Interaction of PiB-Derivative Metal Complexes with Beta-Amyloid Peptides: Selective Recognition of the Aggregated Forms

International audienceMetal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ1–40 peptide. We have studied lanthanide(III) chelates of two PiB-derivative ligands (PiB=Pittsburgh compound B), L1 and L2, differing in the length of the spacer between the metal-complexing DO3A macrocycle (DO3A= 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) and the peptide-recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ1–40 (KD=67–160 μM), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the 15N-labeled, monomer Aβ1–40 peptide indicates nonsignificant interaction with monomeric Aβ. Time-dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ1–40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL1, at higher concentrations, stabilizes β-sheets, GdL2 prevents aggregation by promoting α-helical structures. These results give insight into the behavior of amyloid-targeted metal complexes in general and contribute to a more rational design of metal-based diagnostic and therapeutic agents for amyloid- associated pathologies

Radiolabelling diverse positron emission tomography (PET) tracers using a single digital microfluidic reactor chip

Radiotracer synthesis is an ideal application for microfluidics because only nanogram quantities are needed for positron emission tomography (PET) imaging. Thousands of radiotracers have been developed in research settings but only a few are readily available, severely limiting the biological problems that can be studied in vivo via PET. We report the development of an electrowetting-on-dielectric (EWOD) digital microfluidic chip that can synthesize a variety of (18)F-labeled tracers targeting a range of biological processes by confirming complete syntheses of four radiotracers: a sugar, a DNA nucleoside, a protein labelling compound, and a neurotransmitter. The chip employs concentric multifunctional electrodes that are used for heating, temperature sensing, and EWOD actuation. All of the key synthesis steps for each of the four (18)F-labeled tracers are demonstrated and characterized with the chip: concentration of fluoride ion, solvent exchange, and chemical reactions. The obtained fluorination efficiencies of 90-95% are comparable to, or greater than, those achieved by conventional approaches

Advances in PET Detection of the Antitumor T Cell Response

Positron emission tomography (PET) is a powerful noninvasive imaging technique able to measure distinct biological processes in vivo by administration of a radiolabeled probe. Whole-body measurements track the probe accumulation providing a means to measure biological changes such as metabolism, cell location, or tumor burden. PET can also be applied to both preclinical and clinical studies providing three-dimensional information. For immunotherapies (in particular understanding T cell responses), PET can be utilized for spatial and longitudinal tracking of T lymphocytes. Although PET has been utilized clinically for over 30 years, the recent development of additional PET radiotracers have dramatically expanded the use of PET to detect endogenous or adoptively transferred T cells in vivo. Novel probes have identified changes in T cell quantity, location, and function. This has enabled investigators to track T cells outside of the circulation and in hematopoietic organs such as spleen, lymph nodes, and bone marrow, or within tumors. In this review, we cover advances in PET detection of the antitumor T cell response and areas of focus for future studies

Synthesis and evaluation of 18F-labelled 2-phenylbenzothiazoles as tracer agents for the in vivo diagnosis of Alzheimer's disease

Table of contents Professional career Kim SERDONS i Table of contents vii List of abbreviations xiii CHAPTER I: Introduction 1. ALZHEIMER’S DISEASE 2 1.1. Introduction 2 1.1.1. Early-onset familial Alzheimer’s disease 2 1.1.2. Late-onset sporadic Alzheimer’s disease 3 1.1.3. Other types of dementia 3 1.2. Neuropathological features 4 1.2.1. Amyloid plaques 4 1.2.2. Neurofibrillary tangles 5 1.2.3. Cerebral atrophy 6 1.3. Neurochemical features 7 1.4. Pathogenesis 7 1.4.1. Amyloid cascade hypothesis 7 1.4.2. Tau hypothesis 9 1.4.3. Relation between amyloid and tau 10 1.5. Treatment 10 1.5.1. Current status: symptomatic strategies 11 1.5.2. Future: curative strategies 11 1.6. Animal models 12 2. DIAGNOSIS OF ALZHEIMER’S DISEASE 13 2.1. Clinical diagnosis 13 2.1.1. NINCDS-ADRDA criteria 14 2.1.2. MMSE test 14 2.2. Neuropathological and neurochemical diagnosis 15 2.2.1. Brain staining 15 2.2.2. SPECT and PET 15 2.2.2.1. General 15 2.2.2.2. Amyloid 17 2.2.2.3. Tau 19 2.2.2.4. Brain perfusion 19 2.2.2.5. Glucose metabolism 19 2.2.2.6. Inflammation 20 2.2.2.7. Neurochemical changes 20 2.2.3. CT and MRI 21 2.2.4. CSF and blood analysis 21 3. AIM OF THE STUDY 22 CHAPTER II: Synthesis of 18F-labelled 2-(4’-fluorophenyl)-1,3-benzothiazole and evaluation as amyloid imaging agent in comparison with [11C]PIB 1. Abstract 26 2. Key words 26 3. References and Notes 32 CHAPTER III: Synthesis and evaluation of 18F-labelled 2-phenylbenzothiazoles as positron emission tomography imaging agents for amyloid plaques in Alzheimer’s disease 1. Abstract 38 2. Introduction 38 3. Results and Discussion 40 3.1. Chemistry 40 3.2. Radiochemistry 42 3.3. Affinity 44 3.4. Distribution coefficient 45 3.5. Biodistribution in normal mice 45 3.6. µPET study in a normal rat 49 3.7. Biostability of [18F]7 in normal mice 49 3.8. Biostability of [18F]7 in brain of a normal rat 51 4. Conclusion 51 5. Experimental Section 52 5.1. Chemicals and reagents 52 5.2. Apparatus, instruments and general conditions 52 5.3. Synthesis 54 5.4. Production of [18F]fluoride and radiosynthesis of 6-methoxy-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]5a, 6-hydroxy-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]5b and 6-methyl-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]7 61 5.5. Binding studies 62 5.6. Distribution coefficient determination 63 5.7. Biodistribution in normal mice 63 5.8. µPET study in a normal rat 64 5.9. Plasma radiometabolite analysis after injection of [18F]7 in normal mice 64 5.10. Brain radiometabolite analysis after injection of [18F]7 in normal mice 65 5.11. Brain radiometabolite analysis after injection of [18F]7 in normal rat 65 6. Supporting information 66 7. References 66 CHAPTER IV: Toxicity studies, biodistribution, radiation dosimetry and clinical evaluation of the amyloid imaging agent [18F]KS28 1. Abstract 70 2. Key words 70 3. Introduction 70 4. Results 72 4.1. Radiochemistry 72 4.2. Toxicity study in rats 73 4.3. Whole-body biodistribution and radiation dosimetry 73 4.4. Comparison between [11C]PIB and [18F]KS28 in AD patients 77 5. Discussion 82 6. Conclusions 84 7. Materials and methods 85 7.1. Radiochemistry 85 7.2. Toxicity study in rats 86 7.2.1. Animals 86 7.2.2. Dose formulation and calculation and procedure of injection 86 7.2.3. Observation protocol 86 7.2.4. Clinical pathology 87 7.2.5. Histopathologic examination 87 7.2.6. Genotoxicity 87 7.3. Whole-body biodistribution and radiation dosimetry 87 7.3.1. Healthy subjects 88 7.3.2. Whole-body PET data acquisition and study protocol 88 7.3.3. Data analysis 89 7.3.4. Metabolite analysis 89 7.4. Evaluation in AD patients 89 7.4.1. AD patients 89 7.4.2. Data acquisition and study protocol 90 7.4.3. Data analysis 91 7.4.4. Metabolite analysis 91 8. Acknowledgements 92 9. References 92 CHAPTER V: Synthesis and evaluation of three 18F-labelled aminophenylbenzothiazoles as amyloid imaging agents 1. Abstract 98 2. Introduction 98 3. Results and Discussion 100 3.1. Chemistry 100 3.2. Radiochemistry 102 3.3. Affinity 103 3.4. Distribution coefficient 104 3.5. Biodistribution in normal mice 104 3.6. µPET study in a normal rat 108 3.7. µPET study in a normal rhesus monkey 109 3.8. Biostability of [18F]12, [18F]13 and [18F]16 in normal mice 112 3.9. Plasma radiometabolite analysis after injection of [18F]12, [18F]13 and [18F]16 in a normal rhesus monkey 114 4. Conclusion 115 5. Experimental Section 116 5.1. Chemicals and reagents 116 5.2. Apparatus, instruments and general conditions 117 5.3. Synthesis 118 5.4. Production of [18F]fluoride and radiosynthesis of 6-amino-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]12, 6-methylamino-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]13 and 6-dimethylamino-2-(4’-[18F]fluorophenyl)-1,3-benzothiazole [18F]16 123 5.5. Binding studies 124 5.6. Distribution coefficient determination 125 5.7. Biodistribution in normal mice 125 5.8. µPET study in a normal rat 126 5.9. µPET study in a normal rhesus monkey 126 5.10. Plasma radiometabolite analysis after injection of [18F]12, [18F]13 and [18F]16 in normal mice 127 5.11. Brain radiometabolite analysis after injection of [18F]12, [18F]13 and [18F]16 in normal mice 127 5.12. Plasma radiometabolite analysis after injection of [18F]12, [18F]13 and [18F]16 in a normal rhesus monkey 128 6. References………………………………………….…………………………………...128 Chapter VI: General discussion 1. The amyloid cascade theory: valid hypothesis or just one of the possibilities? 132 2. Usefulness of amyloid imaging in the preclinical phase of Alzheimer’s disease 134 3. Amyloid imaging in drug development 136 4. Amyloid imaging in other forms of dementia 137 5. PET using a fluorine-18 labelled tracer as an ideal non-invasive imaging technique 138 6. Promising characteristics of 18F-labelled KS-compounds 139 7. Preclinical evaluation of PET amyloid tracers in animals 144 8. Clinical evaluation of PET amyloid tracers 144 Summary 147 Samenvatting 151 References 155nrpages: 162status: publishe

Synthesis and Evaluation of Three (18)F-Labeled Aminophenylbenzothiazoles as Amyloid Imaging Agents

We have developed three fluorine-18 labeled 6-(methyl)amino-2-(4'-fluorophenyl)-1,3-benzothiazoles, which display high in vitro binding affinity for human amyloid beta plaques (K(i) < or = 10 nM). The radiolabeled probes were synthesized by aromatic nucleophilic substitution of the corresponding nitro precursor with (18)F-fluoride, followed by deprotection of the BOC group if required. Determination of the octanol/water partition coefficient, biodistribution studies in mice, and in vivo muPET studies in rats and a rhesus monkey showed that initial brain uptake was high and brain washout was fast in normal animals. Radiometabolites were quantified in plasma and brain of mice and in monkey plasma using HPLC. Of the tested compounds, [(18)F]2 (6-amino-2-(4'-[(18)F]fluorophenyl)-1,3-benzothiazole) shows the most favorable brain kinetics in mice, rats, and a monkey. Its polar plasma radiometabolites do not cross the blood-brain barrier. The preliminary results strongly suggest that this new fluorinated compound is a promising candidate as a PET brain amyloid imaging agent.status: publishe

Whole-Body Biodistribution and Radiation Dosimetry of the Cannabinoid Type 2 Receptor Ligand [(11)C]-NE40 in Healthy Subjects

PURPOSE: The type 2 cannabinoid receptor (CB2R) is part of the human endocannabinoid system and is involved in central and peripheral inflammatory processes. In vivo imaging of the CB2R would allow study of several (neuro)inflammatory disorders. In this study we have investigated the safety and tolerability of [(11)C]-NE40, a CB2R positron emission tomography (PET) ligand, in healthy human male subjects and determined its biodistribution and radiation dosimetry. PROCEDURE: Six healthy male subjects (age 20-65 years) underwent a dynamic series of nine whole-body PET/CT scans for up to 140 min, after injection of an average bolus of 286 MBq of [(11)C]-NE40. Organ absorbed and total effective doses were calculated through OLINDA. RESULTS: [(11)C]-NE40 showed high initial uptake in the spleen and a predominant hepatobiliary excretion. In the brain, rapid uptake and swift washout were seen. Organ absorbed doses were largest for the small intestine and liver, with 15.6 and 11.5 μGy/MBq, respectively. The mean effective dose was 3.64 ± 0.81 μSv/MBq. There were no changes with aging observed. No adverse events were encountered. CONCLUSIONS: This first-in-man study of [(11)C]-NE40 showed an expected biodistribution compatible with lymphoid tissue uptake and appropriate fast brain kinetics in the healthy human brain, underscoring the potential of this tracer for further application in central and peripheral inflammation imaging. The effective dose is within the typical expected range for (11)C ligands.status: publishe

Synthesis and Evaluation of F-18-Labeled 2-Phenylbenzothiazoles as Positron Emission Tomography Imaging Agents for Amyloid Plaques in Alzheimer's Disease

Imaging agents targeting amyloid beta(A beta) may be useful for early diagnosis and follow-up of treatment of patients with Alzheimer's disease (AD). Three of five tested 2-(4'-fluorophenyl)-1,3-benzothiazoles displayed high binding affinities for A beta plaques in AD human brain homogenates (K-i between 2.2 and 22.5 nM). They all contained the F-18-label directly attached to the aromatic ring and were synthesized starting from the nitro precursor. Determination of the partition coefficient, biodistribution studies in normal mice, and in vivo mu PET studies in normal rats showed that their initial brain uptake was high and brain washout was fast. The most promising compound [F-18]5, or 6- methyl-2-(4'-[F-18]fluorophenyl)-1,3-benzothiazole, seemed to be metabolically stable in the brain, and its plasma radiometabolites, which do not cross the blood-brain barrier, were determined. The preliminary results strongly suggest that this new fluorinated compound is a promising candidate as an A beta plaque imaging agent for the study of patients with AD.status: publishe

Validation of Parametric Methods for [C-11]UCB-J PET Imaging Using Subcortical White Matter as Reference Tissue

PURPOSE: The aim of this study was to evaluate different non-invasive methods for generating (R)-1-((3-([11C]methyl)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) ([11C]UCB-J) parametric maps using white matter (centrum semi-ovale-SO) as reference tissue. PROCEDURES: Ten healthy volunteers (8 M/2F; age 27.6 ± 10.0 years) underwent a 90-min dynamic [11C]UCB-J positron emission tomography (PET) scan with full arterial blood sampling and metabolite analysis before and after administration of a novel chemical entity with high affinity for presynaptic synaptic vesicle glycoprotein 2A (SV2A). A simplified reference tissue model (SRTM2), multilinear reference tissue model (MRTM2), and reference Logan graphical analysis (rLGA) were used to generate binding potential maps using SO as reference tissue (BPSO). Shorter dynamic acquisitions down to 50 min were also considered. In addition, standard uptake value ratios (SUVR) relative to SO were evaluated for three post-injection intervals (SUVRSO,40-70min, SUVRSO,50-80min, and SUVRSO,60-90min respectively). Regional parametric BPSO + 1 and SUVRSO were compared with regional distribution volume ratios of a 1-tissue compartment model (1TCM DVRSO) using Spearman correlation and Bland-Altman analysis. RESULTS: For all methods, highly significant correlations were found between regional, parametric BPSO + 1 (r = [0.63;0.96]) or SUVRSO (r = [0.90;0.91]) estimates and regional 1TCM DVRSO. For a 90-min dynamic scan, parametric SRTM2 and MRTM2 values presented similar small bias and variability (- 3.0 ± 2.9 % for baseline SRTM2) and outperformed rLGA (- 10.0 ± 5.3 % for baseline rLGA). Reducing the dynamic acquisition to 60 min had limited impact on the bias and variability of parametric SRTM2 BPSO estimates (- 1.0 ± 9.9 % for baseline SRTM2) while a higher variability (- 1.83 ± 10.8 %) for baseline MRTM2 was observed for shorter acquisition times. Both SUVRSO,60-90min and SUVRSO,50-80min showed similar small bias and variability (- 2.8 ± 4.6 % bias for baseline SUVRSO,60-90min). CONCLUSION: SRTM2 is the preferred method for a voxelwise analysis of dynamic [11C]UCB-J PET using SO as reference tissue, while reducing the dynamic acquisition to 60 min has limited impact on [11C]UCB-J BPSO parametric maps. For a static PET protocol, both SUVRSO,60-90min and SUVRSO,50-80min images are an excellent proxy for [11C]UCB-J BPSO parametric maps.status: publishe