Cognitive and Brain Profiles Associated with Current Neuroimaging Biomarkers of Preclinical Alzheimer's Disease.

Neuroimaging biomarkers, namely hippocampal volume loss, temporoparietal hypometabolism, and neocortical ß-amyloid (Aß) deposition, are included in the recent research criteria for preclinical Alzheimer's disease (AD). However, how to use these biomarkers is still being debated, especially regarding their sequence. Our aim was to characterize the cognitive and brain profiles of elders classified as positive or negative for each biomarker to further our understanding of their use in the preclinical diagnosis of AD. Fifty-four cognitively normal individuals (age = 65.8 ± 8.3 years) underwent neuropsychological tests (structural MRI, FDG-PET, and Florbetapir-PET) and were dichotomized into positive or negative independently for each neuroimaging biomarker. Demographic, neuropsychological, and neuroimaging data were compared between positive and negative subgroups. The MRI-positive subgroup had lower executive performances and mixed patterns of lower volume and metabolism in AD-characteristic regions and in the prefrontal cortex. The FDG-positive subgroup showed only hypometabolism, predominantly in AD-sensitive areas extending to the whole neocortex, compared with the FDG-negative subgroup. The amyloid-positive subgroup was older and included more APOE ε4 carriers compared with the amyloid-negative subgroup. When considering MRI and/or FDG biomarkers together (i.e., the neurodegeneration-positive), there was a trend for an inverse relationship with Aß deposition such that those with neurodegeneration tended to show less Aß deposition and the reverse was true as well. Our findings suggest that: (1) MRI and FDG biomarkers provide complementary rather than redundant information and (2) relatively young cognitively normal elders tend to have either neurodegeneration or Aß deposition, but not both, suggesting additive rather than sequential/causative links between AD neuroimaging biomarkers at this age. Significance statement: Neuroimaging biomarkers are included in the recent research criteria for preclinical Alzheimer's disease (AD). However, how to use these biomarkers is still being debated, especially regarding their sequence. Our findings suggest that MRI and FDG-PET biomarkers should be used in combination, offering an additive contribution instead of reflecting the same process of neurodegeneration. Moreover, the present study also challenges the hierarchical use of the neuroimaging biomarkers in preclinical AD because it suggests that the neurodegeneration observed in this population is not due to ß-amyloid deposition. Rather, our results suggest that ß-amyloid- and tau-related pathological processes may interact but not necessarily appear in a systematic sequence.

Multimodal imaging based on MRI and PET reveals [(18)F]FLT PET as a specific and early indicator of treatment efficacy in a preclinical model of recurrent glioblastoma.

PURPOSE: The primary objective of this study was to compare the ability of PET and MRI biomarkers to predict treatment efficacy in a preclinical model of recurrent glioblastoma multiforme. METHODS: MRI (anatomical, diffusion, vasculature and oxygenation) and PET ([(18)F]FDG and [(18)F]FLT) parameters were obtained 3 days after the end of treatment and compared with late tumour growth and survival. RESULTS: Early after tumour recurrence, no effect of treatment with temozolomide combined with bevacizumab was observed on tumour volume as assessed by T2-W MRI. At later times, the treatment decreased tumour volume and increased survival. Interestingly, at the earlier time, temozolomide + bevacizumab decreased [(18)F]FLT uptake, cerebral blood volume and oedema. [(18)F]FLT uptake, oedema and cerebral blood volume were correlated with overall survival but [(18)F]FLT uptake had the highest specificity and sensitivity for the early prediction of treatment efficacy. CONCLUSION: The present investigation in a preclinical model of glioblastoma recurrence underscores the importance of multimodal imaging in the assessment of oedema, tumour vascular status and cell proliferation. Finally, [(18)F]FLT holds the greatest promise for the early assessment of treatment efficacy. These findings may translate clinically in that individualized treatment for recurrent glioma could be prescribed for patients selected after PET/MRI examinations.

Region-specific hierarchy between atrophy, hypometabolism, and ß-amyloid (Aß) load in Alzheimer's disease dementia.

Gray matter atrophy, glucose hypometabolism, and ß-amyloid Aß deposition are well-described hallmarks of Alzheimer's disease, but their relationships are poorly understood. The present study aims to compare the local levels of these three alterations in humans with Alzheimer's disease. Structural magnetic resonance imaging, (18)F-fluorodeoxyglucose positron emission tomography (PET), and (18)F-florbetapir PET data from 34 amyloid-negative healthy controls and 20 demented patients with a high probability of Alzheimer's disease etiology (attested using neuroimaging biomarkers as recently recommended) were analyzed. For each patient and imaging modality, age-adjusted Z-score maps were computed, and direct between-modality voxelwise comparison and correlation analyses were performed. Significant differences in the levels of atrophy, hypometabolism, and Aß deposition were found in most brain areas, but the hierarchy differed across regions. A cluster analysis revealed distinct subsets of regions: (1) in the hippocampus, atrophy exceeded hypometabolism, whereas Aß load was minimal; (2) in posterior association areas, Aß deposition was predominant, together with high hypometabolism and lower but still significant atrophy; and (3) in frontal regions, Aß deposition was maximal, whereas structural and metabolic alterations were low. Atrophy and hypometabolism significantly correlated in the hippocampus and temporo-parietal cortex, whereas Aß load was not significantly related to either atrophy or hypometabolism. These findings provide direct evidence for regional variations in the hierarchy and relationships between Aß load, hypometabolism, and atrophy. Altogether, these variations probably reflect the differential involvement of region-specific pathological or protective mechanisms, such as the presence of neurofibrillary tangles, disconnection, as well as compensation processes.

Evaluation of Specific Binding of [11C]RTI-97 to Kappa Opioid Receptor by Autoradiography and PET Imaging in Rat.

Kappa opioid receptor (KOR) PET imaging remains attractive to understand the role of KOR in health and diseases and to help the development of drugs especially for psychiatric disorders such as depression, anxiety, and addiction. The potent and selective KOR antagonist RTI-97 labeled with carbon-11 was previously demonstrated to display specific KOR binding in mouse brain by ex vivo autoradiography studies. Herein, we evaluated [11C]RTI-97 in rat by in vitro autoradiography and by in vivo PET imaging. The radiosynthesis of [11C]RTI-97 was optimized to obtain high molar activities. Despite a low cerebral uptake, the overall results showed a heterogeneous repartition and specific KOR binding of [11C]RTI-97 in brain and a high and specific accumulation of [11C]RTI-97 in pituitary in accordance with KOR expression.

Multimodal evaluation of hypoxia in brain metastases of lung cancer and interest of hypoxia image-guided radiotherapy.

Lung cancer patients frequently develop brain metastases (BM). Despite aggressive treatment including neurosurgery and external-radiotherapy, overall survival remains poor. There is a pressing need to further characterize factors in the microenvironment of BM that may confer resistance to radiotherapy (RT), such as hypoxia. Here, hypoxia was first evaluated in 28 biopsies from patients with non­small cell lung cancer (NSCLC) BM, using CA-IX immunostaining. Hypoxia characterization (pimonidazole, CA-IX and HIF-1α) was also performed in different preclinical NSCLC BM models induced either by intracerebral injection of tumor cells (H2030-Br3M, H1915) into the cortex and striatum, or intracardial injection of tumor cells (H2030-Br3M). Additionally, [18F]-FMISO-PET and oxygen-saturation-mapping-MRI (SatO2-MRI) were carried out in the intracerebral BM models to further characterize tumor hypoxia and evaluate the potential of Hypoxia-image-guided-RT (HIGRT). The effect of RT on proliferation of BM ([18F]-FLT-PET), tumor volume and overall survival was determined. We showed that hypoxia is a major yet heterogeneous feature of BM from lung cancer both preclinically and clinically. HIGRT, based on hypoxia heterogeneity observed between cortical and striatal metastases in the intracerebrally induced models, showed significant potential for tumor control and animal survival. These results collectively highlight hypoxia as a hallmark of BM from lung cancer and the value of HIGRT in better controlling tumor growth.

Chemical Delivery System of MIBG to the Central Nervous System: Synthesis, 11C-Radiosynthesis, and in Vivo Evaluation.

The norepinephrine transporter (NET) plays an important role in neurotransmission and is involved in a multitude of psychiatric and neurodegenerative diseases. [123I/131I]meta-iodobenzylguanidine (MIBG) is a widely used radiotracer in the diagnosis and follow-up of peripheral neuroendocrine tumors overexpressing the norepinephrine transporter. MIBG does not cross the blood-brain barrier (BBB), and we have demonstrated the "proof-of-concept" that 1,4-dihydroquinoline/quinolinium salt as chemical delivery system (CDS) is a promising tool to deliver MIBG to the brain. To improve BBB passage, various substituents on the 1,4-dihydroquinoline moiety and a linker between CDS and MIBG were added. A series of CDS-MIBG 1a-d was synthesized, labeled with carbon-11, and evaluated in vivo into rats. The in vivo results demonstrated that, although adding substituents on CDS in 1a-c is of no benefit for brain delivery of MIBG, the presence of a linker in CDS-MIBG 1d greatly improved both brain penetration and the release rate of MIBG in the central nervous system.

64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study.

BACKGROUND: Diacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression. METHODS: µPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels. RESULTS: In vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages. CONCLUSION: In the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

[18F]Fludarabine-PET as a promising tool for differentiating CNS lymphoma and glioblastoma: Comparative analysis with [18F]FDG in human xenograft models.

This paper investigated whether positron emission tomography (PET) imaging with [18F]fludarabine ([18F]FDB) can help to differentiate central nervous system lymphoma (CNSL) from glioblastoma (GBM), which is a crucial issue in the diagnosis and management of patients with these aggressive brain tumors. Multimodal analyses with [18F]fluorodeoxyglucose ([18F]FDG), magnetic resonance imaging (MRI) and histology have also been considered to address the specificity of [18F]FDB for CNSL. Methods: Nude rats were implanted with human MC116 lymphoma-cells (n = 9) or U87 glioma-cells (n = 4). Tumor growth was monitored by MRI, with T2-weighted sequence for anatomical features and T1-weighted with gadolinium (Gd) enhancement for blood brain barrier (BBB) permeability assessment. For PET investigation, [18F]FDB or [18F]FDG (~11 MBq) were injected via tail vein and dynamic PET images were acquired up to 90 min after radiotracer injection. Paired scans of the same rat with the two [18F]-labelled radiotracers were investigated. Initial volumes of interest were manually delineated on T2w images and set on co-registered PET images and tumor-to-background ratio (TBR) was calculated to semi-quantitatively assess the tracer accumulation in the tumor. A tile-based method for image analysis was developed in order to make comparative analysis between radiotracer uptake and values extracted from immunohistochemistry staining. Results: In the lymphoma model, PET time-activity curves (TACs) revealed a differential response of [18F]FDB between tumoral and healthy tissues with average TBR varying from 2.45 to 3.16 between 5 to 90 min post-injection. In contrast, [18F]FDG demonstrated similar uptake profiles for tumoral and normal regions with TBR varying from 0.84 to 1.06 between these two time points. In the glioblastoma (GBM) model, the average TBRs were from 2.14 to 1.01 for [18F]FDB and from 0.95 to 1.65 for [18F]FDG. Therefore, inter-model comparisons showed significantly divergent responses (p < 0.01) of [18F]FDB between lymphoma and GBM, while [18F]FDG demonstrated overlap (p = 0.04) between the groups. Tumor characterization with histology (based mainly on Hoechst and CD79), as well as with MRI was overall in better agreement with [18F]FDB-PET than [18F]FDG with regard to tumor selectivity. Conclusions: [18F]FDB-PET demonstrated considerably greater specificity for CNSL when compared to [18F]FDG. It also permitted a more precise definition of target volume compared to contrast-enhanced MRI. Therefore, the potential of [18F]FDB-PET to distinguish CNSL from GBM is quite evident and will be further investigated in humans.

18F-Fludarabine PET for Lymphoma Imaging: First-in-Humans Study on DLBCL and CLL Patients.

This was the first-in-humans clinical study of 18F-fludarabine, which is a radiopharmaceutical for PET imaging in lymphoma, for which many issues remain controversial with the standard radiotracer 18F-FDG. Methods:18F-fludarabine PET or PET/CT was performed on 10 patients: 5 with diffuse large B-cell lymphoma (DLBCL) and 5 with chronic lymphocytic leukemia. The tumor uptake, biodistribution, and radiation dosimetry of 18F-fludarabine were evaluated. Six successive partial-body PET scans were acquired for 250 min after an intravenous 4 MBq/kg bolus of 18F-fludarabine. SUVs were recorded for each involved lymph node territory and for several extranodal sites, with particular reference to the liver. To assess the time-related uptake profile of 18F-fludarabine, PET images were analyzed by delineating volumes of interest over the uptake sites on the optimal scan for visual observation and were projected onto all coregistered scans of the same subject. Physical examination, laboratory studies, and contrast-enhanced CT were performed on all patients. For the DLBCL group, 18F-FDG PET was also considered. Results: In DLBCL patients, increased 18F-fludarabine uptake was observed in sites considered abnormal by CT or 18F-FDG, with SUVs significantly higher in involved lesions than in physiologic nontarget sites. Nonetheless, the comparison of 18F-fludarabine and 18F-FDG PET showed discrepancies in 2 patients. In chronic lymphocytic leukemia patients, the uptake of 18F-fludarabine coincided with sites expected to be involved (including splenic invasion) according to conventional clinical and CT staging and was significant in hematopoietic bone marrow. No uptake was observed, whatever the disease group, in cardiac muscle or brain. The mean effective dose from a mean injected 18F-fludarabine activity of 305 ± 76 MBq was 3.07 ± 0.81 mSv. Conclusion:18F-fludarabine PET might well be a promising tool for lymphoproliferative diseases. The radiation dose of this radiopharmaceutical is below that of 18F-FDG. The specificity of this PET probe for lymphoid cells, its absence of accumulation in reactive tissues, and its feasibility for detection of bone marrow infiltration might play an innovative role in lymphoma imaging.

Delivering FLT to the Central Nervous System by Means of a Promising Targeting System: Synthesis, [11C]Radiosynthesis, and in Vivo Evaluation.

The development of delivery systems to transport some specific radiotracers across the blood-brain barrier (BBB) needs to be investigated for brain imaging. [18F]FLT (3'-deoxy-3'-18F-fluoro-l-thymidine), an analogue substrate of the nucleoside thymidine, has been developed as a proliferation tracer for oncological PET studies. Unfortunately, low-grade brain tumors are poorly visualized due to the low uptake of [18F]FLT in brain tissue, preventing its use in PET imaging to detect brain tumors at an early stage. Based on our previous work, a redox chemical delivery system (CDS) related to Bodor's strategy was developed to enable the penetration of FLT into the brain. To this end, FLT was covalently linked to a series of lipophilic carriers based on a 1,4-dihydroquinoline structure. To determine the best carrier, various sets of [11C]CDS-FLT were prepared and injected into rats. Pleasingly, in vivo results let us suggest that this CDS is a promising approach to overcome the BBB to target low-grade brain tumors for PET imaging.

Dihydroquinoline Carbamate Derivatives as "Bio-oxidizable" Prodrugs for Brain Delivery of Acetylcholinesterase Inhibitors: [¹¹C] Radiosynthesis and Biological Evaluation.

With the aim of improving the efficiency of marketed acetylcholinesterase (AChE) inhibitors in the symptomatic treatment of Alzheimer's disease, plagued by adverse effects arising from peripheral cholinergic activation, this work reports a biological evaluation of new central AChE inhibitors based on an original "bio-oxidizable" prodrug strategy. After peripheral injection of the prodrug 1a [IC50 > 1 mM (hAChE)] in mice, monitoring markers of central and peripheral cholinergic activation provided in vivo proof-of-concept for brain delivery of the drug 2a [IC50 = 20 nM (hAChE)] through central redox activation of 1a. Interestingly, peripheral cholinergic activation has been shown to be limited in time, likely due to the presence of a permanent positive charge in 2a promoting rapid elimination of the AChE inhibitor from the circulation of mice. To support these assumptions, the radiosynthesis with carbon-11 of prodrug 1a was developed for additional ex vivo studies in rats. Whole-body biodistribution of radioactivity revealed high accumulation in excretory organs along with moderate but rapid brain uptake. Radio-HPLC analyses of brain samples confirm rapid CNS penetration of [(11)C]1a, while identification of [(11)C]2a and [(11)C]3a both accounts for central redox activation of 1a and pseudoirreversible inhibition of AChE, respectively. Finally, Caco-2 permeability assays predicted metabolite 3a as a substrate for efflux transporters (P-gp inter alia), suggesting that metabolite 3a might possibly be actively transported out of the brain. Overall, a large body of evidence from in vivo and ex vivo studies on small animals has been collected to validate this "bio-oxidizable" prodrug approach, emerging as a very promising strategy in the rational design of selective central AChE inhibitors.

Precuneus and Cingulate Cortex Atrophy and Hypometabolism in Patients with Alzheimer's Disease and Mild Cognitive Impairment: MRI and (18)F-FDG PET Quantitative Analysis Using FreeSurfer.

OBJECTIVE: The objective of this study was to compare glucose metabolism and atrophy, in the precuneus and cingulate cortex, in patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI), using FreeSurfer. METHODS: 47 individuals (17 patients with AD, 17 patients with amnestic MCI, and 13 healthy controls (HC)) were included. MRI and PET images using (18)F-FDG (mean injected dose of 185 MBq) were acquired and analyzed using FreeSurfer to define regions of interest in the hippocampus, amygdala, precuneus, and anterior and posterior cingulate cortex. Regional volumes were generated. PET images were registered to the T1-weighted MRI images and regional uptake normalized by cerebellum uptake (SUVr) was measured. RESULTS: Mean posterior cingulate volume was reduced in MCI and AD. SUVr were different between the three groups: mean precuneus SUVr was 1.02 for AD, 1.09 for MCI, and 1.26 for controls (p < 0.05); mean posterior cingulate SUVr was 0.96, 1.06, and 1.22 for AD, MCI, and controls, respectively (p < 0.05). CONCLUSION: We found graduated hypometabolism in the posterior cingulate cortex and the precuneus in prodromal AD (MCI) and AD, whereas atrophy was not significant. This suggests that the use of (18)F-FDG in these two regions could be a neurodegenerative biomarker.

Detection of glioblastoma response to temozolomide combined with bevacizumab based on µMRI and µPET imaging reveals [18F]-fluoro-L-thymidine as an early and robust predictive marker for treatment efficacy.

The individualized care of glioma patients ought to benefit from imaging biomarkers as precocious predictors of therapeutic efficacy. Contrast enhanced MRI and [(18)F]-fluorodeoxyglucose (FDG)-PET are routinely used in clinical settings; their ability to forecast the therapeutic response is controversial. The objectives of our preclinical study were to analyze sensitive µMRI and/or µPET imaging biomarkers to predict the efficacy of anti-angiogenic and/or chemotherapeutic regimens. Human U87 and U251 orthotopic glioma models were implanted in nude rats. Temozolomide and/or bevacizumab were administered. µMRI (anatomical, diffusion, and microrheological parameters) and µPET ([(18)F]-FDG and [(18)F]-fluoro-l-thymidine [FLT]-PET) studies were undertaken soon (t(1)) after treatment initiation compared with late anatomical µMRI evaluation of tumor volume (t(2)) and overall survival. In both models, FDG and FLT uptakes were attenuated at t(1) in response to temozolomide alone or with bevacizumab. The distribution of FLT, reflecting intratumoral heterogeneity, was also modified. FDG was less predictive for treatment efficacy than was FLT (also highly correlated with outcome, P < .001 for both models). Cerebral blood volume was significantly decreased by temozolomide + bevacizumab and was correlated with survival for rats with U87 implants. While FLT was highly predictive of treatment efficacy, a combination of imaging biomarkers was superior to any one alone (P < .0001 in both tumors with outcome). Our results indicate that FLT is a sensitive predictor of treatment efficacy and that predictability is enhanced by a combination of imaging biomarkers. These findings may translate clinically in that individualized glioma treatments could be decided in given patients after PET/MRI examinations.

Chemical delivery system of metaiodobenzylguanidine (MIBG) to the central nervous system.

The aim of the present investigation was to apply a chemical delivery system (CDS) to MIBG (4) with the purpose of delivering this drug to the CNS. Compound 4 has been linked to a 1,4-dihydroquinoline moiety in order to achieve its CNS penetration, and here we report the synthesis to link 4 to the chemical delivery system and the radiosynthesis with carbon-11 of the "CDS-4 entity". After iv injection into rats of the [(11)C]CDS-4, the follow-up study of the radioactivity distribution in blood samples and brain homogenates and the analysis by HPLC and LC-MS/MS have confirmed the release of 4 into the CNS.

Debenzylation of tertiary amines using phosgene or triphosgene: an efficient and rapid procedure for the preparation of carbamoyl chlorides and unsymmetrical ureas. Application in carbon-11 chemistry.

Efficient and rapid preparations of carbamoyl chlorides and unsymmetrical ureas from tertiary amines and phosgene or its safe equivalent triphosgene [bis(trichloromethyl)carbonate, BTC] are described. First, the reaction of stoichiometric amounts of phosgene with secondary amines was revisited, and it was shown that the formation of carbamoyl chlorides in high yields required careful adjustments of experimental conditions and the use of pyridine as an HCl scavenger. A phosgene-mediated dealkylation of triethylamine was observed when this base was used instead of pyridine. Taking advantage of this observation, a strategy of synthesis of carbamoyl chlorides from tertiary amines and phosgene has been developed. N-Alkyl-N-benzyl(substituted)tetrahydroisoquinolines, -piperazines, -piperidines, or -anilines were treated with stoichiometric amounts of phosgene (or BTC) in CH(2)Cl(2). Tertiary amines bearing electron-enriched benzyl group(s) afforded carbamoyl chlorides in excellent yields and without any contamination by symmetrical ureas. Subsequent additions of primary or secondary amines to these carbamoyl chlorides produced unsymmetrical ureas in single-pot and high-yielding operations. This methodology was applied in (11)C-chemistry. From [(11)C]phosgene, a common precursor used in the preparation of radiotracers for positron emission tomography, a rapid and efficient synthesis of (11)C-carbamoyl chlorides and (11)C-unsymmetrical ureas derived from tetrahydroisoquinoline and piperazine is described. The first example of (11)C-amide formation from the reaction of a (11)C-carbamoyl chloride and an organometallic (cyanocuprate or a Grignard reagent in the presence of a nickel catalyst) is also presented.