Transcriptomic Analyses of Neurotoxic Astrocytes Derived from Adult Triple Transgenic Alzheimer's Disease Mice.

Neurodegenerative diseases such as Alzheimer's disease have been classically studied from a purely neuronocentric point of view. More recent evidences support the notion that other cell populations are involved in disease progression. In this sense, the possible pathogenic role of glial cells like astrocytes is increasingly being recognized. Once faced with tissue damage signals and other stimuli present in disease environments, astrocytes suffer many morphological and functional changes, a process referred as reactive astrogliosis. Studies from murine models and humans suggest that these complex and heterogeneous responses could manifest as disease-specific astrocyte phenotypes. Clear understanding of disease-associated astrocytes is a necessary step to fully disclose neurodegenerative processes, aiding in the design of new therapeutic and diagnostic strategies. In this work, we present the transcriptomics characterization of neurotoxic astrocytic cultures isolated from adult symptomatic animals of the triple transgenic mouse model of Alzheimer's disease (3xTg-AD). According to the observed profile, 3xTg-AD neurotoxic astrocytes show various reactivity features including alteration of the extracellular matrix and release of pro-inflammatory and proliferative factors that could result in harmful effects to neurons. Moreover, these alterations could be a consequence of stress responses at the endoplasmic reticulum and mitochondria as well as of concomitant metabolic adaptations. Present results support the hypothesis that adaptive changes of astrocytic function induced by a stressed microenvironment could later promote harmful astrocyte phenotypes and further accelerate or induce neurodegenerative processes.

Isolation and characterization of neurotoxic astrocytes derived from adult triple transgenic Alzheimer's disease mice.

Alzheimer's disease has been considered mostly as a neuronal pathology, although increasing evidence suggests that glial cells might play a key role in the disease onset and progression. In this sense, astrocytes, with their central role in neuronal metabolism and function, are of great interest for increasing our understanding of the disease. Thus, exploring the morphological and functional changes suffered by astrocytes along the course of this disorder has great therapeutic and diagnostic potential. In this work we isolated and cultivated astrocytes from symptomatic 9-10-months-old adult 3xTg-AD mice, with the aim of characterizing their phenotype and exploring their pathogenic potential. These "old" astrocytes occurring in the 3xTg-AD mouse model of Alzheimer's Disease presented high proliferation rate and differential expression of astrocytic markers compared with controls. They were neurotoxic to primary neuronal cultures both, in neuronal-astrocyte co-cultures and when their conditioned media (ACM) was added into neuronal cultures. ACM caused neuronal GSK3ß activation, changes in cytochrome c pattern, and increased caspase 3 activity, suggesting intrinsic apoptotic pathway activation. Exposure of neurons to ACM caused different subcellular responses. ACM application to the somato-dendritic domain in compartmentalised microfluidic chambers caused degeneration both locally in soma/dendrites and distally in axons. However, exposure of axons to ACM did not affect somato-dendritic nor axonal integrity. We propose that this newly described old 3xTg-AD neurotoxic astrocytic population can contribute towards the mechanistic understanding of the disease and shed light on new therapeutical opportunities.

Dementia in Latin America: Paving the way toward a regional action plan.

Across Latin American and Caribbean countries (LACs), the fight against dementia faces pressing challenges, such as heterogeneity, diversity, political instability, and socioeconomic disparities. These can be addressed more effectively in a collaborative setting that fosters open exchange of knowledge. In this work, the Latin American and Caribbean Consortium on Dementia (LAC-CD) proposes an agenda for integration to deliver a Knowledge to Action Framework (KtAF). First, we summarize evidence-based strategies (epidemiology, genetics, biomarkers, clinical trials, nonpharmacological interventions, networking, and translational research) and align them to current global strategies to translate regional knowledge into transformative actions. Then we characterize key sources of complexity (genetic isolates, admixture in populations, environmental factors, and barriers to effective interventions), map them to the above challenges, and provide the basic mosaics of knowledge toward a KtAF. Finally, we describe strategies supporting the knowledge creation stage that underpins the translational impact of KtAF.

Quantitative comparison between single-photon emission computed tomography and positron emission tomography imaging of lung ventilation with 99mTc-technegas and 68Ga-gallgas in patients with chronic obstructive pulmonary disease: A pilot study.

The aim of this study was quantitative comparison between 68Ga-Gallgas positron emission tomography (PET) and 99mTc-Technegas single photon emission computed tomography (SPECT) for lung ventilation function assessment in patients with moderate-to-severe obstructive pulmonary disease and to identify image-derived texture features correlating to the physiologic parameters. Five patients with moderate-to-severe chronic obstructive pulmonary disease with PET and SPECT lung ventilation scans were selected for this study. Threshold-based segmentations were used to compare ventilated regions between both imaging techniques. Histograms of both scans were compared to reveal main differences in distributions of radiotracers. Volumes of segmentation as well as 50 textural features measured in the pulmonary region were correlated to the forced expiratory volume in 1 s (FEV1) as the relevant physiological variable. A better peripheral distribution of the radiotracer was observed in PET scans for three out of five patients. A segmentation threshold of 27% and 31% for normalized scans, for PET and SPECT respectively, was found optimal for volume correlation with FEV1. A high correlation (Pearson correlation coefficient >0.9) was found between 16 texture features measured from SPECT and 7 features measured from PET and FEV1. Quantitative measurements revealed different tracer distribution in both techniques. These results suggest that tracer distribution patterns may depend on the cause of the pulmonary obstruction. We found several texture features measured from SPECT to correlate to FEV1.

Biological Assessment of a 18F-Labeled Sulforhodamine 101 in a Mouse Model of Alzheimer's Disease as a Potential Astrocytosis Marker.

Neurodegenerative diseases have mainly been associated with neuronal death. Recent investigations have shown that astroglia may modulate neuroinflammation in the early and late stages of the disease. [11C]Deuterodeprenyl ([11C]DED) is a tracer that has been used for reactive astrocyte detection in Alzheimer's disease, Creutzfeldt-Jakob disease and amyotrophic lateral sclerosis, among others, with some limitations. To develop a new radiotracer for detecting astrocytosis and overcoming associated difficulties, we recently reported the synthesis of a sulfonamide derivative of Sulforhodamine 101 (SR101), labeled with 18F, namely SR101 N-(3-[18F]Fluoropropyl) sulfonamide ([18F]2B-SRF101). The red fluorescent dye SR101 has been used as a specific marker of astroglia in the neocortex of rodents using in vivo models. In the present work we performed a biological characterisation of the new tracer including biodistribution and micro-PET/computed tomography (CT) images. PET/CT studies with [11C]DED were also done to compare with [18F]2B-SRF101 in order to assess its potential as an astrocyte marker. Biodistribution studies with [18F]2B-SRF101 were carried out in C57BL6J black and transgenic (3xTg) mice. A hepatointestinal metabolization as well as the pharmacokinetic profile were determined, showing appropriate characteristics to become a PET diagnostic agent. Dynamic PET/CT studies were carried out with [18F]2B-SRF101 and [11C]DED to evaluate the distribution of both tracers in the brain. A significant difference in [18F]2B-SRF101 uptake was especially observed in the cortex and hippocampus, and it was higher in 3xTg mice than it was in the control group. These results suggested that [18F]2B-SRF101 is a promising candidate for more extensive evaluation as an astrocyte tracer. The difference observed for [18F]2B-SRF101 was not found in the case of [11C]DED. The comparative studies between [18F]2B-SRF101 and [11C]DED suggest that both tracers have different roles as astrocytosis markers in this animal model, and could provide different and complementary information at the same time. In this way, by means of a multitracer approach, useful information could be obtained for the staging of the disease.

Cocaine treatment before pregnancy differentially affects the anxiety and brain glucose metabolism of lactating rats if performed during adulthood or adolescence.

Cocaine exposure disrupts the maternal behavior of lactating rats, yet it is less known whether it alters the affective changes that accompany motherhood. As the long-term action of cocaine on anxiety varies according to the developmental stage of the individuals, this study aimed to compare the effect of a chronic treatment with cocaine to adult and adolescent non-pregnant females on their anxiety-like behavior and basal brain metabolic activity during lactation. Thus, adult and adolescent virgin rats were exposed to cocaine (0.0 or 15.0 mg/kg ip) during 10 days and were mated four days later. Anxiety behavior was evaluated on postpartum days 3-4 in the elevated plus maze test, and the basal brain glucose metabolism was determined on postpartum days 7-9 by means of [18F] fluorodeoxyglucose positron emission tomography. Cocaine treatment during adulthood increased the anxiety-like behavior of lactating females whereas its administration during adolescence decreased it. Also, the basal glucose metabolism of the medial prefrontal cortex differed between lactating females treated with cocaine during adulthood and adolescence. These differential effects of cocaine, according to the age at which the drug was administered, support the idea that the adolescent and adult brains have a distinct susceptibility to this drug, which leads to divergent long-term changes in the neural circuits that regulate anxiety during lactation.

[18F]Amylovis as a Potential PET Probe for ß-Amyloid Plaque: Synthesis, In Silico, In vitro and In vivo Evaluations.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. Neuroimaging methods have widened the horizons for AD diagnosis and therapy. The goals of this work are the synthesis of 2-(3-fluoropropyl)-6-methoxynaphthalene (5) and its [18F]-radiolabeled counterpart ([18F]Amylovis), the in silico and in vitro comparative evaluations of [18F]Amylovis and [11C]Pittsburg compound B (PIB) and the in vivo preclinical evaluation of [18F]Amylovis in transgenic and wild mice. METHODS: Iron-catalysis cross coupling reaction, followed by fluorination and radiofluorination steps were carried out to obtain 5 and 18F-Amylovis. Protein/Aß plaques binding, biodistribution, PET/CT Imaging and immunohistochemical studies were conducted in healthy/transgenic mice. RESULTS: The synthesis of 5 was successful obtained. Comparative in silico studies predicting that 5 should have affinity to the Aß-peptide, mainly through π-π interactions. According to a dynamic simulation study the ligand-Aß peptide complexes are stable in simulation-time (ΔG = -5.31 kcal/mol). [18F]Amylovis was obtained with satisfactory yield, high radiochemical purity and specific activity. The [18F]Amylovis log Poct/PBS value suggests its potential ability for crossing the blood brain barrier (BBB). According to in vitro assays, [18F]Amylovis has an adequate stability in time. Higher affinity to Aß plaques were found for [18F]Amylovis (Kd 0.16 nmol/L) than PIB (Kd 8.86 nmol/L) in brain serial sections of 3xTg-AD mice. Biodistribution in healthy mice showed that [18F]Amylovis crosses the BBB with rapid uptake (7 %ID/g at 5 min) and good washout (0.11±0.03 %ID/g at 60 min). Comparative PET dynamic studies of [18F]Amylovis in healthy and transgenic APPSwe/PS1dE9 mice, revealed a significant high uptake in the mice model. CONCLUSION: The in silico, in vitro and in vivo results justify that [18F]Amylovis should be studied as a promissory PET imaging agent to detect the presence of Aß senile plaques.

Dosimetry and Toxicity Studies of the Novel Sulfonamide Derivative of Sulforhodamine 101([18F]SRF101) at a Preclinical Level.

BACKGROUND: The SR101 N-(3-[18F]Fluoropropyl) sulfonamide ([18F]SRF101) is a Sulforhodamine 101 derivative that was previously synthesised by our group. The fluorescent dye SR101 has been reported as a marker of astroglia in the neocortex of rodents in vivo. OBJECTIVE: The aim of this study was to perform a toxicological evaluation of [18F]SRF101 and to estimate human radiation dosimetry based on preclinical studies. METHODS: Radiation dosimetry studies were conducted based on biokinetic data obtained from a mouse model. A single-dose toxicity study was carried out. The toxicological limit chosen was <100 µg, and allometric scaling with a safety factor of 100 for unlabelled SRF101 was selected. RESULTS: The absorbed and effective dose estimated using OLINDA/EXM V2.0 for male and female dosimetric models presented the same tendency. The highest total absorbed dose values were for different sections of the intestines. The mean effective dose was 4.03 x10-3 mSv/MBq and 5.08 x10-3 mSv/MBq for the male and female dosimetric models, respectively, using tissue-weighting factors from ICRP-89. The toxicity study detected no changes in the organ or whole-body weight, food consumption, haematologic or clinical chemistry parameters. Moreover, lesions or abnormalities were not found during the histopathological examination. CONCLUSION: The toxicological evaluation of SRF101 verified the biosafety of the radiotracer for human administration. The dosimetry calculations revealed that the radiation-associated risk of [18F]SRF101 would be of the same order as other 18F radiopharmaceuticals used in clinical applications. These study findings confirm that the novel radiotracer would be safe for use in human PET imaging.

Image-Guided Development of Heterocyclic Sulfoxides as Ligands for Tau Neurofibrillary Tangles: From First-in-Man to Second-Generation Ligands.

Positron emission tomography (PET) imaging of misfolded protein aggregates that form in neurodegenerative processes of the brain is key to providing a robust marker for improved diagnosis and evaluation of treatments. We report the development of advanced radiotracer candidates based on the sulfoxide scaffold found in proton pump inhibitors (lansoprazole, prevacid) with inherent affinity to neurofibrillary tangles in Alzheimer's disease and related disorders (e.g., dementia with Lewy bodies and the frontotemporal degeneration syndrome). First-in-man results obtained with [18F]lansoprazole and N-methyl-[18F]lansoprazole were used to guide the design of a set of 24 novel molecules with suitable properties for neuroimaging with PET. Compounds were synthesized and characterized pharmacologically, and the binding affinity of the compounds to synthetic human tau-441 fibrils was determined. Selectivity of binding was assessed using α-synuclein and ß-amyloid fibrils to address the key misfolded proteins of relevance in dementia. To complete the pharmacokinetic profiling in vitro, plasma protein binding and lipophilicity were investigated. Highly potent and selective new radiotracer candidates were identified for further study.

68Ga-PSMA and 11C-Choline comparison using a tri-modality PET/CT-MRI (3.0 T) system with a dedicated shuttle.

BACKGROUND: The aim of this study was to prospectively compare the detection rate of 68Ga-PSMA versus 11C-Choline in men with prostate cancer with biochemical recurrence and to demonstrate the added value of a tri-modality PET/CT-MRI system. METHODS: We analysed 36 patients who underwent both 11C-Choline PET/CT and 68Ga-PSMA PET/CT scanning within a time window of 1-2 weeks. Additionally, for the 68Ga-PSMA scan, we used a PET/CT-MRI (3.0 T) system with a dedicated shuttle, acquiring MRI images of the pelvis. RESULTS: Both scans were positive in 18 patients (50%) and negative in 8 patients (22%). Nine patients were positive with 68Ga-PSMA alone (25%) and one with 11C-Choline only (3%). The median detected lesion per patient was 2 for 68Ga-PSMA (range 0-93) and 1 for 11C-Choline (range 0-57). Tumour to background ratios in all concordant lesions (n = 96) were higher for 68Ga-PSMA than for 11C-Choline (110.3 ± 107.8 and 27.5 ± 17.1, mean ± S.D., for each tracer, respectively P = 0.0001). The number of detected lesions per patient was higher for 11C-Choline in those with PSA ≥ 3.3 ng/mL, while the number of detected lesions was independent of PSA levels for 68Ga-PSMA using the same PSA cut-off value. Metastatic pelvic lesions were found in 25 patients (69%) with 68Ga-PSMA PET/CT, in 18 (50%) with 11C-Choline PET/CT and in 21 (58%) with MRI (3.0 T). MRI was very useful in detecting recurrence in cases classified as indeterminate by means of PET/CT alone at prostate bed. CONCLUSIONS: In patients with prostate cancer with biochemical recurrence 68Ga-PSMA detected more lesions per patient than 11C-Choline, regardless of PSA levels. PET/CT-MRI (3.0 T) system is a feasible imaging modality that potentially adds useful relevant information with increased accuracy of diagnosis.

Synthesis of an Al18F radiofluorinated GLU-UREA-LYS(AHX)-HBED-CC PSMA ligand in an automated synthesis platform.

BACKGROUND: Overexpression of prostatic membrane antigen (PSMA) is associated with the progression and prognosis of prostate cancer. There are numerous studies using this peptide with the 68Ga radionuclide. Previous methods to synthetize 18F-labeled PSMA ligands with complexes [18F]AlF2+ have been achieved. However, these reported syntheses were performed manually, using small volumes. Therefore it is only possible to have the radiopharmaceutical on a small scale, for use in preclinical studies. 18F-labelled tracers allow higher doses increasing the number of examined patients. In addition, late images can be acquired in the case of uptake in lymph nodes, to discard inflammation. It is important to transfer the manual synthesis to an automatic module, producing a batch of the radiopharmaceutical with high activity in a safe and effective way. The aim of this work was to optimize the labeling of [18F]AlF-[GLU-UREA-LYS(AHX)-HBED-CC] in a Tracerlab FXFN® (GE) platform. RESULTS: The labeling up to the reactor corroborates the formation of the complex [18F]AlF-PSMA. After purification by HPLC, the radiopharmaceutical was achieved with a radiochemical purity higher than 90%. The quality control of the final product fulfilled all the requirements in agreement with USP, such as radiochemical purity (greater than 90%) and residual solvents. [18F]AlF-PSMA was obtained with a yield of 18 ± 3% (n = 7), not decay corrected (NCD) starting off from 500 to 2000 mCi the 18F and with a radiochemical purity of 95 ± 3% (n = 7). The product verified stability in the final formulation vial during 4 hs and in human plasma up to 1 h. CONCLUSION: The proposed method allowed the production of [18F]AlF-PSMA with suitable radiochemical purity in a commercial platform. High activities were achieved, with a simple and robust methodology appropriate for clinical purposes.

Comparative In Vitro Study of 11C-Methionine and 11C-Deuterodeprenyl Uptake in Three Human Glioma Cell Lines.

AIM: To compare the uptake of 11C-deuterodeprenyl (11C-DED) and 11C-methionine (11C-MET) in three human glioma cell lines and study the relationship with glial fibrillary acid protein (GFAP) and monoamine oxidase B (MAO B) expression. 11C-DED is used in positron emission tomography imaging as a marker of astrocytosis in various central nervous system pathologies. It binds irreversibly to MAO B, a glial dimeric enzyme with increased activity in some neurological pathologies. MATERIALS AND METHODS: Binding and internalization studies of 11C-MET and 11C-DED were performed in astrocytoma grade III, glioblastoma grade IV, and radio-resistant glioblastoma grade IV cells. Immunofluorescence was used. RESULTS: 11C-MET specific activity bound to membrane was 9.0%-11.1% and that internalized was 88.9%-91.0%. 11C-DED specific activity bound to membrane was 34.8%-58.0% and that internalized was 38.7%-65.2%. Immunocytochemistry revealed GFAP and MAO B expression. CONCLUSIONS: The expression of MAO B measured by 11C-DED uptake or immunocytochemistry was not significantly different in grade III or IV cells. The GFAP signal was higher for grade IV compared to grade III. 11C-MET uptake was high in all the tumor cells. 11C-DED is a dopamine analogue and the transport across cell membranes is expected to be mediated by DAT receptors present in astrocytes. Reactive astrocytes surround tumor lesions; so the authors suggest that the 11C-DED uptake might be caused by the reactive astrocytosis and not by MAO B expression in tumor cells.

Synthesis of [18F]2B-SRF101: A Sulfonamide Derivative of the Fluorescent Dye Sulforhodamine 101.

BACKGROUND: The red fluorescent dye Sulforhodamine 101 (SR101) has been used in neuroscience research as a useful tool for staining of astrocytes, since it has been reported as a marker of astroglia in the neocortex of rodents in vivo. The aim of this work is to label SR101 with positron emission radionuclides, in order to provide a radiotracer to study its biological behavior. This is the first attempt to label SR101 by [18F], using a chemical derivatization via a sulfonamidelinker and a commercially available platform. METHODS: The synthesis of SR101 N-(3-Bromopropyl) sulfonamide and SR101 N-(3- Fluoropropyl) sulfonamide (2B-SRF101) was carried out. The radiosynthesis of SR101 N-(3- [18F]Fluoropropyl) sulfonamide ([18F]2B-SRF101) was performed in a TRACERlab® FX-FN. Different labeling conditions were tested. Three pilot batches were produced and quality control was performed. Lipophilicity, plasma protein binding and radiochemical stability of [18F]2BSRF101 in final formulation and in plasma were determined. RESULTS: SR101 N-(3-Bromopropyl) sulfonamide was synthetized as a precursor for radiolabeling with [18F]. 2B-SRF101 was prepared for analytical purpose. [18F]2B-SRF101 was obtained with radiochemical purity of (97.0 ± 0.6%). The yield of the whole synthesis was (11.9 ± 1.7 %), nondecay corrected. [18F]2B-SRF101 was found to be stable in final formulation and in plasma. The octanol-water partition coefficient was (Log POCT = 1.88 ± 0.14). The product showed a high percentage of plasma protein binding. CONCLUSIONS: The derivatization of SR101 via sulfonamide-linker and the first radiosynthesis of [18 F]2B-SRF101 were performed. It was obtained in accordance with quality control specifications. In vitro stability studies verified that [18F]2B-SRF101 was suitable for preclinical evaluations.

Automated One-pot Radiosynthesis of [11C]S-adenosyl Methionine.

BACKGROUND: Glycine N-methyltransferase is an enzyme overexpressed in some neoplastic tissues. It catalyses the methylation of glycine using S-adenosyl methionine (SAM or AdoMet) as substrate. SAM is involved in a great variety of biochemical processes, including transmethylation reactions. Thus, [11C]SAM could be used to evaluate transmethylation activity in tumours. The only method reported for [11C]SAM synthesis is an enzymatic process with several limitations. We propose a new chemical method to obtain [11C]SAM, through a one-pot synthesis. METHOD: The optimization of [11C]SAM synthesis was carried out in the automated TRACERlab® FX C Pro module. Different labelling conditions were performed varying methylating agent, precursor amount, temperature and reaction time. The compound was purified using a semipreparative HPLC. Radiochemical stability, lipophilicity and plasma protein binding were evaluated. RESULTS: The optimum labelling conditions were [11C]CH3OTf as the methylating agent, 5 mg of precursor dissolved in formic acid at 60 °C for 1 minute. [11C]SAM was obtained as a diastereomeric mixture. Three batches were produced and quality control was performed according to specifications. [11C]SAM was stable in final formulation and in plasma. Log POCT obtained for [11C]SAM was (-2,01 ± 0,07) (n=4), and its value for plasma protein binding was low. CONCLUSION: A new chemical method to produce [11C]SAM was optimized. The radiotracer was obtained as a diastereomeric mixture with a 53:47 [(R,S)-isomer: (S,S)-isomer] ratio. The compound was within the quality control specifications. In vitro stability was verified. This compound is suitable to perform preclinical and clinical evaluations.

3-(Benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole: a PET radiotracer to measure acetylcholinesterase in brain.

AIM: Noninvasive studies of the acetylcholinesterase (AChE) level in Alzheimer's disease (AD) patients can contribute to a better understanding of the disease and its therapeutic. We propose 3-(benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole, [18F]-IND1, structurally related to the AChE-inhibitor CP126,998, as a new positron emission tomography-radiotracer. EXPERIMENTAL: Radiosynthesis, with 18F, stability, lipophilicity and protein binding of [18F]-IND1 were studied. In vivo behavior, in normal mice and on AD mice models, were also analyzed. RESULTS: [18F]-IND1 was obtained in good radiochemical yield, was stable for at least 2 h in different conditions, and had adequate lipophilicity for blood-brain barrier penetration. Biodistribution studies, in normal mice, showed that [18F]-IND1 was retained in the brain after 1 h. In vivo tacrine-blocking experiments indicated this uptake could be specifically due to AChE interaction. Studies in transgenic AD mice showed differential, compared with normal mice, binding in many brain regions. CONCLUSION: [18F]-IND1 can be used to detect AChE changes in AD patients.

Early Detection of Bone Metastasis in Small Cell Neuroendocrine Carcinoma of the Cervix by 68Ga-DOTATATE PET/CT Imaging.

The neuroendocrine small cell carcinoma of the cervix is a rare malignancy that has a poor prognosis due to early lymphatic and hematogenous spread. We herein report a case of a 27- year-old woman who was referred for initial staging of a neuroendocrine small cell carcinoma with previous unremarkable structural imaging. Ga-DOTATATE PET/CT revealed focal uptake at the primary tumor and in a solitary pelvic bone lesion suggestive of metastases that was further confirmed by CT-guided biopsy. Somatostatin receptor PET/CT may be a useful image modality for early detection of metastases to guide treatment in these patients.

An efficient preparation of labelling precursor of [11C]L-deprenyl-D2 and automated radiosynthesis.

BACKGROUND: The synthesis of [11C]L-deprenyl-D2 for imaging of astrocytosis with positron emission tomography (PET) in neurodegenerative diseases has been previously reported. [11C]L-deprenyl-D2 radiosynthesis requires a precursor, L-nordeprenyl-D2, which has been previously synthesized from L-amphetamine as starting material with low overall yields. Here, we present an efficient synthesis of L-nordeprenyl-D2 organic precursor as free base and automated radiosynthesis of [11C]L-deprenyl-D2 for PET imaging of astrocytosis. The L-nordeprenyl-D2 precursor was synthesized from the easily commercial available and cheap reagent L-phenylalanine in five steps. Next, N-alkylation of L-nordeprenyl-D2 free base with [11C]MeOTf was optimized using the automated commercial platform GE TRACERlab® FX C Pro. RESULTS: A simple and efficient synthesis of L-nordeprenyl-D2 precursor of [11C]L-deprenyl-D2 as free base has been developed in five synthetic steps with an overall yield of 33%. The precursor as free base has been stable for 9 months stored at low temperature (-20 °C). The labelled product was obtained with 44 ± 13% (n = 12) (end of synthesis, decay corrected) radiochemical yield from [11C]MeI after 35 min synthesis time. The radiochemical purity was over 99% in all cases and specific activity was (170 ± 116) GBq/µmol. CONCLUSIONS: A high-yield synthesis of [11C]L-deprenyl-D2 has been achieved with high purity and specific activity. L-nordeprenyl-D2 precursor as free amine was applicable for automated production in a commercial synthesis module for preclinical and clinical application.

A novel non-Hodgkin lymphoma murine model closer to the standard clinical scenario.

BACKGROUND: Non-Hodgkin lymphomas (NHL) are the most frequent hemato-oncological malignancies. Despite recent major advances in treatment, a substantial proportion of patients relapses highlighting the need for new therapeutic modalities. Promissory results obtained in pre-clinical studies are usually not translated when moving into clinical trials. Pre-clinical studies are mainly conducted in animals with high tumor burden; instead patients undergo chemotherapy as first line of treatment and most likely are under remission when immunotherapies are applied. Thus, an animal model that more closely resembles patients' conditions would be a valuable tool. METHODS: BALB/c mice were injected subcutaneously with A20 lymphoma cells and after tumor development different doses of chemotherapy were assessed to find optimal conditions for minimal residual disease (MRD) establishment. Tumor growth and survival, as well as drugs side effects, were all evaluated. Complete lymphoma remission was monitored in vivo using positron emission tomography (PET), and the results were correlated with histology. Immunological status was assessed by splenocytes proliferation assays in NHL-complete remission mice and by analyzing tumor cell infiltrates and chemokines/cytokines gene expression in the tumor microenvironment of animals with residual lymphoma. RESULTS: Two cycles of CHOP chemotherapy at days 25 and 35 post-tumor implantation induced complete remission for around 20 days. PET showed to be a suitable follow-up technique for MRD condition with 85.7 and 75% of sensibility and specificity respectively. Proliferative responses upon mitogen stimulation were similar in animals that received chemotherapy and wild type mice. Tumors from animals with residual lymphoma showed higher numbers of CD4+ and CD8+ and similar numbers of NK, neutrophils and Tregs infiltrating cells as compared with non-treated animals. Gene expression of several cytokines as well as an array of chemokines associated with migration of activated T cells to tumor sites was upregulated in the tumor microenvironment of animals that received chemotherapy treatment. CONCLUSIONS: We established a NHL-B pre-clinical model using standard chemotherapy to achieve MRD in immunocompetent animals. The MRD condition is maintained for approximately 20 days providing a therapeutic window of time where new immunotherapies can be tested in conditions closer to the clinics.

Decreased Brain Neurokinin-1 Receptor Availability in Chronic Tennis Elbow.

Substance P is released in painful and inflammatory conditions, affecting both peripheral processes and the central nervous system neurokinin 1 (NK1) receptor. There is a paucity of data on human brain alterations in NK1 expression, how this system may be affected by treatment, and interactions between central and peripheral tissue alterations. Ten subjects with chronic tennis elbow (lateral epicondylosis) were selected out of a larger (n = 120) randomized controlled trial evaluating graded exercise as a treatment for chronic tennis elbow (lateral epicondylosis). These ten subjects were examined by positron emission tomography (PET) with the NK1-specific radioligand 11C-GR205171 before, and eight patients were followed up after treatment with graded exercise. Brain binding in the ten patients before treatment, reflecting NK1-receptor availability (NK1-RA), was compared to that of 18 healthy subjects and, longitudinally, to the eight of the original ten patients that agreed to a second PET examination after treatment. Before treatment, patients had significantly lower NK1-RA in the insula, vmPFC, postcentral gyrus, anterior cingulate, caudate, putamen, amygdala and the midbrain but not the thalamus and cerebellum, with the largest difference in the insula contralateral to the injured elbow. No significant correlations between brain NK1-RA and pain, functional severity, or peripheral NK1-RA in the affected limb were observed. In the eight patients examined after treatment, pain ratings decreased in everyone, but there were no significant changes in NK1-RA. These findings indicate a role for the substance P (SP) / NK1 receptor system in musculoskeletal pain and tissue healing. As neither clinical parameters nor successful treatment response was reflected in brain NK1-RA after treatment, this may reflect the diverse function of the SP/NK1 system in CNS and peripheral tissue, or a change too small or slow to capture over the three-month treatment.

68Ga-NOTA-UBI-29-41 as a PET Tracer for Detection of Bacterial Infection.

UNLABELLED: The cationic peptide (68)Ga-NOTA-UBI-29-41 was synthesized and characterized. Biodistribution and PET/CT examinations were performed for evaluation of its biologic behavior. Differentiation of infection from sterile inflammation was investigated using microbiology methods at the sites of bacterial infections. METHODS: Labeling of UBI-29-41 conjugated with NOTA with (68)Ga was optimized at 20°C-100°C and pH 3.5-5.5. Radiochemical purity, stability up to 260 min, and binding to serum proteins were determined. In vitro binding to Staphylococcus aureus was evaluated from 9.14 × 10(7) to 1.17 × 10(10) cfu/mL. Of 3 groups of Mus musculus Swiss male mice, the first was inoculated intramuscularly with 1.2 × 10(8) cfu of S. aureus to provoke infection, and the second, with 1.2 × 10(8) cfu of heat shock-treated S. aureus to generate sterile inflammation. The third mouse was not treated and served as a control. After 24 h, (68)Ga-NOTA-UBI-29-41 was administrated intravenously, and biodistribution was performed at 30, 60, and 120 min. PET/CT dynamic studies (120 min) were acquired. Sinograms were reconstructed using 3D maximum-likelihood expectation maximization and analyzed with software. Infected or inflamed muscles were dissected, homogenized, and cultured in tryptic soy agar medium. Recovered S. aureus was calculated as cfu/g. RESULTS: (68)Ga-NOTA-UBI-29-41 showed high renal excretion (83.2% ± 7.3%) of injected dose and rapid blood clearance. More than 95% was bound in vitro to 5 × 10(9) cfu/mL. A significantly higher (P< 0.05) accumulation of (68)Ga-NOTA-UBI-29-41 was observed at sites of S. aureus inoculation in infected mice (ratio of target to nontarget, 5.0 at 60 min and 4.1 at 120 min) compared with animals with inflammation (ratio of target to nontarget, 1.6 at 60 min and 1.2 at 120 min). CONCLUSION: The difference in uptake of (68)Ga-NOTA-UBI-29-41 in the infected muscles compared with the inflamed muscles was clearly observed in the PET/CT images and positively correlated with the degree of infection.