Donepezil in Alzheimer’s disease: From conventional trials to pharmacogenetics
EuroEspes Biomedical Research Center, Institute for CNS Disorders, Coruña, Spain; EuroEspes Chair of Biotechnology and Genomics, Camilo José Cela University, Madrid, Spain
Correspondence: Ramón Cacabelos, EuroEspes Biomedical Research Center, Institute for CNS Disorders, 15166-Bergondo, Coruña, Spain, Tel +34 981 780505, Fax +34 981 780511, Email moc.sepseorue@solebacacr
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Neuropsychiatr Dis Treat. 2007 Jun; 3(3): 303–333.
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Donepezil is the leading compound for the treatment of Alzheimer’s disease (AD) in more than 50 countries. As compared with other conventional acetylcholinesterase inhibitors (AChEIs), donepezil is a highly selective and reversible piperidine derivative with AChEI activity that exhibits the best pharmacological profile in terms of cognitive improvement, responders rate (40%–58%), dropout cases (5%–13%), and side-effects (6%–13%) in AD. Although donepezil represents a non cost-effective treatment, most studies convey that this drug can provide a modest benefit on cognition, behavior, and activities of the daily living in both moderate and severe AD, contributing to slow down disease progression and, to a lesser exetnt, to delay institutionalization. Patients with vascular dementia might also benefit from donepezil in a similar fashion to AD patients. Some potential effects of donepezil on the AD brain, leading to reduced cortico-hippocampal atrophy, include the following: AChE inhibition, enhancement of cholinergic neurotransmission and putative modulation of other neurotransmitter systems, protection against glutamate-induced excitotoxicity, activation of neurotrophic mechanisms, promotion of non-amyloidodgenic pathways for APP processing, and indirect effects on cerebrovascular function improving brain perfusion. Recent studies demonstrate that the therapeutic response in AD is genotype-specific. Donepezil is metabolized via CYP-related enzymes, especially CYP2D6, CYP3A4, and CYP1A2. Approximately, 15%–20% of the AD population may exhibit an abnormal metabolism of AChEIs; about 50% of this population cluster would show an ultrarapid metabolism, requiring higher doses of AChEIs to reach a therapeutic threshold, whereas the other 50% of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. In AD patients treated with a multifactorial therapy, including donepezil, the best responders are the CYP2D6-related extensive (EM)(*1/*1, *1/*10) (57.47%) and intermediate metabolizers (IM)(*1/*3, *1/*5, *1/*6, *7/*10) (25.29%), and the worst responders are the poor (PM) (*4/*4)(9.20%) and ultra-rapid metabolizers (UM) (*1×N/*1) (8.04%). Pharmacogenetic and pharmacogenomic factors may account for 75%–85% of the therapeutic response in AD patients treated with donepezil and other AChEIs metabolized via enzymes of the CYP family. The implementation of pharmacogenetic protocols can optimize AD therapeutics.
Keywords: donepezil, Alzheimer’s disease, vascular dementia, CNS disorders, pharmacokinetics, pharmacodynamics, CYP2D6, pharmacogenetics
Donepezil is the number one member of the second generation of acetylcholinesterase inhibitors (AChEIs) (ie, donepezil, rivastigmine, galantamine) (Table 1) developed for the treatment of Alzheimer’s disease (AD) after the postulation in the early 1980s that AD was associated with a central cholinergic deficit (Bartus et al 1982; Whitehouse et al 1982). The first generation of AChEIs was represented by physostigmine, tacrine, velnacrine, and metrifonate of which only tacrine reached the marked in 1993 with an ephemeral life due to pharmacokinetic and pharmacodynamic problems (Giacobini 2006). After the closure of tacrine production, donepezil became the mainstay of AD therapeutics from 1996 up to now. More than 1000 papers have been published on the properties of donepezil during the past decade (1996–2006). About 800 papers deal with donepezil in dementia (>300 clinical trials worldwide) (Table 2), and approximately 100 papers refer to the role of donepezil in other central nervous system (CNS) disorders. At the present time, donepezil is the leading compound for AD treatment in the world (marketed in 56 countries) (Sugimoto et al 2002).
Pharmacological properties of selected acetylcholinesterase inhibitors for the treatment of Alzheimer’s disease
Selected studies with donepezil in Alzheimer’s disease and vascular dementia
Major issues for a drug to be successful include efficacy, safety, and at least some pharmacoeconomic benefit. On average, most studies with AChEIs reported by the pharmaceutical industry showed a cognitive enhancement of 2–3 points (vs placebo) in the ADAS-Cog score in clinical trials of 12–30 weeks’ duration, with improvement in 12%–58% of patients, 5%–73% of drop-outs, and side-effects in 2%–58% of cases (Giacobini 2006). As compared with other AChEIs, donepezil exhibits the best pharmacological profile in terms of cognitive improvement (2.8–4.6 vs 0.7–1 points of difference with placebo in the ADAS-Cog scale), responders rate (40%–58%), drop-out cases (5%–13%), and side-effects (6%–13%) (Giacobini 2006). Most studies agree that donepezil is a safe drug, although important adverse drug reactions (ADRs) have been reported in the international literature (Table 3). However, when evaluating efficacy and safety issues with AChEIs in AD, methological limitations in some studies reduce the confidence of independent evaluators in the validity of the conclusions drawn in published reports (Clegg et al 2001; Lanctôt et al 2003; Hogan et al 2004; Kaduszkiewicz et al 2005; Loveman et al 2006). For pharmacoeconomic aspects, some studies assessing the cost-effectiveness of ChEIs suggest that AChEI therapy provides benefit at every stage of disease, with better outcomes resulting from persistent, uninterrupted treatment (Fillit 2000; Sano 2004; Feldman et al 2004), whereas other studies indicate that AChEIs are not cost-effective, with benefits below minimally relevant thresholds or cost-neutral (Clegg et al 2001; Wimo 2004; Wimo et al 2004; Loveman et al 2006). The average annual cost per person with dementia ranges from US$15,000 to US$50,000, depending upon disease stage and country, with a lifetime cost per patient of more than US$175,000. Approximately, 80% of the global costs of dementia (direct and indirect costs) are assumed by the patients and/or their families, and 10%–20% of the costs of dementia are attributed to pharmacological treatment (Cacabelos 2005a, 2005b). Considering an average survival time (from diagnosis to death) of 10 years in optimal conditions, receiving 4–6 different drugs/day, a patient with dementia expends about US$4500–6000 per year (≈US$50,000 in a decade) in medicines.
Adverse drug reactions reported in clinical trials with donepezil in Alzheimer’s disease and other CNS disorders
Since the past experience in AD therapeutics was regrettably unsuccessful, donepezil is a good paradigm to interpret the past and to plan ahead future pharmacological challenges in order to optimize the treatment of dementia, incorporating novel data about the impact of pharmacogenetics on AD therapeutics and the influence of genetic factors on efficacy and safety issues.
Molecular pathology of Alzheimer’s disease
AD is a polygenic/multifactorial complex disorder characterized by the premature death of neurons. More than 200 different genes distributed across the human genome have been potentially involved in the pathogenesis of AD (Cacabelos et al 2005). The genetic defects identified in AD during the past 25 years can be classified into 3 main categories: (a) Mendelian or mutational defects in genes directly linked to AD, including (i) 18 mutations in the amyloid beta (Aβ) precursor protein (APP) gene (21q21); (ii) 142 mutations in the presenilin 1 (PS1) gene (14q24.3); and (iii) 10 mutations in the presenilin 2 (PS2) gene (1q31–q42). (b) Multiple polymorphic variants of risk characterized in more than 200 different genes distributed across the human genome can increase neuronal vulnerability to premature death (Cacabelos et al 2005). Among these genes of susceptibility, the apolipoprotein E (APOE) gene (19q13.2) is the most prevalent as a risk factor for AD, especially in those subjects harbouring the APOE-4 allele, whereas carriers of the APOE-2 allele might be protected against dementia. APOE-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of AD. (c) Diverse mutations located in mitochondrial DNA (mtDNA) through heteroplasmic transmission can influence aging and oxidative stress conditions, conferring phenotypic heterogeneity. It is also likely that defective functions of genes associated with longevity may influence premature neuronal survival, since neurons are potential pacemakers defining life span in mammals. All these factors may interact in as yet unknown genetic networks leading to a cascade of pathogenic events characterized by abnormal protein processing and misfolding with subsequent accumulation of abnormal proteins (conformational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport and chaperone misoperation (Bossy-Wetzel et al 2004; Cacabelos 2005a, b; Cacabelos et al 2005). Some of these mechanisms are common to several neurodegenerative disorders which differ depending upon the gene(s) affected and the involvement of specific genetic networks, together with cerebrovascular factors, epigenetic factors, oxidative stress phenomena, and environmental conditions (eg, nutrition, toxicity, social factors) (Bossy-Wetzel et al 2004; Cacabelos et al 2005; Mattson and Magnus 2006). The higher the number of defective genes involved in AD pathogenesis, the earlier the onset of the disease, the faster its clinical course and the poorer its therapeutic outcome (Cacabelos 2005a, b; Cacabelos et al 2005).
Although the amyloid hypothesis is recognized as the primum movens of AD pathogenesis (Selkoe and Podlisny 2002; Suh and Checler 2002; Cacabelos et al 2005), mutational genetics associated with amyloid precursor protein (APP) and presenilin (PS) genes alone (<10% of AD cases) does not explain in full the neuropathologic findings present in AD, represented by amyloid deposition in senile plaques and vessels (amyloid angiopathy), neurofibrillary tangle (NFT) formation due to hyperphosphorylation of tau protein, synaptic and dendritic desarborization and neuronal loss (Goedert and Spillantini 2006). These findings are accompanied by neuroinflammatory reactions, oxidative stress, and free radical formation probably associated with mitochondrial dysfunction, excitotoxic reactions, alterations in cholesterol metabolism and lipid rafts, deficiencies in neurotransmitters (especially acetylcholine) and neurotrophic factor function, defective activity of the ubiquitin-proteasome, and chaperone systems and cerebrovascular dysregulation (Cacabelos et al 2005). All these neurochemical events are potential targets for treatment; however, it is very unlikely that a single drug be able alone to neutralize the complex mechanisms involved in neurodegeneration (Cacabelos 2005a, b; Cacabelos et al 2005; Cacabelos and Takeda 2006).
The cholinergic hypothesis
Before the understanding of the complex pathology of AD, in the late 1970s and early 1980s it was believed that AD-related memory dysfunction was in part due to a cholinergic deficit in the brain of affected people due to a loss of neurons in the basal forebrain, this giving rise to the cholinergic hypothesis of AD (Bartus et al 1982; Whitehouse et al 1982; Francis et al 1999). The role of acetylcholine on memory function had been postulated many years before, and it was reasonable to think that a cholinergic deficit associated with an age-related decline in the number of neurons (50%–87%) of the nucleus basalis of Meynert accompanied by a reduced number of cholinergic synapses in cortical fronto-parietal-temporal regions and in the entorhinal cortex, might justify the memory deficit present in AD patients (Bartus et al 1982). From the 1950s to the 1980s “the amyloid hypothesis” and “the tau hypothesis” were elaborated, and both theories became the dominant and confronted pathogenic mechanisms potentially underlying AD-related neurodegeneration (Goedert and Spillantini 2006). However, recent genomic studies suggest that amyloid deposition in senile plaques, NFT and cholinergic deficits are but the phenotypic expression of the disease, and that the causative mechanism of premature neuronal death should be upstream of all these pathogenic events (Cacabelos et al 2005).
Since choline donors (precursors) and acetylcholine itself were substances of difficult pharmacological management (or useless to increase brain cholinergic neurotransmission), and, paradoxically, considering that acetylcholinesterase activity progressively decreased in AD brains in parallel with cognitive deterioration, AChEIs were proposed as an option to inhibit acetylcholine degradation in the synaptic cleft and to increase choline reuptake at the presynaptic level with the aim of enhancing acetylcholine synthesis in presynaptic terminals, this facilitating cholinergic neurotransmission (Giacobini 2006). The first candidate to fulfil this criteria was tacrine (tetrahydroaminoacridine) (Summers et al 1986), which after its introduction in the market in 1993 soon fell out of favor due to its hepatotoxicity and poor tolerability; 3 years later, in 1996, donepezil was approved by the FDA for the treatment of mild-to-moderate cases of AD. The other AChEIs, rivastigmine and galantamine, were introduced several years later (Giacobini 2006).
Pharmacological properties of donepezil
Donepezil, 1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methylpiperidine hydrochloride (E2020), is an indanone benzylpiperidine derivative (Sugimoto et al 1995) with selective reversible AChEI activity in the CNS and other tissues (Nochi et al 1995; Giacobini et al 1996; Sugimoto et al 2002). Donepezil is approximately 10 times more potent than tacrine as an inhibitor of acetylcholinesterase (AChE), and 500–1000-fold more selective for AChE over butyrylcholinesterase (BuChE). This compound is slowly absorbed from the gastrointestinal tract and has a terminal elimination half-life of 50–70 hours in young volunteers (>100 hours in elderly subjects) (Ohnishi et al 1993). After extensive metabolization in the liver, the parent compound is 93% bound to plasma proteins (Heydorn 1997).
AChEIs exhibit different affinities and selectivity for AChE and BuChE; however, most of them display a similar potency and clinical efficacy at conventional doses, this fact suggesting that these compounds may exert their therapeutic effects via collateral mechanisms unrelated to or indirectly linked with cholinesterase inhibition. Their chemical structures are also responsible for their pharmacokinetic and pharmacodynamic properties (Nordberg and Svensson 1999). For instance, physostigmine and rivastigmine are carbamates with pseudo-irreversible AChE-BuChE inhibition; tacrine is an acridine with reversible inhibition on the AChE-BuChE substrates; metrifonate is an organophosphate with irreversible inhibition of AChE-BuChE; donepezil is a piperidine with highly specific, reversible AChE inhibition; galantamine is a phenanthrene with reversible inhibition of AChE-BCh; and huperzine A is a pyridine with specific, reversible AChE inhibition (Giacobini 2006) (Table 1). The order of inhibitory potency (IC50) towards AChE activity under optimal assay conditions for each AChEI is the following: physostigmine (0.67 nM) > rivastigmine (4.4 nM) > donepezil (6.7 nM) > TAK-147 (12 nM) > tacrine (77 nM) > ipidacrine (270 nM). According to this study performed by Eisai scientists, the benzylpiperidine derivatives donepezil and TAK-147 showed high selectivity for AChE over BuChE; the carbamate derivatives showed moderate selectivity, while the 4-aminopyridine derivatives tacrine and ipidacrine showed no selectivity (Ogura et al 2000). More recent studies indicate that donepezil is 40–500-fold more potent than galantamine in inhibiting AChE. Clearance of galantamine from the brain is faster than donepezil. Ki values of brain AChE inhibition for galantamine and donepezil, respectively, are 7.1–19.1 and 0.65–2.3 μg/g in different species, suggesting that for a similar degree of brain AChE inhibition, 3–15 times higher galantamine than donepezil doses are needed (Geerts et al 2005).
The pharmacokinetic properties of AChEIs are also different. Tacrine, donepezil, and galantamine are metabolized in the liver via the cytochrome P450 system (CYP1A2-, CYP2D6-, CYP3A4-related enzymes), whereas rivastigmine is metabolized through carbomoylation (Table 1). Donepezil potentially may interact with drugs metabolized via CYP1A2-, CYP2D6-, and CYP3A4-related enzymes; however, formal pharmacokinetic studies have revealed no clinically meaningful interactions with memantine, risperidone, sertraline, carbidopa/levodopa, theophylline, furosemide, cimetidine, warfarin, and digoxin (Tiseo et al 1998a, b, c, d, e, f; Seltzer 2005). Their half-life also differ from 2–4 hours (tacrine, metrifonate, phenserine) to 4–6 hours (rivastigmine, galantamine), and 73 hours (donepezil). Bioavailability is maximum for galantamine (100%) and metrifonate (90%), both substances showing the lowest plasma protein binding (10–20%) in contrast to donepezil (96%) (Nordberg and Svensson 1999; Farlow 2001; Bentué-Ferrer et al 2003; Farmow 2003; Giacobini 2006). In animals, donepezil is found unchanged in brain, and no metabolites are detected in the nervous tissue. In plasma, urine, and bile, most donepezil metabolites are O-glucuronides (Matsui et al 1999). In healthy volunteers, donepezil is hepatically metabolized and the predominant route for the elimination of both parent drug and its metabolites is renal, as 79% of the recovered dose was found in the urine with the remining 21% found in feces. Moreover, the parent compound is the predominant elimination product in urine. The major metabolites of donepezil include M1 and M2 (via O-dealkylation and hydroxylation), M11 and M12 (via glucuronidation of M1 and M2, respectively), M4 (via hydrolysis) and M6 (via N-oxidation) (Tiseo et al 1998f).
After 14 days administration of donepezil, the cerebral acetylcholine level is increased by 35% and the AChE activity is decreased by 66% and 32% in rat brain and blood, respectively. No changes are detected in choline acetyltransferase activity, or the levels of vesicular acetylcholine transporter, choline transporter, or muscarinic receptors. The expression of various cholinergic genes is not affected by donepezil. Donepezil increases acetylcholine concentration in the synaptic cleft of the hippocampus mostly through AChE inhibition (Kosasa et al 1999) and produces a dose-dependent increase in hippocampal theta rhythm amplitude elicited by stimulation of the brainstem reticular formation (Kinney et al 1999). AChE activity in human blood shows 60%–97% and 43%–89% of pre-exposed level after 1 and 3 days of donepezil administration at a daily dose of 5 mg, respectively (Haug et al 2005). The current doses of donepezil at the clinical setting are 5 and 10 mg/day. Above 10 mg, AChE inhibition is assumed to reach a plateau (Jann et al 2002).
It is likely that the modest (and variable) therapeutic effects of AChEIs are related to their pharmacological properties and individual capacity to inhibit AChE activity in AD brains. Rakonczay (2003) compared the effects of 8 AChEIs on AChE and BuChE activity in normal human brain cortex. The most selective AChEIs, in decreasing order were: TAK-147, donepezil, and galantamine. For BuChE, the most specific was rivastigmine; however, none of these AChEIs was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate, and galantamine inhibited both the G1 and G4 AChE forms equally well (Rakonczay 2003).
The cognitive effects of AChEIs have been studied under different paradigms. The most frequent experiments have been performed in animals with cholinergic deficits or with lesions of the nucleus basalis of Meynert, as well as in animal models of AD and transgenic animals.
Donepezil can also act on targets other than cholinesterases in the brain. Among possible indirect actions of AChEIs to protect AD neurons, several options have been postulated. In dissociated hippocampal neurons, donepezil reversibly inhibits voltage-activated Na+ currents, and delays rectifier K+ current and fast transient K+ current. The inhibition of donepezil on rectifier K+ currents is voltage-dependent, whereas that on fast transient currents is voltage-independent. The blocking effects of donepezil on the voltage-gated ion channels are unlikely to contribute to its clinical effects in AD (Yu and Hu 2005).
Donepezil up-regulates nicotinic receptors in cortical neurons, this probably contributing to enhance neuroprotection (Kume et al 2005). It has also been suggested that AChEIs might promote non-amyloidogenic pathways of APP processing by stimulation of α-secretase mediated through protein kinase C (PKC) (Pakaski et al 2001). In the transgenic Tg2576 mouse model of AD, which exhibits age-dependent β-amyloid deposition in the brain as well as abnormalities in the sleep-wakefulness cycle probably due to a cholinergic deficit, the wake-promoting efficacy of donepezil is lower in plaque-bearing Tg2576 mice than in controls (Wisor et al 2005). In AD cases, donepezil increases the percentage of REM (rapid eye movements) sleep to total sleep time, improving sleep efficiency and shortening sleep latency (Mizuno et al 2004; Moraes et al 2006). In healthy volunteers, donepezil specifically enhances the duration of REM sleep (% sleep period time) and the number of REMs (Nissen et al 2005). The activation of the visual association cortex during REM sleep by donepezil might be responsible for the development of abnormal dreams and nightmares in AD (Singer et al 2005).
The influence of AChEIs on APP processing and inhibition of β-amyloid formation, at least in the case of some AChEIs (eg, phenserine), does not appear to be associated with cholinesterase inhibition but with a novel mechanism regulating translation of APP mRNA by a putative interleukin-1 or TGF-β responsive element which has been proposed as a target for drug development (Shaw et al 2001). Donepezil and other AChE noncovalent inhibitors are able to inhibit AChE-induced β-amyloid aggregation (Tumiatti et al 2004). AChEIs may also protect against vascular damage and amyloid angiopathy. In mild–moderate AD patients, increased levels of markers of endothelial dysfunction, such as thrombomodulin and sE-selectin have been observed. After treatment with AChEIs for 1 month, the levels of both parameters are markedly reduced, with values approaching normal ranges (Borroni et al 2005). In the Tg2576-transgenic mouse model in which, at 9–10 months of age, Tg+ mice develop amyloid plaques and impairments on paradigms related to learning and memory as compared to transgene-negative (Tg−) mice, physostigmine and donepezil improve deficits in contextual and cued memory in Tg+, but neither drug alter the deposition of amyloid plaques (Dong et al 2005). In contrast, donepezil protects against the neurotoxic effects induced by β-amyloid(1–40) in primary cultures of rat septal neurons (Kimura et al 2005). In another transgenic model of AD, the AD11 anti-nerve growth factor (anti-NGF) mice, oral administration of ganstigmine (CHF2819) and donepezil reverses the cholinergic and behavioral deficit in AD11 mice but not the amyloid and phosphotau accumulation, uncovering different mechanisms leading to neurodegeneration in AD11 mice (Capsoni et al 2004).
Probably via cholinergic modulation at the hypothalamic level, donepezil is able to reverse the age-related down-regulation of the GH/IGF-1 axis in elderly males in basal conditions and after GHRH stimulation. GHRH-induced GH response is magnified by more than 50% after treatment with donepezil in healthy elderly subjects (Obermayr et al 2005). The enhancement of the somatotropinergic sistem (GRF-GH-IGF axis) associated with donepezil treatment might contribute to activate GRF/GH-related neurotrophic mechanisms (Cacabelos et al 1988a, b). AChEIs also influence pro-inflammatory cytokines released from peripheral blood mononuclear cells, increasing oncostatin M, IL-1β, and IL-6 levels in AD patients after treatment (Reale et al 2005).
Glutamate-related excitotoxicity is an additional deleterious mechanism secondarily contributing to AD neuropathology (Cacabelos et al 1999). The neuroprotective properties of AChEIs on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons. Exposure of neurons to glutamate results in neuronal apoptosis. In this model, bis(7)-tacrine, a novel dimeric AChEI markedly reduces glutamate-induced apoptosis in a time- and dose-dependent manner; however, donepezil and other conventional AChEIs do not show any effect (Li et al 2005). Donepezil blocks the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade is voltage-dependent, suggesting a channel blocker mechanism of action which is not competitive at either the L-glutamate or glycine binding sites. The low potency of donepezil indicates that NMDA receptor blockade does not contribute to its therapeutic effect in AD; however, donepezil binds to the sigma1 receptor with high affinity and shows antidepressant-like activity in the mouse forced-swimming test as does the sigma1 receptor agonist igmesine. All AChEIs attenuate dizocilpine-induced learning impairments, but only the donepezil and igmesine effects are blocked by BD1047 or the antisense treatment, suggesting that donepezil behaves as an effective sigma1 receptor agonist and that interaction with sigma1 protein, but not NMDA receptor, might be involved in the pharmacological activity of donepezil (Maurice et al 2006). Other studies indicate that donepezil has a neuroprotective effect against oxygene-glucose deprivation injury and glutamate toxicity in cultured cortical neurons, and that this neuroprotection may be partially mediated by inhibition of the increase of intracellular calcium concentration (Akasofu et al 2006).
Donepezil influences cells viability and proliferation events in SH-SY5Y human neuroblastoma cells. Short- and long-exposure of these cells to donepezil induced a concentration-dependent inhibition of cell proliferation unrelated to muscarinic or nicotinic receptor blockade or apoptosis. Donepezil reduces the number of cells in the S-G2/M phases of the cell cycle, increases the G0/G1 population, and reduces the expression of two cyclins of the G1/S and G2/M transitions, cyclin E and cyclin B, in parallel with an increase in the expression of the cell cycle inhibitor p21 (Sortino et al 2004). Using the same in vitro model, others have reported that galantamine, donepezil, and rivastigmine afford neuroprotection through a mechanism that is likely unrelated to AChE inhibition, suggesting that at least donepezil and galantamine, but not rivastigmine, may exert their potential neuroproptective effects via α7 nicotinic receptors and the PI3K-Akt pathway (Arias et al 2005). In addition, donepezil increases action potential-dependent dopamine release (Zhang et al 2004) and modulates nicotinic receptors of substantia nigra dopaminergic neurons (Di Angelantonio et al 2004).
Donepezil in Alzheimer’s disease
Most clinical trials with donepezil in AD during the past 10 years have been performed in patients with mild-to-moderate dementia (mAD) (Rogers et al 1996, 1998a, 1998b; Burns et al 1999; Homma et al 2000; Doody et al 2001a, b; Mohs et al 2001; Winblad et al 2001) (Table 2), and a small number of studies have been carried out in severe cases of AD (sAD) (Feldman et al 2001; Bullock et al 2005; Winblad et al 2006a) (Table 2). More than 10,000 patients recruited from 26 different countries have been included in major clinical trials with AChEIs (24–30 weeks’ duration) during the past decade (Giacobini 2006). The typical outcome measures in most trials include psychometric assessment, behavior and function with different scales. Although the differences in the design of clinical trials is obvious, in patients treated with donepezil the differences with placebo range from 0.7–1.2 to 2.8–4.6 points in the ADAS-Cog (Giacobini 2006; Whitehead et al 2004). Despite this optimistic view resulting from the observation of selected trials (Table 2), many other studies and meta-analyses (Glegg et al 2001; Lanctôt et al 2003; Kaduszkiewicz et al 2005; Loveman et al 2006; Birks 2006; Birks et al 2006) indicate that AChEIs in general and donepezil in particular are of poor efficacy in AD. In 16 trials with 5159 treated patients (placebo = 2795 patients) the pooled mean proportion of global responders to AChEIs in excess of that of placebo was 9%. The rate of adverse events, dropout for any reason and dropout because of adverse events were also higher among patients receiving AChEIs than among those receiving placebo, with an excess proportion of 7%–8% (Lanctôt et al 2003). In this meta-analysis, including 8 trials with donepezil, 2 trials with rivastigmine and 5 trials with galantamine, the cognitive response was positive in 23%–35% of patients treated with donepezil, in 30% of patients treated with rivastigmine, and in 20–32% of patients treated with galantamine. The dropout rate was 20%–60% due to adverse events (Lanctôt et al 2003). In a meta-analysis including 22 trials published from 1989 to 2004, 12 of 14 studies showed an improvement of 1.5–3.9 points in the ADAS-Cog; however, because of flawed methods and small clinical benefits, the German evaluators established that the scientific basis for recommendations of AChEIs in AD was questionable (Kaduszkiewicz et al 2005). In a 10-study meta-analysis of donepezil in AD and two-study combined analysis of donepezil in vascular dementia (VD), an Irish group concluded that although there are differences between AD and VD patients in comorbid conditions and concomitant medications, donepezil is effective and well tolerated in both types of dementia (Passmore et al 2005). In the AD2000 clinical trial of Courtney et al (2004), no significant benefits were seen with donepezil compared with placebo in institutionalization or progression of disability. Similarly, no significant differences were seen between donepezil and placebo in behavioral and psychological symptoms, carer psychopathology, formal care costs, unpaid caregiver time, adverse events or deaths, or between 5 mg and 10 mg donepezil (Courtney et al 2004). In a critical appraisal of the AD2000 study, the first long-term RCT not sponsored by the pharmaceutical industry, a German group led by Kaiser et al (2005) concluded that the widespread use of AChEIs in AD is not supported by current evidence, and that long-term-randomized controlled trials focusing on patient-relevant outcomes instead of cognitive scores are urgently needed (Kaiser et al 2005).
Recent studies in early-stage AD suggest significant treatment benefits of donepezil, supporting the initiation of therapy early in the disease course to improve daily cognitive functioning (Seltzer et al 2004). During the past decade more than 100 papers dealt with the use of AChEIs or memantine in severe AD (sAD), but only a few studies provide evidence in favor of a positive therapeutic intervention with donepezil in sAD (Feldman et al 2001, 2003, 2005; Bullock et al 2005; Forchetti 2005; Winblad et al 2006a). In the international literature there are 13 articles related to donepezil in sAD, but only 3 fulfil strict criteria for further consideration (Rawls 2005)
In one study, to evaluate efficacy and safety of donepezil in sAD, Feldman et al (2001) found that donepezil had significant benefits over placebo on global, cognitive, functional, and behavioral measures in patients with sAD (Feldman et al 2001, 2005). In another study of Feldman et al (2003), donepezil demonstrated a significantly slower decline than placebo in instrumental and basic ADLs in patients with m/sAD. Bullock et al (2005) found similar effects of donepezil and rivastigmine on cognition and behavior in m/sAD. Winblad et al (2006a) have studied 248 patients with severe AD (sAD) (MMSE score: 1–10) living in nursing homes of Sweden for 6 months. The patients (n = 128) received 5 mg/day of donepezil for 30 days and then 10 mg/day thereafter. The primary end points in this study were change from baseline to month 6 in the severe impairment battery (SIB) and modified Alzheimer’s Disease Cooperative Study activities of daily living inventory for severe AD (ADCS-AD-severe). Under this protocol, 95 patients assigned donepezil and 99 patients assigned placebo (n = 120) completed the study. AD patients treated with donepezil improved more in SIB scores and declined less in ADCS-ADL-severe scores after 6 months of treatment compared with baseline than did the patients enrolled in the placebo group (Winblad et al 2006a).
To evaluate the representation of frail older adults in randomized controlled trials (RCTs), and to assess consequences of under representation by analyzing drug discontinuation rates, Gill et al (2004) studied a cohort of older adults newly dispensed donepezil (n = 6424) in Ontario between September 2001 and March 2002, and compared patients dispensed donepezil to clinical trials subjects. In this interesting study, between 51% and 78% of the Ontario cohort would have been ineligible for RCT enrolment. Patients dispensed donepezil were older (>80 years) and more likely to be in long-term care than RCT subjects. Overall, 27.8% of the Ontario cohort discontinued donepezil within 7 months of initial prescription, and the discontinuation rates were significantly higher for patients with a history of obstructive lung disease, active cardiovascular disease, or parkinsonism (Gill et al 2004).
It would be highly recommendable that outcome measures of efficacy in the long-term incorporate specific AD-related biomarkers (eg, serum markers, cerebro-spinal fluid [CSF] markers, neuroimaging biomarkers [MRI, fMRI, PET, SPECT], brain atrophy rate, brain perfusion, optical topography) (McMahon et al 2000; Jagust 2004; Dickerson and Sperling 2005). In this regard, PET studies have demonstrated that donepezil-induced inhibition of cortical AChE is modest (19%–24%) in patients with mAD. In the brain of AD patients assessed with an AChE tracer by PET scanning, treatment with donepezil for 3 months reduced AChE activity by 39% in the frontal cortex, 29% in the temporal cortex, and 28% in the parietal cortex (Kaasinen et al 2002). The degree of cortical AChE inhibition correlates with changes in excutive and attentional functions (Bohnen et al 2005). Long-term treatment with donepezil can lead to a lesser deterioration in qEEG, paralleling a milder neuropsychological decline (Rodríguez et al 2002), with reduction of slow-wave activity in frontal and temporo-parietal areas (Kogan et al 2001). Mean P300 ERPs are also improved in dementia after donepezil treatment (Werber et al 2001). By using the rate of hippocampal atrophy as a surrogate marker of disease progression, Hashimoto et al (2005) found that treatment with donepezil slows the progression of hippocampal atrophy in AD (mean annual rate of hippocampal volume loss: 3.82%) as compared with untreated patients (5.04%). Smaller hippocampal volume and inward variation of the lateral and inferomedial portions of the hippocampal surface were correlated with a poorer response to donepezil therapy in dementia (Csernansky et al 2005). AD patients who show more severe cholinergic dysfunction and less severe structural damage of the hippocampus and parahippocampus are likely to respond to donepezil treatment (Tanaka et al 2003a). Atrophy of the substantia innominata was more pronounced in transiently and continuously responding groups than in non-responders. Logistic regression analysis revealed that the overall discrimination rate with the thickness of the substantia innominata was 70% between responders and non-responders, suggesting that atrophy of the substantia innominata on MRI helps to predict response to donepezil treatment in AD (Tanaka et al 2003b). Krishnan et al (2003) have found that donepezil treated patients had significantly smaller mean decreases in total and right hippocampal volumes and a smaller, nearly significant mean decrease in left hippocampal volume, compared with the placebo-treated patients. Other studies revealed that the diversity of clinical responses to donepezil therapy in AD is associated with regional cerebral blood flow (rCBF) changes, mainly in the frontal lobe (Shimizu et al 2006). Furthermore, there is a parallelism between cognitive improvement and increase in brain M1 muscarinic receptor binding after treatment with donepezil in AD (Kemp et al 2003). AChE activity also decreases in the CSF of patients treated with donepezil, but changes in other biomarkers, such as BuChE activity, β-amyloid (1–42), tau and phosphorylated tau proteins are not affected by donepezil treatment (Parnetti et al 2002).
In summary, it appears that donepezil is beneficial (in a dose-dependent manner) when assessed using global and cognitive outcome measures in AD; however, by finding the mean effect sizes of the treatment on the outcome measures of cognition from 8 empirical studies, it was determined that neither donepezil nor other AChEIs were greatly efficacious (Harry and Zakzanis 2005). Over 770 million days of patient use and an extensive publication database demonstrate that donepezil has a good tolerability and safety profile (Jackson et al 2004). The use of AChEIs in AD is currently appraised by the National Institute for Clinical Evidence (NICE). In a recent review providing the latest, best quality evidence of the effects of AChEIs on cognition, quality of life and adverse events in people with mild to moderately-severe AD (m/sAD), Takeda et al (2006) stated (on a systemtic review of 26 RCTs) that AChEIs can delay cognitive impairment in m/sAD for at least 6 months duration; however, results from head to head comparisons are limited by the low number of studies and the study quality. The Cochrane Database Reviewers conclude that people with mAD or sAD treated for periods of 12, 24, or 52 weeks with donepezil experienced benefits in cognitive function, activities of the daily living and behavior. Study clinicians rated global clinical state more positively in treated patients, and measured less decline in measures of global disease severity (Birks and Harvey 2006). In general terms, there is not robust support for any AChEI because the treatment effects are small and are not always apparent in practice (Birks and Harvey 2006; Takeda et al 2006). Donepezil treatment may be associated with reduced mortality in nursing home residents with dementia (Gasper et al 2005) and with delayed nursing home placement (Geldmacher et al 2003), although some authors denied that donepezil was able to reduced the rate of institutionalization or disability in mAD (Courtney et al 2004; Standridge 2004). The meta-analysis of caregiver-specific outcomes in antidementia clinical trials revealed that AChEIs have a small beneficial effect on burden and active time use among caregivers of persons with AD (Lingler et al 2005).
Mild cognitive impairment
Mild cognitive impairment (MCI) is the postulated transitional state between the cognitive changes of normal aging and early AD (Petersen et al 1999, 2005). The rate of progression to clinically diagnosable AD is 10%–15%/year among persons who meet the criteria for the amnestic form of MCI, in contrast to a rate of 1–2%/year among normal elderly persons (Petersen et al 1999). This is a clinical concept and instrumental aid invented to substitute the lack of accurate biological markers able to predict the risk of suffering AD. Despite its questionable value, it is important to keep in mind that neurodegeneration starts many years before the onset of the disease (Cacabelos et al 2005). It is very likely that AD neurons begin their deceasing process 20–40 years prior to the appearance of the first symptoms (eg, memory deficit, behavioral changes, functional decline, subtle praxis-related psychomotor alterations). In some patients with a specific genetic profile, it is possible to detect, by means of sensitive brain imaging techniques, a progressive brain dysfunction after the age of 30 years (Cacabelos 2003, 2005b). In this regard, it is clear that an early therapeutic intervention could be of some benefit for these patients precluding the possibility of a premature neuronal death or delaying the onset of the disease for several years. AChEIs have been proposed as feasible candidate drugs for the treatment of MCI (Stirling Meyer et al 2002; Salloway et al 2003; Gauthier 2005).
Few studies have been performed with donepezil in MCI (Jelic et al 2005). In a double-blind, placebo-controlled, multicenter trial in US with 270 cases, a mild benefit in cognitive function has been reported (Salloway et al 2004). A Chinese group has performed a clinical trial with donepezil (2.5 mg/day for 3 months) in patients with amnestic MCI and found a significant improvement in cognitive performance as well as changes in the hippocampus as assessed by magnetic resonance spectroscopy (MRS) (Wang et al 2004).
In a recent study, Petersen et al (2005) evaluated 769 subjects with the amnestic subtype of MCI in a randomized, double-blind study with donepezil (10 mg/day) or vitamin E (2000 IU/day) for 3 years. The overall rate of progression from MCI to AD was 16%/year (212 patients evolved into the AD condition). As compared with the placebo group, there were no significant differences in the probability of progression to AD in the vitamin E group or the donepezil group during the 3 years of treatment. The donepezil group had a reduced likelihood of progression to AD during the first 12 months of the study, with better results among APOE-4 carriers (Petersen et al 2005).
The proposed benefit of AChEI therapy as a preventive strategy in MCI or as a regular option for people requesting some medication for memory improvement is far from clear and probably poses some underestimated dangers, despite the optimistic position of some authors (Jelic et al 2005). It has been observed that neuropsychological test performance deteriorates in healthy elderly volunteers receiving donepezil for 2 weeks; worsening is significant on tests of speed, attention, and short-term memory as compared with the placebo group, suggesting a perturbation of an already optimized cholinergic system in healthy subjects (Beglinger et al 2005). If the rate of conversion form MCI into AD is about 10%–15% per year, it is probably irresponsible to sacrifice 80% MCI cases to pyrrhically protect only 10% assuming that AChEIs in healthy subjects may induce undesirable cognitive effects.
The postulated long-lasting effects of AChEIs for 1–5 years (Doody et al 2001b; Bullock and Dengiz 2005; Giacobini 2006) were never clearly documented in well-controlled trials. In a recent study, Winblad et al (2006b) provide some support to the long-lasting efficacy and safety of donepezil after 3 years of treatment. In a cohort of 286 patients, there was a trend for patients receiving continuous therapy to have less global deterioration on the Gottfries-Brane-Steen scale than those who had delayed treatment. Small but statistically significant differences between the groups were observed for the secondary measures of cognitive function (MMSE scores) and cognitive and functional abilities (GDS) in favor of continuous donepezil therapy (Winblad et al 2006b).
On a pathogenic basis, there is no evidence that AChEIs protect neurons against AD-related premature death. It might occur – as demonstrated with multifactorial therapies in AD (Cacabelos et al 2004c) – that cognitive enhancement induced by AChEI administration is the result of forcing surviving neurons to overwork for a period of time after which neurons become exhausted with the subsequent acceleration of their metabolic decline. This phenomenon has been demonstrated after administration of a combination therapy with CDP-choline, piracetam, and metabolic supplementation (Cacabelos et al 2004c). Under this therapeutic protocol, AD patients clearly improved for the first 9 months of treatment, and a progressive decline in therapeutic efficacy has been observed thereafter (Cacabelos et al 2004c; Cacabelos 2003, 2005a, b). The study of Petersen et al (2005) might be a good paradigm to illustrate the same phenomenon with donepezil in MCI patients who showed a positive response during the first year of treatment and no effect after 3 years. Taking into account these observations, we should be very cautious with the administration of pharmaceuticals (as a preventive strategy) to patients with MCI until a clear long-lasting efficacy of the therapeutic options can be demonstrated. This is especially important when some studies reveal that chronic administration of AChEIs (eg, galantamine) may even increase mortality (Scheltens et al 2004; Kirshner 2005).
Combination drug therapy is the standard of care for treating many neuropsychiatric disorders and other medical conditions (eg, cardiovascular disease, hypertension, cancer, AIDS, diabetes). For the past 20 years, the pharmaceutical industry and the medical community have made a show of reluctance to treat AD with a combination therapy, but in fact most patients with dementia have been receiving an average of 6–9 different drugs per day in an attempt to control the multifaceted expressions of dementia. Multifactorial therapy, combining several types of drugs with potential neuroprotective effect on the CNS, has been tried in AD and other forms of dementia with promising results (Cacabelos et al 2000a, b; Cacabelos 2002a, b; Cacabelos 2003; Cacabelos et al 2004c; Cacabelos 2005a, b; Cacabelos et al 2006). Donepezil has been given in combination with other substances to patients with AD. Probably the best evidence-based combination strategy is the addition of memantine to stable donepezil therapy in m/sAD (Tariot et al 2004; van Dyck et al 2006). This combination was found to benefit cognition, behavior, and activities of daily living. It appears that memantine in combination with donepezil is significantly better than donepezil alone in the management of behavioral symptoms (Tariot et al 2004; Xiong and Doraiswamy 2005; Doody 2005). Combination therapy with donepezil and memantine in healthy subjects did not show any significant alteration in pharmacokinetic or pharmacodynamic parameters of both drugs, suggesting that donepezil and memantine may be safely and effectively used in combination (Periclou et al 2004). According to basic studies using the whole-cell patch-clamp technique with multipolar neurons, the combination of donepezil and memantine might be a contradiction since donepezil potentiates NMDA currents (Moriguchi et al 2005) and memantine acts as a partial NMDA antagonist (Cacabelos et al 1999).
Combination therapy of donepezil (5 mg/day) with ginkgo biloba (90 mg/day) for 30 days did not show any significant difference in cognitive performance, pharmacokinetics and pharmacodynamics of donepezil, indicating that ginkgo supplementation does not have major impact on donepezil therapy (Yasui-Furukori et al 2004). Donepezil has also been given in combination with acetyl-L-carnitine (ALC) in AD. The addition of ALC to donepezil increased the response rate from 38% (AChEI alone) to 50% (AChEI + ALC) (Bianchetti et al 2003). Initial data resulting from combination studies of donepezil and vitamin E indicated that this long-term combination might be beneficial for AD (Klatte et al 2003).
Donepezil plus sertraline did not show any advantage over donepezil alone in AD, although the combination appeared to be beneficial in a subgroup of patients with moderate-to-severe behavioral and psychological symptoms (Finkel et al 2004). In patients with psychotic symptoms and lack of improvement of their delusions/hallucinations during perphenazine treatment, donepezil may reduce psychotic symptoms, suggesting that donepezil augmentation of neuroleptics (risperidone, olanzapine, quetiapine) may be appropriate for those patients for whom neuroleptic monotherapy either does not lead to symptom remission or is associated with intolerable side-effects (Bergman et al 2003). In some cases the combination of donepezil and neuroleptics may exacerbate extrapyramidal side-effects (Liu et al 2002). In general, combination therapy tends to show better results than monotherapy with one AChEI or any other single drug for dementia (Cacabelos 2002a, b; Cacabelos et al 2004c; Cacabelos 2005a, b). Similar results can be seen in animal models when donepezil is given in combination with other compounds (Sonkusare et al 2005).
Comparative studies with different AChEIs did not show any significant difference or traces of superiority among them in AD patients (Wilkinson et al 2002; Ritchie et al 2004; Aguglia et al 2004; Bullock et al 2005; Harry and Zakzanis 2005). In independent studies, there are apparent differences in ADAS-Cog changes, improvement rate, dropouts, and incidence of side-effects among different classes of AChEIs; however, since the clinical protocols vary from one study to another, these results are not comparable and unreliable. In a number of studies analysed by Giacobini (2006) comparing 7 AChEIs, the ADAS-Cog variation vs placebo (AD/P) was 4.0–5.3/0.8–2.8 with tacrine, 4.7/1.83 with eptastigmine, 2.8–4.6/0.7–1.2 with donepezil, 1.9–4.9/0.7–1.2 with rivastigmine, 2.8–3.2/0.5–0.75 with metrifonate, and 3.1–3.9/1.73 with galantamine. About 30%–50% of patients improved with tacrine, 40%–58% with donepezil, 25%–37% with rivastigmine, 35%–40% with metrifonate, and 10%–23% with galantamine. The drop-out rate was 55%–73% in patients treated with tacrine, 35% with eptastigmine, 5%–13% with donepezil, 15%–36% with rivastigmine, 2%–28% with metrifonate, and 10%–13% with galantamine. Side-effects were more prevalent in patients treated with tacrine (405–58%) than with the other AChEIs (donepezil, 6%–13%; rivastigmine, 15%–28%; metrifonate, 2%–12%; galantamine, 13%–16%) (Giacobini 2006). In clinical terms, according to Birks (2006), despite the slight variations in the mechanism of action of donepezil, rivastigmine, and galantamine, there is no evidence of any substantial differences between them with respect to efficacy; even though there appears to be less adverse effects associated with donepezil compared with rivastigmine (Birks 2006).
Effects on behavioral symptoms and functional deficits
Dementia is clinically characterized by memory disorders, behavioral changes, and progressive functional decline. The estimated prevalence of psychiatric symptoms in AD accounts for 40%–60% of the cases (Cacabelos et al 1996; Cacabelos et al 1997). In cross-sectional studies it has been reported that several psychiatric symptoms are associated with lower total MMSE scores and overall cognitive deterioration. Psychotic symptoms, especially delusions, hallucinations and misidentifications, are positively correlated with aggressive behavior and institutionalization. Agitation and wandering are also associated with rapid cognitive decline in dementia. In general, psychotic symptoms parallel an accelerated cognitive deterioration, partially induced by psychotropic drugs in some cases or by many other classes of drugs currently used by patients in geriatric long-term settings (Arinzon et al 2006). In other cases, behavioral changes do not seem to be associated with exogenous factors and might be intrinsic to cortical atrophy and selective brain damage in dementia. The prevalence of psychotic symptoms in AD ranges from 15% to 50%, constituting a very common problem which is not always reduced by conventional anti-psychotic or antianxiety drugs. In addition, antypsychotics may increase the risk of cerebrovascular accidents and also contribute to cortical atrophy after years of chronic treatment (Cacabelos et al 1996, 1997; Gill et al 2005). Furthermore, behavioral symptoms in AD significantly increase direct costs of care (US$10,670–16,141 higher annual costs in high-NPI as compared with low-NPI) (Murman et al 2002). Several studies with AChEIs indicate that these compounds may exert a beneficial effect on some neuropsychiatric symptoms such as delusions, hallucinations, apathy, psychomotor agitation, depression and anxiety (
Inhibitors of AChE (EC 22.214.171.124.) and BChE (EC 126.96.36.199) are neurotoxic compounds capable of causing central, peripheral or both central and peripheral cholinergic crises. A number of these compounds have also found application as drugs developed for the treatment of Alzheimer’s disease (AD) and myasthenia gravis [1,2]. These are based on the premise that increasing the availability of acetylcholine (ACh) at acetylcholine receptors in the brain, results in better neuron to neuron transport that will improve cognitive function. Cholinergic nerves, however, can be found in both the central (CNS) and peripheral (PNS) nervous systems and disparate body tissues . Drugs that cross the blood brain barrier do not have dissociable groups as can be seen in the case of AD drugs . Some of these drugs cannot penetrate the CNS and this property makes them suitable for use in myasthenia gravis . For a long time, regulation of immunity was not considered an effect of AChE inhibitors. However, recent evidence casts new light on the subject. In this review, we explore the link between immunity and the AChE inhibitors as currently available AChE inhibiting drugs for AD.
2. The Cholinergic Anti-Inflammatory Pathway
Ach is a ubiquitous neurotransmitter [6,7] and found even in the roundworm Caenorhabditis elegans, one of the simplest organisms with a nervous system [8,9]. In the roundworm one third of the nervous system is cholinergic . Humans have a large percentage of nervous system that is cholinergic including the CNS. Cholinergic nerves also form a major part of the parasympathetic and sympathetic nervous systems [11,12]. The wider significance of Ach is in understanding the biological effects of tested toxins and/or medical drugs: as any immunological effects of AChE inhibitors can involve both CNS and PNS, this has to be taken into consideration in interpreting any findings. For this reason, vagotomy, used to study the cholinergic anti-inflammatory pathway in animal experiments or selecting compounds that do not cross the blood brain barrier should be considered carefully before drawing any conclusions as to which pathway is involved in the proposed mechanism.
The cholinergic system is tightly associated with the cholinergic anti-inflammatory pathway dominantly located in blood and mucosa. This pathway is a regulatory link between nerve terminations in blood and macrophages expressing the α7 nicotinic acetylcholine receptor (α7 nAChR) on their surface [11,13,14]. For a long time, the mechanisms of inflammatory regulation remained unclear. Discovery of the cholinergic anti-inflammatory pathway, however, allowed us to understand how the CNS is involved in the regulation of innate immunity [15,16,17,18,19,20,21,22,23,24]. AChE bound on erythrocytes plays an important role in termination of cholinergic anti-inflammatory pathway activation [11,25]. AChE activity is typically low in AD patients treated with AChE inhibitors . Compared to AChE, BChE is constituted in the liver and secreted into the plasma where the enzyme is dissolved . Apart from the fact that the conversion rate of Ach by BChE is lower than the conversion by AChE, BChE can substitute for AChE and split the neurotransmitter once they make contact [28,29]. The effect of BChE became relevant once the cholinergic anti-inflammatory pathway was studied as BChE plays a greater role in the blood than in the nervous system.
The cholinergic anti-inflammatory pathway is one-way: the CNS can attenuate inflammation mediated by macrophages or any other immune cells having α7 nAChR. Ach released from the vagus nerve termination, agonizes α7 nAChR, which responds by opening a central channel allowing an influx of Ca2+ into macrophages [11,30,31]. Increased levels of Ca2+ activate the nuclear factor κ B (NF κB) resulting in suppression of inflammatory cytokine production including tumor necrosis factor α (TNFα), high mobility group box of proteins and interleukin 6 (IL-6) [32,33]. These blood AChE and plasma BChE are able to terminate the stimulation of the cholinergic anti-inflammatory pathway due to splitting ACh. The principle of the pathway is depicted in Figure 1.
Figure 1. Principle of the cholinergic anti-inflammatory pathway; abbreviations: ACh-acetylcholine; AChE-acetylcholinesterase; BChE-butyrylcholinesterase; HMGB-high mobility group box; IL-6-interleukin 6; NFκB-nuclear factor kappa B; TNFα-tumor necrosis factor alpha.
Figure 1. Principle of the cholinergic anti-inflammatory pathway; abbreviations: ACh-acetylcholine; AChE-acetylcholinesterase; BChE-butyrylcholinesterase; HMGB-high mobility group box; IL-6-interleukin 6; NFκB-nuclear factor kappa B; TNFα-tumor necrosis factor alpha.
The primary purpose of using AChE inhibitors in pharmacology is not modulation of immunity related pathologies. However, recent studies indicate that these inhibitors can cause a significant modulation of immunity as a side effect [29,34,35]. As seen from Figure 1, they can modulate the cholinergic anti-inflammatory pathway via protection of Ach from splitting by cholinesterases and thus enhancing the pathway. The mechanism of action is probably less effective than the standard mode but it becomes relevant when someone is using an inhibitor of cholinesterases in large amounts and/or for a long time such as patients suffering from AD.
Apart from the regulation processes, some inhibitors can influence immunity via forming antigens by reaction with e.g., plasma proteins. The immune system is thus activated and the stimulation counteracts the anti-inflammatory action. This effect is, however, very weak but it can play a role in forming antibody proteins modified by nerve agents .
3. Division of Inhibitors
The structure of AChE and BChE has been extensively reviewed in the following publications [7,29,37,38]. In brief, AChE has a more developed peripheral anionic site and narrower aromatic gorge than BChE. Aromatic compounds have higher affinity for AChE than BChE. Some aromatic inhibitors of AChE do not inhibit BChE, for example aflatoxins [39,40]. When the role of the cholinesterases is evaluated in humans, their genomic diversity and posttranslational modifications have to be taken into account [41,42].
Organophosphorus compounds are irreversible inhibitors of both AChE and BChE. They bind to the active site of the cholinesterases and easily cross the blood brain barrier [6,43]. Nerve agents, e.g., sarin, soman, tabun, and some highly toxic compounds, formerly used as pesticides (paraoxon, parathion, malaoxon, malathion), are examples [44,45,46,47]. High toxicity characterizes organophosphorus inhibitors that are used in chemical warfare or as pesticides. Their pharmacological importance is relatively small; Metrifonate (trichlorfon) was chosen for AD treatment and became an exception but it was withdrawn because of adverse effects [48,49,50]. Though organophosphorus compounds typically inhibit both AChE and BChE, tetraisopropyl pyrophosphoramide, also known as iso-OMPA, is dissimilar to the other inhibitors. It does not penetrate to the active site of AChE and it inhibits BChE only. It is typically used as a reagent for rapid distinction between AChE and BChE activity in biological samples [51,52].
Carbamate inhibitors bind to the active site of both cholinesterases like organophosphorus inhibitors; however, the covalent bound is not stable and the carbamate moiety is hydrolytically split from the active site after some time [29,53,54]. The mechanism of carbamate binding is sometimes called pseudo-irreversible because of carbamate moiety spontaneous hydrolysis and resurrection of cholinesterase activity. From a pharmacological point of view, there is a big difference between carbamates and organophosphorus inhibitors. Many carbamates do not cross the blood brain barrier and the carbamate moiety has to be modified or encapsulated to cross it [55,56,57]. Quarternary ammonium containing pyridostigmine and neostigmine are examples. On the other hand, the blood brain barrier is not impenetrable by all carbamates e.g., rivastigmine (see further text) and physostigmine easily reach AChE in the brain [58,59,60].
AChE and BChE can be inhibited by a group of secondary metabolites from plants and fungi. Galantamine and huperzine are examples of plant alkaloids used in pharmacology. Alkaloids α-chaconine, α-solanine, tomatine, berberine, palmatine and jatrorrhizine are other metabolites that inhibit cholinesterases [29,61,62,63,64,65]. Aflatoxins too, can be introduced as secondary metabolites from fungi.
Disparate synthetic drugs not belonging to carbamates and organophosphates can be mentioned last but not least. Donepezil is the most relevant compound of this group. (See the next chapter). Tacrine (1,2,3,4-tetrahydroacridin-9-amine) is another synthetic drug which easily crosses the blood brain barrier and is used as a highly effective drug for ameliorating Alzheimer disease manifestation by inhibition AChE and by lower but still effective inhibition of BChE [53,66,67]. It was withdrawn from clinical use because of adverse effects. Hepatotoxicity was the main pathological consequence of tacrine intake [68,69]. The basic facts about groups of inhibitors are summarized in Table 1.
In considering the role of the inhibitors in modulating immunity, other factors such as the environment and genetic disposition should be taken into account. This conclusion is based on blood cholinesterase activity in male volunteers working with pesticides . The activity varied in infected patients with proven bacterial meningitis . The sensitivity of humans to the inhibitors can also significantly be affected because detoxification mechanisms have unequal efficacy. This idea was, e.g., demonstrated by Sonali et al.on AD patients treated with rivastigmine . Variability in inhibitor effects should be considered when conclusions are drawn from animal models and cell lines, extrapolated to humans.
Table 1. Summarization of facts about cholinesterases’ inhibitors.
|Group of Compounds||Compounds (Examples)||Mechanism of Inhibition||Inhibition of AChE and BChE||Penetration through Blood Brain Barrier||Importance as Drugs||References|
|Organophosphates||sarin, soman, tabun, malaoxon||irreversible||equal to AChE and BChE||Good||Low||[6,43,44,45,46,47,48,49,50]|
|Carbamates||pyridostigmine, physostigmine neostigmine, rivastigmine||pseudo-irreversible||equal to AChE and BChE||low (pyridostigmine, neostigmine), good (physostigmine, rivastigmine)||High||[29,53,54,55,56,57,58,59,60]|
|-||Tacrine||non-competitive||AChE > BChE||Good||former drug, discontinued now||[53,66,68,69]|
|-||huperzine A||non-competitive||AChE >> BChE||Good||will increase||[29,75,77,78,79]|
4. Inhibitors that Cross the Blood Brain Barrier
The following text focuses on galantamine, donepezil, huperzine and rivastigmine. Galantamine (Figure 2) is a drug used for treating AD and related dementias. Currently, galantamine is an alternative to rivastigmine and can be given to patients with similar stages of dementia. On the market, it is sold under the trade names Razadyne™, Razadyne™ER, Reminyl™ER, and Reminyl® The drug was firstly isolated by soviet scientists Mashkovsky and Kruglikova-Lvova from bulbs of Caucasian snowdrops Galanthus sp. in the early 1950s and chemical synthesis was introduced in the following decades . After marketing of the drug, it drove out the more toxic tacrine  and has become one of the best drugs for Alzheimer disease treatment .
Figure 2. Structure of cited compounds that cross the blood brain barrier.
Figure 2. Structure of cited compounds that cross the blood brain barrier.
Galantamine is a competitive inhibitor of AChE and an allosteric modulator of nAChR . Its dual action on both AChE and nAChR is an advantage and unlike other marketed drugs that inhibit AChE. It is believed that galantamine may interact with the cholinergic anti-inflammatory pathway via direct modulation of the α7 nAChR . The anti-inflammatory pathway modulation may explain the activation of microglia followed by amyloid beta clearance . In a rat model, galantamine was also approved as effective in reducing circulating TNFα which was, in the past, initiated by administration of bacterial lipopolysaccharide . For this reason, galantamine could act not only as a drug for symptomatic treatment but as a compound for slowing Alzheimer disease progression. This conclusion is not, however, commonly accepted and more detailed evidence of the process is needed.
Donepezil is a selective, noncompetitive inhibitor of AChE. The inhibition is quite effective as the equilibrium constant is reported to be 12.5 nmol/L for AChE from rat erythrocytes . Donepezil is available under the trade name Aricept as a highly effective drug for Alzheimer disease, originally developed by Eisai and Pfizer. The structure of donepezil is depicted in Figure 3. Clinical experience with donepezil is good: it is well tolerated and slowly eliminated so that the drug can be taken over long periods . Compared to other drugs for Alzheimer disease, donepezil works via a simple pathway based on AChE inhibition. It is not involved in other pathways and does not involve BChE inhibition .
Figure 3. Structure of carbamates that cross the blood brain barrier.
Figure 3. Structure of carbamates that cross the blood brain barrier.
We can assume that donepezil activates the cholinergic anti-inflammatory pathway via inhibition of AChE and increased availability of ACh. However, Hwang and coworkers found anti-inflammatory effects on microglia cell lines where no AChE was present [85