M1 Agonist Research Articles

Lu 25-109, a combined m1 agonist and m2 antagonist, modulates regulated processing of the amyloid precursor protein of Alzheimer's disease.

To examine the effects of the combined muscarinic ml-agonist/m2-antagonist Lu 25-109 on regulated processing of the amyloid protein precursor (APP), we used both transfected cells expressing human muscarinic m1 or m2 acetylcholine receptors, and fresh rat hippocampal slices. Lu 25-109 readily stimulated APPs secretion from HEK 293 cells overexpressing m1, but not m2, receptors, as well as from the hippocampal brain slices. Time-course analyses revealed a rapid (5-35 minutes), and a delayed (55-75 minutes) secretory response to Lu 25-109 with distinct concentration profiles suggesting two distinct cell biological mechanisms. Both responses appeared to reflect post-translational mechanisms because levels of APP message were unchanged after 60 minutes of stimulation with Lu 25-109. In comparison to carbachol, Lu 25-109 had a significantly lower intrinsic activity at muscarinic m1 receptors, compatible with a pharmacological profile as a partial agonist at recombinantly expressed m1 receptors. In as much as stimulation of APPs secretion is associated with reduced formation of A beta peptides, Lu 25-109 may be useful to reduce A beta generation, and thus, slow amyloid plaque formation. Moreover, Lu 25-109 may be useful in promoting the known neurotrophic and neuroprotective biological functions of secreted APPs.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: An unifying hypothesis

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimer's disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimer's disease.

Identification of side chains on 1,2,5-thiadiazole-azacycles optimal for muscarinic M1 receptor activation

Series of analogs to the functional m1 selective agonist, xanomeline (hexyloxy-TZTP), were evaluated for their in vitro m1 efficacy in cell lines transfected with the human m1 receptor. Systematic variation of the side chain and the azacyclic ring led to the discovery of potent muscarinic agonists with robust m1 efficacy, all having the phenylpropargyloxy/thio as the side chain. The most selective compound was the phenylpropargylthio-[3.2.1] endo analog 28, which is a potent and efficacious m1 agonist with no m2 activity.

Alzheimer's disease: relationship between muscarinic cholinergic receptors, beta-amyloid and tau proteins.

Senile dementia is one of the most important health problems in developed countries. The main disease causing dementia is Alzheimer's disease that is characterized by the progressive deterioration of the cholinergic system, beta-amyloid production and deposition, and neurofibrillary tangle formation. Most of the reviewed data, along with data from experiments performed in our laboratory, suggest that there are no changes in the number of muscarinic receptors between Alzheimer and control brains, although the receptors expressed in Alzheimer's disease brains can be anomalous in their function. The muscarinic receptor-G-protein interaction also seems to be impaired in Alzheimer's disease compared with control brains, as well as the G-protein system, with an important decrease in the function of the Gq/11, the most important G-protein stimulating phosphoinositide hydrolysis in human brain; in addition, the second messenger system is also impaired, with a decrease in the synthesis of phosphoinositides and in the number of IP3 receptors. Muscarinic cholinergic receptors are also linked to beta-amyloid production, stimulation of the M1 subtype with agonists results in the processing of the beta-amyloid precursor protein to non-amyloidogenic products and administration of a fraction of the beta-amyloid (beta-amyloid 25-35) to rats, results in a decrease in the number of muscarinic receptors in brain. M1 agonists also decrease the phosphorylation of tau proteins, playing again a modulatory role in the pathogenesis of Alzheimer's disease. The existence of a link between beta-amyloid and tau proteins also has been reported; treatment of hippocampal neurones with beta-amyloid, or the 25-35 residue fragment, resulted in an increase in tau protein phosphorylation. The particular contribution of muscarinic receptors, beta-amyloid and tau proteins in the pathogenesis of Alzheimer's disease remains still unclear. Probably Alzheimer's disease could be due to a progressive degeneration in the relationship between the three components covered in this review.

Xanomeline compared to other muscarinic agents on stimulation of phosphoinositide hydrolysis in vivo and other cholinomimetic effects

Activation of muscarinic m1 receptors which are coupled to the phosphoinositide (PI) second messenger transduction system is the initial objective of cholinergic replacement therapy in Alzheimer's disease. Thus, we evaluated the ability of the selective muscarinic receptor agonist (SMRA) xanomeline to stimulate in vivo phosphoinositide (PI) hydrolysis and compared it to a number of direct acting muscarinic agonists, two cholinesterase inhibitors and a putative m1 agonist/muscarinic m2 antagonist. Using a radiometric technique, it was determined that administration of xanomeline robustly stimulated in vivo PI hydrolysis and the effect was blocked by muscarinic antagonists, demonstrating mediation by muscarinic receptors. The non-selective muscarinic agonists pilocarpine, oxotremorine, RS-86, S-aceclidine, but not the less active isomer R-aceclidine, also effectively stimulated PI hydrolysis in mice. Amongst the putative m1 agonists, thiopilocarpine, hexylthio-TZTP as well as xanomeline effectively stimulated PI hydrolysis, but milameline, WAL 2014, SKB 202026 and PD 142505 did not significantly alter PI hydrolysis. Furthermore, WAL 2014 and SKB 202026 inhibited agonist-induced PI stimulation, suggesting that they act as antagonists at PI-coupled receptors in vivo. The cholinesterase inhibitors, tacrine and physostigmine, and the mixed muscarinic m1 agonist/m2 antagonist LU25-109 did not activate in vivo PI hydrolysis. Xanomeline, hexylthio-TZTP and thiopilocarpine were relatively free of cholinergic side effects, whereas milameline, WAL 2014 and SKB 202026 produced non-selective effects. Therefore, these data demonstrate that xanomeline selectively activates in vivo PI hydrolysis, consistent with activation of biochemical processes involved in memory and cognition and xanomeline's beneficial clinical effects on cognition in Alzheimers patients.

Selective modulation of muscarinic receptor subtypes: therapeutic potential

Muscarinic receptors comprise five subtypes, each of which has a different distribution and function. The therapeutic utility of either activating or antagonising muscarinic receptors has long been recognised, although the clinical use of existing compounds is limited by their side-effects. Since these effects usually emanate from a lack of subtype discrimination, a concerted effort is underway to identify selective muscarinic receptor agonists and antagonists. Functionally-selective M1 agonists are now in clinical trials for cognitive disorders, some of which demonstrate an improvement in function. In terms of antagonists, several compounds are in advanced clinical evaluation for the treatment of smooth muscle disorders including urinary incontinence, irritable bowel syndrome and chronic obstructive pulmonary disease.

The effects of the acetylcholinesterase inhibitor ENA713 and the M1 agonist AF150(S) on apolipoprotein E deficient mice

Apolipoprotein E (apoE)-deficient and control mice were treated chronically with either the acetylcholinesterase (AChE) inhibitor ENA713, or the M1 muscarinic agonist AF150(S). Both treatments reversed the spatial working memory impairment of apoE-deficient mice but they differed in their effects on the levels of brain AChE activity. AF150(S) enhanced the brain AChE activity of apoE-deficient mice and rendered it similar to that of the untreated controls, whereas ENA713 reduced the brain AChE activity of control mice but had no effect on that of apoE-deficient mice. These findings suggest that AChE inhibition and M1 muscarinic activation have similar beneficial cognitive effects on apoE-deficient mice, but that the cellular and molecular mechanisms underlying these effects differ.

Muscarinic M1 and M2 receptor subtype selective drugs modulate neocortical EEG via thalamus.

The present study was designed to investigate the effects of infusions into reticular nucleus of thalamus (NRT) or intracerebroventricular (i.c.v.) infusions of muscarinic M1 and M2 receptor subtype selective drugs on thalamocortically generated neocortical high voltage spindles (HVSs) in awake immobile rats. NRT administration of 2.0 and 20.0 microg McN-A-343, a muscarinic M1 agonist, and 20.0 microg methoctramine, a muscarinic M2 antagonist, suppressed HVSs. The results suggest that the blockade of presynaptic M2 receptors and activation of postsynaptic M1 receptors in the NRT suppress thalamocortical oscillations and increase neocortical electrical arousal.

Comparison of the effects of M1 and M2 muscarinic receptor activation in the absence of GABAergic inhibition in immature rat hippocampal CA3 area

We have reported previously that addition of carbachol (CCh, 25 μM) in the presence of the GABA A antagonist bicuculline (BMI, 10 μM), depresses evoked synaptic responses and significantly increases the frequency of spontaneous synchronous discharges in immature (postnatal days 10–20, P10–P20) hippocampus in vitro. The present experiments were designed in order to compare the activity of CCh with that of the selective muscarinic agonists McN-A-343 (M1 agonist) and oxotremorine-M (M2 agonist), in inducing the above-mentioned effects. Cholinergic agonists were tested in concentrations ranging from 2.5 nM to 5 μM and synaptic field potentials (evoked, spontaneous) were recorded from the CA3 pyramidal layer of hippocampal slices in the presence of BMI (10 μM). Depression of the evoked responses was measured as decrease (i) in their duration, (ii) in the number of population spikes/response, and (iii) in the amplitude of the first population spike of each sequence. Sigmoid dose–response curves were plotted, and the IC 50s were calculated. For all three indexes, the profile of effectiveness was Oxotremorine-M>carbachol=McN-A-343. CCh and oxotremorine-M induced or increased the frequency of spontaneous synchronous discharges in the presence of BMI in a concentration-dependent manner. At the range of concentrations tested, McN-A-343 did not mimic this effect. In conclusion, our results suggest that M2 muscarinic receptors play a major role in both the depression of synaptic responses and the increase in the frequency of spontaneous synchronous discharges observed in the absence of GABAergic inhibition.

Cholinergic regulation of the rat adrenal zona glomerulosa.

Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells. To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M1 muscarinic receptor antagonist pirenzepine (10 and 100 microM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M1-M3 muscarinic receptor antagonist 4-DAMP, nor the selective M2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.

Effects of the M1 Agonist Xanomeline on Processing of Human β-Amyloid Precursor Protein (FAD, Swedish Mutant) Transfected into Chinese Hamster Ovary-m1 Cells

Complimentary DNA (cDNA) encoding human β-amyloid precursor protein familial Alzheimer's disease (FAD) Swedish mutant (βAPPSM) form was cloned into a mammalian expression vector (PK255) containing the CMV promoter. The vector was transfected into Chinese hamster ovary cells containing human muscarinic m1 receptors (CHO-m1), and clonal cells stably expressing βAPPSMwere isolated. The effects of m1-receptor activation by the selective m1 agonist xanomeline and the non-selective muscarinic agonist carbachol on processing of βAPPSMto release soluble APP (APPs) and β-amyloid peptide (Aβ) were compared. Xanomeline stimulated APP release with a potency 1000-fold greater than that observed for carbachol. Concentrations of carbachol and xanomeline producing maximal effects on APPs release reduced the secretion of Aβ by 28 and 46%, respectively. These results extend previous studies with xanomeline and suggest that cholinergic replacement therapy for Alzheimer's disease may reduce amyloid deposition.

The involvement of muscarinic M1 receptor in the regulation of action potentials in mouse isolated right atria

We investigated the involvement of muscarinic M1 receptors in the regulation of action potentials, and its modulation by adrenergic signaling and its change by aging in mouse isolated right atria using a conventional glass microelectrode technique. In adult mice, acetylcholine (ACh) (3-10 microM) reduced the maximum upstroke velocity of action potential (Vmax) followed by an increase. In electrically driven atria, similar effects of ACh on Vmax were observed. McN-A-343 (100-300 microM), a M1 agonist, reduced Vmax, while M2 agonist oxotremorine (0.1-0.3 microM), increased it. Isoproterenol (3 nM), antagonized ACh- and McN-A-343-induced reduction of Vmax, and potentiated the ACh- and oxotremorine-induced increase. The effects of isoproterenol were mimicked by cholera toxin, a Gs-protein activator, and forskolin, a direct activator of adenylyl cyclase. H-89, a selective protein kinase-A inhibitor, abolished the antagonism by isoproterenol of ACh-induced reduction in Vmax. Calphostin C, a selective protein kinase-C inhibitor, but not pertussis toxin attenuated ACh-induced reduction in Vmax. These results show that 1) ACh-induced reduction of Vmax and its subsequent increase are mediated by the activation of muscarinic M1 and M2 receptors, respectively, 2) the M1 and M2 subtypes may exert a balancing action on each other, and 3) the beta-adrenergic activation antagonizes M1-mediated effects, and enhances M2-mediated effects, on Vmax. In young mice, ACh (5-10 microM) increased Vmax, which was abolished by AF-DX 116 (0.3 microM), a M2 antagonist. In aged mice, ACh did not affect Vmax up to a concentration of 10 microM. The present findings may be of importance in the occurrence of cardiac disfunction in aging.

M1 agonist CS-932 ameliorates learning deficits in experimental models

M1 agonist CS-932 ameliorates learning deficits in experimental models

Central activation by CS-932, a functionally relative M1 agonist

1.The present studies evaluated the participation of central muscarinic receptors in the cardiovascular effects of centrally injected neostigmine, a quaternary anticholinesterase, in conscious, sham-operated rats and in sinoaortic denervated animals.2.The dose-dependent pressor effect of neostigmine (0.1 to 1 μg ICV) was greater in sinoaortic denervated rats than in sham-operated animals, but only a dose-dependent bradycardic effect was seen in sham-operated rats.3.Doses of 3.3 nmol (ICV) of both the M1 muscarinic antagonist, pirenzepine, and the M3 muscarinic antagonist, 4-DAMP, prevented the pressor response to 1 μg of neostigmine in sham-operated rats and in sinoaortic denervated animals; however, the M2 muscarinic antagonist, AF-DX116, partially blocked this response in sham-operated rats while failing to do so in sinoaortic denervated rats. In sham rats, doses of 3.3 nmol (ICV) of both pirenzepine and 4-DAMP prevented the bradycardic response to 1 μg (ICV) of neostigmine, whereas AF-DX116 induced a partial blockade.4.4-DAMP, at the dose of 0.3 nmol (ICV), but not pirenzepine at the same dose, prevented the pressor effect of neostigmine (0.1 to 1 μg ICV) in both groups of rats. Both muscarinic antagonists at this dose prevented the bradycardia elicited by the anticholinesterase (0.1 to 1 μg ICV), but 4-DAMP showed a greater antagonistic action on this cardiac effect than pirenzepine. In sham-operated rats, ICV injection of 0.3 nmol of AF-DX116 failed to modify the cardiovascular responses to 0.3 μg of neostigmine.5.Results suggest mainly an involvement of brain M3-subtype muscarinic receptors in the cardiovascular effect of intracerebroventricular administration of anticholinesterase neostigmine in both groups of rats.

1,2,5-Thiadiazole analogues of aceclidine as potent m1 muscarinic agonists.

The acetyl group of the muscarinic agonist aceclidine 4 was replaced by various 1,2,5-thiadiazoles to provide a new series of potent m1 muscarinic agonists 17 and 18. Optimal m1 muscarinic agonist potency was achieved when the 1,2,5-thiadiazole substituent was either a butyloxy, 17d, or butylthio, 18d, group. Although 1,2,5-oxadiazole 37 and pyrazine 39 are iso-pi-electronic with 1,2,5-thiadiazole 17d, both analogues were substantially less active than 17d. Compounds with high muscarinic affinity and/or m1 muscarinic agonist efficacy were also obtained when the 3-oxyquinuclidine moiety of 17d or 18c was replaced by ethanolamines, hydroxypyrrolidines, hydroxyazetidine, hydroxyisotropanes, or hydroxyazanorbornanes. The structure-activity data support the participation of the oxygen or sulfur atom in the substituent on the 1,2,5-thiadiazole in the activation of the m1 receptor. Several of these new 1,2,5-thiadiazoles have m1 agonist efficacy, potency, and selectivity comparable to those of xanomeline 2 in the muscarinic tests investigated.

5-HT3 antagonists derived from aminopyridazine-type muscarinic M1 agonists.

A conformational analysis, performed on muscarinic M1 agonists, identified four structural features characteristic of the muscarinic M1 pharmacophore: (i) a protonable basic or quaternary nitrogen acting as a cationic head; (ii) an electronegative dipole usually part of a planar mesomeric ester, amide, or amidine function which can be replaced by an ether (muscarine) or a dioxolane (AF 30); (iii) an intercharge distance of 5 +/- 0.5 A between the cationic head and the electronegative atom of the dipole; (iv) an elevation of 0.5 +/- 0.03 A of the cationic head over the plane containing the electronegative dipole. During a reinvestigation of the conformational behavior of published structures of 5-HT3 antagonists, similar features were observed for the 5-HT3 pharmacophore. However many 5-HT3 antagonists possess additional aromatic planes not present in the muscarinic M1 agonists. These observations brought us to predict the chemical modifications that would change muscarinic M1 agonists into 5-HT3 antagonists. Four of the predicted aminopyridazines were actually synthesized and submitted to testing. The observed IC50 values for 5-HT3 receptor binding ([3H] BRL 43694) ranged from 10 to 425 nM, whereas the affinities for the muscarinic receptor preparations ([3H] pirenzepine) layed over 10,000 nM. In electrophysiological studies the two most active compounds 10 and 13 produced antagonist-like effects on the 5-HT receptor channel complexes responsible for the generation of the rapidly desensitizing ionic currents, and agonist-like effects on those responsible for the slowly desensitizing components.

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci.

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 >> AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.

Acetylcholine-induced vasoconstrictor response of coronary vessels in rats: a possible contribution of M2 muscarinic receptor activation.

A mechanism by which acetylcholine (ACh) may elicit vasoconstrictor response in coronary vessels was studied in rat hearts perfused at a constant flow rate. In spontaneously beating hearts, bolus injections of ACh and carbachol (CCh) produced biphasic changes in coronary perfusion pressure (CPP): a transient increase at the initial period followed by a sustained decrease. In KCl-arrested hearts, ACh and CCh produced a monophasic increase in CPP, which was attenuated by either removal of endothelial cells by saponin or cyclooxygenase inhibition by diclofenac sodium. In the spontaneously beating heart, ACh-induced vasoconstriction was almost abolished by atropine (0.1 microM) and was markedly attenuated by an M2 antagonist, methoctramine (0.1 microM), but not by an M1 antagonist, pirenzepine (1 microM). Arecaidine propargyl ester (APE), an M2 agonist, produced coronary artery constriction which was attenuated by methoctramine (0.1 microM) but not by pirenzepine (0.1 microM) in both spontaneously beating and KCl-arrested hearts. McN-A-343, an M1 agonist, increased CPP in both beating and KCl-arrested hearts, but to a lesser degree than APE. These results suggest that the release of vasoconstrictor prostaglandins from endothelial cells contributes to the vasoconstrictor response to ACh in perfused rat coronary vessels, and the response to ACh appears to be mediated, in part, via the M2 subtype of muscarinic receptors.

Synthesis and biological characterization of 1,4,5,6-tetrahydropyrimidine and 2-amino-3,4,5,6-tetrahydropyridine derivatives as selective m1 agonists.

Previous studies identified several novel tetrahydropyrimidine derivatives exhibiting muscarinic agonist activity in rat brain. Such compounds might be useful in treating cognitive and memory deficits associated with low acetylcholine levels, as found in Alzheimer's disease. To determine the molecular features of ligands important for binding and activity at muscarinic receptor subtypes, the series of tetrahydropyrimidines was extended. Several active compounds were examined further for functional selectivity through biochemical studies of muscarinic receptor activity using receptor subtypes expressed in cell lines. Several amidine derivatives displayed high efficacy at m1 receptors and lower activity at m3 receptors coupled to phosphoinositide (PI) metabolism in A9 L cells. Four ligands, including 1b, 1f, 2b, and 7b, exhibited marked functional selectivity for m1 vs m3 receptors. Compound 1f also exhibited low activity at m2 receptors coupled to the inhibition of adenylyl cyclase in A9 L cells. Molecular modeling studies also were initiated to help understand the nature of the interaction of muscarinic agonists with the m1 receptor using a nine amino model of the m1 receptor. Several important interactions were identified, including interactions between the ester moiety and Thr192. Additional interactions were found for oxadiazoles and alkynyl derivatives with Asn382, suggesting that enhanced potency and selectivity may be achieved by maximizing interactions with Asp105, Thr192, and Asn382. Taken together, the data indicate that several amidine derivatives display functional selectivity for m1 muscarinic receptors, warranting further evaluation as therapeutic agents for the treatment of Alzheimer's disease. In addition, several amino acid residues were identified as potential binding sites for m1 agonists. These data may be useful in directing efforts to develop even more selective m1 agonists.

Muscarinic agonists in Alzheimer's disease.

As therapeutic agents, M1 agonists in the short-term may palliate symptoms of AD and improve memory function. In the long-term, M1 agonists have the potential to modify the underlying pathophysiology of AD, and thereby prevent or retard the course of dementia.