Selective Muscarinic Agonist Research Articles

Agonist-selective activation of individual G-proteins by muscarinic receptors

Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

Xanomeline-Trospium and Muscarinic Involvement in Schizophrenia.

Schizophrenia is a severe mental illness that has its onset in late adolescence or early adulthood and is associated with significant dysfunction across multiple domains. The pathogenesis of schizophrenia remains unknown, but physiologic understanding of the illness has been driven by the dopamine hypothesis. However, acetylcholine (ACh) clearly plays a role with mixed results regarding effect on psychosis. Selective muscarinic M1 and M4 agonists, such as xanomeline, originally developed to aid in cognitive loss with Alzheimer's, showed promise in proof-of-concept study in 20 patients with schizophrenia. Unfortunately, tolerability problems made muscarinic agonists impractical in either condition. However, coadministration of trospium, a lipophobic, non-selective muscarinic antagonist previously used for the treatment of overactive bladder, with xanomeline resulted in a significant reduction of cholinergic adverse effects. A recent randomized, placebo-controlled study of the antipsychotic effects of this combination in 182 patients with acute psychosis revealed improved tolerability with 80% of subjects staying to the end of the 5 weeks study. At the end of the trial, the treatment group saw a -17.4 change in the positive and negative symptom scale (PANSS) score from baseline compared to a -5.9 change in the placebo arm (P < 0.001). Furthermore, the negative symptom subscore, was also superior in the active arm (P < 0.001). These early studies are exciting because they suggest that the cholinergic system may be recruited to treat a severe and disabling disorder with suboptimal treatment options. Xanomeline-trospium combination is currently in phase III studies.

PS-B03-5: HIGH-DOSES OF LOSARTAN RATHER ENHANCE CATECHOLAMINE SECRETION FROM THE PERFUSED ADRENAL GLAND

Objective: Previously, we have found that Losartan (5∼50 μM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 μM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 μM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 μM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 μM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 μM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 μM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusion: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

S-13-4: INHIBITORY IMPACTS OF CHLOROGENIC ACID ON ADRENAL CATECHOLAMINE SECRETION

(Objective) Chlorogenic acid (CGA) is one of the most available phenolic acid compounds in foods, such as coffee and tea. Thus, the present study was aimed to determine the characteristics of CGA on secretion of catecholamines (CA) in the perfused model of the rat adrenal medulla, and also to validify its mechanism of action. (Design and Methods) The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. (Results) CGA (30∼300 μM), administered into an adrenal vein for 90 min, reduced ACh-induced CA secretion in a dose- and time-dependent manner. CGA also time-dependently depressed the CA secretion induced by McN-A-343 (a selective muscarinic M1 receptor agonist), DMPP (a selective neuronal nicotinic receptor agonist), and angiotensin II. CGA itself failed to influence spontaneous CA secretion. Also, During the perfusion of CGA for 90 min, the CA secretory responses induced by high K+ (56 mM, a direct membrane depolarizer), veratridine (a voltage-dependent Na+ channel activator, cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator were suppressed. However, in the simultaneous presence of CGA and L-NAME (an inhibitor of NO synthase), the CA secretory responses induced by ACh, Ang II, Bay-K-8644 and veratridine was restored nearly to the control level compared to those of the inhibitory effects of CGA-treatment alone. Virtually, the adrenal NO release was significantly enhanced by CGA-treatment. Also, in the coexistence of CGA and olmesartan, ACh-induced CA secretion was markedly reduced compared to that of olmesartan-treatment alone. (Conclusion) Taken together, we present the first evidence that CGA suppresses the CA secretion induced by activation of cholinergic receptors as well as AT1-receptors. This CGA-induced inhibitory effect seems to be exerted by reducing both the influx of Na+ and Ca2+ through their ionic channels into the adrenomedullary chromaffin cells as well as by suppressing the Ca2+ release from the cytoplasmic calcium store, at least through the elevated NO release by the activation of NO synthase, which is associated to the blockade of neuronal cholinergic receptors and AT1-receptors. Based on these results, the ingestion of CGA may be helpful to alleviate or prevent the cardiovascular diseases, via reduction of adrenal CA secretion and consequent decreased CA level in the circulation. Co-administration of CGA and Olmesartan (ARB) may be clinically beneficial for treatment of cardiovascular diseases, including hypertension and angina pectoris.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan &gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

P491. Rationale for Selective Muscarinic Receptor Agonists as Candidates for Antipsychotic Drug Development

Clinical trials of muscarinic receptor agonists for schizophrenia are underway and, if successful, hold the potential to change the current treatment landscape of therapies that directly interact with dopamine (DA) receptors. Muscarinic acetylcholine receptors (mAChRs) have important regulatory effects on dysfunctional DA circuits associated with psychosis and may provide an indirect way to normalize these circuits.

A phase 1b/2a multicenter study of the safety and preliminary pharmacodynamic effects of selective muscarinic M1 receptor agonist HTL0018318 in patients with mild-to-moderate Alzheimer's disease.

IntroductionThis study examined the safety and pharmacodynamic effects of selective muscarinic M1 receptor orthosteric agonist HTL0018318 in 60 patients with mild‐to‐moderate Alzheimer's disease (AD) on background donepezil 10 mg/day.MethodsA randomized, double‐blind, placebo‐controlled 4‐week safety study of HTL0018318 with up‐titration and maintenance phases, observing exploratory effects on electrophysiological biomarkers and cognition.ResultsTreatment‐emergent adverse events (TEAEs) were mild and less frequently reported during maintenance versus titration. Headache was most commonly reported (7–21%); 0 to 13% reported cholinergic TEAEs (abdominal pain, diarrhea, fatigue, nausea) and two patients discontinued due to TEAEs. At 1 to 2 hours post‐dose, HTL0018318‐related mean maximum elevations in systolic and diastolic blood pressure of 5 to 10 mmHg above placebo were observed during up‐titration but not maintenance. Postive effects of HTL0018318 were found on specific attention and memory endpoints.DiscussionHTL0018318 was well tolerated in mild‐to‐moderate AD patients and showed positive effects on attention and episodic memory on top of therapeutic doses of donepezil.

Safety and Pharmacokinetics of HTL0018318, a Novel M1 Receptor Agonist, Given in Combination with Donepezil at Steady State: A Randomized Trial in Healthy Elderly Subjects.

IntroductionHTL0018318 is a selective muscarinic M1 receptor partial agonist under development for the symptomatic treatment of dementias, including Alzheimer’s disease. Clinically, HTL0018318 would likely be used alone or in conjunction with cholinesterase inhibitors (e.g. donepezil).ObjectiveWe investigated the safety, tolerability, and pharmacokinetics of HTL0018318 given alone and in combination with donepezil.MethodsThis was a randomized, double-blind, placebo-controlled trial in 42 (to deliver 36 with combination treatment) healthy elderly subjects investigating the effects of oral HTL0018318 15 and 25 mg given alone and combined with donepezil 10 mg at steady state on adverse events (AEs), vital signs, saliva production, sleep quality, pulmonary function, subjective feelings, and pharmacokinetics.ResultsAEs were reported by lower percentages of subjects after HTL0018318 alone than after donepezil alone. There was no increase in the percentage of subjects reporting AEs after co-administration than after donepezil alone. Supine systolic blood pressure was 1.6 mmHg (95% confidence interval [CI] −3.1 to −0.1) lower after HTL0018318 alone than after combination treatment. This was comparable with results from placebo alone: 1.7 mmHg (95% CI −3.2 to 0.2) lower than with combination treatment. Supine pulse rate was 3.3 bpm (95% CI 1.5–5.1) higher after HTL0018318 alone than with co-administration. HTL0018318 and donepezil did not meaningfully affect each other’s pharmacokinetics.ConclusionHTL0018318 was well tolerated when given alone and in combination with donepezil. HTL0018318 and donepezil do not demonstrate pharmacokinetic or pharmacodynamic interactions, indicating that HTL0018318 can be safely administered in combination with donepezil.Clinical trial registrationNetherlands Trial register identifier NL5915, registered on 28 October 2016.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40268-021-00352-5.

COMPARISON OF INHIBITORY EFFECTS EVOKED BY OLMESARTAN AND ENALAPRIL ON CATECHOLAMINE SECRETION

Objective: The present study was designed to compare enalapril, an angiotensin converting enzyme inhibitor (ACEI) with olmesartan an angiotensin II receptor blocker (ARB) in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril (50 microM) and olmesartan (50 microM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 microM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM), high K+ (a direct membrane-depolarizer, 56 mM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 microM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced. Based on the same concentration (50 microM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan&gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

HIGH-DOSES OF LOSARTAN RATHER ACCELERATE THE SECRETION OF ADRENAL CATECHOLAMINES

Objective: Previously, we have found that Losartan (5∼50 microM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 microM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 microM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 microM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 microM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 microM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 microM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusions: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

Reactivity of human labial glands in response to cevimeline treatment.

Among the pathologies affecting the salivary glands, the Sjögren's syndrome (SS), an autoimmune disease, causes progressive destruction of the glandular tissue. The effect of SS is particularly evident on the labial glands and the morphological analysis of these minor glands is considered useful for diagnosis. Cevimeline hydrochloride (SNI), a selective muscarinic agonist drug, is one of the elective treatments for the hyposalivation due to SS, acting not only on major salivary glands, but also on labial glands since their secretion is primarily under parasympathetic control. Aim of this study is to describe the morphology of human labial glands treated with SNI by light, transmission, and high-resolution scanning electron microscopy. Moreover, a morphometric analysis was applied to the light and transmission electron microscopy micrographs to obtain data that were then compared with analogous data collected on control and carbachol-treated labial glands. Following SNI administration, the mucous tubules exhibited enlarged lumina, which were filled with a dense mucous secretion. Occasionally, small broken debris of the cells were retrieved into the lumen. In the mucous secretory cells, some mucous droplets fused to form a large vacuole-like structure. Similarly, the seromucous acini showed both dilated lumina and canaliculi. These above reported signs of secretion were confirmed through morphometric analysis and a milder action of SNI than carbachol on labial parenchyma was observed. This study confirmed that SNI also evoked secretion on labial glands and that its effect is more physiologic than that of the pan-muscarinic agonists.

Ethnopharmacological basis for folkloric claims of Anagallis arvensis Linn. (Scarlet Pimpernel) as prokinetic, spasmolytic and hypotensive in province of Punjab, Pakistan

Ethnopharmacological relevanceThe conventional naturopaths of Punjab Province (Pakistan) have trivial usage of Anagallis arvensis Linn.(Primulaceae) for cure of diarrhea, constipation, asthma as well as hypertension. AimPresent research was focused to discover comprehensive mechanism of spasmogenic, spasmolytic, bronchorelaxant and hypotensive folkloric usage of Anagallis arvensis Linn.. MethodologyThe crude extract of Anagallis arvensis Linn. (Aa.Cr) & its (aqueous & organic) portions tested in-vitro on isolated jejunum, ileum, trachea, aorta, paired atria preparations as well as in-vivo in mice & normotensive anaesthetized rats. The responses have been noted by transducers (isotonic & isometric) coupled to Power Lab. ResultAnagallis arvensis Linn. (Aa.Cr; crude aqueous-alcoholic extract) produced contractile action at low concentrations but relaxant action was observed by increasing concentrations on spontaneous contractions of isolated jejunum of rabbit. But, pre-treatment of tissue with atropine prior extract caused suppression of contractile effect indicating presence of cholinergic muscarinic response of Aa.Cr. It also triggered relaxation of high Potassium -stimulated contractions of jejunum with subsequent non-parallel right move in Ca++ CRCs. Moreover, Aa.Cr relaxed carbachol - & high Potassium - stimulated contractions in trachea of rabbit but observed relaxant effect was powerful against CCh (1 μM)- stimulated contractions with rightside parallel move of CCh-curves succeeded by non-parallel move, like Dicyclomine, having dual activities. The Aa.Cr also showed relaxant result on Phenylephrine and High Potassium -prompted contractions in endothelium intact aorta. The fractionation revealed segregations of contractile & relaxant effects in relevant aqueous & organic portions. The Intravenous administration of Aa.Cr to ketamine-diazepam anaesthetized normo-tensive albino rats resulted in decreased MABP, SBP & DBP. The Aa.Cr applied negative (−) inotropic & chronotropic action on paired atria. The Aa.Cr also exhibited anti-diarrheal action in mice against castor oil prompted diarrhea and also mitigated distance covered by charcoal meal in gastrointestinal tract in a manner comparable with loperamide. ConclusionThese results revealed presence of CCB and selective muscarinic agonist activity in Aa.Cr, hence validating folkloric practice of Anagallis arvensis Linn. in diarrhea, constipation, asthma & hypertension.

M3 but not M4 muscarinic receptors in the rostromedial tegmental nucleus are involved in the acquisition of morphine-induced conditioned place preference

Opioids strongly inhibit GABAergic neurons in the rostromedial tegmental nucleus (RMTg) that expresses μ-opioid receptors to induce rewarding and psychomotor effects. M3 and M4 muscarinic receptors are co-localized with μ-opioid receptors at these GABAergic neurons. This study explored whether RMTg M3 and M4 muscarinic receptors are involved in regulating opioid-induced reward and locomotion via a conditioned place preference (CPP) paradigm. Selective muscarinic receptor agonists and antagonists were both singly and combinatorically injected into the RMTg to examine their effects on the acquisition of systemic morphine-induced CPP and locomotor activity. The M3 muscarinic receptor agonist, pilocarpine, inhibited the acquisition of morphine-induced CPP, whereas its antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP, 1 μg/side), reversed the inhibitory effect of pilocarpine (30 μg/side). Additionally, 4-DAMP increased locomotor activity while pilocarpine (30 μg/side) partially decreased locomotor activity when combined with morphine. In contrast, the M4 muscarinic receptor agonist, LY2033298 (0.1 and 0.2 μg/side), and antagonist, tropicamide (20 and 40 μM/side), did not affect the acquisition of morphine-induced CPP or locomotor activity. Taken together, our findings suggest that RMTg M3 muscarinic receptors are involved in opioid-induced rewarding and psychomotor effects. Therefore, RMTg M3 muscarinic receptors may represent a promising target for the treatment of opioid addiction.

Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors.

We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.

Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission.

BackgroundCholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods.ResultsWe found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex.ConclusionsThese results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.

Activation of M1/4 receptors phase advances the hamster circadian clock during the day

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) can be reset by the cholinergic agonist carbachol. In hamsters, intraSCN carbachol produces phase advances during the day. This phenomenon has previously been attributed to the muscarinic receptors, as carbachol-induced phase shifts are blocked by pretreatment with the muscarinic antagonist atropine. The SCN contains all five muscarinic receptors, leaving open the question as to which muscarinic receptors mediate these shifts. Here we test two selective muscarinic agonists, the M1/4 agonist McN-A-343 and the M2/3 agonist bethanechol, in addition to the non-selective cholinergic agonist carbachol. Consistent with previous reports, carbachol produced significant phase advances when injected to the SCN during the mid-subjective day. At the doses used here, McN-A-343, but not bethanechol, also produced significant phase shifts when injected to the SCN during the mid-subjective day. Phase shifts to McN-A-343 were as large as those produced by carbachol, suggesting that activation of the M1/4 receptors alone can fully account for the daytime phase advances produced by cholinergic agonists. Given acetylcholine’s role in arousal, and the similarity between phase advances to carbachol/McN-A-343 and to exercise and arousal manipulations, it is possible that acetylcholine may contribute to non-photic resetting of the circadian clock.

Stereoselective synthesis of oxazolidinonyl-fused piperidines of interest as selective muscarinic (M1) receptor agonists: a novel M1 allosteric modulator.

Syntheses of (1RS,2SR,6SR)-2-alkoxymethyl-, 2-hetaryl-, and 2-(hetarylmethyl)-7-arylmethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones, of interest as potential muscarinic M1 receptor agonists, are described. A key step in the synthesis of (1RS,2SR,6SR)-7-benzyl-6-cyclobutyl-2-methoxymethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-one, was the addition of isopropenylmagnesium bromide to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal. This gave the 4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-isopropenyloxazolidinone with the 5-isopropenyl and 4-tert-butyldimethylsilyloxymethyl groups cis-disposed about the five-membered ring by chelation controlled addition and in situ cyclisation. This reaction was useful for a range of organometallic reagents. The hydroboration-oxidation of (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-(1-methoxyprop-2-en-2-yl)-1,3-oxazolidin-2-one gave (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-[(SR)-1-hydroxy-3-methoxyprop-2-yl]-1,3-oxazolidin-2-one stereoselectively. 4,7-Diaza-9-oxabicyclo[4.3.0]nonan-8-ones with substituents at C2 that could facilitate C2 deprotonation were unstable with respect to oxazolidinone ring-opening and this restricted both the synthetic approach and choice of 2-heteroaryl substituent. The bicyclic system with a 2-furyl substituent at C2 was therefore identified as an important target. The addition of 1-lithio-1-(2-furyl)ethene to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal gave (4SR,5RS)-4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-[1-(2-furyl)ethenyl]-1,3-oxazolidinone after chelation controlled addition and in situ cyclisation. Following oxazolidinone N-benzylation, hydroboration at 35 °C, since hydroboration at 0 °C was unexpectedly selective for the undesired isomer, followed by oxidation gave a mixture of side-chain epimeric alcohols that were separated after SEM-protection and selective desilylation. Conversion of the neopentylic alcohols into the corresponding primary amines by reductive amination, was followed by N-nosylation, removal of the SEM-groups and cyclisation using a Mitsunobu reaction. Denosylation then gave the 2-furyloxazolidinonyl-fused piperidines, the (1RS,2SR,6SR)-epimer showing an allosteric agonistic effect on M1 receptors. Further studies resulted in the synthesis of other 2-substituted 4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones and an analogous tetrahydropyran.

Approaches to 3,4,5-substituted piperidines via 1,2,5,6-tetrahydropyridines prepared by ring-closing metathesis

Synthetic approaches to (1RS,2SR,6SR)-7-arylmethyl-2-alkoxymethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones, potentially selective muscarinic M1 receptor agonists, by hydration of 1,2,5,6-tetrahydropyridines were investigated. 3-Substituted N-tosyl-1,2,5,6-tetrahydropyridines were prepared by ring-closing metathesis (RCM). The direct hydration of these by hydroboration-oxidation was not usefully selective, but cis-3-hydroxymethyl-4-tert-butyldimethylsilyloxy-N-tosylpiperidine was prepared from 3-hydroxymethyl-N-tosyl-1,2,5,6-tetrahydropyridine by epoxidation, mesylation, reductive elimination, silylation and hydroboration-oxidation. Problems were encountered during attempts to prepare 3-alkoxymethyl-1,2,5,6-tetrahydropyridines with protected amino and cyclobutyl substituents at C5 by ring-closing metathesis, perhaps because of steric hindrance. Nevertheless interesting chemistry was encountered during the synthesis of the RCM precursors including a novel coupling via a 2-ethenyl-N-nosylaziridine and the formation of an oxaazathiocin by an intramolecular substitution of the nitro group of an N-nosyl protected amine by a proximate hydroxyl substituent.

A Stereoselective Synthesis of a 3,4,5-Substituted Piperidine of Interest as a Selective Muscarinic (M1) Receptor Agonist

Syntheses of (1RS,2SR,6SR)-2-alkoxymethyl-, 2-hetaryl-, and 2-(hetarylmethyl)-7-arylmethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones, of interest as potential muscarinic M1 receptor agonists, are described. A key step in the synthesis of (1RS,2SR,6SR)-7-benzyl-6-cyclobutyl-2-methoxymethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-one, was the addition of isopropenylmagnesium bromide to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal. This gave the 4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-isopropenyloxazolidinone with the 5-isopropenyl and 4-tert-butyldimethylsilyloxymethyl groups cis-disposed about the five-membered ring by chelation controlled addition and in situ cyclisation. This reaction was useful for a range of organometallic reagents. The hydroboration–oxidation of (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-(1-methoxyprop-2-en-2-yl)-1,3-oxazolidin-2-one gave (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-[(SR)-1-hydroxy-3-methoxyprop-2-yl]-1,3-oxazolidin-2-one stereoselectively. 4,7-Diaza-9-oxabicyclo[4.3.0]nonan-8-ones with substituents at C2 that could facilitate C2 deprotonation were unstable with respect to oxazolidinone ring-opening and this restricted both the synthetic approach and choice of 2-heteroaryl substituent. The bicyclic system with a 2-furyl substituent at C2 was therefore identified as an important target. The addition of 1-lithio-1-(2-furyl)ethene to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal gave (4SR,5RS)-4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-[1-(2-furyl)ethenyl]-1,3-oxazolidinone after chelation controlled addition and in situ cyclisation. Following oxazolidinone N-benzylation, hydroboration at 35 °C, since hydroboration at 0 °C was unexpectedly selective for the undesired isomer, followed by oxidation gave a mixture of side-chain epimeric alcohols that were separated after SEM-protection and selective desilylation. Conversion of the neopentylic alcohols into the corresponding primary amines by reductive amination, was followed by N-nosylation, removal of the SEM-groups and cyclisation using a Mitsunobu reaction. Denosylation then gave the 2-furyloxazolidinonyl-fused piperidines, the (1RS,2SR,6SR)-epimer showing an allosteric agonistic effect on M1 receptors. Further studies resulted in the synthesis of other 2-substituted 4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones and an analogous tetrahydropyran

Acute Effects of Muscarinic M1 Receptor Modulation on AβPP Metabolism and Amyloid-β Levels in vivo: A Microdialysis Study.

Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-β (Aβ) production by shifting endoproteolytic amyloid-β protein precursor (AβPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aβ production in awake and freely moving AβPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aβ concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aβ levels while treatment with the M1 antagonist dicyclomine increased ISF Aβ levels reaching significance within 120 minutes of treatment. The reduction in Aβ levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AβPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of β-secretase levels associated with increased amyloidogenic AβPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aβ and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AβPP/Aβ metabolism.