the SEP responses recorded from both hemispheres of ischemic rats without affecting the P2, N2, and interpeak N2-P2 latencies. The iopromide effect was coupled with high concentrations of the contrast agent in the brain. Iopromide had no effect on healthy rats. In the comparative study, gadobenate dimeglumine did not induce any alteration in SEP components of either the lesioned or unlesioned hemisphere of ischemic rats. In fact, no significant difference was found between responses obtained before and after gadobenate dimeglumine injection. Gadobutrol, administered at the same dose, on the whole showed the same behavior as gadobenate dimeglumine, although a slight but significant decrease in the P2 latency, a sign of excitatory activity, was observed 2 hours after injection. Analytic data indicated higher levels of contrast agent in the lesioned hemisphere versus the unlesioned hemisphere 2 hours after injection. CONCLUSIONS: Based on these results, three conclusions can be drawn: (1) the evaluation of SEPs in ischemic rats is a useful tool for assessing the potential neurological effects of a new contrast agent because ischemic and contrast agent effects can be clearly differentiated; (2) the experimental conditions used allow the contrast agents to penetrate into the brain, where their activity can be manifested and evaluated; and (3) the complete absence of neurological activity of gadobenate dimeglumine shows its safety profile and confirms its suitability for use in neurological diseases for which contrast-enhanced magnetic resonance imaging is indicated.

Neurotolerability of contrast agents in rats with brain ischemia induced by transient middle cerebral artery occlusion: EEG evaluation.

RATIONALE AND OBJECTIVES: Because contrast agent (CA) formulations are injected intravenously to patients who may have a disrupted blood-brain barrier, their neurotolerability should be tested by using appropriate animal models. In the present study, a model of rat brain ischemia evaluated in terms of the electroencephalogram (EEG) was validated and then used to compare the neurotolerability of gadobenate dimeglumine to that of gadodiamide, a well-documented CA for brain MRI. METHODS: Rats were prepared for EEG recording about 15 days before ischemia induction. Ischemia was induced in the right hemisphere by 2-hour middle cerebral artery (MCA) occlusion and 3-day reperfusion. Model validation in terms of EEG, on day 3 after MCA occlusion, was performed by using iopromide, a poorly neurotolerated iodinated CA in rats, intravenously injected at 7 g iodine/kg. The EEG recording was analyzed for pathological tracings and for changes in spectral content in terms of the frequency index (FI) at 1, 2, and 3 hours after test compound injection. The comparative study between gadobenate dimeglumine and gadodiamide was performed at 2.0 mmol/kg. D-Mannitol was used as a control compound. The presence of CA in the rat brain was verified by measuring the total gadolinium content by using inductively coupled plasma-atomic emission spectrometry analysis. Given the absence of metabolism for both CAs, the values of gadolinium content can be interpreted as representing unmetabolized CA. RESULTS: On days 1, 2, and 3 after transient MCA occlusion, the lesioned hemisphere of rats presented a decreased FI value with respect to the basal value. The unlesioned hemisphere, after a slight, nonsignificant decrease in the FI value on the first 2 days, presented a normal FI value on day 3. Thus, ischemic rats on day 3 after transient MCA occlusion were chosen for these neurotolerability studies. Iopromide injected intravenously into ischemic rats at a dose 10 times higher than the maximum clinical dose caused bilateral spikes on the EEG and increases in FI values for the unlesioned hemisphere without affecting the lesioned hemisphere. Gadobenate dimeglumine, like gadodiamide when injected into ischemic rats, did not cause spikes or further changes in the FI value of the lesioned hemisphere and did not modify the normal FI value of the unlesioned hemisphere. Furthermore, no significant differences between gadobenate dimeglumine, gadodiamide, and D-mannitol were found when postinjection FI values were compared. Finally, higher levels of gadolinium were found in the lesioned hemisphere with respect to the unlesioned hemisphere after both gadobenate dimeglumine and gadodiamide administration. CONCLUSIONS: We can therefore conclude that (1) on the EEG, ischemia induced by transient MCA occlusion is an appropriate model for evaluating CA neurotolerability because ischemic and CA effects can be clearly differentiated; (2) the higher level of CA in the lesioned hemisphere compared with the unlesioned one (two to three times), even 3 hours after injection, demonstrates that the CA effectively penetrated the brain; if it were neurotoxic, any negative effects would have been detected; and (3) gadobenate dimeglumine, like gadodiamide, injected intravenously at a dose 20 times higher than the intended clinical dose for brain MRI is well tolerated and, also like gadodiamide, is suitable for use in neurological diseases for which contrast-enhanced MRI is indicated.

Neurotolerability of gadobenate dimeglumine in a rat model of focal brain ischemia: EEG evaluation.

RATIONALE AND OBJECTIVES: The neurologic pathologies for which contrast-enhanced MRI is indicated are often accompanied by a disruption of the blood-brain barrier (BBB), which allows the contrast agent to come into contact with the nervous tissue. Thus, assessment of the neurologic safety for a new contrast agent is of crucial importance. The objective of this study was to assess the neurotolerability of the new MRI contrast agent gadobenate dimeglumine using EEG in the presence of focal lesions of the BBB. METHODS: Lesions of the BBB were obtained inducing a photochemical ischemia in rats. Gadobenate dimeglumine was intravenously administered at 4.0 mmol/kg. An EEG was recorded during sleep/awake behavior and was analyzed for pathologic tracing and for changes in spectral content in terms of total power and frequency index. The presence of the BBB lesions was verified using high-performance liquid chromatography measurement of the gadobenate ion content in the brain. RESULTS: Gadobenate dimeglumine did not have any epileptogenic effect in ischemic rats. However, it caused a transitory shift of the EEG power spectrum toward the 0.5 to 9 Hz frequency bands of the lesioned hemisphere during quiet wake. In the lesioned cortex, higher levels of gadobenate ion were found until 3 hours after administration. CONCLUSIONS: In experimental conditions of focal brain ischemia associated with BBB lesions, gadobenate dimeglumine was well tolerated up to doses even 10 times higher than the maximum clinical dose (0.3 mmol/kg) intended for brain imaging procedures.

An experimental study on the neurotolerability of gadobenate dimeglumine in a rat model of focal brain ischemia.

RATIONALE AND OBJECTIVES: The neurologic safety of the new magnetic resonance imaging contrast agent gadobenate dimeglumine has been assessed in rats suffering from localized blood-brain barrier damage. METHODS: The adopted experimental model was the photochemically induced brain ischemia in the rat. Flash-evoked visual potentials were recorded simultaneously from the damaged area and from the corresponding contralateral position in conscious rats before and after the administration of the contrast medium. RESULTS: The intravenous administration of 0.9 mmol/kg of gadobenate dimeglumine did not induce any significant changes in the visual responses recorded from the ischemic side compared with the intact side. Rats treated with saline or with the reference compounds gadopentetate dimeglumine (Magnevist) and gadoteridol (ProHance) gave comparable results. CONCLUSIONS: The results are in favor of the use of gadobenate dimeglumine in patients suffering from a localized damage to blood-brain barrier.