Author Report for: Fisher A

Presently, enhancement of cholinergic neurotransmission via cholinesterase inhibitors represents the main available approach to treat cognitive and behavioral symptoms of early as well as late stages of Alzheimer's disease. Restoring the cholinergic system has been a primary means of improving cognition in Alzheimer's disease, as four of the six approved therapies are acetylcholinesterase inhibitors (Fig 1). Memantine is an NMDA antagonist with well documented clinical effect on behavioral symptoms which is often added to cholinesterase inhibitors to potentiate their effect and Aducanumab, targeting the amyloid pathology, has recently been approved. The early, progressive, and selective degeneration of the cholinergic system together and its close relation to cognitive deficits supports the use of cholinergic therapy for Alzheimer's disease. The Review provides an updated view of the basal forebrain cholinergic system, its relation to cognition, and its relevance for therapy of Alzheimer's disease. It deals with the three main aspects that form the basis of the cholinergic-oriented therapy of Alzheimer's disease, its origin, its mechanism of action, its clinical effects, advantages, and limits of a cholinergic therapeutic approach. It includes a new and updated overview of the involvement of muscarinic receptors in Alzheimer's disease as well as the recent development of new and highly selective M1 muscarinic receptor agonists with disease-modifying potential. It also addresses the discovery of a novel Nerve Growth Factor metabolic pathway responsible for the trophic maintenance of the basal forebrain system and its deregulation in Alzheimer's disease. It discusses new clinical studies and provides evidence of the long-term efficacy of cholinesterase-inhibitors therapy suggesting a disease- modifying effect of these drugs. The classical symptomatic cholinergic therapy based on cholinesterase inhibitors is judiciously discussed for its maximal efficacy and best clinical application. The review proposes new alternatives of cholinergic therapy that should be developed to amplify its clinical effect and supplement the disease-modifying effect of new treatments to slow down or arrest disease progression.

PURPOSES: To assess the impact of a government-sponsored reimbursement policy for cholinesterase inhibitors (ChEIs) on trends in physician visits with a diagnosis of Alzheimer's disease (AD). METHODS: Longitudinal population-based study using interrupted time series methods. British Columbia outpatient claims data for individuals aged 65 and older were used to compute monthly AD visit rates and examine the impact of the ChEI reimbursement policy on the coding of AD. We examined trends in the number of patients with AD visits, the number of AD visits per patient, and visits with "competing" diagnoses (mental, neurological, and cerebrovascular disorders and accidental falls). Finally, we described demographic and clinical features of diagnosed patients. RESULTS: We analyzed 1.9 million AD visits. Faster growth in recorded AD visits was observed after the policy was implemented, from monthly growth of 7.5 visits per 100 000 person-months before the policy (95% confidence interval [CI], 6.1-8.9) to monthly growth of 16.5 per 100 000 person-months after the policy (95% CI, 14.8-18.3). After the implementation of the policy, we observed increased growth in the number of patients with recorded AD visits and the number of AD visits per patient, as well as a shift in diagnoses away from mental diseases and accidental falls to AD (diagnosis substitution). CONCLUSIONS: British Columbia's reimbursement policy for ChEIs was associated with a significant acceleration in Alzheimer's visits. Evaluations of health services utilization and clinical outcomes following drug policy changes need to consider policy-induced influences on the reliability of the data used in the analysis.

BACKGROUND: Cholinesterase inhibitors (ChEIs) are prescribed to dementia patients despite their poor tolerance. Low tolerability potentially reduces persistence and adherence, while inducing switching between medications. Comparisons of these utilization measures contribute to knowledge of the relative tolerability of these medications. AIM: The aim was to compare persistence, adherence, and switching between donepezil, galantamine, oral rivastigmine, and rivastigmine patch.
METHODS: A population-based cohort study, using British Columbia claims data (2009-2013), assessed ChEI new users aged 40 and older. We conducted survival analysis to compare persistence and Poisson regression to estimate switching rates. Good adherence, defined as a medication possession ratio of >/=80%, was modeled using log-binomial regression. Analyses were adjusted using propensity scores.
RESULTS: Patients on galantamine had longer mean persistence and better adherence compared with patients on donepezil, with a hazard ratio for discontinuation of 0.91 [95% confidence interval (CI) 0.87-0.96] and a relative risk for good adherence of 1.01 (95% CI 1.002-1.03). Rivastigmine was associated with the shortest mean persistence [3.6 months (95% CI 3.0-4.2) and 5.0 (95% CI 4.7-5.3) for oral and patch, respectively] and the highest mean switching rates. Comparing the two rivastigmine preparations, the patch was associated with decreased discontinuation compared with oral [hazard ratio 0.79 (95% CI 0.71-0.89)] and decreased switching [relative risk 0.63 (95% CI 0.46-0.87) during the first 6 months of treatment]. Paradoxically, the patch was also associated with poorer adherence [relative risk for good adherence 0.94 (95% CI 0.91-0.98)] than the oral formulation.
CONCLUSIONS: Based on estimates of persistence, adherence, and switching, galantamine was the best tolerated ChEI and rivastigmine the least.

BACKGROUND: In October 2007, British Columbia started to cover the cost of cholinesterase inhibitors (ChEIs)-donepezil, galantamine, and rivastigmine-for patients with mild to moderate dementia and prominent Alzheimer's disease. OBJECTIVES: To examine the impact of this policy on persistence with ChEIs.
METHODS: A population-based cohort study was conducted using British Columbia administrative health data. We examined 45,537 new ChEI users aged 40 years and older between 2001 and 2012; 20,360 (45%) started the treatment after the coverage policy was launched. Patients were followed until treatment discontinuation, defined as a ChEI-free gap of 90 days, death, or December 2013. Persistence on ChEIs was estimated using survival analysis and competing risk approach. Hazards of discontinuation were compared using competing risk Cox regression with propensity adjustment.
RESULTS: Patients who started ChEI therapy after the introduction of the coverage policy had a significantly longer persistence. Median ChEI persistence until discontinuation or death was 9.37 months (95% confidence interval [CI] 9.0-39.7) and 17.6 months (95% CI 16.9-18.3) in patients who started therapy before and after the new policy, respectively. The propensity-adjusted hazard ratio for discontinuing therapy was 0.91 (95% CI 0.88-0.94). Similar patterns were observed for persistence with the first ChEI (propensity-adjusted hazard ratio of 0.94; 95% CI 0.91-0.98). In rivastigmine users, the hazard ratio was insignificant (0.98; 95% CI 0.92-1.02).
CONCLUSIONS: The British Columbia ChEI coverage policy was associated with significantly prolonged persistence with donepezil and galantamine, but not rivastigmine.

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-beta (Abeta) production by shifting endoproteolytic amyloid-beta protein precursor (AbetaPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Abeta production in awake and freely moving AbetaPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Abeta 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) Abeta levels while treatment with the M1 antagonist dicyclomine increased ISF Abeta levels reaching significance within 120 minutes of treatment. The reduction in Abeta levels was associated with PKCalpha and ERK activation resulting in increased levels of the alpha-secretase ADAM17 and a shift in AbetaPP 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 PKCalpha, and led to an elevation of beta-secretase levels associated with increased amyloidogenic AbetaPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Abeta and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AbetaPP/Abeta metabolism.

The prescribed drugs for treatment of cognitive deficits in Alzheimer's disease (AD) patients are regarded as symptomatic drugs. Effective disease modifying therapies are not yet prescribed in AD patients. Three major hallmarks of AD (e.g. cholinergic hypofunction, Abeta and tau neuropathologies) are closely linked raising the expectation that restoring the cholinergic hypofunction to normal, in particular via selective activation of M1 muscarinic receptors, may alter the onset or progression of AD dementia. This review is focused mainly on modulation of amyloid precursor processing and Abeta levels in the brain via cholinergic treatment strategies based on M1 muscarinic agonists versus other cholinergic treatments (e.g. cholinesterase inhibitors prescribed for treatment of AD, M2 antagonists and nicotinic agonists). Advantages and potential drawbacks of these treatment modalities are reviewed versus the notion that due to an elusive etiology of AD, future disease modifiers should address comprehensively most of these AD hallmarks (e.g. Abeta pathology, tau and tangle pathologies, as well as the cholinergic hypofunction and cognitive impairments). This major requirement may be fulfilled with M1-selective muscarinic agonists and less with other reviewed cholinergic treatments.

The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.

The only prescribed drugs for treatment of Alzheimer's disease (AD) are acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine, and tacrine) and memantine, an NMDA antagonist. These drugs ameliorate mainly the symptoms of AD, such as cognitive impairments, rather than halting or preventing the causal neuropathology. There is currently no cure for AD and there is no way to stop its progression, yet there are numerous therapeutic approaches directed against various pathological hallmarks of AD that are extensively being pursued. In this context, the three major hallmark characteristics of AD (i.e., the CNS cholinergic hypofunction, formation of beta-amyloid plaques, and tangles containing hyperphosphorylated tau proteins) are apparently linked. Such linkages may have therapeutic implications, and this review is an attempt to analyze these versus the advantages and drawbacks of some cholinergic compounds, such as acetylcholinesterase inhibitors, M1 muscarinic agonists, M2 antagonists, and nicotinic agonists. Among the reviewed treatments, M1 selective agonists emerge, in particular, as potential disease modifiers.

The cellular prion protein (PrP(c)) undergoes a physiological processing yielding the N-terminal fragment referred to as N1, the production of which can be constitutive or protein kinase C regulated. We show that activation of endogenous muscarinic receptors by carbachol and by the M1-selective agonist AF267B increases N1 recovery in an atropine-sensitive manner, in mouse embryonic primary neurons. To identify the muscarinic receptor subtype involved, we used human embryonic kidney HEK293 (HEK) cells stably overexpressing M1, M2, M3, or M4 receptor subtype. Carbachol and the selective M1 agonist AF267B dose dependently increased N1 release by HEK-M3 and HEK-M1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M2 or HEK-M4 cells. We demonstrate that the increase of N1 was not attributable to modified trafficking to the membrane of either PrP(c) or the disintegrin metalloproteases ADAM10 or ADAM17. Furthermore, we establish that carbachol affects the overall phosphorylation of ADAM17 on its threonine and tyrosine but not serine residues, whereas levels of phosphorylated ADAM9 were not affected. Interestingly, carbachol also increases the hydrolysis of the fluorimetric substrate JMV2770, which mimicked the sequence encompassing the N1 site cleavage and was shown previously to behave as an ADAM protease substrate. Mutations of threonine 735 but not of tyrosine 702 of the ADAM17 cytoplasmic tail abolishes the carbachol-induced increase of N1, ADAM17 phosphorylation, and JMV2770-hydrolyzing activity in M1- and M3-expressing HEK293 cells. Thus, our data provide strong evidence that muscarinic receptor activation increases the physiological processing of PrP(c) by upregulating the phosphorylation state and activity of ADAM17 protease.

This study was designed to study the in vitro metabolism of indiplon, a novel hypnotic agent, and to assess its potential to cause drug interactions. In incubations with pooled human liver microsomes, indiplon was converted to two major, pharmacologically inactive metabolites, N-desmethyl-indiplon and N-desacetyl-indiplon. The N-deacetylation reaction did not require NADPH, and appeared to be catalyzed by organophosphate-sensitive microsomal carboxylesterases. The N-demethylation of indiplon was catalyzed by CYP3A4/5 based on the following observations: (1) the sample-to-sample variation in N-demethylation of indiplon ([S] = 100 microM) in a bank of human liver microsomes was strongly correlated with testosterone 6beta-hydroxylase (CYP3A4/5) activity (r(2) = 0.98), but not with any other CYP enzyme; (2) recombinant CYP1A1, CYP1A2, CYP3A4, CYP3A5 and CYP3A7 had the ability to catalyze this reaction; (3) the N-demethylation of indiplon was inhibited by CYP3A4/5 inhibitors (ketoconazole and troleandomycin), but not by a CYP1A2 inhibitor (furafylline). In pooled human liver microsomes, indiplon exhibited a weak capacity to inhibit CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4/5 and carboxylesterase (p-nitrophenylacetate hydrolysis) activities (IC50 >/= 20 microM). Clinical data available on indiplon support the conclusions of this paper that the in vitro metabolism of indiplon is catalyzed by multiple enzymes, and indiplon is a weak inhibitor of human CYP enzymes.

M1 muscarinic receptors (M1 mAChRs) play a role in an apparent linkage of three major hallmarks of Alzheimer's disease (AD): beta-amyloid (Abeta) peptide; tau hyperphosphorylation and paired helical filaments (PHFs); and loss of cholinergic function conducive to cognitive impairments. We evaluated the M1 muscarinic agonists AF102B (Cevimeline, EVOXAC trade mark : prescribed for Sjogren's syndrome), AF150(S), and AF267B on some of these hallmarks of AD. Activation of M1 mAChRs with these agonists leads, inter alia, to enhanced secretion of amyloid precursor protein (alpha-APP), (via alpha-secretase activation), to decreased Abeta (via gamma-secretase inhibition), and to inhibition of Abeta- and/or oxidative stress-induced cell death. In several animal models mimicking different aspects of AD, these drugs restored cognitive impairments, and in select cases induced a decrease in brain Abeta elevation, with a high safety margin, following po administration. Notably, in mice with small hippocampi, unlike rivastigmine and nicotine, AF150(S) and AF267B restored cognitive impairments also on escape latency in a Morris water maze paradigm, in reversal learning. Studies from other labs showed that AF102B and talsaclidine (another M1 agonist) decreased cerbrospinal fluid (CSF) Abeta in AD patients following chronic treatment, being the first reported drugs with such a profile. The clinical significance of these studies remains to be elucidated, yet based on in vivo (rabbits) and in vitro studies (cell cultures), our M1 agonists can decrease brain Abeta, owing to a novel and dual complementary effect (e.g., inhibition of gamma-secretase and activation of alpha-secretase). Remarkably, although M1 agonists can decrease CSF Abeta in AD patients, an increased AD-type pathology in Parkinson's disease was recently been associated with chronic antimuscarinic treatment. In another aspect, these agonists decreased tau hyperphosphorylation in vitro and in vivo. Notably, nicotinic agonists or cholinesterase inhibitors increased tau hyperphosphorylation. In summary, the M1 agonists tested are effective on cognition and behavior and show unique disease-modifying properties owing to beneficial effects on major hallmarks of AD. This may place such drugs in the first line of modern AD therapies (e.g., beta- or gamma-secretase inhibitors, vaccines against Abeta, statins, and inhibitors of tau hyperphosphorylation).

Distractibility in primates may be influenced by central cholinergic systems. Two cholinomimetics, the m-1 muscarinic agonist (+/-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3')quinuclidine (AF102B, civemeline) and the cholinesterase inhibitor tetrahydroaminoacridine (THA, tacrine), were compared to vehicle controls for effects on distractibility in an automated visuospatial attention task. The task required visual pursuit of a moving target amongst distractor stimuli that acted to impair performance and was executed by seven healthy adult bonnet macaque monkeys. Task accuracy and reaction time were measured 1.5 h after systemic administration of each substance. For the seven-subject group at individually titrated best doses, accuracy increased significantly relative to vehicle for both drugs. Reaction time at best dose decreased for both drugs, but not significantly. Muscarinic agonists and cholinesterase inhibitors may reduce distractibility in primates.

The cholinergic hypofunction in Alzheimer's disease (AD) appears to be linked with two other major hallmarks of this disease, beta-amyloid and hyperphosphorylated tau protein. Formation of beta-amyloids might impair the coupling of M1 muscarinic acetylcholine receptors (mAChR) with G-proteins. This can lead to decreased signal transduction, a decrease of trophic and non-amyloidogenic amyloid precursor protein (APPs) and generation of more beta-amyloids, aggravating further the cholinergic deficiency. This review is an attempt to explore the M1 mAChR regulation of beta-amyloid metabolism, tau hyperphosphorylation and cognitive functions. The therapeutic potential of M1-selective muscarinic agonists including AF102B, AF150(S), AF267B (the AF series) is evaluated and compared, when possible, with several FDA-approved acetylcholinesterase inhibitors. These M1 agonists can elevate APPs, decrease tau protein phosphorylation/hyperphosphorylation in vitro and in vivo and restore cognitive impairments in several animal models for AD. Except for the M1 agonists, no other compounds were reported yet with combined effects; e.g., amelioration of cognition dysfunction and beneficial modulation of APPs/beta-amyloid together with tau hyperphosphorylation/phosphorylation. This property of M1 agonists to alter different aspects associated with AD pathogenesis could represent the most remarkable clinical value of such drugs.

A cholinergic hypofunction in Alzheimer's disease (AD) may lead to formation of beta-amyloids that might impair the coupling of M1 muscarinic ACh receptors (mAChRs) with G proteins. This disruption in coupling can lead to decreased signal transduction, to a reduction in levels of trophic amyloid precursor proteins (APPs), and to generation of more beta-amyloids that can also suppress ACh synthesis and release, aggravating further the cholinergic deficiency. These "vicious cycles," a presynaptic and a postsynaptic one, may be inhibited, in principle, by M1 selective agonists. Such properties can be detected in the functionally selective M1 agonists from the AF series [e.g., project drugs, AF102B, AF150(S)]. These M1 agonists promote the nonamyloidogenic APP processing pathways and decrease tau protein phosphorylation. The effects on tau proteins suggest a link between M1 mAChR-mediated signal transduction system(s) and the neuronal cytoskeleton via regulation of phosphorylation of tau microtubule-associated protein. This may indicate a dual role for M1 agonists: as inhibitors of two "vicious cycles," one induced by beta-amyloids, and the other due to overactivation of certain kinases (e.g., glycogen synthase kinase-3, GSK-3) or downregulation of phosphatases, respectively. Prolonged administration of AF150(S) in apolipoprotein E-knockout mice restored cognitive impairments, cholinergic hypofunction, and tau hyperphosphorylation, and unveiled a high-affinity binding site to M1 mAChRs. Except M1 agonists, there are no reports of compounds having such combined effects, for example, amelioration of cognition dysfunction and beneficial modulation of APPs together with tau phosphorylation. This unique property of M1 agonists to alter different aspects of AD pathogenesis could represent the most remarkable, yet unexplored, clinical value of such compounds.

The cholinesterase inhibitor tacrine (THA) and the M1 muscarinic agonist AF102B (cevimeline), both reported to enhance cognition in animals and humans, were tested in 5 macaques for reduction of spontaneous, random movements. Monkeys were videotaped 1 hour after administration of normal saline vehicle, after low- and high-dose intramuscular AF102B, and after low- and high-dose oral THA. Two independent blind judges counted numbers of spontaneous movements made by each monkey over 12 consecutive 15-second segments for each drug condition. Both THA and AF102B reduced movement significantly at high doses without overt side effects, warranting further research on the agitation-reducing potential of cognition-enhancing cholinomimetic drugs.

Apolipoprotein E (apoE)-deficient mice have memory deficits that are associated with synaptic loss of basal forebrain cholinergic projections and with hyperphosphorylation of distinct epitopes of the microtubule-associated protein tau. Furthermore, treatment of apoE-deficient mice with the M1 selective agonist 1-methylpiperidine-4-spiro-(2'-methylthiazoline) [AF150(S)] abolishes their memory deficits and results in recovery of their brain cholinergic markers. In the present study, we used a panel of anti-tau monoclonal antibodies to further map the tau epitopes that are hyperphosphorylated in apoE-deficient mice and examined the effects of prolonged treatment with AF150(S). This revealed that tau of apoE-deficient mice contains a distinct, hyperphosphorylated "hot spot" domain which is localized N-terminally to the microtubule binding domain of tau, and that AF150(S) has an epitope-specific tau dephosphorylating effect whose magnitude is affected by apoE deficiency. Accordingly, epitopes which reside in the hyperphosphorylated "hot spot" are dephosphorylated by AF150(S) in apoE-deficient mice but are almost unaffected in the controls, whereas epitopes which flank this tau domain are dephosphorylated by AF150(S) in both mice groups. In contrast, epitopes located at the N and C terminals of tau are unaffected by AF150(S) in both groups of mice. These findings suggest that apoE deficiency results in hyperphosphorylation of a distinct tau domain whose excess phosphorylation can be reduced by muscarinic treatment.

The effects of cholinergic drugs proposed for treatment of cognitive impairment in normal aging and dementia on divided attention have been little studied in non-human primates. We tested the hypothesis that cholinergic drugs improve spatial divided attention in primates via a computer task requiring simultaneous tracking of two visual targets in three young and two aged healthy bonnet macaques. Task accuracy (number of correct responses) and reaction time (RT) were measured 2 h after administration of either the m1 agonist +/- -cis-2-methyl-spiro(1,3-oxathiolane-5,3')quinuclidine (AF102B; 0.1-2.1 mg/kg IM) or the cholinesterase inhibitor 9-amino-1,2,3,4-tetrahydroamino-acridine (THA; 0.5-2.0 mg/kg orally). Accuracy increased for four of five monkeys at appropriate doses of one or both cholinomimetics, accompanied in two monkeys by a drop in RT. Responses were less uniform to THA than to AF102B. For the five-monkey group at Best dose, accuracy increased 34% (THA) or 43% (AF102B) above baseline (P<0.05 for both drugs), respectively, with no significant change in RT and with minimal untoward effects. Cholinotherapy may improve divided attention in young and aged healthy primates.

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.

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.

Recent studies suggest that apolipoprotein E (apoE) plays a specific role in brain cholinergic function and that the E4 allele of apoE (apoE4), a major risk factor for Alzheimer's disease (AD), may predict the extent of cholinergic dysfunction and the efficacy of cholinergic therapy in this disease. Animal model studies relevant to this hypothesis revealed that apoE-deficient (knockout) mice have working memory impairments that are associated with distinct dysfunction of basal forebrain cholinergic neurons. Cholinergic replacement therapy utilizing M1-selective muscarinic agonists has been proposed as effective treatment for AD patients. In the present study, we examined whether the memory deficits and brain cholinergic deficiency of apoE-deficient mice can be ameliorated by the M1-selective agonist 1-methylpiperidine-4-spiro-(2'-methylthiazoline), [AF150(S)]. Treatment of apoE-deficient mice with AF150(S) for 3 weeks completely abolished their working memory impairments. Furthermore, this reversal of cognitive deficit was associated with a parallel increase of histochemically determined brain choline acetyltransferase and acetylcholinesterase levels and with the recovery of these cholinergic markers back to control levels. These findings show that apoE deficiency-related cognitive and cholinergic deficits can be ameliorated by M1-selective muscarinic treatment. They also provide a novel model system for development and evaluation of therapeutic strategies directed specifically at the AD patients whose condition is attributed to the apoE genotype.

1. Object working memory, a function which declines in aging and dementia, was tested in young and aged pretrained monkeys using a delayed match-to-sample task. 2. During drug treatment, monkeys were given the m 1 muscarinic agonist AF102B (0.1-2.1 mg/kg i.m.), the cholinesterase inhibitor tacrine (0.5-2.0 mg/kg p.o.), or vehicle controls in a repeated measures design to assess putative cognitive enhancement. 3. Both agents improved task performance in both young and aged monkeys, AF102B yielding equivalent or greater, and less variable, improvement than tacrine. 4. AF102B may represent a low-toxicity alternative to tacrine for the treatment of age-related memory disorders.

Dehydroepiandrosterone (DHEA), the major secretory product of the human adrenal cortex, significantly declines with advanced age. We have previously demonstrated that DHEA prevents the reduction in non-amyloidogenic APP processing, following prolonged stimulation of the muscarinic receptor, in PC12 cells that express the ml acetylcholine-receptor. The present study examined whether this effect may be mediated via modulation of APP metabolism. It was found that DHEA treatment increases the content of membrane-associated APP holoprotein by 24%, and the accumulation of secreted APP in the medium by 63%. No increase in viable cell number nor in nonspecific protein production was observed in DHEA-treated cells. Thus, DHEA seems to increase specifically both APP synthesis and secretion. We propose that the age-associated decline in DHEA levels may be related to the pathological APP metabolism observed in Alzheimer's disease.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151 (S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single-blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales.

It was previously shown by us and by others that activation of muscarinic acetylcholine receptors evoke amyloid precursor protein (APP) secretion in various cell lines. Here we examined if such muscarinic control of APP secretion occurs also in normal brain tissues. We found that the secretion of APP from rat cerebrocortical slices (rich in M1 receptors) was significantly increased by K+ depolarization, the non-selective agonist, carbachol (CCh), and the M1-selective agonist, AF102B. CCh also increased APP secretion from cerebellar slices (rich in M2 receptors) while AF102B had no significant effect in this brain region. Despite of its stimulatory effect on APP release in the cerebellum, CCh had no effect on phosphoinositide (PI) metabolism in this brain region. In the cerebral cortex PI metabolism was significantly increased by CCh but only partially increased by AF102B. These results suggest that APP secretion in the brain is mediated via muscarinic receptors. In the cerebral cortex APP secretion seems to be regulated via M1 receptors. Our results also suggest that PI metabolism is not a pronounced step in mediating APP processing.

Epidemiologic studies suggest that the age-related decline in dehydroepiandrosterone (DHEA) levels may be associated with Alzheimer's disease (AD). Cholinergic markers also decline with age, and are associated with AD pathology. Activation of m1AChR-transfected PC12 cells (PC12M1) with cholinergic agonists results in secretion of Alzheimer's beta-amyloid precursor protein (APP) which in turn reduces beta-amyloid production. This study examined whether DHEA affects APP processing in m1AChR-transfected PC12 cells. DHEA treatment did not significantly alter basal or m1AChR-stimulated APP secretion. However, DHEA (0.1 microM) significantly diminished the desensitization of APP secretion in cells exposed to carbachol for 24 h. The effect of DHEA on APP processing is probably not related to up-regulation of m1AChR or increased m1AChR-activated phosphoinositide hydrolysis since these parameters did not change following DHEA treatment. These findings imply a possible involvement of DHEA in APP processing. Thus, the age-associated decline in DHEA levels may contribute to decreased APP secretion and a consecutive increase in beta-amyloid deposition, which in turn may play a role in the development of AD.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. Notably, the selectivity of the new m1 agonists is reflected also by activation of select second messenger systems via distinct G-proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand-selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor subtypes. AF102B, AF125, AF150 and AF151 are such rigid analogs of ACh. Whilst AF125 is an M2 > M1 agonist, AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia, only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRs in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. In various animal models for Alzheimer's disease (AD) all three agonists (AF102B, AF150 and AF151), and in particular AF102B, exhibited positive effects on mnemonic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy in AD. Here we review some current features of these compounds, which may be relevant to a rational treatment strategy in AD. Comparison is made, whenever possible, with some new and old muscarinic agonists.

The effects of muscarinic agonists (AF102B, pilocarpine, oxotremorine) and anticholinesterases (physostigmine, tetrahydroaminoacridine) were investigated on the incidence of thalamically generated rhythmic high voltage spindles and on scopolamine (0.2 mg/kg)-induced neocortical slow wave activity (i.e. increased sum amplitude value of the 1-20 Hz band in a quantitative electroencephalography (qEEG) analysis). AF102B and pilocarpine decreased high voltage spindles and scopolamine increased sum amplitude values at 3 and 9 mg/kg, but not at 1 mg/kg. Oxotremorine was less potent than AF102B or pilocarpine in suppressing high voltage spindles. Oxotremorine had no effect on the scopolamine-induced qEEG changes. Tetrahydroaminoacridine decreased high voltage spindles at 1, 3 and 9 mg/kg and slow waves at 9 mg/kg. Physostigmine decreased high voltage spindles and slow waves at 0.12 and 0.36 mg/kg. Based on the present results we propose that agonists possessing muscarinic M1 receptor activity are effective in decreasing high voltage spindles and scopolamine-induced slow wave activity, but agonists showing predominant muscarinic M2 receptor activity may be less effective in decreasing high voltage spindles and slow waves. Furthermore, tetrahydroaminoacridine decreased high voltage spindles at doses lower than those required to decrease scopolamine-induced slow waves. Physostigmine decreased high voltage spindles and slow waves over the same dose range. This result may indicate that non-cholinergic mechanisms are involved in the tetrahydroaminoacridine-induced decrease in high voltage spindles.

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 microliters/side) or vehicle (saline). Synaptosomes were preloaded with [3H]choline ([3H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [3H]Ch and [3H]acetylcholine ([3H]ACh) and for their ability to synthesize and release [3H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [3H]Ch was reduced to 60% of control; (ii) residing [3H]ACh levels were 43% of control while residing [3H]Ch levels were 72% of control; (iii) basal and K(+)-induced [3H]ACh release were 77% and 73% of control, respectively; (iv) high K(+)-induced synthesis of [3H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.

The putative muscarinic M1 receptor agonist, AF102B, was applied to rat hippocampal slices and the responses of intracellularly recorded pyramidal cells were examined. AF102B mimicked some effects of acetylcholine on these cells as follows: at low concentration, AF102B attenuated a slow after-hyperpolarization in response to a long depolarizing current pulse. This effect was blocked by the M1 antagonist, pirenzepine. At higher concentrations, AF102B also depolarized the cells and caused an increase in their input resistance. AF102B did not affect local excitatory postsynaptic potentials or reactivity to topically applied excitatory amino acid substances. These experiments indicate that AF102B acts as an agonist at some muscarinic M1 receptor subtypes in mammalian brain.

AF102B [(+-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3')quinuclidine], a structurally rigid analog of acetylcholine, was investigated in a number of neurochemical, pharmacological and behavioral tests related to cholinergic functions. AF102B induced atropine-sensitive contractions of isolated guinea pig ilea and trachea preparations with EC50 values of 3.5 and 3 microM being 87- and 1.3-fold less potent than acetylcholine, respectively. Binding studies using the radioligands pirenzepine, cis-dioxolane and quinuclidinyl benzilate in rat cerebral cortex and quinuclidinyl benzilate in cerebellar homogenates indicated that AF102B was a potent and highly selective M1-type muscarinic probe, being more selective for M1 receptors than oxotremorine, carbachol and AF102A (the trans-isomer of AF102B). AF102B had a 3-fold higher apparent affinity for M1 receptors than the prototype M1 agonist, McN-A-343, cis- and trans-AF30 (other rigid analogs of acetylcholine). Treatment of rat cortical homogenates with Cu++ ions did not modify the affinity observed for the muscarinic antagonists atropine, scopolamine and pirenzepine, whereas increasing the proportion of high affinity sites for the agonists oxotremorine-M, carbachol and McN-A-343. The apparent affinity of AF102B also increased by Cu++ treatment suggesting that this compound interacts with rat cerebral cortex muscarinic receptors as an agonist. AF102B did not affect high affinity choline transport, choline acetyltransferase and acetylcholinesterase activities in rat brain preparations. In rats treated with AF64A (the cholinotoxin ethylcholine aziridinium ion; 3 nmol/2 microliters/side i.c.v.), AF102B (1 mg/kg p.o. or i.p.), AF102A (1 mg/kg i.p.), cis-AF30 (1 mg/kg, i.p.) and physostigmine (0.06 mg/kg i.p.), each reversed cognitive impairments in a step-through passive avoidance task. Both AF102B and AF102A (1 mg/kg i.p.), but not physostigmine (0.1 mg/kg i.p.), were effective also in reversing reference memory impairments in a Morris water maze test. Repetitive administrations of AF102B (0.2 mg/kg/day i.p.) improved AF64A-induced working memory deficits in the Morris water maze test, but did not affect open field behavior. The data show that the selective M1 agonist AF102B can restore AF64A-induced cognitive impairments, without producing adverse central and peripheral side effects at the effective doses and this can indicate its potential use for the treatment of Alzheimer's disease.

Binding assays involving various antagonists, including N-[3H] methylscopolamine, [3H]quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat "brainstem" (medulla, pons, and colliculi). However, competition curves between N-[3H]methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of [3H]-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with [3H]quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.

Postsynaptic potentials elicited by various muscarinic agonists and by preganglionic stimuli in the presence of such agonists were recorded from rabbit superior cervical ganglia using sucrose-gap and air-gap methods. While methacholine and bethanechol (both at 10(-4) M) induced biphasic potential changes, McN-A-343 and a novel synthetic compound AF-102B (10(-7) M-10(-5) M) produced only a depolarizing response which was depressed by the M1-antagonist pirenzepine (10(-7) M), but not by the M2 antagonist AF-DX 116 (same concentration), indicating that these compounds act purely as M1-muscarinic agonists in this system. These agonists selectively depressed the orthodromic slow excitatory postsynaptic potential (EPSP) in a dose-dependent manner without substantially affecting the fast EPSP; this is in accord with the view that their depolarizing action is on the same postsynaptic muscarinic receptor that mediates the slow EPSP. The slow inhibitory post synaptic potential (IPSP), on the other hand, was found potentiated in the presence of these agonists. This potentiation was antagonized not only by pirenzepine but also by yohimbine; the potentiation was itself enlarged by nomifensine (a dopamine-uptake inhibitor). We postulate that M1-muscarinic receptors are present not only on the postganglionic principal cells but also on the interneurons; the former were already known to be responsible for the generation of slow EPSP, but the latter may be on terminals of dopamine-containing small intensely fluorescent cells and regulate the orthodromic release of dopamine and are to be distinguished from the M2-receptors.

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.

AF64A is a toxin which can diminish irreversibly cholinergic transmission in vivo (1, 2). Disruption of neurotransmitter function in vivo is specific to the cholinergic system when AF64A is administered in nanomolar quantities (3, 4). The mechanisms involved appear to be mediated presynaptically (g). The neurochemical and behavioral consequences of AF64A administration are reminiscent of similar measures in patients with Alzheimer's disease (5, 6). Consequently, we have suggested tentatively that the AF64A treated animal may be explored as a potential animal model of this debilitating disease state (7). In this report we provide a brief overview of our recent findings using this compound in vivo, attempt to correlate these findings with those of others with similar aziridinium agnts in vitro, and propose a possible mechanism of action of AF64A in vivo, based on recent observations made in our laboratories.

The selective neurotoxic effects of the aziridinium ion of ethylcholine (AF64A) have been examined after stereotaxic injection into the rat striatum. In a dose-response study (2-26 nmol), 8 nmol caused a 46% decrease in striatal choline acetyltransferase (CAT) activity with minimal effects on the activities of glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH) at 7 days. Maximal CAT reductions of 78-82% occurred with doses of 16-26 nmol which also caused dose-related decreases in GAD and TH activities that paralleled the progressive decrements in CAT. A time course study with 8 nmol indicated a rapid 20% reduction of CAT activity by 12 h and an additional gradual fall of 20% over the next week; TH and GAD activities were not significantly reduced. The selective inhibition of CAT activity persisted for at least 3 months. Histological examination of Nissl stained sections revealed an area of nonspecific damage at the injection site with an abrupt border surrounded by apparently normal striatal neuropil; however; neuronal perikarya staining intensely for acetylcholinesterase were not reduced. These preliminary findings strongly suggest that AF64A has selective neurotoxic effects against striatal cholinergic neurons while relatively sparing striatal GABAergic intrinsic neurons or dopaminergic afferents.

Ethylcholine mustard aziridinium ion (AF64A) is a neurotoxin which is specific for cholinergic nerve terminals. Besides its effects on elements of the acetylcholine system, we observed that, after 2 and 8 days, a single 20-nmol intracerebroventricular dose altered the Timm's staining of certain regions of the central nervous system and reduced the tissue levels of trace metals. In the hippocampal formation, there was a considerable decrease in the staining of the neuropil of the stratum radiatum and stratum oriens, which contain cholinergic nerve terminals. A reduction in staining was also demonstrated in the perikarya of cortical pyramidal cells. The diminished trace-metal level in both regions was confirmed by quantitative measurements of zinc and copper levels. A similar reduction was not observed at a lower dose (8 nmol) of the cholinotoxin. The results led to the conclusion that AF64A may cause the decrease of the trace-metal content of the postsynaptic neurons through an indirect mechanism.

The behavioral and biochemical effects of AF64A, a presynaptic cholinergic neurotoxin, were investigated. Bilateral administration of this compound into the lateral cerebral ventricles produced transient and dose-related effects on sensorimotor function and long-term impairments of cognitive behavior. Male Fischer-F344 rats dosed with either 15 or 30 nmol of AF64A reacted 29-62% faster than CSF-injected controls in a hot-plate test 14 (but not 1, 7, 21 or 28) days following dosing. The group administered 15 nmol of AF64A was also significantly more active (41%) than controls 28 days following dosing. The activity level of this group was comparable to that of controls at other times and hyperactivity was never observed in the 30 nmol group. Retention of a step-through passive avoidance task, assessed 35 days after dosing, was impaired in both the 15 and the 30 nmol groups. Their step-through latencies were significantly shorter than the control latencies, and they exhibited more partial entries during the 24-h retention test. Radial-arm maze performance, measured 60-80 days following treatment, was markedly impaired in the treated groups. Animals treated with AF64A made fewer correct responses in their first 8 choices, required more total selections to complete the task, and had an altered pattern of spatial responding in the maze. The neurochemical changes produced by AF64A, determined 120 days after dosing, were specific to the cholinergic system and consisted of decreases of ACh in both the hippocampus (15 and 30 nmol groups) and the frontal cortex (30 nmol group). The concentrations of catecholamines, indoleamines, their metabolites and choline in various brain regions were not affected by AF64A. Furthermore, histological analysis revealed that the doses of AF64A used in the present study did not damage the hippocampus, the fimbria-fornix, the septum or the caudate nucleus. These data support the contention that cholinergic processes in the hippocampus and/or frontal cortex play an important role in learning and memory processes. Furthermore, based upon the behavioral and biochemical data presented, it is suggested that AF64A could be a useful pharmacological tool for examining the neurobiological substrates of putative cholinergic disorders such as senile dementia of the Alzheimer's type.

The neuropharmacologic effects of ethylcholine aziridinium ion, AF64A, were studied in cats, using various physiological techniques, to ascertain its synaptic site of action and to determine whether it may act as a cholinergic specific neurotoxin in vivo. Nictitating membrane contractions elicited by preganglionic nerve stimulation (1-16 Hz) were diminished in a dose-dependent manner after injection of AF64A into the carotid artery. Contractions due to injection of l-norepinephrine, tetramethylammonium or acetylcholine were not changed. Postganglionic action potentials from the superior cervical ganglion evoked by preganglionic stimulation were also abolished by AF64A, whereas the postganglionic firing produced by tetramethylammonium was unchanged. Neither the nictitating membrane nor ganglionic responses on the contralateral side of the animal were affected by AF64A treatment. In the same animals, twitch tension in the tongue produced by stimulation of the ipsilateral hypoglossal nerve (1-16 Hz) was gradually reduced and in most experiments completely blocked by AF64A. Repetitive stimulation of either the autonomic or somatic nerves at high frequencies (greater than 10 Hz) magnified and accelerated the onset of neurotoxic effects of AF64A. The suppression of ganglionic and neuromuscular transmission by AF64A was irreversible during the course of the experiments (12-18 hr). From these results, we can conclude that AF64A produces in the peripheral nervous system a longlasting inhibition of cholinergic transmission, without interfering with adrenergic transmission. Moreover, because AF64A did not block the postganglionic responses elicited by cholinergic nicotinic and muscarinic agonists, the inhibitory effects of AF64A must be mediated by a presynaptic action on cholinergic nerve terminals.

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.

Ethylcholine mustard aziridinium ion (AF64A), a neurotoxic choline analog, was evaluated for its interactions with the cholinergic system in mice. Parenterally administered AF64A was lethal (LD50 = 32.6 mumol/kg i.v.) and the lethality could be antagonized even at 8 LD50 doses by pretreatment with choline (714 mumol/kg i.p.) 2 min earlier. Mice that were protected by choline slowly developed neurological motor disturbances such as ataxia and hypokinesia, and lost weight. Intracerebroventricular administration of 65 nmol of AF64A was not acutely lethal, but produced similar delayed behavioral effects similar to those found after parenteral administration of AF64A. Seven days after a single injection of 65 nmol of AF64A i.c.v., there was a significant decrease in acetylcholine content in the cortex, striatum and hippocampus, but no change in choline levels. Acetylcholine content was still significantly reduced in the hippocampus at 3 weeks after this treatment. The reduction in activity of choline acetyltransferase and high-affinity choline transport paralleled the reduction in acetylcholine measured at 7 days post AF64A treatment, whereas muscarinic receptors in all three brain areas were unchanged. These combined data indicate that AF64A is a presynaptic chemical neurotoxin, capable of inducing a persistent deficiency in central cholinergic transmission.

The present work is an attempt to elucidate: (1) whether highly rigid structural analogs of acetylcholine are still capable of activating the muscarinic receptor; (2) whether such analogs, be they agonists or antagonists, discriminate among the various ACh-mediated functions, thereby providing a tool for the study of a possible receptor heterogeneity; (3) whether structural rigidity is a significant factor in the kinetics of drug-receptor interaction. To this end, we investigated some properties of drugs in the spiro-(1,3-dioxolane-4,3')-quinuclidine system (SDQ) which embodies the muscarinic pharmacophore in a framework of utmost rigidity. Wherever possible, these properties were compared with those of a closely related but more flexible analog. Variation in effect between members of a rigid-flexible pair or among drugs of varying rigidity is considered to reflect varying affinities towards various sites of action. 2-Methyl-spiro-(1,3-dioxolane-4,3')-quinuclidine (AF-30) is a weak but selective muscarinic agonist. It can be viewed as a highly rigid version of 3-acetoxyquinuclidine (3-AcQ) and it can be used as a probe for detection of heterogeneity among muscarinic receptors. AF-30 is equipotent with 3-AcQ in causing tremors (mice), but has 1/17th the activity of 3-AcQ in the guinea-pig ileum, 1/30th in lowering blood pressure (cats) and 1/10th in inducing analgesia (mice). 2-Diphenylmethyl-spiro(1,3-dioxolane-4',3)-quinuclidine (AF-41) and 2.2-diphenyl-spiro-(1,3-dioxolane-4,3')-quinuclidine (AF-32 are potent antagonists and possess KD values in the same range as those of the more flexible congener 3-diphenylacetoxy-quinuclidine (AF-43) and atropine (0.6--2 nM) but with koff = 0.1 msec-1 (AF-41) and koff = 1 msec-1 (AF-43) (carp atrium). Thus, duration of drug action of drug action at the receptor is a function of structural rigidity in the drug molecule, termination of action being fastest with the flexible molecules. Differences in rigidity among various antagonists also find expression in an unequal distribution of potencies in various tests; thus the rigid antagonists differentiate between two central effects in mice, viz., prevention of oxotremorine-induced tremors and fall from the rotating rod by a factor of 1:20 (especially AF-41 versus AF-43), whereas the more flexible antagonists (AF-43, atropine or even 3-quinuclidinyl-benzilate) do not show such as a selectivity. The existence of heterogenous muscarinic receptors can be inferred from data presented. Both theoretical and practical implications are discussed.

Time course measurements of the action of muscarinic antagonists were performed in the spontaneously beating carp atrium. Several high affinity drugs, which embody the quinuclidine structure were examined. The structural flexibility of these molecules was reflected in the dissociation of the drugs from the muscarinic receptor. The dissociation of rigid drugs was very much prolonged as compared to flexible drugs of the same affinity.

A study of heterogeneity among muscarinic receptors was carried out with new rigid molecules, comprising structures in the fused quinuclidine-valerolactone, quinuclidine-cyclohexenone, quinuclidine-cyclohexanone and quinuclidine-cyclohexane derivatives. These are structurally related to the potent muscarinic agent, 3-acetoxyquinuclidine but substantially different from in it conformation. All proved to antagonize acetylcholine-like activity, but to a different extent in different systems. The equipotent molar ratio with respect to atropine (as 1) was: isolated guinea pig ileum, 10,000-1,000; salivary gland (mouse), 1,000-100; superior cervical ganglion (cat), 100-10; CNS (mouse), approximately 10. It is suggested that the rigid structure induces a three-point constrained fit in the receptor (onium group, hydrophobic moiety and carbonyl group), but that not all muscarinic receptors are capable of responding equally. In this case, receptor specificity of the drug is a direct consequence of its graded departure from the preferred conformation of acetycholine and, therefore, is necessarily associated with partial loss of potency.