Anti-Neurodegenerative Benefits of Acetylcholinesterase Inhibitors in Alzheimer's Disease: Nexus of Cholinergic and Nerve Growth Factor Dysfunction.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is increasingly viewed as a complex multi-dimensional disease without effective treatments. Recent randomized, placebo-controlled studies have shown volume losses of ~0.7% and ~3.5% per year, respectively, in the basal cholinergic forebrain (CBF) and hippocampus in untreated suspected prodromal AD. One year of donepezil treatment reduced these annualized rates of atrophy to about half of untreated rates. Similar positive although variable results have also been found in volumetric measurements of the cortex and whole brain in patients with mild cognitive impairment as well as more advanced AD stages after treatments with all three currently available acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, and galantamine). Here we review the anti-neurodegenerative benefits of AChE inhibitors and the expected parallel disease-accelerating impairments caused by anticholinergics, within a framework of the cholinergic hypothesis of AD and AD-associated loss of nerve growth factor (NGF). Consistent with the "loss of trophic factor hypothesis of AD," we propose that AChE inhibitors enhance acetylcholine-dependent release and uptake of NGF, thereby sustaining cholinergic neuronal viability and thus slowing AD-associated degeneration of the CBF, to ultimately delay dementia progression. We propose that improved cholinergic therapies for AD started early in asymptomatic persons, especially those with risk factors, will delay the onset, progression, or emergence of dementia. The currently available competitive and pseudo- irreversible AChE inhibitors are not CNS-selective and thus induce gastrointestinal toxicity that limits cortical AChE inhibition to ~30% (ranges from 19% to 41%) as measured by in vivo PET studies in patients undergoing therapy. These levels of inhibition are marginal relative to what is required for effective symptomatic treatment of dementia or slowing AD-associated neurodegeneration. In contrast, because of the inherently slow de novo synthesis of AChE in the CNS (about one-- tenth the rate of synthesis in peripheral tissues), irreversible AChE inhibitors produce significantly higher levels of inhibition in the CNS than in peripheral tissues. For example, methanesulfonyl fluoride, an irreversible inhibitor reduces CNS AChE activity by ~68% in patients undergoing therapy and ~80% in cortical biopsies of non-human primates. The full therapeutic benefits of AChE inhibitors, whether for symptomatic treatment of dementia or disease-slowing, thus would benefit by producing high levels of CNS inhibition. One way to obtain such higher levels of CNS AChE inhibition would be by using irreversible inhibitors.

Improving Anti-Neurodegenerative Benefits of Acetylcholinesterase Inhibitors in Alzheimer's Disease: Are Irreversible Inhibitors the Future?

Decades of research have produced no effective method to prevent, delay the onset, or slow the progression of Alzheimer's disease (AD). In contrast to these failures, acetylcholinesterase (AChE, EC 3.1.1.7) inhibitors slow the clinical progression of the disease and randomized, placebo-controlled trials in prodromal and mild to moderate AD patients have shown AChE inhibitor anti-neurodegenerative benefits in the cortex, hippocampus, and basal forebrain. CNS neurodegeneration and atrophy are now recognized as biomarkers of AD according to the National Institute on Aging-Alzheimer's Association (NIA-AA) criteria and recent evidence shows that these markers are among the earliest signs of prodromal AD, before the appearance of amyloid. The current AChE inhibitors (donepezil, rivastigmine, and galantamine) have short-acting mechanisms of action that result in dose-limiting toxicity and inadequate efficacy. Irreversible AChE inhibitors, with a long-acting mechanism of action, are inherently CNS selective and can more than double CNS AChE inhibition possible with short-acting inhibitors. Irreversible AChE inhibitors open the door to high-level CNS AChE inhibition and improved anti-neurodegenerative benefits that may be an important part of future treatments to more effectively prevent, delay the onset, or slow the progression of AD.

Is Combining an Anticholinergic with a Cholinesterase Inhibitor a Good Strategy for High-Level CNS Cholinesterase Inhibition?

The currently approved cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) produce gastrointestinal toxicity which limits dosing to that which produces only about 25% to 35% CNS cholinesterase inhibition in Alzheimer's disease patients undergoing treatment, below the minimum therapeutic target of about 40% to 50% CNS inhibition considered necessary to treat cognitive impairment. A recent strategy for producing high-level CNS acetylcholinesterase (AChE) inhibition (50% or higher) is to co-administer a muscarinic anticholinergic with the AChE inhibitor to block the dose-limiting cholinergic overstimulation of the gastrointestinal system, allow more robust AChE inhibition in the CNS, and improve efficacy in the treatment of Alzheimer's disease. Unfortunately, most common muscarinic anticholinergics, including solifenacin, readily penetrate the CNS and are directly associated with long-term exacerbation of the underlying neuropathology of Alzheimer's disease and increased brain atrophy. The co-administration of an anticholinergic with an AChE inhibitor is a rational strategy for improving efficacy in the symptomatic treatment of dementia, but there are significant long-term risks that have not yet been considered. For long-term safety against accelerating the underlying disease processes in Alzheimer's disease, anticholinergics used to increase the tolerability of AChE inhibitors should not penetrate, or have very limited penetration, of the blood-brain barrier. Neurotrophic-mediated mechanisms by which cholinergic drugs may affect neurodegeneration in Alzheimer's disease are explored and improved treatment options are suggested.

Cholinesterase Inhibitor Therapy in Alzheimer's Disease: The Limits and Tolerability of Irreversible CNS-Selective Acetylcholinesterase Inhibition in Primates.

Irreversible acetylcholinesterase (AChE) inhibition accumulates to high levels in the central nervous system (CNS) because AChE turnover in the brain is much slower than in peripheral tissues. As expected from this CNS selectivity, the irreversible AChE inhibitor methanesulfonyl fluoride (MSF) produces significant cognitive improvement in Alzheimer's disease patients without the gastrointestinal toxicity that plagues other AChE inhibitors. However, without dose-limiting gastrointestinal toxicity, one shortcoming of the prior human studies of MSF is that the upper limits of CNS AChE inhibition that might be tolerated could not be tested. Therefore, in this study, monkeys were treated with escalating intramuscular (IM) doses of MSF that culminated with several weeks of 1.5 mg/kg dosing, more than eight times the prior human clinical dose, still without signs of toxicity. Brain biopsies showed that ∼80% AChE inhibition had been produced and that the new synthesis of cortical AChE had a half-time (t1/2) of ∼12 days. A single IM dose of 1.5 mg/kg MSF produced ∼59% inhibition in cerebrospinal fluid (CSF) AChE as measured one day later. This corresponds to a peak of ∼80% inhibition in CSF AChE at the time of the injection, recovering with a t1/2 of 2.4 days. Computational analyses suggest that MSF at clinically relevant doses could theoretically produce a steady-state AChE inhibition between 65% and 85% in the CNS. These data suggest that the full therapeutic advantage of AChE inhibition therapy can be realized without interference from dose-limiting gastrointestinal toxicity if an irreversible inhibitor is employed.

Cholinergic transmission during nicotine withdrawal is influenced by age and pre-exposure to nicotine: implications for teenage smoking.

Adolescence is a unique period of development characterized by enhanced tobacco use and long-term vulnerability to neurochemical changes produced by adolescent nicotine exposure. In order to understand the underlying mechanisms that contribute to developmental differences in tobacco use, this study compared changes in cholinergic transmission during nicotine exposure and withdrawal in naïve adult rats compared to (1) adolescent rats and (2) adult rats that were pre-exposed to nicotine during adolescence. The first study compared extracellular levels of acetylcholine (ACh) in the nucleus accumbens (NAc) during nicotine exposure and precipitated withdrawal using microdialysis procedures. Adolescent (postnatal day, PND, 28-42) and adult rats (PND60-74) were prepared with osmotic pumps that delivered nicotine for 14 days (adolescents 4.7 mg/kg/day; adults 3.2 mg/kg/day; expressed as base). Another group of adults was exposed to nicotine during adolescence and then again in adulthood (pre-exposed adults) using similar methods. Control rats received a sham surgery. Following 13 days of nicotine exposure, the rats were implanted with microdialysis probes in the NAc. The following day, dialysis samples were collected during baseline and following systemic administration of the nicotinic receptor antagonist mecamylamine (1.5 and 3.0 mg/kg, i.p.) to precipitate withdrawal. A second study compared various metabolic differences in cholinergic transmission using the same treatment procedures as the first study. Following 14 days of nicotine exposure, the NAc was dissected and acetylcholinesterase (AChE) activity was compared across groups. In order to examine potential group differences in nicotine metabolism, blood plasma levels of cotinine (a nicotine metabolite) were also compared following 14 days of nicotine exposure. The results from the first study revealed that nicotine exposure increased baseline ACh levels to a greater extent in adolescent versus adult rats. During nicotine withdrawal, ACh levels in the NAc were increased in a similar manner in adolescent versus adult rats. However, the increase in ACh that was observed in adult rats experiencing nicotine withdrawal was blunted in pre-exposed adults. These neurochemical effects do not appear to be related to nicotine metabolism, as plasma cotinine levels were similar across all groups. The second study revealed that nicotine exposure increased AChE activity in the NAc to a greater extent in adolescent versus adult rats. There was no difference in AChE activity in pre-exposed versus naïve adult rats. In conclusion, our results suggest that nicotine exposure during adolescence enhances baseline ACh in the NAc. However, the finding that ACh levels were similar during withdrawal in adolescent and adult rats suggests that the enhanced vulnerability to tobacco use during adolescence is not related to age differences in withdrawal-induced increases in cholinergic transmission. Our results also suggest that exposure to nicotine during adolescence suppresses withdrawal-induced increases in cholinergic responses during withdrawal. Taken together, this report illustrates important short- and long-term changes within cholinergic systems that may contribute to the enhanced susceptibility to tobacco use during adolescence.

A randomized phase I study of methanesulfonyl fluoride, an irreversible cholinesterase inhibitor, for the treatment of Alzheimer's disease.

AIMS: To ascertain the tolerability profile of single and repeated oral doses of methanesulfonyl fluoride (MSF, SNX-001) in healthy aged subjects, and to determine the degree of erythrocyte acetylcholinesterase (AChE) inhibition induced by MSF after single and repeated oral doses. METHODS: To calculate properly the kinetics and the duration of AChE inhibition, the effects of MSF were also studied in rodents. These experiments suggested that MSF administered three times per week should provide safe and efficacious AChE inhibition. In a randomized placebo-controlled phase I study, 3.6 mg, 7.2 mg or 10.8 mg MSF were then orally administered to 27 consenting healthy volunteers (aged 50 to 72 years). After a single dose phase and a 1 week wash-out period, the subjects received the same doses three times per week for 2 weeks. RESULTS: Twenty-two out of the 27 subjects completed the study. Four patients withdrew due to adverse events (AEs) and one for non-compliance. Erythrocyte AChE was inhibited by a total of 33%, 46%, and 62% after 2 weeks of 3.6 mg, 7.2 mg and 10.8 mg MSF, respectively. No serious AEs occurred. The most frequent AEs were headache (27%), nausea (11%) and diarrhoea (8%). CONCLUSIONS: MSF proved to be well tolerated even with repeated oral dosing. It is estimated that MSF provided a degree of AChE inhibition that should effectively enhance memory. This molecule deserves to be tested for efficacy in a pilot randomized controlled study in patients with Alzheimer's disease.

In utero methanesulfonyl fluoride differentially affects learning and maze performance in the absence of long-lasting cholinergic changes in the adult rat.

There is increasing evidence that acetylcholinesterase (AChE) may have various specific developmental roles in brain development. Nevertheless, specific effects of AChE inhibition during early brain development have not been adequately described. Therefore, methanesulfonyl fluoride (MSF), an irreversible AChE inhibitor that shows high selectivity for the CNS was used to produce AChE inhibition in utero to study subsequent adult behaviors, sleep, and cholinergic markers. Rats exposed to MSF in utero showed a deficit in spatial learning tasks using appetitive motivation but, surprisingly, they performed equally well or better than controls when aversive motivation was used. One hypothesis was that MSF treatment in utero affected the response to stress. Tests of anxiety however showed no differences in basal levels of anxiety. Studies of sleep behavior, however, indicated a higher level of REM sleep which is only seen during the light phase of male rats exposed to MSF in utero as compared to controls. No differences in cholinergic markers in the brains of adults were found except that females exposed to MSF in utero had a higher level of ChAT activity in the synaptosomal fraction of the hippocampus. Even so, whether cholinergic alterations accompany the in utero MSF exposure remains to be determined. The failure to find widespread changes in cholinergic markers in the adult brains suggests changes in behaviors should be further investigated by testing the participation of postsynaptic mechanisms, measuring of cholinergic markers during earlier development periods and the possible participation of other neurotransmitter systems to clearly reveal the role of the cholinergic system following in utero MSF exposure.

Prenatal exposure to the acetylcholinesterase inhibitor methanesulfonyl fluoride alters forebrain morphology and gene expression.

Methanesulfonyl fluoride (MSF) is a CNS-selective acetylcholinesterase (AChE) inhibitor, currently being developed and tested for the treatment of symptoms of Alzheimer's disease. We have previously confirmed that a single in utero exposure to MSF at clinically appropriate doses inhibits AChE activity in fetal rat brain by 20%, and when administered throughout gestation, MSF achieves a 40% level of inhibition. Here, we show that rats chronically exposed in utero to MSF display marked sex-specific differences in morphological development of the cerebral cortical layers compared with controls at 7 days of age. Forebrain size and cortical thickness were increased in females and decreased in males. An analysis of gene expression in neonate brain on the day of birth revealed sex-specific differential expression of over 25 genes, including choline acetyltransferase (ChAT), which were affected by prenatal MSF exposure. Many of these genes are associated with sexual differentiation and brain development, while others are involved in more generalized cellular and metabolic processes. The changes observed in cortical morphology and gene expression suggest a critical developmental role for AChE in the fetal nervous system, most likely through its effect on cholinergic neurotransmission.

Methanesulfonyl fluoride, an acetylcholinesterase inhibitor, attenuates simple learning and memory deficits in ischemic rats.

Methanesulfonyl fluoride (MSF), a highly selective CNS inhibitor of acetylcholinesterase, has been recently demonstrated to promote improvement in cognitive performance in patients with senile dementia of Alzheimer type. Because a similar cognitive impairment may accompany stroke, we investigated in the present study whether treatment with MSF could produce beneficial effects in adult rats subjected to an experimental stroke model. Sprague-Dawley rats received transient 60 min intraluminal occlusion of the right middle cerebral artery (MCAo) and were given i.p. injections of either MSF (1 mg/kg at 24 and 48 h post-MCAo and 0.3 mg/kg thereafter every other day) or the vehicle, peanut oil, for 4 weeks. Behavioral tests and biochemical assays were performed at 28 days post-surgery. MSF treatment produced about 90% inhibition of acetylcholinesterase in the brain. Ischemic animals that received the vehicle displayed significant elevated body swing biased activity (84.8 +/- 10%) and significantly prolonged acquisition (398 +/- 62 s) and shortened retention (79 +/- 26 s) of the passive avoidance task. Interestingly, while the ischemic animals that received the MSF exhibited elevated body swing biased activity (87.7 +/- 8%), they performed significantly better in the passive avoidance task (255 +/- 36 s and 145 +/- 18 s in acquisition and retention) than the vehicle-treated animals. Moreover, whereas brains from both groups of animals revealed similar extent and degree of cerebral infarction, the MSF-treated ischemic animals showed more intense immunoreactivity, as well as a significantly higher number (10-15% increase) of septal choline acetyltransferase-positive cells than the vehicle-treated ischemic animals. These results show that MSF, possibly by preserving a functional cholinergic system, attenuated stroke-induced deficits in a simple learning and memory task.