Antonio Aliaga

Oxalic Acid Supported Si–18F-Radiofluorination: One-Step Radiosynthesis of N-Succinimidyl 3-(Di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for Protein Labeling

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)⊂2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)⊂2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.

In vivo and in vitro validation of reference tissue models for the mGluR5 ligand [11C]ABP688

The primary objective of this study was to verify the suitability of reference tissue-based quantification methods of the metabotropic glutamate receptor type 5 (mGluR5) with [11C]ABP688. This study presents in vivo (Positron Emission Tomography (PET)) and in vitro (autoradiography) measurements of mGluR5 densities in the same rats and evaluates both noninvasive and blood-dependent pharmacokinetic models for the quantification of [11C]ABP688 binding. Eleven rats underwent [11C]ABP688 PET scans. In five animals, baseline scans were compared with blockade experiments with the antagonist 1,2-methyl-6-(phenylethynyl)-pyridine (MPEP), and arterial blood samples were drawn and corrected for metabolites. Afterward, saturation-binding autoradiography was performed. Blocking with MPEP resulted in an average decrease of the total distribution volume (VT) between 43% and 58% (thalamus and caudate-putamen, respectively) but had no significant effect on cerebellar VT (mean reduction: −0.01%). Comparing binding potential (BPND) based on the VT with noninvasively determined BPND revealed an average negative bias of 0.7% in the caudate-putamen and an average positive bias of 3.1% in the low-binding regions. Scan duration of 50 minutes is required. The cerebellum is a suitable reference region for the quantification of mGluR5 availability as measured with [11C]ABP688 PET in rats. Blood-based and reference region-based PET quantification shows a significant linear relationship to autoradiographic determinations.

Intact memory in TGF-β1 transgenic mice featuring chronic cerebrovascular deficit: recovery with pioglitazone

The roles of chronic brain hypoperfusion and transforming growth factor-beta 1 (TGF-β1) in Alzheimers disease (AD) are unresolved. We investigated the interplay between TGF-β1, cerebrovascular function, and cognition using transgenic TGF mice featuring astrocytic TGF-β1 overexpression. We further assessed the impact of short, late therapy in elderly animals with the antioxidant N-acetyl–cysteine (NAC) or the peroxisome proliferator-activated receptor-γ agonist pioglitazone. The latter was also administered to pups as a prophylactic 1-year treatment. Elderly TGF mice featured cerebrovascular dysfunction that was not remedied with NAC. In contrast, pioglitazone prevented or reversed this deficit, and rescued the impaired neurovascular coupling response to whisker stimulation, although it failed to normalize the vascular structure. In aged TGF mice, neuronal and cognitive indices—the stimulus-evoked neurometabolic response, cortical cholinergic innervation, and spatial memory in the Morris water maze—were intact. Our findings show that impaired brain hemodynamics and cerebrovascular function are not accompanied by memory impairment in this model. Conceivably in AD, they constitute aggravating factors against a background of aging and underlying pathology. Our data further highlight the ability of pioglitazone to protect the cerebrovasculature marked by TGF-β1 increase, aging, fibrosis, and antioxidant resistance, thus of high relevance for AD patients.

Metformin abolishes increased tumor 18F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by 18F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

Test-retest stability of cerebral mGluR5 quantification using [11C]ABP688 and positron emission tomography in rats

This study evaluates the reproducibility of the quantification of metabotropic glutamate receptor type 5 (mGluR5) densities in rats using the PET radiotracer [11C]ABP688 and pharmacokinetic models that are based on an input function, which is derived from a reference tissue. Seven rats underwent dynamic PET scans (60 min) after bolus injection of [11C]ABP688. Kinetic analyses included: binding potential (BPND) determined by calculating (a) the simplified reference tissue model (SRTM) and (b) its two‐steps simplified version (SRTM2); (c) multilinear reference tissue model (MRTM) and (d) its 2‐parameter version (MRTM2); (e) noninvasive graphical analysis (NIGA). Parametric images were generated representing BPND by the MRTM2 model. BPND determinations were reproducible with low to acceptable variability ranging from 5 to 10% and reproducibility scores (intraclass correlation coefficient) between 0.51 and 0.88. The pharmacokinetic model that showed lowest overall variability was the SRTM. In contrast, the use of the NIGA was associated with significantly lower reproducibility scores. Comparison of parametric images revealed no significant bias between test and retest measurements and is therefore suitable to compare groups at voxel levels. In conclusion, our results suggest that noninvasive quantification of [11C]ABP688 imaging is reproducible and reliable for PET studies of the cerebral mGluR5 in rats. Synapse, 2012.

Imaging in Vivo Glutamate Fluctuations with [11C]ABP688: A GLT-1 Challenge with Ceftriaxone

Molecular imaging offers unprecedented opportunities for investigating dynamic changes underlying neuropsychiatric conditions. Here, we evaluated whether [11C]ABP688, a positron emission tomography (PET) ligand that binds to the allosteric site of the metabotropic glutamate receptor type 5 (mGluR5), is sensitive to glutamate fluctuations after a pharmacological challenge. For this, we used ceftriaxone (CEF) administration in rats, an activator of the GLT-1 transporter (EAAT2), which is known to decrease extracellular levels of glutamate. MicroPET [11C]ABP688 dynamic acquisitions were conducted in rats after a venous injection of either saline (baseline) or CEF 200 mg/kg (challenge). Binding potentials (BPND) were obtained using the simplified reference tissue method. Between-condition statistical parametric maps indicating brain regions showing the highest CEF effects guided placement of microdialysis probes for subsequent assessment of extracellular levels of glutamate. The CEF administration increased [11C]ABP688 BPND in the thalamic ventral anterior (VA) nucleus bilaterally. Subsequent microdialysis assessment revealed declines in extracellular glutamate concentrations in the VA. The present results support the concept that availability of mGluR5 allosteric binding sites is sensitive to extracellular concentrations of glutamate. This interesting property of mGluR5 allosteric binding sites has potential applications for assessing the role of glutamate in the pathogenesis of neuropsychiatric conditions.

PET imaging of cholinergic deficits in rats using [18F]fluoroethoxybenzovesamicol ([18F]FEOBV).

[(18)F]fluoroethoxybenzovesamicol ([(18)F]FEOBV) is one of the most promising radioligands for imaging the vesicular ACh transporter (VAChT) with positron emission tomography (PET). We report here that this method can detect subtle cholinergic terminals losses such as those associated with aging, or those following a partial lesion of the nucleus basalis magnocellularis (NBM). Twenty-one adult rats were evenly distributed in three groups including 1) aged rats (18 months); 2) young rats (3 months); and 3) rats with unilateral lesion of the NBM, following a local stereotaxic infusion of 192 IgG-saporin. In both normal and lesioned rats, our results revealed the highest [(18)F]FEOBV binding to be in the striatum, followed by similar values in both frontal cortex and thalamus, while lower values were observed in both hippocampus and temporo-parietal cortex. This binding distribution is consistent with the known anatomy of brain cholinergic systems. In the lesioned rats, [(18)F]FEOBV binding was found to be reduced mostly in the ventral frontal cortex on the side of the lesion, but some reductions were also observed in the homologous region of the contralateral hemisphere. Aging was found to be associated with a [(18)F]FEOBV binding reduction limited to the hippocampus of both hemispheres. [(18)F]FEOBV appears to be a very promising marker for the in vivo quantification of the brain VAChT; PET imaging of this agent allows in vivo detection of both physiological and pathological reductions of cholinergic terminals density.

Metabolic brain activity suggestive of persistent pain in a rat model of neuropathic pain.

Persistent pain is a central characteristic of neuropathic pain conditions in humans. Knowing whether rodent models of neuropathic pain produce persistent pain is therefore crucial to their translational applicability. We investigated the spared nerve injury (SNI) model of neuropathic pain and the formalin pain model in rats using positron emission tomography (PET) with the metabolic tracer [18F]fluorodeoxyglucose (FDG) to determine if there is ongoing brain activity suggestive of persistent pain. For the formalin model, under brief anesthesia we injected one hindpaw with 5% formalin and the FDG tracer into a tail vein. We then allowed the animals to awaken and observed pain behavior for 30min during the FDG uptake period. The rat was then anesthetized and placed in the scanner for static image acquisition, which took place between minutes 45 and 75 post-tracer injection. A single reference rat brain magnetic resonance image (MRI) was used to align the PET images with the Paxinos and Watson rat brain atlas. Increased glucose metabolism was observed in the somatosensory region associated with the injection site (S1 hindlimb contralateral), S1 jaw/upper lip and cingulate cortex. Decreases were observed in the prelimbic cortex and hippocampus. Second, SNI rats were scanned 3weeks post-surgery using the same scanning paradigm, and region-of-interest analyses revealed increased metabolic activity in the contralateral S1 hindlimb. Finally, a second cohort of SNI rats was scanned while anesthetized during the tracer uptake period, and the S1 hindlimb increase was not observed. Increased brain activity in the somatosensory cortex of SNI rats resembled the activity produced with the injection of formalin, suggesting that the SNI model may produce persistent pain. The lack of increased activity in S1 hindlimb with general anesthetic demonstrates that this effect can be blocked, as well as highlights the importance of investigating brain activity in awake and behaving rodents.

FDG-PET imaging for the evaluation of antiglioma agents in a rat model

The increasing development of novel anticancer agents demands parallel advances in the methods used to rapidly assess their therapeutic efficacy (TE) in the preclinical phase. We evaluated the ability of small-animal PET, using the (18)F-fluoro-deoxy-D-glucose (FDG) radiotracer, to predict the TE of a number of anticancer agents in the rat C6 glioma model following 3 days of treatment. Semi-quantitative measurements of changes in FDG uptake during the course of treatment (standardized uptake value response [SUV(r)]) were found to be significantly lower in tumors treated with the hypoxia-inducible factor-1alpha inhibitor YC-1 (15 mg/kg) than in tumors in the control group. No significant SUV(r) change was observed following a similar 3-day regimen with the proapoptotic agent NS1619 (20 microg/kg), the combination of YC-1 and NS1619, or the alkylating agent temozolomide (7.5 mg/kg). Quantitative immunohistochemical studies demonstrated significantly lower levels of glucose transporter-1 (GLUT-1) expression in the YC-1-treated tumors, thereby correlating with the low SUV(r) observed in this group. The ability of SUV(r) to predict gold-standard outcomes of TE was further validated as YC-1-treated tumors had decreased volumes compared to control tumors. As such, we successfully demonstrated the ability of FDG-PET to rapidly determine the TE of novel agents for the treatment of glioma in the preclinical phase of evaluation.

Syntheses and Evaluation of Carbon-11- and Fluorine-18-Radiolabeled pan-Tropomyosin Receptor Kinase (Trk) Inhibitors: Exploration of the 4-Aza-2-oxindole Scaffold as Trk PET Imaging Agents

Tropomyosin receptor kinases (TrkA/B/C) are critically involved in the development of the nervous system, in neurological disorders as well as in multiple neoplasms of both neural and non-neural origins. The development of Trk radiopharmaceuticals would offer unique opportunities toward a more complete understanding of this emerging therapeutic target. To that end, we first developed [(11)C]GW441756 ([(11)C]9), a high affinity photoisomerizable pan-Trk inhibitor, as a lead radiotracer for our positron emission tomography (PET) program. Efficient carbon-11 radiolabeling afforded [(11)C]9 in high radiochemical yields (isolated RCY, 25.9% ± 5.7%). In vitro autoradiographic studies in rat brain and TrkB-expressing human neuroblastoma cryosections confirmed that [(11)C]9 specifically binds to Trk receptors in vitro. MicroPET studies revealed that binding of [(11)C]9 in the rodent brain was mostly nonspecific despite initial high brain uptake (SUVmax = 2.0). Modeling studies of the 4-aza-2-oxindole scaffold led to the successful identification of a small series of high affinity fluorinated and methoxy derivatized pan-Trk inhibitors based on our lead compound 9. Out of this series, the fluorinated compound 10 was selected for initial evaluation and radiolabeled with fluorine-18 (isolated RCY, 2.5% ± 0.6%). Compound [(18)F]10 demonstrated excellent Trk selectivity in a panel of cancer relevant kinase targets and a promising in vitro profile in tumors and brain sections but high oxidative metabolic susceptibility leading to nonspecific brain distribution in vivo. The information gained in this study will guide further exploration of the 4-aza-2-oxindole scaffold as a lead for Trk PET ligand development.