Sangram Nag

Activated MAO-B in the brain of Alzheimer patients, demonstrated by [11C]-L-deprenyl using whole hemisphere autoradiography

In the human brain the monoaminooxidase-B enzyme or MAO-B is highly abundant in astrocytes. As astrocyte activity and, consequently, the activity of the MAO-B enzyme, is up-regulated in neuroinflammatory processes, radiolabelled analogues of deprenyl may serve as an imaging biomarker in neuroinflammation and neurodegeneration, including Alzheimers disease. In the present study [(11)C]-L-deprenyl, the PET radioligand version of L-deprenyl or selegiline®, a selective irreversible MAO-B inhibitor was used in whole hemisphere autoradiographic experiments in human brain sections in order to test the radioligands binding to the MAO-B enzyme in human brain tissue, with an eye on exploring the radioligands applicability as a molecular imaging biomarker in human PET studies, with special regard to diagnostic detection of reactive astrogliosis. Whole hemisphere brain sections obtained from Alzheimer patients and from age matched control subjects were examined. In control brains the binding of [(11)C]-L-deprenyl was the highest in the hippocampus, in the basal ganglia, the thalamus, the substantia nigra, the corpus geniculatum laterale, the nucleus accumbens and the periventricular grey matter. In Alzheimer brains significantly higher binding was observed in the temporal lobes and the white matter. Furthermore, in the Alzheimer brains in the hippocampus, temporal lobe and white matter the binding negatively correlated with Braak stages. The highest binding was observed in Braak I-II, whereas it decreased with increasing Braak grades. The increased regional binding in Alzheimer brains coincided with the presence of an increased number of activated astrocytes, as demonstrated by correlative immunohistochemical studies with GFAP in adjacent brain slices. Deprenyl itself as well as the MAO-B antagonist rasagiline did effectively block the binding of the radioligand, whereas the MAO-A antagonist pirlindole did not affect it. Compounds with high affinity for the PBR system did not block the radioligand binding either, providing evidence for the specificity of [(11)C]-L-deprenyl for the MAO-B enzyme. In conclusion, the present observations indicate that [(11)C]-L-deprenyl may be a promising and selective imaging biomarker of increased MAO-B activity in the human brain and can therefore serve as a prospective PET tracer targeting neuroinflammation and neurodegeneration.

In vivo TSPO imaging in patients with multiple sclerosis: a brain PET study with [18F]FEDAA1106

AbstractBackgroundThe activation of microglia, in general, and the upregulation of the translocator protein (18 kDa) (TSPO) system, in particular, are key features of neuroinflammation, of which the in vivo visualization and quantitative assessment are still challenging due to the lack of appropriate molecular imaging biomarkers. Recent positron emission tomography (PET) studies using TSPO radioligands such as [11C]PK11195 and [11C]PBR28 have indicated the usefulness of these PET biomarkers in patients with neuroinflammatory diseases, including multiple sclerosis (MS). [18F]FEDAA1106 is a recently developed PET radioligand for the in vivo quantification of TSPO. In the present study, we aimed at investigating the diagnostic usefulness of [18F]FEDAA1106 in patients with MS.MethodsNine patients (three on the interferon beta therapy and six without immunomodulatory therapy; seven females/two males; age 34.2 ± 9.1 years old) with relapsing-remitting MS in acute relapse and with gadolinium (Gd)-enhancing lesion(s) in the magnetic resonance imaging (MRI) scans and five healthy controls (four females/one male, age 38.0 ± 9.7 years old) were investigated in this study. Genetic information about the TSPO binding could not be obtained because knowledge about the importance of genetic background for TSPO binding was not available at the time the study was performed. Dynamic PET measurements were performed using an ECAT EXACT HR system (CTI/Siemens, Knoxville, TN, USA) for a total of 150 min, with a 30-min break after the injection of 153.4 ± 10.2 MBq of [18F]FEDAA1106. Metabolite-corrected arterial plasma samples were used to calculate the input function. PET data were analyzed in the following ways: (1) region-of-interest analysis for cortical and subcortical regions was performed using a two-tissue compartment kinetic model in order to estimate binding potentials (BPND) and distribution volume (VT), (2) the feasibility of the estimation of BPND and VT was investigated for MS lesions, and (3) VT parametric images by a Logan plot and standard uptake value (SUV) images were visually compared with the corresponding MRI, focusing on MRI-identified MS lesions.ResultsThere were no significant differences in the BPND or VT values between patients with MS and healthy controls. Robust BPND and VT values could not be obtained for most MS lesions due to noisy time-activity curves. Visual inspection of VT and SUV images in all nine patients did not reveal high uptake of the radioligand inside and beyond MRI-identified active MS lesions with the exception of one Gd-enhanced MS lesion in the whole patient population.ConclusionsIn our study, [18F]FEDAA1106 as a PET radioligand could neither differentiate patients with MS from healthy controls nor detect active plaques in the brain of MS patients. Stratification with respect to genetics and binder status might help to uncover the differences between the groups, which could not be detected here.Trial registrationClinicalTrials.gov, NCT01031199

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimers disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[(18)F]FMeNER-D(2). (S,S)-[(18)F]FMeNER-D(2) appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[(18)F]FMeNER-D(2) in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[(18)F]FMeNER-D(2) as a molecular imaging biomarker in AD.

Development of a novel fluorine-18 labeled deuterated fluororasagiline ([18F]fluororasagiline-D2) radioligand for PET studies of monoamino oxidase B (MAO-B)

The objective of this study was to synthesize and evaluate a novel fluorine-18 labeled deuterium substituted analogue of rasagiline (9, [(18)F]fluororasagiline-D2) as a potential PET radioligand for studies of monoamine oxidase B (MAO-B). The precursor compound (6) and reference standard (7) were synthesized in multi-step syntheses. Radiolabeling of 9 was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulfamidate group. The incorporation radiochemical yield from fluorine-18 fluoride was higher than 30%, the radiochemical purity was >99% and the specific radioactivity was >160GBq/μmol at the time of administration. In vitro compound 7 inhibited the MAO-B activity with an IC50 of 173.0±13.6nM. The MAO-A activity was inhibited with an IC50 of 9.9±1.1μM. The fluorine-18 version 9 was characterized in the cynomolgus monkey brain where a high brain uptake was found (275% SUV at 4min). There was a higher uptake in the striatum and thalamus compared to the cortex and cerebellum. A pronounced blocking effect (50% decrease) was observed in the specific brain regions after administration of l-deprenyl (0.5mg/kg) 30min prior to the administration of 9. Radiometabolite studies demonstrated 40% of unchanged radioligand at 90min post injection. An efficient radiolabeling of 9 was successfully established and in the monkey brain 9 binds to MAO-B rich regions and its binding is blocked by the selective MAO-B compound l-deprenyl. The radioligand 9 is a potential candidate for human PET studies.

Synthesis and evaluation of [18F]fluororasagiline, a novel positron emission tomography (PET) radioligand for monoamine oxidase B (MAO-B)

The aim of this study was to synthesize and evaluate a novel fluorine-18 labeled analogue of rasagiline (6) as a PET radioligand for monoamine oxidase B (MAO-B). The corresponding non-radioactive fluorine-19 ligand, (1S,2S)-2-fluoro-N-(prop-2-yn-1-yl)indan-1-amine (4), was characterized in in vitro assays. The precursor compound (3aS,8aR)-3-(prop-2-yn-1-yl)-3,3a,8,8a-tetrahydroindeno[1,2-d][1,2,3]oxathiazole 2,2-dioxide (3) and reference standard 4 were synthesized in multi-step syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used in order to determine IC(50) values for compound 4 by use of an enzymatic assay employing kynuramine as substrate. Radiolabeling was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulphamidate group. Human whole hemisphere autoradiography (ARG) was performed with [(18)F]fluororasagiline. Blocking experiments with pirlindole (MAO-A), L-deprenyl and rasagiline (MAO-B) were conducted to demonstrate the specificity of the binding. A positron emission tomography (PET) study was carried out in a cynomolgus monkey where time activity curves for whole brain and regions with high and low MAO-B activity were recorded. Radiometabolites were measured in monkey plasma using gradient HPLC. Compound 4 inhibited MAO-B with an IC(50) of 27 nM and MAO-A with an IC(50) of 2.3 μM. Radiolabeling of precursor 3 and subsequent hydrolysis of the protecting group towards (1S,2S)-2-[(18)F]fluoro-N-(prop-2-yn-1-yl)indan-1-amine (6) was successfully accomplished with an radiochemical yield of 40-70%, a radiochemical purity higher than 99% and a specific radioactivity higher than 200GBq/μmol. ARG demonstrated selective binding for [(18)F]fluororasagiline (6) to MAO-B containing brain regions, for example, striatum. The initial uptake in the monkey brain was 250% SUV at 4 min post injection. The highest amounts of radioactivity were observed in the striatum and thalamus as expected whereas in the cortex and cerebellum lower levels were observed. Metabolite studies demonstrated 30% unchanged radioligand at 90 min post injection. Our investigations demonstrated that the new ligand [(18)F]fluororasagiline (6) binds specifically to MAO-B in vitro and has a MAO-B specific binding pattern in vivo. Thus, it could serve as a novel potential candidate for human PET studies.

Synthesis of Three Novel Fluorine-18 Labeled Analogues of l-Deprenyl for Positron Emission Tomography (PET) studies of Monoamine Oxidase B (MAO-B)

The aim in this project was to synthesize and to study fluorine-18 labeled analogues of l-deprenyl which bind selectively to the enzyme monoamine oxidase B (MAO-B). Three fluorinated l-deprenyl analogues have been generated in multistep organic syntheses. The most promising fluorine-18 compound N-[(2S)-1-[(18)F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography on human brain tissue sections demonstrated specific binding for compound 4c to brain regions known to have a high content of MAO-B. In addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited recombinant human MAO-B with an IC(50) of 170.5 ± 29 nM but did not inhibit recombinant human MAO-A (IC(50) > 2000 nM), demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ± 0.04%ID/g after 2 min post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a candidate for further investigation in PET.

Quantitative analysis of [18F]FE-PE2I binding to the dopamine transporter in Parkinson’s disease

18F-(E)-N-(3-iodoprop-2-enyl)-2β-carbofluoroethoxy-3β-(4′-methyl-phenyl) nortropane (18F-FE-PE2I) is a recently developed radioligand for the in vivo quantification of the dopamine transporter (DAT) in the striatum and substantia nigra (SN). The aim of this study was to examine the suitability of 18F-FE-PE2I as a tool for imaging the nigrostriatal pathway in Parkinson disease (PD) with PET. Methods: Ten PD patients (9 men and 1 woman; mean age ± SD, 60 ± 9 y; Hoehn and Yahr, 1–2; Unified Parkinson Disease Rating Scale motor, 18.9 ± 6.7) and 10 controls (9 men and 1 woman; mean age ± SD, 60 ± 7 y) were included. PET measurements with 18F-FE-PE2I were conducted for 93 min using the High-Resolution Research Tomograph. Venous blood was drawn to compare protein binding, parent fraction, and radiometabolite composition in PD patients and controls. Regions of interest for the caudate, putamen, ventral striatum, SN, and cerebellum were drawn on coregistered MR images. The outcome measure was the binding potential (BPND) estimated with the simplified reference tissue model and the Logan graphical analysis, using the cerebellum as a reference region. Time stability of BPND was examined to define the shortest acquisition protocol for quantitative studies. The wavelet-aided parametric imaging method was used to obtain high-resolution BPND images to compare DAT availability in the striatum and SN in PD patients and control subjects. Group differences were assessed with the unpaired t test (P < 0.05). Results: Parent, radiometabolite fractions, plasma concentration, and cerebellar uptake of 18F-FE-PE2I did not differ significantly between PD patients and controls. Stable estimates of BPND (<8% of the 93-min value) were obtained with the simplified reference tissue model using approximately 66 min of data. BPND values in PD patients were significantly lower than those in controls (P < 0.05) in the caudate (2.54 ± 0.79 vs. 3.68 ± 0.56), putamen (1.39 ± 1.04 vs. 4.41 ± 0.54), ventral striatum (2.26 ± 0.93 vs. 3.30 ± 0.46), and SN (0.46 ± 0.20 vs. 0.68 ± 0.15). Conclusion: 18F-FE-PE2I is clearly a suitable radioligand for DAT quantification and imaging of the nigrostriatal pathway in PD. Similar metabolism in controls and PD patients, suitability of the cerebellum as a reference region, and accuracy of quantification using approximately 66 min of PET data are advantages for noninvasive and simplified imaging protocols for PD studies. Finally, DAT loss in PD can be measured in both the striatum and the SN, supporting the utility of 18F-FE-PE2I as an imaging tool of the nigrostriatal pathway.

In vivo evaluation in cynomolgus monkey brain and metabolism of [18F]fluorodeprenyl: A new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO‐B) radioligand, [18F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [18F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [11C]deprenyl, an established MAO‐B radioligand. The highest uptake was observed in the striatum, one of the MAO‐B‐rich regions, with a peak at approximately 2–3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [18F]fluorodeprenyl was largely inhibited by preadministration of the MAO‐B inhibitor, L‐deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO‐B. [18F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [11C]deprenyl. These results suggest that [18F]fluorodeprenyl is a potential PET radioligand for visualization of MAO‐B activity. Synapse, 2012.

Comparative binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains

BackgroundThe aim of this study was to compare the binding properties of several tau positron emission tomography tracers—THK5117, THK5351, T807 (also known as AV1451; flortaucipir), and PBB3—head to head in the same human brain tissue.MethodsBinding assays were performed to compare the regional distribution of 3H-THK5117 and 3H-THK5351 in postmortem tissue from three Alzheimer’s disease (AD) cases and three control subjects in frontal and temporal cortices as well as in the hippocampus. Competition binding assays between THK5351, THK5117, PBB3, and T807, as well as off-target binding of THK5117 and T807 toward monoamine oxidase B (MAO-B), were performed using binding assays in brain homogenates and autoradiography of three AD cases.ResultsRegional binding of 3H-THK5117 and 3H-THK5351 was similar, except in the temporal cortex, which showed higher 3H-THK5117 binding. Saturation studies demonstrated two binding sites for 3H-THK5351 (Kd1 = 5.6 nM, Bmax = 76 pmol/g; Kd2 = 1 nM, Bmax = 40 pmol/g). Competition studies in the hippocampus between 3H-THK5351 and unlabeled THK5351, THK5117, and T807 revealed super-high-affinity sites for all three tracers (THK5351 Ki = 0.1 pM; THK5117 Ki = 0.3 pM; T807 Ki = 0.2 pM) and an additional high-affinity site (THK5351 Ki = 16 nM; THK5117 Ki = 20 nM; T807 Ki = 78nM). 18F-T807, 11C-THK5351, and 11C-PBB3 autoradiography of large frozen sections from three AD brains showed similar regional binding for the three tracers, with lower binding intensity for 11C-PBB3. Unlabeled THK5351 and T807 displaced 11C-THK5351 to a similar extent and a lower extent, respectively, compared with 11C-PBB3. Competition with the MAO-B inhibitor 3H-l-deprenyl was observed for THK5117 and T807 in the hippocampus (THK5117 Ki = 286 nM; T807 Ki = 227 nM) and the putamen (THK5117 Ki = 148 nM; T807 Ki = 135 nM). 3H-THK5351 binding was displaced using autoradiography competition with unlabeled THK5351 and T807 in cortical areas by 70–80% and 60–77%, respectively, in the basal ganglia, whereas unlabeled deprenyl displaced 3H-THK5351 binding by 40% in the frontal cortex and 50% in the basal ganglia.ConclusionsTHK5351, THK5117, and T807 seem to target similar binding sites, but with different affinities, whereas PBB3 seems to target its own binding site. Both THK5117 and T807 demonstrated off-target binding in the hippocampus and putamen with a ten times lower binding affinity to the MAO-B inhibitor deprenyl compared with 3H-THK5351.

Synthesis and biological evaluation of novel propargyl amines as potential fluorine-18 labeled radioligands for detection of MAO-B activity

The aim of this project was to synthesize and evaluate three novel fluorine-18 labeled derivatives of propargyl amine as potential PET radioligands to visualize monoamine oxidase B (MAO-B) activity. The three fluorinated derivatives of propargyl amine ((S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)-pent-4-en-2-amine (5), (S)-N-(1-fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (10) and (S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (15)) were synthesized in multi-step organic syntheses. IC(50) values for inhibition were determined for compounds 5, 10 and 15 in order to determine their specificity for binding to MAO-B. Compound 5 inhibited MAO-B with an IC(50) of 664 ± 48.08 nM. No further investigation was carried out with this compound. Compound 10 inhibited MAO-B with an IC(50) of 208.5 ± 13.44 nM and compound 15 featured an IC(50) of 131.5 ± 0.71 nM for its MAO-B inhibitory activity. None of the compounds inhibited MAO-A activity (IC(50) > 2 μM). The fluorine-18 labeled analogues of the two higher binding affinity compounds (10 and 15) (S)-N-(1-[(18)F]fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (16) and (S)-1-[(18)F]fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (18) were both prepared from the corresponding precursors 9A, 9B and 14A, 14B by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography experiments on human postmortem brain tissue sections were performed with 16 and 18. Only compound 18 demonstrated a high selectivity for MAO-B over MAO-A and was, therefore, chosen for further examination by PET in a cynomolgus monkey. The initial uptake of 18 in the monkey brain was 250% SUV at 4 min post injection. The highest uptake of radioactivity was observed in the striatum and thalamus, regions with high MAO-B activity, whereas lower levels of radioactivity were detected in the cortex and cerebellum. The percentage of unchanged radioligand 18 was 30% in plasma at 90min post injection. In conclusion, compound 18 is a selective inhibitor of MAO-B in vitro and demonstrated a MAO-B specific binding pattern in vivo by PET in monkey. It can, therefore, be considered as a candidate for further investigation in human by PET.