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It is widely accepted that the world is facing increasing risk of environmental emergency yet at the same time, there is also increasing vulnerability.

The first two models of the Sea and Land Surface Temperature Radiometers (SLSTR) for the European Copernicus Sentinel-3 missions were tested prior to launch at the Rutherford Appleton Laboratory space instrument calibration facility. The pre-launch tests provide an essential reference that ensures that the flight data of SLSTR are calibrated to the same standards needed for surface temperature measurements and to those used by shipborne radiometers for Fiducial Reference Measurement (FRM). The radiometric calibrations of the thermal infrared channels were validated against accurate and traceable reference BB sources under flight representative thermal vacuum environment. Measurements were performed in both earth views for source temperatures covering the main operating range, for different instrument configurations and for the full field-of-view of the instruments. The data were used to derive non-linearity curves to be used in the level-1 processing. All results showed that the measured brightness temperatures and radiometric noise agreed within the requirements for the mission. An inconsistency that particularly affected SLSTR-A was observed which has been attributed to an internal stray light error. A correction for the stray light has been proposed to reduce the error. The internal stray light error was reduced for SLSTR-B by replacing the coating on the main aperture stop. We present a description of the test methodology and the key results.

Introduction Several psychiatric and neurodegenerative diseases are associated with malfunction of brain norepinephrine transporter (NET). However, current clinical evaluations of NET function are limited by the lack of sufficiently sensitive methods of detection. To this end, we have synthesized exo-3-[(6-[18F]fluoro-2-pyridyl)oxy]-8-azabicyclo[3.2.1]-octane ([18F]NS12137) as a radiotracer for positron emission tomography (PET) and have demonstrated that it is highly specific for in vivo detection of NET-rich regions of rat brain tissue. Methods We applied two methods of electrophilic, aromatic radiofluorination of the precursor molecule, exo-3-[(6-trimethylstannyl-2-pyridyl)oxy]-8-azabicyclo-[3.2.1]octane-8-carboxylate: (1) direct labeling with [18F]F2, and (2) labeling with [18F]Selectfluor, a derivative of [18F]F2, using post-target produced [18F]F2. The time-dependent distribution of [18F]NS12137 in brain tissue of healthy, adult Sprague–Dawley rats was determined by ex vivo autoradiography. The specificity of [18F]NS12137 binding was demonstrated on the basis of competitive binding by nisoxetine, a known NET antagonist of high specificity. Results [18F]NS12137 was successfully synthesized with radiochemical yields of 3.9% ± 0.3% when labeled with [18F]F2 and 10.2% ± 2.7% when labeled with [18F]Selectfluor. The molar activity of radiotracer was 8.8 ± 0.7 GBq/μmol with [18F]F2 labeling and 6.9 ± 0.4 GBq/μmol with [18F]Selectfluor labeling at the end of synthesis of [18F]NS12137. Uptake of [18F]NS12137 in NET-rich areas in rat brain was demonstrated with the locus coeruleus (LCoe) having the highest regional uptake. Prior treatment of rats with nisoxetine showed no detectable [18F]NS12137 in the LCoe. Analyses of whole brain samples for radiometabolites showed only the parent compound [18F]NS12137. Uptake of 18F-radioactivity in bone increased with time. Conclusions The two electrophilic 18F-labeling methods proved to be suitable for synthesis of [18F]NS12137 with the [18F]Selectfluor method providing an approximate three-fold higher yield than the [18F]F2 method. As an electrostatically neutral radiotracer [18F]NS12137 crosses the blood–brain barrier and enabled specific labeling of NET-rich regions of rat brain tissue with the highest concentration in the LCoe.

Providing uncertainties in satellite datasets used for Earth observation can be a daunting prospect because of the many processing stages and input data required to convert raw detector counts to calibrated radiances. The Sea and Land Surface Temperature Radiometer (SLSTR) was designed to provide measurements of the Earth’s surface for operational and climate applications. In this paper the authors describe the traceability chain and derivation of uncertainty estimates for the thermal infrared channel radiometry. Starting from the instrument model, the contributing input quantities are identified to build up an uncertainty effects tree. The characterisation of each input effect is described, and uncertainty estimates provided which are used to derive the combined uncertainties as a function of scene temperature. The SLSTR Level-1 data products provide uncertainty estimates for fully random effects (noise) and systematic effects that can be mapped for each image pixel, examples of which are shown.

The &alpha, 7 nicotinic acetylcholine receptor (&alpha, 7 nAChR) is involved in several cognitive and physiologic processes, its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer&rsquo, s disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]&alpha, bungarotoxin, specific binding was absent in &alpha, 7 nAChR gene-deficient mice and binding was blocked by a range of &alpha, 7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in &beta, 2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% &plusmn, 2.7%) than [125I]&alpha, bungarotoxin (23% &plusmn, 0.2%), potentially indicating different binding properties to heteromeric &alpha, 7&beta, 2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for &alpha, 7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of &alpha, 7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of &alpha, 7 nAChR, comparable to previously published data.

Introduction The α7 nicotinic acetylcholine receptor (α7nAChR) has been previously shown to have an essential role in angiogenesis1. A number of positron emission tomography (PET) radiotracers, such as18F-NS144902, are now available for the non-invasive imaging of α7nAChR. This highly sensitive imaging approach has the potential to be a powerful tool to study the angiogenic process in a number of pathologies. However, as part of the validation of this target for angiogenesis, a better understanding of the α7nAChR in vascular cells under angiogenic conditions is needed. Objective This study aims to investigate the in vitro expression of α7nAChR in human vascular-associated cells under angiogenic conditions in order to evaluate its suitability as a marker for angiogenesis. Methods Various human cell types were cultured, Human Umbilical Vein Endothelial cells (HUVEC), THP-1 derived Macrophages (PMA stimulated for 72 hour), Human Cardiac Microvascular Endothelial cells (CMVEC), Human Coronary Artery Smooth Muscle cells (CVSMC). Sub-confluent cultures were exposed to hypoxia (1% O2) over a time course (0–16 hours). The protein content of lysed cells was quantified by spectrophotometry before western blotting was carried out to assess α7nAChR expression relative to GAPDH using the Licor system of detection. α7nAChR expression was assessed in the treatment groups vs. a basal control, which for endothelial and smooth muscle cells was normoxic, non-proliferating confluent cultures, and for the macrophage was a normoxic culture. Results HUVEC demonstrated a significant increase in α7nAChR expression under proliferation and hypoxia, with α7nAChR expression peaking at 2 hours post hypoxia (p Conclusion This study demonstrates that all investigated cells expressed α7nAChR, with the highest expression found in macrophages, followed by endothelial cells, and then CVSMC. Importantly, only the endothelial cells exhibited a hypoxia-driven increase in α7nAChR expression. The results of this study suggest that α7nAChR may be a useful marker of angiogenesis, warranting further investigation. References Heeschen, C. et al. (2002). J Clin Investig. 110(4):527–536. Rotering, S. et al. (2013). Bioorganic Med Chem. 21(9):2635–2642.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6β2-containing (α6β2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6β2* agonist exhibiting functional selectivity toward other nAChRs, including α4β2, α3β4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6β2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6β2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

By structure–activity relationship studies on the tilorone scaffold, the ‘one armed’ substituted dibenzothiophenes and the fluoren-9-ones were identified as the most potential α7 nAChR ligands. While the suitability of dibenzothiophene derivatives as PET tracers is recognized, the potential of fluoren-9-ones is insufficiently investigated. We herein report on a series of fluoren-9-one based derivatives targeting α7 nAChR with compounds 8a and 8c possessing the highest affinity and selectivity. Accordingly, with [18F]8a and [18F]8c we designed and initially evaluated the first fluoren-9-one derived α7 nAChR selective PET ligands. A future application of these radioligands is facilitated by the herein presented successful implementation of fully automated radiosynthesis.

Introduction NS9531, NS9762 and NS6417 are nitroquinolinyl-diazabicyclo-alkane derivatives that have been developed as inhibitors of serotonin reuptake transporters (SERT) by NeuroSearch A/S. Methods IC50 was measured on the up-take of serotonin, dopamine and noradrenaline in synaptosomes prepared from selected rat brain regions. For the pre-clinical evaluation in pigs, [ 11 C]NS9531, [ 11 C]NS9762 and [ 11 C]NS6417 were prepared by N -methylation using [ 11 C]methyl iodide. These syntheses were later on optimized regarding: 1) choice of labelled precursor; 2) HPLC purification conditions; and 3) formulation using SPE columns. The synthesis protocols were then fully automated on a GE FX c Pro. Preclinical evaluation was performed by PET studies in landrace pigs before and after treatment with citalopram. Results IC50 measurements showed that all three compounds have low nanomolar affinity for SERT, and micromolar affinity for DAT and NET. The radiochemical yield (r.y.) of all three ligands from [ 11 C]methyl iodide was higher than 30%. From [ 11 C]methyl triflate, the r.y. of [ 11 C]NS9531 and [ 11 C]NS9762 were higher than 80% whereas the r.y. of [ 11 C]NS6417 was 65%. Residual precursor amounts in final products could be significantly reduced by the use of [ 11 C]methyl triflate, Conclusion The three 11 C labelled PET tracers could be prepared in medium to high yield and high purity. IC50 measurements showed that the three NS compounds were highly selective, high affinity SERT inhibitors. PET studies in pig showed high brain uptake that could be blocked by citalopram pre-treatment.

Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4–α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4–α4 interface. Given that epibatidine shows large functional potency differences at α4–β2 vs. α4–α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [3H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2HQT), which converts the β(−) face to resemble that of an α4(−) face, was utilized to create (α4)3(β2HQT)2 receptors harboring three α4–α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2HQT)2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4–β2 vs. α4–α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.

Gifted children with coexisting disabilities—the twice exceptional or 2e—exhibit complex patterns of strengths and weaknesses. Either exceptional ability or disability may mask the other, or combined exceptionalities may appear average. A case study illustrates how interpretation of changes in special education law may overlook 2e students who perform too well to qualify for services yet have disabilities significant enough to undermine success as the demands of their education increase. Department of Education policy letters are discussed, especially regarding a state’s right to condition service eligibility upon below-grade-level performance and reduce access to assessment. The student’s comprehensive diagnostic testing is explored. A triaged approach, using observed characteristics of 2e learners, is recommended to determine when comprehensive evaluation is needed to interpret a complex 2e student’s learning challenges, guide interventions/accommodations through an Individualized Education Program or Section 504 Plan, and ensure a Free Appropriate Public Education.

[18F]NS10743, a promising and highly competitive α7 nAChR radioligand has been synthesized so far by microwave irradiation using a manual single-mode device followed by a palladium-catalyzed reduction of remaining nitro-precursor for HPLC separation purposes. For further preclinical and clinical use, regulated production of [18F]NS10743 by fully automated radiosynthesis is a crucial requirement. Therefore, we chose a commercial synthesis module and developed the automated radiosynthesis of [18F]NS10743. Besides evaluation of several radiosynthesis procedures, we performed an extensive HPLC study for quantitative separation of [18F]NS10743 from the corresponding nitro precursor. After implementation of the optimized procedure on a TRACERlabTM FX F-N synthesis module, [18F]NS10743 was obtained in high radiochemical purity (≥99%) with an overall radiochemical yield of 32.2±7% (n=3). The specific activities at the end of the synthesis were 571±17 GBq/µmol (n=3).

As clinical psychologists, our role is to provide psychological and educational testing, and counseling for children and their families. Our job is to understand the developmental trajectories and expectations across areas of functioning (i.e., behavior, self-regulation, cognitive, academic, and social), differentiate typical versus atypical development, and provide recommendations for accommodation, intervention, and growth in the home, at school, and in the community. As psychologists who work primarily with gifted and talented individuals, we see many with profound strengths as well as disabilities who are not easily categorized. These are twice-exceptional students. With twice-exceptionality, the diagnostic category, although important, is not the ultimate focus—identifying the needs and the strengths is equally, if not more, important.

Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6 receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3(V9'S):β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3β2β3(V9'S) to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3(V9'S) replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts.

Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3β4 and α4β2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with β2- and β4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing β4- but not β2-subunits, NS3861 displays the opposite β-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the β-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the β-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (β) affect agonist efficacy.

BACKGROUND AND PURPOSE Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

Molecular imaging of brain structures by highly sensitive non-invasive techniques offers unique possibilities in the understanding of physiological and pathological processes in the central nervous system. In particular, the quantitative analysis by positron emission tomography (PET) of α7 nicotinic acetylcholine receptors (α7 nAChR), which are involved in different signalling pathways in the brain, is assumed to provide important information on the relation between receptor dysfunction and the pathogeneses of neuropsychiatric brain diseases, but the applicability of this imaging approach is still hampered due to insufficient imaging agents. This paper presents the recent efforts made to develop PET radiotracers targeting α7 nAChR as well as the current state of the evaluation of the most promising radiolabelled compounds in animal models and humans.

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21–76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters (Z'≈0.7 and Z≈0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 µM to ~193 µM. Binding to the PICK1 PDZ domain was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 µM) in the FP assay, and was according to fluorescence resonance energy transfer (FRET) experiments capable of inhibiting the interaction between the C-terminus of the GluR2 subunit of the AMPA-type glutamate receptor and PICK1 in live cells. Additional experiments suggested that CSC-03 most likely is an irreversible inhibitor but with specificity for PICK1 since it did not bind three different PDZ domains of PSD-95. Summarized, our data suggest that FP based screening assays might be a widely applicable tool in the search for small molecule inhibitors of PDZ domain interactions.

To conduct a quantitative PET assessment of the specific binding sites in the brain of juvenile pigs for [18F]NS10743, a novel diazabicyclononane derivative targeting α7 nicotinic acetylcholine receptors (α7 nAChRs). Dynamic PET recordings were made in isoflurane-anaesthetized juvenile pigs during 120 min after administration of [18F]NS10743 under baseline conditions (n = 3) and after blocking of the α7 nAChR with NS6740 (3 mg·kg−1 bolus + 1 mg·kg−1·h−1 continuous infusion; n = 3). Arterial plasma samples were collected for determining the input function of the unmetabolized tracer. Kinetic analysis of regional brain time–radioactivity curves was performed, and parametric maps were calculated relative to arterial input. Plasma [18F]NS10743 passed readily into the brain, with peak uptake occurring in α7 nAChR-expressing brain regions such as the colliculi, thalamus, temporal lobe and hippocampus. The highest SUVmax was approximately 2.3, whereas the lowest uptake was in the olfactory bulb (SUVmax 1.53 ± 0.32). Administration of NS6740 significantly decreased [18F]NS10743 binding late in the emission recording throughout the brain, except in the olfactory bulb, which was therefore chosen as reference region for calculation of BPND. The baseline BPND ranged from 0.39 ± 0.08 in the cerebellum to 0.76 ± 0.07 in the temporal lobe. Pretreatment and constant infusion with NS6740 significantly reduced the BPND in regions with high [18F]NS10743 binding (temporal lobe −29%, p = 0.01; midbrain: −35%, p = 0.02), without significantly altering the BPND in low binding regions (cerebellum: −16%, p = 0.2). This study confirms the potential of [18F]NS10743 as a target-specific radiotracer for the molecular imaging of central α7 nAChRs by PET.

(−)-3-(2-Benzothienyl)-8-H-8-azabicyclo[3,2,1]oct-2-ene acetate, NS9544 acetate, is a candidate drug intended to treat pain and other CNS disorders. In the synthetic route tropinone was enantiosele...

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Background and purpose: Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved. Experimental approach: Two structurally related α7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm. Key results: NS6784 activated human and rat α7 nAChR with EC50s of 0.72 and 0.88 µM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at α7 nAChR (

A series of 5-(pyridine-3-yl)octahydropyrrolo[3,4-c]pyrroles have been prepared that exhibit high affinity to alpha4beta2 and/or alpha7 nicotinic acetylcholine receptors (nAChRs). Simple substitution patterns have been identified that allow construction of ligands that are highly selective for either nAChR subtype. The effects of substitution on subtype selectivity provide some insight into the differences in the ligand binding domains of the alpha4beta2 and alpha7 receptors, especially in regions removed from the cation binding pocket.

The outstanding diversity of cellular properties mediated by neuronal and nonneuronal alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) points to the diagnostic potential of quantitative nuclear molecular imaging of alpha7 nAChR in neurology and oncology. It was our goal to radiolabel the alpha7 nAChR agonist 4-[5-(4-fluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-1,4-diaza-bicyclo[3.2.2]nonane (NS10743) and to assess the selectivity of [(18)F]NS10743 binding site occupancy in animal experiments.[(18)F]NS10743 was synthesized by nucleophilic substitution of the nitro precursor. In vitro receptor affinity and selectivity were assessed by radioligand competition and autoradiography. The radiotracer properties were evaluated in female CD-1 mice by brain autoradiography and organ distribution. Target specificity was validated after treatment with SSR180711 (10 mg/kg, intraperitoneal), and metabolic stability was investigated using radio-HPLC.The specific activity of [(18)F]NS10743 exceeded 150 GBq/micromol at a radiochemical purity99%. In vitro, NS10743 and [(18)F]NS10743 showed high affinity and specificity towards alpha7 nAChR. The brain permeation of [(18)F]NS10743 was fast and sufficient with values of 4.83 and 1.60% injected dose per gram and brain to plasma ratios of 3.83 and 2.05 at 5 and 60 min after radiotracer administration. Brain autoradiography and organ distribution showed target-specific accumulation of [(18)F]NS10743 in brain substructures and various alpha7 nAChR-expressing organs. The radiotracer showed a high metabolic stability in vivo with a single polar radiometabolite, which did not cross the blood-brain barrier.The good in vitro and in vivo features of [(18)F]NS10743 make this radioligand a promising candidate for quantitative in vivo imaging of alpha7 nAChR expression and encourage further investigations.

Small conductance Ca2+-activated K+ channels (SK channels) participate in the control of neuronal excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic transmission.SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and neurological diseases such as depression, cognition impairment, and Parkinson's disease. In the present study we describe the structure-activity relationship (SAR) of a class of 2-(N-substituted)-2-aminobenzimidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished by point mutations. These novel SK inhibitors shift the concentration-response curve for Ca2+ toward higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated currents with a K(d) value of 9 nM.

Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.

Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

Radiopharmacological investigations of [(18)F]NS14490 have proven that this radiotracer could be a potential PET radiotracer for imaging of alpha7 nicotinic acetylcholine receptor particularly with regard to vulnerable plaques of diseased vessels. For further optimisation of the previously automated one-pot radiosynthesis of [(18)F]NS14490 using a tosylate precursor, precursors with other leaving groups (nosylate and mosylate) were synthesized and compared with the tosylate with respect to their reactivities towards [(18)F]fluoride. The use of these different precursors resulted in comparable labelling yields of [(18)F]NS14490. A novel mosylate precursor was synthesized and evaluated, which has revealed a higher stability during a storage period of five months compared to the corresponding tosylate and nosylate.

NS9531, NS9762 and NS6417 are nitroquinolinyl-diazabicyclo-alkane derivatives that have been developed as inhibitors of serotonin reuptake transporters (SERT) by NeuroSearch A/S.IC50 was measured on the up-take of serotonin, dopamine and noradrenaline in synaptosomes prepared from selected rat brain regions. For the pre-clinical evaluation in pigs, [(11)C]NS9531, [(11)C]NS9762 and [(11)C]NS6417 were prepared by N-methylation using [(11)C]methyl iodide. These syntheses were later on optimized regarding: 1) choice of labelled precursor; 2) HPLC purification conditions; and 3) formulation using SPE columns. The synthesis protocols were then fully automated on a GE FXc Pro. Preclinical evaluation was performed by PET studies in landrace pigs before and after treatment with citalopram.IC50 measurements showed that all three compounds have low nanomolar affinity for SERT, and micromolar affinity for DAT and NET. The radiochemical yield (r.y.) of all three ligands from [(11)C]methyl iodide was higher than 30%. From [(11)C]methyl triflate, the r.y. of [(11)C]NS9531 and [(11)C]NS9762 were higher than 80% whereas the r.y. of [(11)C]NS6417 was 65%. Residual precursor amounts in final products could be significantly reduced by the use of [(11)C]methyl triflate,0.2 μg compared with10 μg, calculated for a 300 MBq injection at 20 minutes EOS. The optimized conditions gave 2.5-4.5 GBq of products with a specific radioactivity of 20-70 MBq/nmol, residual acetonitrile 15-30 ppm, and pH 6.5-7.1. All three compounds showed a rapid and comparable high pig brain uptake of about 3%, producing PET images of good contrast, and uptake was reduced after pre-administration with citalopram.The three (11)C labelled PET tracers could be prepared in medium to high yield and high purity. IC50 measurements showed that the three NS compounds were highly selective, high affinity SERT inhibitors. PET studies in pig showed high brain uptake that could be blocked by citalopram pre-treatment.

Binding studies initially suggested that the muscarinic agonist, xanomeline, was a subtype selective muscarinic M (1) receptor agonist, and a potential new treatment for Alzheimer's disease. However, later in vitro and in vivo functional studies suggest that this compound is probably better described as a subtype selective M(1)/M(4) muscarinic receptor agonist. This subtype selectivity profile has been claimed to explain the limited classical cholinomimetic side effects, particularly gastrointestinal, seen with xanomeline in animals. However, in both healthy volunteers and Alzheimer's patients many of these side effects have been reported for xanomeline and in the patient population this led to a>50% discontinuation rate. Clearly, the preclinical studies have not been able to predict this adverse profile of xanomeline, and this suggests that either xanomeline is not as subtype selective as predicted from preclinical research or that there are differences between humans and animals with regard to muscarinic receptors. Nevertheless, in Alzheimer's patients xanomeline dose‐dependently improves aspects of behavioral disturbance and social behavior including a reduction in hallucinations, agitation, delusions, vocal outbursts and suspiciousness. The effects on cognition are not as robust and mainly seen at the highest doses tested. These effects in Alzheimer's patients have given impetus to the suggestion that muscarinic agonists have potential antipsychotic effects. The current review assesses the antipsychotic profile of xanomeline within the framework of the limited clinical studies with cholinergic agents in man, and the preclinical research on xanomeline using various models commonly used for the assessment of new antipsychotic drugs. In general, xanomeline has an antipsychotic‐like profile in various dopamine models of psychosis and this agrees with the known interactions between the cholinergic and dopaminergic systems in the brain. Moreover, current data suggests that the actions of xanomeline at the M(4) muscarinic receptor subtype might mediate its antidopaminergic effects. Particularly intriguing are studies showing that xanomeline, even after acute administration, selectively inhibits the firing of mesolimbic dopamine cells relative to dopamine cell bodies projecting to the striatum. This data suggest that xanomeline would have a faster onset of action compared to current antipsychotics and would not induce extrapyramidal side effects. The preclinical data on the whole are promising for an antipsychotic‐like profile. If in a new formulation (i.e., transdermal) xanomeline has less adverse effects, this drug may be valuable in the treatment of patients with psychosis.

The synthesis of unsaturated unnatural amino acids according to the Heck-Jeffery protocol using a ball-milling procedure is presented. NOE data recorded on the products confirm that the Z-isomer is formed. This type of mechano-chemistry provides an efficient, "solvent free" and reliable method for the synthesis of substituted dehydroalanines. These conditions provide patterns of reactivity complementary to conventional procedures. (C) 2004 Elsevier B.V. All rights reserved.

A series of 2-arylureidobenzoic acids (AUBAs) was prepared by a short and effective synthesis, and the pharmacological activity at glutamate receptors was evaluated in vitro and in vivo. The compounds showed noncompetitive antagonistic activity at the kainate receptor subtype GluR5. The most potent compounds showed more than 50-fold selectivity for GluR5 compared to GluR6 and the AMPA receptor subtypes GluR1-4. The structure-activity relationships for the AUBAs showed distinct structural requirements for the substituents on the two aromatic ring systems. Only para-substituents were tolerated on the benzoic acid moiety (ring A), whereas ring B tolerated a variety of substituents, but with a preference for lipophilic substituents. The most potent compounds had a 4-chloro substituent on ring A and 3-chlorobenzene (6b), 2-naphthalene (8h), or 2-indole (8k) as ring B and had IC(50) values of 1.3, 1.2, and 1.2 microM, respectively, in a functional GluR5 assay. Compound 6c (IC(50) = 4.8 microM at GluR5) showed activity in the in vivo ATPA rigidity test, indicating that 6c has better pharmacokinetic properties than 8h, which was inactive in this test. The AUBAs are the first example of a series of noncompetitive GluR5-selective antagonists and may prove to be important pharmacological tools and leads in the search for therapeutic glutamatergic agents.

The serotonin transporter ligand (±)-10-[11C]-methyl 3-[6-nitro-(2-quinolinyl)]-3,10-diazabicyclo-[4.3.1]-decane ([11C-methyl]NS 2495) was synthesized via a methylation reaction with [11C]methyl iodide. The radiochemical purity exceeded 99% and the specific radioactivity was found to be 1.8 GBq/μmol at 40 min after the end of bombardment. The uptake of the tracer in the brain of a living pig was recorded by positron emission tomography (PET), first in a baseline condition, and again after treatment with citalopram (1 mg/kg, i.v.) to displace the specific binding. The distribution volume relative to the metabolism-corrected arterial input was high in pig brain, ranging from 75–150 ml g−1; treatment with citalopram uniformly reduced the distribution volume to 75 ml g−1. Binding potential (pB) maps generated using the cerebellum as a reference tissue showed highest binding in the mesencephalon and cingulate cortex, where the magnitude of pB was close to 0.6. Thus, the pattern of binding in vivo agrees with the known pattern of serotonin innervations in pig brain. However, the specific binding was incompletely displaced by pre-treatment with citalopram. Thus, [11C-methyl]NS 2495 can label serotonin transporters in a PET study of the brain of a living pig, but full displacement by cold citalopram was not obtained in vivo, possibly reflecting binding sites which are inaccessible to citalopram. Copyright © 2003 John Wiley & Sons, Ltd.

In vitro, the novel diazabicyclononane NS 4194 has several thousandfold selectivity for blocking the transport into rat brain synaptosomes of [ 3 H]-serotonin in comparison to [3H]-dopamine or [ 3 H]-noradrenaline. We have prepared [ 1 1 C]-NS 4194 in order to test its properties for PET imaging of brain serotonin transporters in comparison with the well-documented tracer [ 1 1 C]-DASB. Both compounds had rapid clearance from blood to brain of living pigs. The apparent equilibrium distribution volumes in cerebellum were 35 ml g - 1 for [ 1 1 C]-NS 4194 and 11 ml g - 1 for [ 1 1 C]-DASB. Pretreatment of pigs with citalopram did not reduce the uptake of either tracer in cerebellum, validating the use of that tissue as a nonbinding reference tissue for kinetic analysis of specific binding. The binding potential (pB) calculated for [ 1 1 C]-NS 4194 using arterial input models was close to 0.5 in the telencephalon, and was 60% displaced by citalopram. However, the reference tissue method of Lammertsma was unsuited to calculate pB for this tracer, apparently due to its excessive nonspecific binding. In contrast to the relatively homogeneous binding of [ 1 1 C]-NS 4194, the pB of [ 1 1 C]-DASB ranged from 0.6 in frontal cortex to 2 in the mesencephalon when calculated by the method of Lammertsma. Parametric maps of the pB of [ 1 1 C]-DASB showed a pattern consistent with the known distribution of serotonin transporters in pig brain in vitro, and there was a uniform displacement of 80% of the specific binding after citalopram treatment in vivo. In conclusion, [ 1 1 C]-DASB is in several respects superior to [ 1 1 C]-NS 4194 for the detection of serotonin uptake sites by PET.

NS 1231 [5-(4-chlorophenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3.2-h]naphthalene-2,3-dione-3-oxime] belongs to a chemical series of compounds, which exhibit neurotrophic-like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)-differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF-induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF-induced signalling events, such as TrkA phosphorylation at the Shc-binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA-induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.