Characterizing the binding of TC-5619 and encenicline on the alpha7 nicotinic acetylcholine receptor using PET imaging in the pig.

The alpha7 nicotinic acetylcholine receptor (α7-nAChR) has has long been considered a promising therapeutic target for addressing cognitive impairments associated with a spectrum of neurological and psychiatric disorders, including Alzheimer's disease and schizophrenia. However, despite this potential, clinical trials employing α7-nAChR (partial) agonists such as TC-5619 and encenicline (EVP-6124) have fallen short in demonstrating sufficient efficacy. We here investigate the target engagement of TC-5619 and encenicline in the pig brain by use of the α7-nAChR radioligand 11C-NS14492 to characterize binding both with in vitro autoradiography and in vivo occupancy using positron emission tomography (PET). In vitro autoradiography demonstrates significant concentration-dependent binding of 11C-NS14492, and both TC-5619 and encenicline can block this binding. Of particular significance, our in vivo investigations demonstrate that TC-5619 achieves substantial α7-nAChR occupancy, effectively blocking approximately 40% of α7-nAChR binding, whereas encenicline exhibits more limited α7-nAChR occupancy. This study underscores the importance of preclinical PET imaging and target engagement analysis in informing clinical trial strategies, including dosing decisions.

A novel reuptake inhibitor, IP2015, induces erection by increasing central dopamine and peripheral nitric oxide release.

BACKGROUND AND PURPOSE: An estimated 40% of patients with erectile dysfunction have a poor prognosis for improvement with currently available treatments. The present study investigated whether a newly developed monoamine transport inhibitor, IP2015, improves erectile function. EXPERIMENTAL APPROACH: We investigated the effects of IP2015 on monoamine uptake and binding, erectile function in rats and diabetic mice and the effect on corpus cavernosum contractility. KEY RESULTS: IP2015 inhibited the uptake of 5-HT, noradrenaline and dopamine by human monoamine transporters expressed in cells and in rat brain synaptosomes. Intracavernosal pressure measurement in anaesthetized rats revealed that IP2015 dose-dependently increased the number and the duration of spontaneous erections. Whereas pretreatment with the dopamine D2-like receptor antagonists, clozapine and (-)-sulpiride, or cutting the cavernosal nerve inhibited IP2015-induced erectile responses, the phosphodiesterase type 5 inhibitor sildenafil further enhanced the IP2015-mediated increase in intracavernosal pressure. IP2015 also increased the number of erections in type 2 diabetic db/db mice. Direct intracavernosal injection of IP2015 increased penile pressure, and in corpus cavernosum strips, IP2015 induced concentration-dependent relaxations. These relaxations were enhanced by sildenafil and blunted by endothelial cell removal, a nitric oxide synthase inhibitor, NG-nitro-l-arginine and a D1-like receptor antagonist, SCH23390. Quantitative polymerase chain reaction (qPCR) showed the expression of the dopamine transporter in the rat corpus cavernosum. CONCLUSION AND IMPLICATIONS: Our findings suggest that IP2015 stimulates erectile function by a central mechanism involving dopamine reuptake inhibition and direct NO-mediated relaxation of the erectile tissue. This novel multi-modal mechanism of action could offer a new treatment approach to erectile dysfunction.

Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490.

BACKGROUND: Cardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (ɑ7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of ɑ7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies. RESULTS: ɑ7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of ɑ7nAChR positive tissue revealed strong associations between ɑ7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed ɑ7nAChR protein under normoxic and hypoxic conditions. CONCLUSION: ɑ7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the ɑ7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by ɑ7nAChR expression in fibroblasts. Overall, these findings support the involvement of ɑ7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with ɑ7nAChR following MI.

In Vitro and In Vivo Characterization of Dibenzothiophene Derivatives [125I]Iodo-ASEM and [18F]ASEM as Radiotracers of Homo- and Heteromeric α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (α7 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'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]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in ß2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% ± 2.7%) than [125I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7ß2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 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 α7 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 α7 nAChR, comparable to previously published data.

Characterization of AN317, a novel selective agonist of α6ß2-containing nicotinic acetylcholine receptors.

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. α6ß2-containing (α6ß2*) 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 α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 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 α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

In vivo characterization of a novel norepinephrine transporter PET tracer [18F]NS12137 in adult and immature Sprague-Dawley rats.

Norepinephrine modulates cognitive processes such as working and episodic memory. Pathological changes in norepinephrine and norepinephrine transporter (NET) function and degeneration of the locus coeruleus produce irreversible impairments within the whole norepinephrine system, disrupting cognitive processes. Monitoring these changes could enhance diagnostic accuracy and support development of novel therapeutic components for several neurodegenerative diseases. Thus, we aimed to develop a straightforward nucleophilic fluorination method with high molar activity for the novel NET radiotracer [18F]NS12137 and to demonstrate the ability of [18F]NS12137 to quantify changes in NET expression. Methods: We applied an 18F-radiolabeling method in which a brominated precursor was debrominated by nucleophilic 18F-fluorination in dimethyl sulfoxide. Radiolabeling was followed by a deprotection step, purification, and formulation of the radiotracer. The [18F]NS12137 brain uptake and distribution were studied with in vivo PET/CT and ex vivo autoradiography using both adult and immature Sprague-Dawley rats because postnatal NET expression peaks at 10-20 days post birth. The NET specificity for the tracer was demonstrated by pretreatment of the animals with nisoxetine, which is well-known to have a high affinity for NET. Results: [18F]NS12137 was successfully synthesized with radiochemical yields of 18.6±5.6%, radiochemical purity of >99%, and molar activity of >500 GBq/µmol at the end of synthesis. The in vivo [18F]NS12137 uptake showed peak standard uptake values (SUV) of over 1.5 (adult) and 2.2 (immature) in the different brain regions. Peak SUV/30 min and peak SUV/60 min ratios were calculated for the different brain regions of the adult and immature rats, with a peak SUV/60 min ratio of more than 4.5 in the striatum of adult rats. As expected, in vivo studies demonstrated uptake of the tracer in brain areas rich in NET, particularly thalamus, neocortex, and striatum, and remarkably also in the locus coeruleus, a quite small volume for imaging with PET. The uptake was significantly higher in immature rats compared to the adult animals. Ex vivo studies using autoradiography showed very strong specific binding in NET-rich areas such as the locus coeruleus and the bed nucleus of the stria terminalis, and high binding in larger grey matter areas such as the neocortex and striatum. The uptake of [18F]NS12137 was dramatically reduced both in vivo and ex vivo by pretreatment with nisoxetine, demonstrating the specificity of binding. Conclusions: [18F]NS12137 was synthesized in good yield and high molar activity and demonstrated the characteristics of a good radiotracer, such as good brain penetration, fast washout, and high specific binding to NET.

Synthesis and radiofluorination of novel fluoren-9-one based derivatives for the imaging of α7 nicotinic acetylcholine receptor with PET.

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.

18F-labeled norepinephrine transporter tracer [18F]NS12137: radiosynthesis and preclinical evaluation.

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.

[¹¹C]NS9531, [¹¹C]NS9762 and [¹¹C]NS6417, specific SERT tracers: pre-clinical evaluation in pigs and optimization of synthesis conditions using [¹¹C]methyl triflate.

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 FXc 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, <0.2 µg compared with <10 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. 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.

A Promising PET Tracer for Imaging of α7 Nicotinic Acetylcholine Receptors in the Brain: Design, Synthesis, and in Vivo Evaluation of a Dibenzothiophene-Based Radioligand.

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.

Radiosynthesis and in vitro validation of (3)H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand.

The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described (11)C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, (3)H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. (3)H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand (125)I-α-bungarotoxin ((125)I-BTX) in autoradiography, and unlabeled NS14492 displaced (125)I-BTX with an inhibition constant (Ki) of 23nM. (3)H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/mg protein. This binding assay further revealed the Ki rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of (3)H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that (3)H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.

Engineered α4ß2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface.

The nicotinic acetylcholine receptor α4ß2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(ß2)3 and (α4)3(ß2)2. While these are similar in many aspects, the (α4)3(ß2)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-ß2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(ß2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered ß2 construct (ß2(HQT)), which converts the ß(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(ß2(HQT))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(ß2(HQT))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-ß2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4ß2 receptors.

A high-yield automated radiosynthesis of the alpha-7 nicotinic receptor radioligand [18F]NS10743.

[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±17GBq/µmol (n=3).

[11C]NS8880, a promising PET radiotracer targeting the norepinephrine transporter.

INTRODUCTION: Positron emission tomography (PET) imaging of the norepinephrine transporter (NET) is still hindered by the availability of useful PET imaging probes. The present study describes the radiosynthesis and pre-clinical evaluation of a new compound, exo-3-(6-methoxypyridin-2-yloxy)-8-H-8-azabicyclo[3.2.1]octane (NS8880), targeting NET. NS8880 has an in vitro binding profile comparable to desipramine and is structurally not related to reboxetine. METHODS: Labeling of NS8880 with [(11)C] was achieved by a non-conventional technique: substitution of pyridinyl fluorine with [(11)C]methanolate in a Boc-protected precursor. The isolated [(11)C]NS8880 was evaluated pre-clinically both in a pig model (PET scanning) and in a rat model (µPET scanning) and compared to (S,S)-[(11)C]-O-methylreboxetine ([(11)C]MeNER). RESULTS: The radiolabeling technique yielded [(11)C]NS8880 in low (<10%) but still useful yields with high purity. The PET in vivo evaluation in pig and rat revealed a rapid brain uptake of [(11)C]NS8880 and fast obtaining of equilibrium. Highest binding was observed in thalamic and hypothalamic regions. Pretreatment with desipramine efficiently reduced binding of [(11)C]NS8880. CONCLUSION: Based on the pre-clinical results obtained so far [(11)C]NS8880 displays promising properties for PET imaging of NET.

Imaging of α7 nicotinic acetylcholine receptors in brain and cerebral vasculature of juvenile pigs with [(18)F]NS14490.

BACKGROUND: The α7 nicotinic acetylcholine receptor (nAChR) is an important molecular target in neuropsychiatry and oncology. Development of applicable highly specific radiotracers has been challenging due to comparably low protein expression. To identify novel ligands as candidates for positron emission tomography (PET), a library of diazabicyclononane compounds was screened regarding affinity and specificity towards α7 nAChRs. From these, [(18)F]NS14490 has been shown to yield reliable results in organ distribution studies; however, the radiosynthesis of [(18)F]NS14490 required optimization and automation to obtain the radiotracer in quantities allowing dynamic PET studies in piglets. METHODS: Automated radiosynthesis of [(18)F]NS14490 has been performed by [(18)F]fluorination with the tosylate precursor in the TRACERlab™ FX F-N synthesis module (Waukesha, WI, USA). After optimization, the radiochemical yield of [(18)F]NS14490 was consistently approximately 35%, and the total synthesis time was about 90 min. The radiotracer was prepared with >92% radiochemical purity, and the specific activity at the end of the synthesis was 226 ± 68 GBq µmol(-1). PET measurements were performed in young pigs to investigate the metabolic stability and cerebral binding of [(18)F]NS14490 without and with administration of the α7 nAChR partial agonist NS6740 in baseline and blocking conditions. RESULTS: The total distribution volume relative to the metabolite-corrected arterial input was 3.5 to 4.0 mL g(-1) throughout the telencephalon and was reduced to 2.6 mL g(-1) in animals treated with NS6740. Assuming complete blockade, this displacement indicated a binding potential (BPND) of approximately 0.5 in the brain of living pigs. In addition, evidence for specific binding in major brain arteries has been obtained. CONCLUSION: [(18)F]NS14490 is not only comparable to other preclinically investigated PET radiotracers for imaging of α7 nAChR in brain but also could be a potential PET radiotracer for imaging of α7 nAChR in vulnerable plaques of diseased vessels.

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells.

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, ß2 and ß3 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 ß2 and ß3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the ß3(V9'S):ß2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type ß3, significant functional expression of α6/α3ß2ß3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3ß2ß3(V9'S) to that of α6/α3ß2ß3 revealed the ß3 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/α3ß2ß3(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.

Two distinct allosteric binding sites at α4ß2 nicotinic acetylcholine receptors revealed by NS206 and NS9283 give unique insights to binding activity-associated linkage at Cys-loop receptors.

Positive allosteric modulators (PAMs) of α4ß2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4ß2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4ß2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4ß2 stoichiometries (2α:3ß and 3α:2ß). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2ß receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2ß receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2ß receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4ß2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.

Structural analysis of the positive AMPA receptor modulators CX516 and Me-CX516 in complex with the GluA2 ligand-binding domain.

Positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) can serve as lead compounds for the development of cognitive enhancers. Several benzamide-type (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor modulators such as aniracetam, CX516 and CX614 have been shown to inhibit the deactivation of AMPA receptors with a less pronounced effect on desensitization. Despite CX516 being an extensively investigated AMPA receptor modulator and one of the few clinically evaluated compounds, the binding mode of CX516 to AMPA receptors has not been reported. Here, the structures of a GluA2 ligand-binding domain mutant in complex with CX516 and the 3-methylpiperidine analogue of CX516 (Me-CX516) are reported. The structures show that the binding modes of CX516 and Me-CX516 are similar to those of aniracetam and CX614 and that there is limited space for substitution at the piperidine ring of CX516. The results therefore support that CX516, like aniracetam and CX614, modulates deactivation of AMPA receptors.