Near-Infrared-Emitting BODIPY-trisDOTA(111) In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations.

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl-BODIPY (BODIPY=boron-dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site. (111) In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY-chelators derivative as a bimodal imaging probe.

BODIPY: A Highly Versatile Platform for the Design of Bimodal Imaging Probes.

In molecular imaging, multimodal imaging agents can provide complementary information, for improving the accuracy of disease diagnosis or enhancing patient management. In particular, optical/nuclear imaging may find important preclinical and clinical applications. To simplify the preparation of dual-labeled imaging agents, we prepared versatile monomolecular multimodal imaging probe (MOMIP) platforms containing both a fluorescent dye (BODIPY) and a metal chelator (polyazamacrocycle). One of the MOMIP was conjugated to a cyclopeptide (i.e., octreotide) and radiolabeled with (111) In. In vitro and in vivo studies of the resulting bioconjugate were conducted, highlighting the potential of these BODIPY-based bimodal probes. This work also confirmed that the biovector and/or the bimodal probes must be chosen carefully, due to the impact of the MOMIP on the overall properties of the resulting imaging agent.

In-vivo metabolic characterization of healthy prostate and orthotopic prostate cancer in rats using proton magnetic resonance spectroscopy at 4.7 T.

BACKGROUND: To assist the development of new anti-cancer drugs, it is important to identify biomarkers of treatment efficacy in the preclinical phases of drug development. In order to improve the predictivity of preclinical experiments, more realistic animal models are needed, for example, tumors xenografted directly on the prostate gland of rodents. PURPOSE: To characterize the in-vivo metabolism of healthy rat prostate and of an orthotopic human prostate cancer model using proton magnetic resonance spectroscopy (MRS). MATERIAL AND METHODS: The highly metastatic and hormone-independent PC3-MM2 human prostate cancer model was implanted into the ventral prostate lobe of three Nude rats. Healthy Nude (n = 6) and Sprague-Dawley (n = 6) rats were also studied for interspecies comparison of normal prostate metabolism. Magnetic resonance imaging and short echo-time (TE 11.2 ms) single voxel PRESS spectroscopy were performed on dorsal (DP) and ventral (VP) prostate as well as tumor at 4.7 T. The metabolic content and volume of dorsal and ventral lobes were characterized as a function of species and age. RESULTS: Slightly lower total creatine (tCr)/water (11.3 ± 2.6 vs. 15.3 ± 3.0, NS), but significantly higher Inositol (Ins)/water (18.9 ± 1.9 vs. 6.6 ± 3.3, P < 0.003) and total choline (tCho)/water (15.0 ± 2.1 vs. 5.6 ± 1.1, P < 0.00007) were observed within healthy DP lobes with respect to VP lobes. No significant variation in metabolic content was seen in healthy DP and VP lobes of Nude rats as a function of age, and no species dependence was observed in their metabolic content. For the orthotopic PC3-MM2 tumor, implanted in VP, the tCr/water ratio was significantly lower (3.1 ± 0.9) than neighboring DP (12.8 ± 1.8, P < 0.00003) and healthy VP (15.3 ± 3.0, P < 0.00006). For Ins, the metabolite ratio in PC3-MM2 was close to that of healthy VP (4.3 ± 2.8 vs. 6.6 ± 3.3, p = NS), but much lower than in neighboring DP (19.1 ± 1.3, P < 0.00005). A similar trend was also observed for tCho, where metabolite ratios in PC3-MM2, healthy VP and neighboring DP were 3.5 ± 0.9, 5.6 ± 1.1, and 15.9 ± 0.8, respectively. CONCLUSION: The in-vivo MRS study of healthy prostate and orthotopic prostate cancer is feasible in rats. Such baseline data could be important when following the modifications in metabolism, including during anti-cancer drug development protocols or following radiotherapy.

Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects.

Early imaging or blood biomarkers of tumor response is needed to customize anti-tumor therapy on an individual basis. This study evaluates the sensitivity and relevance of five potential MRI biomarkers. Sixty nude rats were implanted with human glioma cells (U-87 MG) and randomized into three groups: one group received an anti-angiogenic treatment (Sorafenib), a second a cytotoxic drug [1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU (Carmustine)] and a third no treatment. The tumor volume, apparent diffusion coefficient (ADC) of water, blood volume fraction (BVf), microvessel diameter (vessel size index, VSI) and vessel wall integrity (contrast enhancement, CE) were monitored before and during treatment. Sorafenib reduced tumor CE as early as 1 day after treatment onset. By 4 days after treatment onset, tumor BVf was reduced and tumor VSI was increased. By 14 days after treatment onset, ADC was increased and the tumor growth rate was reduced. With BCNU, ADC was increased and the tumor growth rate was reduced 14 days after treatment onset. Thus, the estimated MRI parameters were sensitive to treatment at different times after treatment onset and in a treatment-dependent manner. This study suggests that multiparametric MR monitoring could allow the assessment of new anti-tumor drugs and the optimization of combined therapies.

Serial in vivo spectroscopic nuclear magnetic resonance imaging of lactate and extracellular pH in rat gliomas shows redistribution of protons away from sites of glycolysis.

The acidity of the tumor microenvironment aids tumor growth, and mechanisms causing it are targets for potential therapies. We have imaged extracellular pH (pHe) in C6 cell gliomas in rat brain using 1H magnetic resonance spectroscopy in vivo. We used a new probe molecule, ISUCA [(+/-)2-(imidazol-1-yl)succinic acid], and fast imaging techniques, with spiral acquisition in k-space. We obtained a map of metabolites [136 ms echo time (TE)] and then infused ISUCA in a femoral vein (25 mmol/kg body weight over 110 min) and obtained two consecutive images of pHe within the tumor (40 ms TE, each acquisition taking 25 min). pHe (where ISUCA was present) ranged from 6.5 to 7.5 in voxels of 0.75 microL and did not change detectably when [ISUCA] increased. Infusion of glucose (0.2 mmol/kg.min) decreased tumor pHe by, on average, 0.150 (SE, 0.007; P < 0.0001, 524 voxels in four rats) and increased the mean area of measurable lactate peaks by 54.4 +/- 3.4% (P < 0.0001, 287 voxels). However, voxel-by-voxel analysis showed that, both before and during glucose infusion, the distributions of lactate and extracellular acidity were very different. In tumor voxels where both could be measured, the glucose-induced increase in lactate showed no spatial correlation with the decrease in pHe. We suggest that, although glycolysis is the main source of protons, distributed sites of proton influx and efflux cause pHe to be acidic at sites remote from lactate production.

The ammonium-induced increase in rat brain lactate concentration is rapid and reversible and is compatible with trafficking and signaling roles for ammonium.

The glutamate-glutamine shuttle requires a flux of fixed N from neurons to astrocytes. The suggestion that some or all of this N is ammonium has received support from reports that ammonium (as NH(4)(+)) rapidly enters astrocytes. Ammonium might also help control astrocyte energy metabolism by increasing lactate production. If ammonium has these functions, then its effect on brain metabolism must be rapid and reversible. To make a minimal test of this requirement, we have followed the time courses of the changes induced by a 4 min venous infusion of 1 mol/L NH(4)Cl, 2.5 mmol/kg body weight, in rat. Extracellular [NH(4)(+)] in cortex, monitored with ion-selective microelectrodes, reached a peak of approximately 0.7 mmol/L 1.65 mins after the end of the infusion, then recovered. Brain metabolites were monitored non-invasively every 4 mins by (1)H magnetic resonance spectroscopy. Lactate peak area during the 3.2 min acquisition starting at the end of the infusion was 1.84+/-0.24 times baseline (+/-s.e.m., P=0.009, n=9). Lactate increased until 13.2+/-2.1 mins after the end of the infusion and recovered halfway to baseline by 31.2 mins. Glutamate decreased by at least 7.1% (P=0.0026). Infusion of NaCl caused no change in lactate signal. Cerebral blood flow, measured by arterial magnetization labeling, more than doubled, suggesting that the lactate increase was not caused by hypoxia. At least three consecutive ammonium-induced increases in lactate signal could be evoked. The results are compatible with an intercellular trafficking/signaling function for ammonium.

Blood-brain barrier permeability to manganese and to Gd-DOTA in a rat model of transient cerebral ischaemia.

Loss of integrity of the blood-brain barrier (BBB) and brain swelling is a potentially lethal complication of reperfusion in human stroke. To assess the time course of BBB modifications, we performed angiography, diffusion-weighted imaging, T1-weighted (T1 W) imaging and T1 mapping, and monitored acute changes after middle cerebral artery occlusion and recanalization in rats (n = 27). The animals were grouped according to the duration of occlusion: 30 min (group A, n = 8), 1 h 30 min (group B, n = 9), and 2 h 30 min (group C, n = 10). For 17 animals (four in group A, six in group B, and seven in group C), MnCl2 and dimeglumine gadoterate (Gd-DOTA) were injected at 13 min and 34 min after recanalization, respectively. The 10 remaining animals (control groups) underwent the same acquisition protocols, but no contrast agents were injected. Cell damage was determined 1 h after recanalization on haematoxylin and eosin-stained sections. Our results indicate that in the middle cerebral artery occlusion model in the rat, changes in BBB permeability assessed by contrast agent extravasation occur within the first hour of reperfusion, even after an occlusion period not exceeding 30 min. No differences between BBB permeability to Gd-DOTA and Mn2+ were detected in our experimental conditions. The reduction in apparent diffusion coefficient during occlusion appears to be a good predictor of BBB modifications after reperfusion in this model.

2D J-resolved spiral spectroscopic imaging at 7 T: application to mobile lipid mapping in a rat glioma.

Lactate (Lac) and mobile lipids (Lip), which are present in rat gliomas, are difficult to map because their 1H resonances overlap in the 1.3 ppm region. 2D J-resolved spectroscopy enables proper separation of the two resonance lines. To obtain high-spatial-resolution mapping of Lac and Lip resonances within a reasonable experiment time, we coupled 2D J-resolved spectroscopy with a fast spectroscopic imaging (SI) method, based on an out-and-in spiral k-space description. The method was applied to a rat glioma at 7 T, and Lac and Lip maps were reconstructed. The duration of SI (2D spatial, 2D spectral) was 64 min for a theoretical in-plane resolution of 1 x 1 mm, and a slice thickness of 2 mm (voxel size 8.2 microl, taking into account the point-spread function (PSF)).