Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases.

The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 µg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.

Guidance on current good radiopharmacy practice for the small-scale preparation of radiopharmaceuticals using automated modules: a European perspective.

This document is meant to complement Part B of the EANM 'Guidelines on current good radiopharmacy practice (cGRPP) in the preparation of radiopharmaceuticals' issued by the Radiopharmacy Committee of the European Association of Nuclear Medicine, covering small-scale in-house preparation of radiopharmaceuticals with automated modules. The aim is to provide more detailed and practice-oriented guidance to those who are involved in the small-scale preparation of radiopharmaceuticals, which are not intended for commercial purposes or distribution.

Are spatial memories strengthened in the human hippocampus during slow wave sleep?

In rats, the firing sequences observed in hippocampal ensembles during spatial learning are replayed during subsequent sleep, suggesting a role for posttraining sleep periods in the offline processing of spatial memories. Here, using regional cerebral blood flow measurements, we show that, in humans, hippocampal areas that are activated during route learning in a virtual town are likewise activated during subsequent slow wave sleep. Most importantly, we found that the amount of hippocampal activity expressed during slow wave sleep positively correlates with the improvement of performance in route retrieval on the next day. These findings suggest that learning-dependent modulation in hippocampal activity during human sleep reflects the offline processing of recent episodic and spatial memory traces, which eventually leads to the plastic changes underlying the subsequent improvement in performance.

Cyclosporine, a P-glycoprotein modulator, increases [18F]MPPF uptake in rat brain and peripheral tissues: microPET and ex vivo studies.

PURPOSE: Pretreatment with cyclosporine, a P-glycoprotein (P-gp) modulator increases brain uptake of 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethylpiperazine ([(18)F]MPPF) for binding to hydroxytryptamine(1A) (5-HT(1A)) receptors. Those increases were quantified in rat brain with in vivo microPET and ex vivo tissue studies. MATERIALS AND METHODS: Each Sprague-Dawley rat (n = 4) received a baseline [(18)F]MPPF microPET scan followed by second scan 2-3 weeks later that included cyclosporine pretreatment (50 mg/kg, i.p.). Maximum a posteriori reconstructed images and volumetric ROIs were used to generate dynamic radioactivity concentration measurements for hippocampus, striatum, and cerebellum, with simplified reference tissue method (SRTM) analysis. Western blots were used to semiquantify P-gp regional distribution in brain. RESULTS: MicroPET studies showed that hippocampus uptake of [(18)F]MPPF was increased after cyclosporine; ex vivo studies showed similar increases in hippocampus and frontal cortex at 30 min, and for heart and kidney at 2.5 and 5 min, without concomitant increases in [(18)F]MPPF plasma concentration. P-gp content in cerebellum was twofold higher than in hippocampus or frontal cortex. CONCLUSIONS: These studies confirm and extend prior ex vivo results (J. Passchier, et al., Eur J Pharmacol, 2000) that showed [(18)F]MPPF as a substrate for P-gp. Our microPET results showed that P-gp modulation of [(18)F]MPPF binding to 5-HT(1A) receptors can be imaged in rat hippocampus. The heterogeneous brain distribution of P-gp appeared to invalidate the use of cerebellum as a nonspecific reference region for SRTM modeling. Regional quantitation of P-gp may be necessary for accurate PET assessment of 5-HT(1A) receptor density when based on tracer uptake sensitive to P-gp modulation.

Thrombolytic therapy based on fucoidan-functionalized polymer nanoparticles targeting P-selectin.

Injection of recombinant tissue plasminogen activator (rt-PA) is the standard drug treatment for thrombolysis. However, rt-PA shows risk of hemorrhages and limited efficiency even at high doses. Polysaccharide-poly(isobutylcyanoacrylate) nanoparticles functionalized with fucoidan and loaded with rt-PA were designed to accumulate on the thrombus. Fucoidan has a nanomolar affinity for the P-selectin expressed by activated platelets in the thrombus. Solid spherical fluorescent nanoparticles with a hydrodynamic diameter of 136 ± 4 nm were synthesized by redox radical emulsion polymerization. The clinical rt-PA formulation was successfully loaded by adsorption on aminated nanoparticles and able to be released in vitro. We validated the in vitro fibrinolytic activity and binding under flow to both recombinant P-selectin and activated platelet aggregates. The thrombolysis efficiency was demonstrated in a mouse model of venous thrombosis by monitoring the platelet density with intravital microscopy. This study supports the hypothesis that fucoidan-nanoparticles improve the rt-PA efficiency. This work establishes the proof-of-concept of fucoidan-based carriers for targeted thrombolysis.

Silver Nanoparticles Impair Retinoic Acid-Inducible Gene I-Mediated Mitochondrial Antiviral Immunity by Blocking the Autophagic Flux in Lung Epithelial Cells.

Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as an alternative to antivirals to treat human infectious diseases, especially influenza virus infections where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of an influenza virus infection of lung epithelial cells, that AgNPs down-regulated influenza induced CCL-5 and -IFN-ß release (two cytokines important in antiviral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the antiviral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent antiviral responses and up-regulation of IL-8-dependent antibacterial responses) may have practical implications for their use in the clinic.

PET/CT of skull base meningiomas using 2-18F-fluoro-L-tyrosine: initial report.

UNLABELLED: Precise delineation of the shape of skull base meningiomas is critical for their treatment and follow-up but is often difficult using conventional imaging such as CT and MRI. We report our results with PET/CT and 2-(18)F-fluoro-L-tyrosine ((18)F-TYR), a marker of amino acid transport, as part of the yearly follow-up of irradiated patients. METHODS: Eleven patients (mean age, 56.5 y) with skull base meningiomas (n=13 lesions) previously irradiated were included. All patients received 300 MBq of (18)F-TYR and were imaged after 30 min of uptake, using a dedicated PET/CT system. The images were first visually examined, and regions of interest (ROI) were then placed over the transaxial PET slice showing the highest uptake. Another ROI was placed over the normal parietal cortex. Tumor-to-cortex activity ratios were obtained by dividing the maximum pixel value in the tumor ROI by the maximum pixel value in the cortex ROI. The PET/CT images were compared with the MR images obtained as part of routine follow-up. RESULTS: Accumulation of the tracer was higher in all meningiomas than in the surrounding tissue. The tumor-to-cortex activity ratio was 2.53 +/- 0.35 (range, 1.3-6). Nonneoplastic tissue such as hyperemic cavernous sinus did not take up the radionuclide and was therefore easily distinguished from the meningioma. The (18)F-TYR anomalies completely overlapped with the MR image in 54% of the tumors, extended beyond the MRI lesion in 38% of the tumors, and were smaller in 8% of the tumors. CONCLUSION: Meningiomas of the skull base are clearly visualized using (18)F-TYR PET/CT, even after irradiation. In addition to MRI, (18)F-TYR PET/CT images may contribute to the evaluation, delineation, and follow-up of these tumors.

Measuring brain synaptic vesicle protein 2A with positron emission tomography and [18F]UCB-H.

INTRODUCTION: Brain distribution of synaptic vesicle protein 2A was measured with fluorine-18 UCB-H ([18F]UCB-H) and positron emission tomography (PET). METHODS: Images of synaptic density were acquired in healthy volunteers (two young participants and two seniors). Input function was measured by arterial blood sampling (arterial input function) and derived from PET images using carotid activity (image-derived input function). Logan graphical analysis was used to estimate regional synaptic vesicle protein 2A distribution volume. RESULTS: [18F]UCB-H uptake was ubiquitous in cortical and subcortical gray matter. Arterial input function and image-derived input function provided regional distribution volume with a high linear relationship. DISCUSSION: The cerebral distribution of [18F]UCB-H is similar to that recently observed with carbon-11 UCB-J ([11C]UCB-J). An accurate [18F]UCB-H quantification can be performed without invasive arterial blood sampling when no suitable reference region is available, using dynamic PET carotid activity. Brain synaptic density can be studied in vivo in normal and pathological aging.

Enabling Efficient Positron Emission Tomography (PET) Imaging of Synaptic Vesicle Glycoprotein 2A (SV2A) with a Robust and One-Step Radiosynthesis of a Highly Potent 18F-Labeled Ligand ([18F]UCB-H).

We herein describe the straightforward synthesis of a stable pyridyl(4-methoxyphenyl)iodonium salt and its [18F] radiolabeling within a one-step, fully automated and cGMP compliant radiosynthesis of [18F]UCB-H ([18F]7), a PET tracer for the imaging of synaptic vesicle glycoprotein 2A (SV2A). Over the course of 1 year, 50 automated productions provided 34 ± 2% of injectable [18F]7 from up to 285 GBq (7.7 Ci) of [18F]fluoride in 50 min (uncorrected radiochemical yield, specific activity of 815 ± 185 GBq/µmol). The successful implementation of our synthetic strategy within routine, high-activity, and cGMP productions attests to its practicality and reliability for the production of large doses of [18F]7. In addition to enabling efficient and cost-effective clinical research on a range of neurological pathologies through the imaging of SV2A, this work further demonstrates the real value of iodonium salts for the cGMP 18F-PET tracer manufacturing industry, and their ability to fulfill practical and regulatory requirements in that field.

Whole-body tumor imaging using PET and 2-18F-fluoro-L-tyrosine: preliminary evaluation and comparison with 18F-FDG.

UNLABELLED: 18F-FDG PET imaging is now established as a valuable tool for evaluating cancer patients. However, a limitation of (18)F-FDG is its absence of specificity for tumor. Both protein synthesis and amino acid transport are enhanced in most tumor cells, but their metabolism is less affected in inflammation. We therefore decided to evaluate the ability of PET with 2-(18)F-fluoro-L-tyrosine ((18)F-TYR) to visualize cancer lesions in patients compared with (18)F-FDG PET. METHODS: (18)F-FDG PET and (18)F-TYR PET were performed on 23 patients with histologically proven malignancies (11 non-small cell lung cancers (NSCLCs), 10 lymphomas, and 2 head and neck carcinomas). Fully corrected, whole-body PET studies were obtained on separate days. (18)F-FDG studies were performed after routine clinical fashion. (18)F-TYR studies were started 36 +/- 6 min after tracer injection and a second scan centered over a reference lesion was acquired after completion of the whole-body survey-on average, 87 min after injection. Standardized uptake values (SUVs) were calculated for all abnormal foci and for various normal structures. Results were compared with pathologic or correlative studies. RESULTS: (18)F-FDG PET correctly identified 54 malignant lesions, among which 36 were also visualized with (18)F-TYR (67%). (18)F-TYR did not detect any additional lesion. Tumor SUVs (SUV(bw), 5.2 vs. 2.5), tumor-to-muscle (7.4 vs. 2.7), and tumor-to-mediastinum activity ratios (3 vs. 1.4) were higher with (18)F-FDG than with (18)F-TYR. Two of 11 NSCLCs and 4 of 10 lymphomas were understaged with (18)F-TYR compared with (18)F-FDG. Although the NSCLC lesions missed by (18)F-TYR PET were small, several large lymphoma lesions did not accumulate the tracer. In 4 patients, (18)F-TYR-positive lesions coexisted with (18)F-TYR-negative lesions. There was a high physiologic (18)F-TYR uptake by the pancreas (average SUV(bw), 10.3) and the liver (average SUV(bw), 6.3). Muscle and bone marrow uptakes were also higher with (18)F-TYR than with (18)F-FDG: average SUV(bw), 1 versus 0.7 and 2.6 versus 1.8, respectively. There was no change over time in the (18)F-TYR uptake by the tumors or the normal structures. CONCLUSION: (18)F-TYR PET is not superior to (18)F-FDG PET for staging patients with NSCLC and lymphomas.

Cerebral correlates of explicit sequence learning.

Using positron emission tomography (PET) and regional cerebral blood flow (rCBF) measurements, we investigated the cerebral correlates of consciousness in a sequence learning task through a novel application of the Process Dissociation Procedure, a behavioral paradigm that makes it possible to separately assess conscious and unconscious contributions to performance. Results show that the metabolic response in the anterior cingulate/mesial prefrontal cortex (ACC/MPFC) is exclusively and specifically correlated with the explicit component of performance during recollection of a learned sequence. This suggests a significant role for the ACC/MPFC in the explicit processing of sequential material.

Brain function in the vegetative state.

Positron emission tomography (PET) techniques represent a useful tool to better understand the residual brain function in vegetative state patients. It has been shown that overall cerebral metabolic rates for glucose are massively reduced in this condition. However, the recovery of consciousness from vegetative state is not always associated with substantial changes in global metabolism. This finding led us to hypothesize that some vegetative patients are unconscious not just because of a global loss of neuronal function, but rather due to an altered activity in some critical brain regions and to the abolished functional connections between them. We used voxel-based Statistical Parametric Mapping (SPM) approaches to characterize the functional neuroanatomy of the vegetative state. The most dysfunctional brain regions were bilateral frontal and parieto-temporal associative cortices. Despite the metabolic impairment, external stimulation still induced a significant neuronal activation (i.e., change in blood flow) in vegetative patients as shown by both auditory click stimuli and noxious somatosensory stimuli. However, this activation was limited to primary cortices and dissociated from higher-order associative cortices, thought to be necessary for conscious perception. Finally, we demonstrated that vegetative patients have impaired functional connections between distant cortical areas and between the thalami and the cortex and, more importantly, that recovery of consciousness is paralleled by a restoration of this cortico-thalamo-cortical interaction.

Evaluation of 18F-UCB-H as a novel PET tracer for synaptic vesicle protein 2A in the brain.

UNLABELLED: Synaptic vesicle protein 2 isoforms are critical for proper nervous system function and are involved in vesicle trafficking. The synaptic vesicle protein 2A (SV2A) isoform has been identified as the binding site of the antiepileptic levetiracetam (LEV), making it an interesting therapeutic target for epilepsy. (18)F-UCB-H is a novel PET imaging agent with a nanomolar affinity for human SV2A. METHODS: Preclinical PET studies were performed with isoflurane-anesthetized rats. The arterial input function was measured with an arteriovenous shunt and a ß-microprobe system. (18)F-UCB-H was injected intravenously (bolus of 140 ± 20 MBq). RESULTS: Brain uptake of (18)F-UCB-H was high, matching the expected homogeneous distribution of SV2A. The distribution volume (Vt) for (18)F-UCB-H was calculated with Logan graphic analysis, and the effect of LEV pretreatment on Vt was measured. In control animals the whole-brain Vt was 9.76 ± 0.52 mL/cm(3) (mean ± SD; n = 4; test-retest), and the reproducibility in test-retest studies was 10.4% ± 6.5% (mean ± SD). The uptake of (18)F-UCB-H was dose dependently blocked by pretreatment with LEV (0.1-100 mg/kg intravenously). CONCLUSION: Our results indicated that (18)F-UCB-H is a suitable radiotracer for the imaging of SV2A in vivo. To our knowledge, this is the first PET tracer for the in vivo quantification of SV2A. The necessary steps for the implementation of (18)F-UCB-H production under good manufacturing practice conditions and the first human studies are being planned.

Evaluation of an unshielded luminescence flow-through radio-HPLC detector for LC quality control and preparation of PET radiopharmaceuticals.

Radio-HPLC is an essential method to assess the purity of PET radiopharmaceuticals. The usual NaI scintillator radiodetector requires heavy, costly and cumbersome lead shielding. The luminescence LB 500 fLumo detector has been developed to tackle these drawbacks and achieve high sensitivity. The fLumo uses a photon counting detector combined with a flow-through cell modified with a solid melt-on scintillator only sensitive to the positron. This study demonstrates the usefulness of the fLumo for analysis and purification of PET radiopharmaceuticals.

Preclinical radiation dosimetry for the novel SV2A radiotracer [18F]UCB-H.

BACKGROUND: [18F]UCB-H was developed as a novel radiotracer with a high affinity for synaptic vesicle protein 2A, the binding site for the antiepileptic levetiracetam. The objectives of this study were to evaluate the radiation dosimetry of [18F]UCB-H in a preclinical trial and to determine the maximum injectable dose according to guidelines for human biomedical research. The radiation dosimetry was derived by organ harvesting and dynamic micro positron emission tomography (PET) imaging in mice, and the results of both methods were compared. METHODS: Twenty-four male C57BL-6 mice were injected with 6.96 ± 0.81 MBq of [18F]UCB-H, and the biodistribution was determined by organ harvesting at 2, 5, 10, 30, 60, and 120 min (n = 4 for each time point). Dynamic microPET imaging was performed on five male C57BL-6 mice after the injection of 9.19 ± 3.40 MBq of [18F]UCB-H. A theoretical dynamic bladder model was applied to simulate urinary excretion. Human radiation dose estimates were derived from animal data using the International Commission on Radiological Protection 103 tissue weighting factors. RESULTS: Based on organ harvesting, the urinary bladder wall, liver and brain received the highest radiation dose with a resulting effective dose of 1.88E-02 mSv/MBq. Based on dynamic imaging an effective dose of 1.86E-02 mSv/MBq was calculated, with the urinary bladder wall and liver (brain was not in the imaging field of view) receiving the highest radiation. CONCLUSIONS: This first preclinical dosimetry study of [18F]UCB-H showed that the tracer meets the standard criteria for radiation exposure in clinical studies. The dose-limiting organ based on US Food and Drug Administration (FDA) and European guidelines was the urinary bladder wall for FDA and the effective dose for Europe with a maximum injectable single dose of approximately 325 MBq was calculated. Although microPET imaging showed significant deviations from organ harvesting, the Pearson's correlation coefficient between radiation dosimetry derived by either method was 0.9666.

Fully automated preparation and conjugation of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) with RGD peptide using a GE FASTlab™ synthesizer.

PURPOSE: The aim of this work was to automate the radiosynthesis of [(18)F]SFB, a widely used reagent for the labeling of biomolecules with (18)F on a new generation commercial synthesis module (FASTLab™, GE Healthcare). PROCEDURES: Two synthesis approaches were implemented on this module: the classical "two-pot radiosynthesis" and the more recently described "one-pot" method. RESULTS: The "two-pot" approach affords [(18)F]SFB with a 42% decay-corrected yield in 57 min (n = 24) with a chemical purity sufficient to avoid an intermediate HPLC purification. The recently established "one-pot" method, afforded a product with a lower chemical purity, in the conditions used in this report. The lower d.c. yield obtained (32% (n = 15)) was related to the low (18)F labeling yields obtained in MeCN compared with DMSO. The subsequent conjugation step with a RGD (PRGD2) peptide was also successfully automated. CONCLUSIONS: The formulated [(18)F]FPRGD2 was obtained without any operator manipulation with a d.c. yield of 13% ± 3% (n = 13) in 130 min, a radiochemical purity >98% and a specific activity of 140 ± 40 TBq/mmol.

Use of a beta microprobe system to measure arterial input function in PET via an arteriovenous shunt in rats.

BACKGROUND: Kinetic modeling of physiological function using imaging techniques requires the accurate measurement of the time-activity curve of the tracer in plasma, known as the arterial input function (IF). The measurement of IF can be achieved through manual blood sampling, the use of small counting systems such as beta microprobes, or by derivation from PET images. Previous studies using beta microprobe systems to continuously measure IF have suffered from high background counts. METHODS: In the present study, a light-insensitive beta microprobe with a temporal resolution of up to 1 s was used in combination with a pump-driven femoral arteriovenous shunt to measure IF in rats. The shunt apparatus was designed such that the placement of the beta microprobe was highly reproducible. The probe-derived IF was compared to that obtained from manual sampling at 5-s intervals and IF derived from a left ventricle VOI in a dynamic PET image of the heart. RESULTS: Probe-derived IFs were very well matched to that obtained by "gold standard" manual blood sampling, but with an increased temporal resolution of up to 1 s. The area under the curve (AUC) ratio between probe- and manually derived IFs was 1.07 ± 0.05 with a coefficient of variation of 0.04. However, image-derived IFs were significantly underestimated compared to the manually sampled IFs, with an AUC ratio of 0.76 ± 0.24 with a coefficient of variation of 0.32. CONCLUSIONS: IF derived from the beta microprobe accurately represented the IF as measured by blood sampling, was reproducible, and was more accurate than an image-derived technique. The use of the shunt removed problems of tissue-background activity, and the use of a light-tight probe with minimal gamma sensitivity refined the system. The probe/shunt apparatus can be used in both microprobe and PET studies.

¹8F-fluoride PET/CT for assessing bone involvement in prostate and breast cancers.

OBJECTIVE: To evaluate the accuracy of ¹8F-fluoride PET/computed tomography (CT) to detect bone metastases (BMs) in a breast and prostate cancer population, using magnetic resonance imaging (MRI) or thin-slice CT as a gold standard. METHODS: We have prospectively included 34 patients with breast (N=24) or prostate cancer (N=10) at high risk of BMs. Whole-body PET/CT (low-dose CT) and bone scintigraphy (BS) with single photon emission CT were obtained for all 34 patients and the results compared with a radiological gold standard. RESULTS: Out of the 386 foci detected by PET/CT, 219 (56.7%) could be verified by CT or MRI. Eighty-six additional foci were detected by BS (n=46) or seen only by CT (n=9), MRI (n=23), or both CT and MRI (n=8). The total number of verified lesions was therefore 274 (58.1%), including 119 (43.4%) benign and 155 (56.6%) BM. The sensitivity, specificity, and accuracy of ¹8F-fluoride PET/CT were 76, 84.2, and 80%, respectively. For BS, they were 44.8, 79.2, and 60%, respectively. Sensitivity significantly decreased for the lytic lesions. The accuracy of PET/CT was significantly superior to BS for pelvic and lumbar lesions. PET/CT provided a correct diagnosis (M+/M0) in 32 of 33 patients (one false positive) compared with 28 of 33 with BS (four false positive, one false positive). CONCLUSION: ¹8F-fluoride PET/CT is significantly more accurate than BS for detecting BMs from breast and prostate cancers.