Pharmacological characterization of 5-iodo-A-85380, a ß2-selective nicotinic receptor agonist, in mice.

BACKGROUND: Because of their implications in several pathological conditions, α4ß2* nicotinic acetylcholine receptors (nAChRs) are potential targets for the treatment of nicotine dependence, pain, and many psychiatric and neurodegenerative diseases. However, they exist in various subtypes, and finding selective tools to investigate them has proved challenging. The nicotinic receptor agonist, 5-iodo-A-85380 (5IA), has helped in delineating the function of ß2-containing subtypes in vitro; however, much is still unknown about its behavioral effects. Furthermore, its effectiveness on α6-containing subtypes is limited. AIMS: To investigate the effects of 5IA on nociception (formalin, hot-plate, and tail-flick tests), locomotion, hypothermia, and conditioned reward after acute and repeated administration, and to examine the potential role of ß2 and α6 nAChR subunits in these effects. Lastly, its selectivity for expressed low sensitivity (LS) and high sensitivity (HS) α4ß2 receptors is investigated. RESULTS: 5IA dose-dependently induced hypothermia, locomotion suppression, conditioned place preference, and antinociception (only in the formalin test but not in the hot-plate or tail-flick tests). Furthermore, these effects were mediated by ß2 but not α6 nicotinic subunits. Finally, we show that 5-iodo-A-85380 potently activates both stoichiometries of α4ß2 nAChRs with differential efficacies, being a full agonist on HS α4(2)ß2(3) nAChRs, and a partial agonist on LS α4(3)ß2(2) nAChRs and α6-containing subtypes as well.

Higher availability of α4ß2 nicotinic receptors (nAChRs) in dorsal ACC is linked to more efficient interference control.

Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the human brain and play an important role in the neuromodulation of brain networks implicated in attentional processes. Previous work in humans showed that heteromeric α4ß2 nAChRs are abundant in the cingulo-insular network underlying attentional control. It has been proposed that cholinergic neuromodulation by α4ß2 nAChRs is involved in attentional control during demanding tasks, when additional resources are needed to minimize interference from task-irrelevant stimuli and focus on task-relevant stimuli. Here we investigate the link between the availability of α4ß2 nAChRs in the cingulo-insular network and behavioral measures of interference control using two versions of the Stroop paradigm, a task known to recruit cingulo-insular areas. We used a previously published PET dataset acquired in 24 non-smoking male subjects in the context of a larger study which investigated the brain distribution of nAChRs in two clinical groups using 2-[(18)F]F-A-85380 PET. We found that higher availability of α4ß2 nAChRs in the dorsal anterior cingulate cortex (ACC) predicted better interference control independently of group and age. In line with animal models, our results support the view that the availability of α4ß2 nAChRs in the dorsal ACC is linked with more efficient attentional control.

Nicotinic Acetylcholine Receptor Density in the "Higher-Order" Thalamus Projecting to the Prefrontal Cortex in Humans: a PET Study.

PURPOSE: The parcellation of the thalamus into different nuclei involved in different corticothalamocortical loops reflects its functional diversity. The connections between the mediodorsal nucleus and the prefrontal cortex play a major role in cognition, particularly in the rapid processing of behaviorally relevant information. The thalamus is the brain region with the highest density in α4ß2 nicotinic acetylcholine receptors, the main human nicotinic acetylcholine receptor subtype. The aim of this study was to investigate the possible role of the nicotinic cholinergic system in the thalamo-cortical loops measuring receptor density in different subregions of the thalamus, based on their cortical connectivity. PROCEDURES: We studied α4ß2 nicotinic acetylcholine receptors using positron emission tomography and [18F]Fluoro-A-85380, a radiotracer specific for this receptor subtype, in 36 non-smoking male subjects, including 12 healthy controls and 24 patients with epilepsy. [18F]Fluoro-A-85380 ratio index of binding potential was compared by a repeated measures general linear model, including the thalamic subregions and the brain hemisphere as within-subject factor and clinical groups as between-subject factor. RESULTS: The "prefrontal" thalamus, the subregion including the mediodorsal nucleus, had a significantly higher nicotinic acetylcholine receptor density than all other thalamic subregions. These findings were confirmed when analyzing solely the 12 healthy controls. CONCLUSIONS: This particular neurochemical organization of the thalamus supports a major role of the cholinergic system in the loops between the thalamus and the prefrontal cortex. The highest nicotinic acetylcholine receptor density in the « higher-order thalamus ¼ could partly explain the beneficial effect of acute nicotine on attentional and executive functions and possibly the pathophysiology of some neuropsychiatric disorders.

Development of 99mTc radiolabeled A85380 derivatives targeting cerebral nicotinic acetylcholine receptor: Novel radiopharmaceutical ligand 99mTc-A-YN-IDA-C4.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been implicated in higher brain functions. To elucidate the functional mechanisms underlying nAChRs and contribute significantly to development of drugs targeting neurological and neuropsychiatric diseases, non-invasive nuclear medical imaging can be used for evaluation. In addition, technetium-99m (99mTc) is a versatile radionuclide used clinically as a tracer in single-photon emission computed tomography. Because A85380 is known as a potent α4ß2-nAChR agonist, we prepared A85380 derivatives labeled with 99mTc using a bifunctional chelate system. A computational scientific approach was used to design the probe efficiently. We used non-radioactive rhenium (Re) for a 99mTc analog and found that one of the derivatives, Re-A-YN-IDA-C4, exhibited high binding affinity at α4ß2-nAChR in both the docking simulation (-19.3 kcal/mol) and binding assay (Ki = 0.4 ±â€¯0.04 nM). Further, 99mTc-A-YN-IDA-C4 was synthesized using microwaves, and its properties were examined. Consequently, we found that 99mTc-A-YN-IDA-C4, with a structure optimized by using computational chemistry techniques, maintained affinity and selectivity for nAChR in vitro and possessed efficient characteristics as a nuclear medicine molecular imaging probe, demonstrated usefulness of computational scientific approach for molecular improvement strategy.

Brainstem cholinergic pathways diminish cardiovascular and neuroinflammatory actions of endotoxemia in rats: Role of NFκB/α7/α4ß2AChRs signaling.

Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4ß2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4ß2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50 µg/kg) abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4ß2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4ß2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-ß-erythroidine (DHßE, α4ß2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4ß2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia.

Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain.

We have designed (S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate ([99mTc]CPTT-A-E) with high affinity for nicotinic acetylcholine receptors (nAChRs) using (2(S)-azetidinylmethoxy)-pyridine (A-85380) as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-technetium (99mTc)-labeled nAChR imaging probe. Because technetium does not contain a stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized by coordinating rhenium, which is a homologous element having the same coordination structure as technetium. Further, the binding affinity to nAChR was evaluated. CPTR-A-E exhibited a high binding affinity to nAChR (Ki = 0.55 nM). Through the radiosynthesis of [99mTc]CPTT-A-E, an objective compound could be obtained with a radiochemical yield of 33% and a radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain exhibited that the local nAChR density strongly correlated with the amount of [99mTc]CPTT-A-E that was accumulated in each region of interest. Further, the in vivo evaluation of biodistribution revealed a higher accumulation of [99mTc]CPTT-A-E in the thalamus (characterized by the high nAChR density) when compared with that in the cerebellum (characterized by the low nAChR density). Although additional studies will be necessary to improve the uptake of [99mTc]CPTT-A-E to the brain, [99mTc]CPTT-A-E met the basic requirements for nAChR imaging.

Nicotinic receptor abnormalities as a biomarker in idiopathic generalized epilepsy.

PURPOSE: Mutations of cholinergic neuronal nicotinic receptors have been identified in the autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), associated with changes on PET images using [18F]-F-85380-A (F-A-85380), an α4ß2 nicotinic receptor ligand. The aim of the present study was to evaluate potential changes in nicotinic receptor availability in other types of epilepsy. METHODS: We included 34 male participants, 12 patients with idiopathic generalized epilepsy (IGE), 10 with non-lesional diurnal focal epilepsy, and 12 age-matched healthy controls. All patients underwent PET/CT using F-A-85380 and [18F]-fluorodeoxyglucose (FDG), 3D T1 MRI and diffusion tensor imaging (DTI). F-A-85380 and FDG images were compared with the control group using a voxel-wise (SPM12) and a volumes of interest (VOI) analysis. RESULTS: In the group of patients with IGE, the voxel-wise and VOI analyses showed a significant increase of F-A-85380 ratio index of binding potential (BPRI, corresponding to the receptor availability) in the anterior cingulate cortex (ACC), without structural changes on MRI. At an individual level, F-A-85380 BPRI increase in the ACC could distinguish IGE patients from controls and from patients with focal epilepsy with good accuracy. CONCLUSIONS: We observed focal changes of density/availability of nicotinic receptors in IGE, namely an increase in the ACC. These data suggest that the modulation of α4ß2 nicotinic receptors plays a role not only in ADNFLE, but also in other genetic epileptic syndromes such as IGE and could serve as a biomarker of epilepsy syndromes with a genetic background.

Competition, Selectivity and Efficacy of Analogs of A-84543 for Nicotinic Acetylcholine Receptors with Repositioning of Pyridine Nitrogen.

Nicotinic acetylcholine receptors (nAChRs) play a crucial role in a number of clinically relevant mental and neurological pathways, as well as autonomic and immune functions. The development of subtype-selective ligands for nAChRs therefore is potentially useful for targeted therapeutic management of conditions where nAChRs are involved. We tested if selectivity for a particular nAChR subtype can be achieved through small structural modifications of a lead compound containing the nicotinic pharmacophore by changing the distance between the electronegative elements. For this purpose, analogs of A-84543 were designed, synthesized and characterized as potentially new nAChR subtype-selective ligands. Compounds were tested for their binding properties in rat cerebral cortical tissue homogenates, and subtype-selectivity was determined using stably transfected HEK cells expressing different nAChR subtypes. All compounds synthesized were found to competitively displace [(3)H]-epibatidine ([(3)H]EB) from the nAChR binding site. Of all the analogues, H-11MNH showed highest affinity for nAChRs compared to a ~ fivefold to tenfold lower affinity of A-84543. All other compounds had affinities >10,000 nM. Both A-84543 and H-11MNH have highest affinity for α2ß2 and α4ß2 nAChRs and show moderate affinity for ß4- and α7-containing receptors. H-11MNH was found to be a full agonist with high potency at α3ß4, while A-84543 is a partial agonist with low potency. Based on their unique pharmacological binding properties we suggest that A-84543 and its desmethylpyrrolidine analog can be useful as pharmacological ligands for studying nAChRs if selective pharmacological and/or genetic tools are used to mask the function of other receptors subtypes.

In vivo PET imaging of the α4ß2 nicotinic acetylcholine receptor as a marker for brain inflammation after cerebral ischemia.

PET imaging of nicotinic acetylcholine receptors (nAChRs) could become an effective tool for the diagnosis and therapy evaluation of neurologic diseases. Despite this, the role of nAChRs α4ß2 receptors after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. To investigate this, we performed in parallel in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 at 1, 3, 7, 14, 21, and 28 d after middle cerebral artery occlusion (MCAO) in rats. In the ischemic territory, PET with 2[(18)F]-fluoro-A85380 and [(11)C]PK11195 showed a progressive binding increase from days 3-7, followed by a progressive decrease from days 14-28 after cerebral ischemia onset. Ex vivo immunohistochemistry for the nicotinic α4ß2 receptor and the mitochondrial translocator protein (18 kDa) (TSPO) confirmed the PET findings and demonstrated the overexpression of α4ß2 receptors in both microglia/macrophages and astrocytes from days 7-28 after experimental ischemic stroke. Likewise, the role played by α4ß2 receptors on neuroinflammation was supported by the increase of [(11)C]PK11195 binding in ischemic rats treated with the α4ß2 antagonist dihydro-ß-erythroidine hydrobromide (DHBE) at day 7 after MCAO. Finally, both functional and behavioral testing showed major impaired outcome at day 1 after ischemia onset, followed by a recovery of the sensorimotor function and dexterity from days 21-28 after experimental stroke. Together, these results suggest that the nicotinic α4ß2 receptor could have a key role in the inflammatory reaction underlying cerebral ischemia in rats.

Nicotinic α4ß2 acetylcholine receptors and cognitive function in Parkinson's disease.

BACKGROUND: Idiopathic Parkinson's disease (IPD) is characterized by the clinical motor symptoms of hypokinesia, rigidity, and tremor. Apart from these motor symptoms, cognitive deficits often occur in IPD. The positive effect of cholinesterase inhibitors on cognitive deficits in IPD and findings of earlier molecular imaging studies suggest that the cholinergic system plays an important role in the origin of cognitive decline in IPD. METHODS: Twenty-five non-demented patients with IPD underwent a 5-[123I]iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380) SPECT to visualize α4ß2 nicotinic acetylcholine receptors (nAchR) and cognitive testing with the CERAD (Consortium to Establish a Registry for Alzheimer's Disease) battery to identify domains of cognitive dysfunction. RESULTS: In the CERAD, the IPD patients exhibited deficits in non-verbal memory, attention, psychomotor velocity, visuoconstructive ability, and executive functions. After Bonferroni correction for multiple comparisons, we found significant correlations between performance of the CERAD subtests Boston Naming Test (a specific test for visual perception and for detection of word-finding difficulties) and Word List Intrusions (a specific test for learning capacity and memory for language information) vs binding of α4ß2 nAchR in cortical (the right superior parietal lobule) and subcortical areas (the left thalamus, the left posterior subcortical region, and the right posterior subcortical region). CONCLUSIONS: These significant correlations between the results of the CERAD subtests and the cerebral α4ß2 nAchR density, as assessed by 5-I-A-85380 SPECT, indicate that cerebral cholinergic pathways are relevant to cognitive processing in IPD.