Elsevier

Brain Research

Volume 795, Issues 1–2, 8 June 1998, Pages 179-190
Brain Research

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

https://doi.org/10.1016/S0006-8993(98)00267-4Get rights and content

Abstract

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.

Introduction

In a large clinical trial the selective muscarinic receptor agonist (SMRA) xanomeline improved cognitive function and clinical global impression in patients with Alzheimer's disease [3]. After six months of treatment with xanomeline, patients with Alzheimer's disease (AD) had significantly better scores on cognitive instruments such as ADAS-Cog than placebo-treated patients. Further, xanomeline dose-dependently reduced the occurrence of problematic behaviors such as hallucinations, delusions, aggressive behavior and mood swings. Thus, this study with xanomeline has demonstrated that muscarinic agonists may be of therapeutic benefit in AD, in agreement with the cholinergic hypothesis [2].

Previous clinical trials with non-selective muscarinic agonists suggested that they were of minimal therapeutic value 14, 33, but these studies were conducted using non-selective muscarinic agonists which were possibly limited by short duration of action, insufficient efficacy and dose-limiting peripheral cholinergic side effects. However, the discovery of subtypes of muscarinic receptors suggested that it may be possible to develop muscarinic agonists for therapeutic uses without necessarily producing undesirable parasympathomimetic side effects. To date, gene products of five distinct muscarinic subtypes have been found 36, 4, 8and have been termed m1–m5. Four subtypes (M1–M4) have been pharmacologically characterized 24, 31, 27, 49and correspond to the m1–m4 genetic receptors. The muscarinic m1, m3 and m5 receptor subtypes are positively coupled to phospholipase C and stimulation of phosphoinositide (PI) hydrolysis, whereas the m2 and m4 subtypes are negatively coupled to adenylyl cyclase [37].

The muscarinic M1 receptor has been suggested as the primary target for selective muscarinic agonist replacement therapy in AD. Although the muscarinic cholinergic neurons that project from the nucleus basalis to the hippocampus and cortex degenerate to a large extent in AD 51, 15, the density of postsynaptic muscarinic receptors in the projection areas are not substantially altered [30]. These postsynaptic receptors have been identified as the M1 subtype and selective activation of these receptors has been hypothesized to be of therapeutic benefit for treating the cognitive dysfunction associated with AD [25]. Further, in vitro evidence suggests that activation of m1 receptors may alter β-amyloid processing and phosphorylation of tau 34, 41, suggesting potential effects on disease progression.

Xanomeline was a potent agonist of M1 receptors in isolated rabbit vas deferens and potently stimulated PI hydrolysis and arachidonic acid release in cell lines transfected with muscarinic m1 receptors 42, 45, 12. Stimulation of PI hydrolysis by xanomeline has been shown in brain slices from hippocampus, cerebral cortex and striatum [38]. Xanomeline was also a potent agonist at muscarinic m4 receptors in clonal cell lines [12]. In isolated tissues, xanomeline had relatively low potency and efficacy at M2 receptors in guinea pig atria and M3 receptors in guinea pig bladder [46]. In vivo xanomeline was shown to activate presynaptic muscarinic M1 heteroreceptors on dopamine nerve terminals in rat striatum resulting in increased concentrations of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), but produced only small increases in tissue levels of acetylcholine, suggesting minimal activation of M2 autoreceptors 46, 10. Moreover, xanomeline did not produce non-selective effects such as tremor and salivation [46]. Recently, activation of PI-coupled receptors in vivo by the muscarinic agonist pilocarpine was shown using a radiometric technique [35]. Activation of PI hydrolysis in vivo by muscarinic agonists demonstrates that compounds penetrate into the brain and have sufficient efficacy to activate target PI-coupled receptors in situ which is the goal of cholinergic augmentation/replacement therapy. The efficacy and selectivity of muscarinic agonists have been in large part determined in cell lines transfected with muscarinic receptors 50, 46. The receptor reserve and coupling efficiency of transfected receptors in cell lines may be significantly different than in mammalian brain and efficacy of agonists may be overestimated [35]. Preliminary studies on xanomeline indicated that it increased PI hydrolysis in vivo in mouse brain [12]. Therefore, we have compared the ability of several non-selective muscarinic agonists, xanomeline and six putative m1 agonists to stimulate PI hydrolysis in vivo in mouse brain. The putative m1 agonists were hexylthio-TZTP 42, 11, milameline 44, 47, SKB 202026 6, 7, WAL 2014 19, 1, PD 142505 [48]and thiopilocarpine 20, 21. The putative m1 agonist/m2 antagonist LU25-109 [32]was also evaluated. The ability of cholinesterase inhibitors to activate PI hydrolysis in vivo was also assessed. Non-selective side effects such as salivation, hypothermia and tremor induced by the cholinergic agents were determined. Additionally, stimulation of PI hydrolysis in vivo in rat brain by xanomeline was studied.

Section snippets

Animals and treatment

Male CF1 (Charles Rivers, Wilmington, MA) mice weighing 18–20 g were injected intracerebroventricularly over 1 min time period with 2 μCi of [3H]−myoinositol (18.6 Ci/mmol, Amersham, Buckinghamshire, England) in 5 μl saline according to the published method [35]. After 24 h the mice were treated subcutaneously (s.c.) with lithium chloride. The effect of muscarinic agonists on PI hydrolysis was determined by administering the agonists s.c. to mice in groups of five 1 h after lithium

Effect of lithium on in vivo PI accumulation

After intraventricular administration of [3H]-myoinositol for 24 h to radioactively label PI stores, 2.6% of the radioactivity in the hippocampus from saline-treated mice was converted to [3H]-IP (Fig. 1). Administration of lithium chloride (1–40 mmol/kg) for 2 h increased conversion of [3H]-IP in a dose related fashion in hippocampus up to 8.5%. The dose of lithium chloride required to produce 50% of the maximal effect (ED50) was 7 mmol/kg. In a duration study, the conversion to [3H]-IP in

Discussion

Using a radiometric technique to determine the production of [3H]-IP in vivo [35], we have compared the ability of muscarinic agonists and cholinesterase inhibitors to activate central muscarinic receptors and stimulate PI hydrolysis in conscious mice. For comparison purposes, we have evaluated the ability of the muscarinic agonists to stimulate PI hydrolysis in A9 L m1 cells, inhibit binding of [3H]-pirenzepine and [3H]-oxotremorine-M to muscarinic receptors and induce undesirable cholinergic

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