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1.  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.
doi:10.1111/bcp.12018
PMCID: PMC3635594  PMID: 23116458
acetylcholinesterase; Alzheimer's disease; cholinesterase inhibition; methanesulfonyl fluoride; MSF; phase I human study
2.  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.
doi:10.1111/bcp.12018
PMCID: PMC3635594  PMID: 23116458
acetylcholinesterase; Alzheimer's disease; cholinesterase inhibition; methanesulfonyl fluoride; MSF; phase I human study
3.  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.
doi:10.1016/j.pbb.2007.09.008
PMCID: PMC2278117  PMID: 17920111
Acetylcholine; Acetylcholinesterase; Methanesulfonyl fluoride (msf); Development; Choline acetyltransferase; Choline uptake; Sleep
4.  Synthesis, docking and acetylcholinesterase inhibitory assessment of 2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione derivatives with potential anti-Alzheimer effects 
Background
Alzheimer’s disease (AD) as neurodegenerative disorder, is the most common form of dementia accounting for about 50-60% of the overall cases of dementia among persons over 65 years of age. Low acetylcholine (ACh) concentration in hippocampus and cortex areas of the brain is one of the main reasons for this disease. In recent years, acetylcholinesterase (AChE) inhibitors like donepezil with prevention of acetylcholine hydrolysis can enhance the duration of action of acetylcholine in synaptic cleft and improve the dementia associated with Alzheimer’s disease.
Results
Design, synthesis and assessment of anticholinesterase activity of 2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione derivatives showed prepared compounds can function as potential acetylcholinesterase inhibitor. Among 12 synthesized derivatives, compound 4a with ortho chlorine moiety as electron withdrawing group exhibited the highest potency in these series (IC50 = 0.91 ± 0.045 μM) compared to donepezil (IC50 = 0.14 ± 0.03 μM). The results of the enzyme inhibition test (Ellman test) showed that electron withdrawing groups like Cl, F and NO2 can render the best effect at position ortho and para of the phenyl ring. But compound 4g with methoxy group at position 3(meta) afforded a favorable potency (IC50 = 5.5 ± 0.7 μM). Furthermore, docking study confirmed a same binding mode like donepezil for compound 4a.
Conclusions
Synthesized compounds 4a-4l could be proposed as potential anticholinesterase agents.
doi:10.1186/2008-2231-21-47
PMCID: PMC3704662  PMID: 23758724
Synthesis; Phthalimide; Acetylcholinesterase (AChE); Anti-Alzheimer; Ellman test; Docking
5.  In vivo visualization of donepezil binding in the brain of patients with Alzheimer's disease 
Aims
The aims of this study were to visualize in vivo binding of donepezil to acetylcholinesterase (AChE) in the brain and to establish a method for measuring the amount of binding of orally administered donepezil.
Methods
[5-11C-methoxy]-donepezil ([11C]-donepezil) was radiolabelled as a positron emission tomography (PET) tracer. The biodistribution of [11C]-donepezil was measured by PET in 10 AD patients and six elderly normal subjects. Two AD patients underwent additional PET measurements after oral administration of donepezil for 6 months.
Results
[11C]-donepezil-PET images demonstrated high densities of tracer distribution in AChE-rich brain regions such as the striatum, thalamus, and cerebellum. Compared with elderly normal subjects, patients with mild AD exhibited about 18–20% reduction of donepezil binding in the neocortex and hippocampus, while patients with moderate AD exhibited about 24–30% reduction of donepezil binding throughout the brain. Orally administered donepezil (5 mg day−1) induced 61.6–63.3% reduction of donepezil binding in AD brains. The distribution volume of [11C]-donepezil in the hippocampus was significantly correlated with MMSE scores in AD patients.
Conclusions
[11C]-donepezil-PET enables quantitative measurement of donepezil binding in the brain. AD patients exhibited reduction of donepezil binding in the brain, even in the early stage of disease. Longitudinal evaluation by this technique enables determination of AChE binding occupancy of orally administered donepezil.
What is already known about this subject Deficit in central cholinergic neurotransmission is a consistent change associated with Alzheimer's disease (AD).Donepezil hydrochloride exhibits selective inhibition of acetylcholinesterase (AChE) and is widely used for the treatment of AD.The biodistribution of donepezil in the brain after administration is not precisely understood in vivo.There is no method to measure the amount of binding of orally administered donepezil to AChE.
What this study adds This study clearly visualizes the distribution of donepezil in human brain using [11C]-donepezil and positron emission tomography.This study demonstrates prominent reduction of the donepezil binding site in the AD brain.This study provides methodology to measure the AChE binding occupancy of orally administered donepezil and provides a new surrogate marker for evaluation and prediction of response to donepezil treatment.
doi:10.1111/j.1365-2125.2007.03063.x
PMCID: PMC2291371  PMID: 18070217
acetylcholinesterase; Alzheimer's disease; donepezil; positron emission tomography (PET)
6.  Donepezil in Alzheimer’s disease: an evidence-based review of its impact on clinical and economic outcomes 
Core Evidence  2006;1(3):195-219.
Introduction:
Donepezil is indicated for the symptomatic treatment of mild to moderate Alzheimer’s disease. It is a specific and reversible inhibitor of acetylcholinesterase (AChE); by increasing levels of available acetylcholine, donepezil may compensate for the loss of functioning cholinergic brain cells.
Aims:
This review evaluates the clinical impact of donepezil by assessing randomized controlled and open-label naturalistic trials, as well as observational studies. A broad perspective is gained of its effectiveness on various outcomes.
Evidence review:
There is strong evidence that donepezil has efficacy against the three major domains of Alzheimer’s disease symptoms, namely functional ability, behavior, and cognition. The strongest evidence is for improvement or less deterioration in global outcomes and cognition in the short to medium term. There is limited evidence that improved global outcomes are maintained in the long term and clear evidence to support long-term maintenance of cognitive benefits. Also, donepezil appears to maintain function in the long term and there is some level 1 and 2 evidence of improved or limited deterioration in behavior or mood in the short to medium term. Despite donepezil’s effects on major symptoms of Alzheimer’s disease, its impact on patients’ quality of life has not been consistently demonstrated, perhaps reflecting the difficulty of assessing this aspect in this patient population. Donepezil may also lessen caregiver burden. Donepezil has some effect on markers of brain function, but more data are needed to confirm a neuroprotective effect. There is limited and conflicting evidence that long-term donepezil treatment delays time to institutionalization. There is some evidence that donepezil may be cost effective, especially when unpaid caregiver costs are considered. Donepezil is generally safe and well tolerated.
Clinical value:
AChE inhibitors are the only agents recommended for the treatment of cognitive decline in patients with mild to moderate Alzheimer’s disease. Donepezil is more effective than placebo and is well tolerated in improving the major symptoms of this disease. Improvements are usually modest, although stabilization of cognitive and functional symptoms with donepezil can also be considered an important clinical outcome. Donepezil may lessen caregiver burden. Donepezil may also be cost effective, especially when unpaid caregiver costs are considered. More data are required from randomized controlled trials with long-term follow-up to confirm its cost effectiveness and impact on quality of life, disease progression, and time to institutionalization.
PMCID: PMC3321665  PMID: 22500154
acetylcholinesterase (AChE) inhibitor; Alzheimer’s disease; dementia; donepezil; evidence; outcomes
7.  ANTICHOLINESTERASE INSECTICIDE RETROSPECTIVE 
Chemico-biological interactions  2012;203(1):221-225.
The anticholinesterase (antiChE) organophosphorus (OP) and methylcarbamate (MC) insecticides have been used very effectively as contact and systemic plant protectants for seven decades. About 90 of these compounds are still in use – the largest number for any insecticide chemotype or mode of action. In both insects and mammals, AChE inhibition and acetylcholine accumulation leads to excitation and death. The cholinergic system of insects is located centrally (where it is protected from ionized OPs and MCs) but not at the neuromuscular junction. Structural differences between insect and mammalian AChE are also evident in their genomics, amino acid sequences and active site conformations. Species selectivity is determined in part by inhibitor and target site specificity. Pest population selection with OPs and MCs has resulted in a multitude of modified AChEs of altered inhibitor specificity some conferring insecticide resistance and others enhancing sensitivity. Much of the success of antiChE insecticides results from a suitable balance of bioactivation and detoxification by families of CYP450 oxidases, hydrolases, glutathione S-transferases and others. Known inhibitors for these enzymes block detoxification and enhance potency which is particularly important in resistant strains. The current market for OPs and MCs of 19% of worldwide insecticide sales is only half of that of 10 years ago for several reasons: there have been no major new compounds for 30 years; resistance has eroded their effectiveness; human toxicity problems are still encountered; the patents have expired reducing the incentive to update registration packages; alternative chemotypes or control methods have been developed. Despite this decline, they still play a major role in pest control and the increasing knowledge on their target sites and metabolism may make it possible to redesign the inhibitors for insensitive AChEs and to target new sites in the cholinergic system. The OPs and MCs are down but not out.
doi:10.1016/j.cbi.2012.08.002
PMCID: PMC3572339  PMID: 22926007
Acetylcholinesterase; Anticholinesterase; Cytochrome; P450 Insecticide; Resistance; Selective toxicity
8.  Effect of oral administration of zanapezil (TAK-147) for 21 days on acetylcholine and monoamines levels in the ventral hippocampus of freely moving rats 
British Journal of Pharmacology  2005;145(8):1035-1044.
Zanapezil (TAK-147 (3-[1benzylpiperdin-4-yl]-1-(2,3,4,5-tetrahydro-1 H-1-benzazepin-8-yl) propan-1-one fumarate)) is a selective acetylcholine (ACh) esterase inhibitor under investigation as a drug for Alzheimer's disease (AD) treatment.In this study, the effects of TAK-147 at 2 mg kg−1 p.o. for 21 days, compared to donepezil (E2020), on the levels of ACh, catecolamines and indoleamines were investigated in the ventral hippocampus (VH) of freely moving rats by microdialysis-high-performance liquid chromatography.The results revealed that the VH contains 92.05±21.97 fmol 20 μl−1 ACh and the following monoamines levels (pg 30 μl−1), norepinephrine (NE) 1.92±0.39, epinephrine (Epi) 1.91±0.183, 3-methoxy-4-hydroxyphenylglycol (MHPG) 11.53±3.22, normetanephrine 3.26±0.61, dopamine (DA) 0.77±0.23, 3,4-dihydroxyphenylacetic acid (DOPAC) 3.37±1.01, homovanillic acid (4-hydroxy-3-methoxyphenylacetic acid; HVA) 4.04±0.93, 3-methoxytyramine 0.64±0.13, serotonin (5-HT) 0.73±0.16 and 5-hydroxyindoleacetic acid (5-HIAA) 313.15±18.42.On the 21st day and prior to the last dose, TAK-147 increased ACh, Epi, DA and 5-HT, whereas E2020 increased MHPG, Epi and DA. Following the last dose, TAK-147 increased NE, whereas E2020 increased NE, ACh and 5-HT in addition to their effects prior to the last dose. TAK-147 decreased HVA : DA ratio, but only marginally decreased DOPAC : DA and 5-HIAA : 5-HT ratios. On the other hand, E2020 decreased ratios of HVA : DA, DOPAC : DA (prior to the last dose), and 5-HIAA : 5-HT (90–180 min after the last dose). Both drugs decreased MHPG : NE only at 180 min after the last dose. The results also showed that TAK-147 increased Epi : NE ratio prior to and for 120 min following the last dose, whereas E2020 increased the ratio only before the last dose. The present results show that TAK-147 at a subthreshold dose could differentially increase ACh and 5-HT, compared to MHPG increased by E2020. The last dose of each drug could extend their effects to other monoamines.The increase of the monoamines levels, in addition to that on the ACh, and decrease of their oxidation could be of value in the treatment of the AD, other dementic diseases and the cohort neurological disorders depending on the type of the monoamine underlying the disorder.
doi:10.1038/sj.bjp.0706288
PMCID: PMC1576235  PMID: 15951830
Zanapezil; donepezil; ventral hippocampus; acetylcholine; monoamines; microdialysis; Alzheimer's disease
9.  Acute but not chronic Donepezil administration increases muscarinic receptor-mediated brain signaling involving arachidonic acid in unanesthetized rats 
Donepezil, an acetylcholinesterase (AChE) inhibitor that is used to treat patients with Alzheimer’s disease, is thought to act by increasing brain extracellular acetylcholine (ACh), and thus ACh binding to cholinergic receptors. Cholinergic muscarinic receptors may be coupled to cytosolic phospholipase A2 (cPLA2) activation and arachidonic acid (AA) release from synaptic membrane phospholipid, and this activation can be imaged in rodents as an AA incorporation coefficient k*, using quantitative autoradiography. To examine acute and chronic effects of Donepezil on the AA signal, k* for AA was measured with quantitative autoradiography in 81 brain regions of unanesthetized rats. Twenty min after a single oral dose (3.0 mg/kg) of Donepezil compared with saline, k* was increased significantly in 37 brain regions, whereas k* did not differ from control 7 h afterwards or following chronic (21 days) of Donepezil. Pretreatment with atropine prevented the 20-min increments in k* following Donepezil. Donepezil also increased the brain ACh concentration and reduced brain AChE activity, but did not change cPLA2 activity, regardless of administration regimen. These results show that Donepezil acutely increases the brain AA signal that is mediated by ACh acting at muscarinic receptors, but that this signal is rapidly desensitized despite continued elevated brain ACh concentration. In contrast, the AA signal in response to arecoline was not altered following Donepezil.
doi:10.3233/JAD-2009-1058
PMCID: PMC2790024  PMID: 19363262
Donepezil; muscarinic receptors; acetylcholine; arachidonic acid; phospholipase A2; acetylcholinesterase; anticholinesterase; Alzheimer disease; imaging; brain; desensitization
10.  Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of α7 nicotinic receptors and internalization of NMDA receptors 
British Journal of Pharmacology  2010;161(1):127-139.
BACKGROUND AND PURPOSE
Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.
EXPERIMENTAL APPROACH
Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca2+ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.
KEY RESULTS
Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepexzil. In addition, glutamate-mediated Ca2+ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of α7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca2+ entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca2+ entry induced by donepezil.
CONCLUSIONS AND IMPLICATIONS
Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of α7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.
doi:10.1111/j.1476-5381.2010.00894.x
PMCID: PMC2962822  PMID: 20718745
donepezil; glutamate; NMDA receptor; α7 nicotinic receptor; neuroprotection
11.  Positive allosteric modulator of alpha 7 nicotinic-acetylcholine receptors, PNU-120596 augments the effects of donepezil on learning and memory in aged rodents and non-human primates 
Neuropharmacology  2012;67C:201-212.
The development of novel therapeutic agents for disorders of cognition such as Alzheimer’s disease (AD) is of paramount importance given the ever-increasing elderly population, however; there is also considerable interest in any strategy that might enhance the clinical efficacy of currently available treatments. The purpose of this study was to evaluate an adjunctive treatment strategy to memory enhancement, namely combining the commonly prescribed acetylcholinesterase inhibitor (AChEI) donepezil, with a positive allosteric modulator (PAM) of α7 nicotinic-acetylcholine receptors (α7-nAChRs), PNU-120596. The treatment strategy was evaluated in a (non-spatial) spontaneous novel object recognition (NOR) task in young rats; a water maze spatial learning and recall procedure in aged, cognitively-impaired rats, and a delayed match to sample (working/short term memory) task in aged rhesus monkeys. In all three experiments a similar drug response was observed, namely that donepezil administered alone improved task performance in a dose-dependent manner; that PNU-120596 administered alone was without significant effect, but that the combination of PNU-120596 with a subthreshold dose of donepezil was effective. The positive effect of the drug combination appeared to be α7-nAChR mediated given that it was blocked in the NOR task by the selective α7-nAChR antagonist methyllycaconitine (MLA). Collectively, these data indicate that PNU-120596 increases the effective dose range of donepezil in learning/memory-related tasks in young and age-impaired animal models. The results suggest that α7-nAChR-selective PAMs like PNU-120596 have potential as adjunctive treatments with acetylcholinesterase inhibitors (e.g., donepezil) for age-related illnesses such as AD as well memory disorders not necessarily associated with advanced age.
doi:10.1016/j.neuropharm.2012.10.019
PMCID: PMC3562411  PMID: 23168113
Alzheimer’s Disease; cholinergic; memory; pro-cognitive; Mild Cognitive Impairment; aging
12.  Acetylcholinesterase inhibitors attenuate angiogenesis 
Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimer's disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to determine whether donepezil affects angiogenesis. A hindlimb ischaemia model was created by unilateral femoral artery ligation. Blood flow recovery examined by laser Doppler perfusion imaging and capillary density by immunohistochemical staining of CD31-positive cells in the ischaemic hindlimb were significantly decreased in donepezil- and physostigmine-treated mice compared with control mice after 2 weeks. Donepezil reduced expression of IL (interleukin)-1β and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1β to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1β expression in C2C12 myoblast cells, which was inhibited by pre-incubation with ACh (acetylcholine) or LY294002, a PI3K (phosphoinositide 3-kinase) inhibitor. Donepezil inhibited phosphorylation of Akt [also known as PKB (protein kinase B)], a downstream kinase of PI3K, in the ischaemic hindlimb. These findings suggest that cholinergic stimulation by acetylcholinesterase inhibitors suppresses angiogenesis through inhibition of PI3K-mediated IL-1β induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.
doi:10.1042/CS20110633
PMCID: PMC3359057  PMID: 22369073
acetylcholinesterase inhibitor; angiogenesis; hindlimb ischaemia; interleukin-1β; ACh, acetylcholine; AD, Alzheimer's disease; bFGF, basic fibroblast growth factor; BP, blood pressure; CNS, central nervous system; COPD, chronic obstructive pulmonary disease; DMEM, Dulbecco's modified Eagle's medium; donepezil, (RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; HPF, high-power field; HR, heart rate; IL, interleukin; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; mAChR, muscarinic ACh receptor; MAPK, mitogen-activated protein kinase; nAChR, nicotinic ACh receptor; NF-κB, nuclear factor κB; PBMC, peripheral blood mononuclear cell; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; qRT–PCR, quantitative reverse transcription–PCR; TNFα, tumour necrosis factor α; VEGF, vascular endothelial growth factor
13.  Donepezil in Alzheimer’s disease: From conventional trials to pharmacogenetics 
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.
PMCID: PMC2654795  PMID: 19300564
donepezil; Alzheimer’s disease; vascular dementia; CNS disorders; pharmacokinetics; pharmacodynamics; CYP2D6; pharmacogenetics
14.  Comparative chemical profiling, cholinesterase inhibitions and anti-radicals properties of essential oils from Polygonum hydropiper L: A Preliminary anti- Alzheimer’s study 
Background
Cholinesterase inhibition is a vital target for the development of novel and mechanism based inhibitors, owing to their role in the breakdown of acetylcholine (ACh) neurotransmitter to treat various neurological disorders including Alzheimer’s disease (AD). Similarly, free radicals are implicated in the progression of various diseases like neurodegenerative disorders. Due to lipid solubility and potential to easily cross blood brain barrier, this study was designed to investigate the anticholinesterase and antioxidant potentials of the standardized essential oils from the leaves and flowers of Polygonum hydropiper.
Methods
Essential oils from the leaves (Ph.LO) and flowers (Ph.FO) of P. hdropiper were isolated using Clevenger apparatus. Oil samples were analyzed by GC-MS to identify major components and to attribute the antioxidant and anticholinesterase activity to specific components. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potentials of the samples were determined following Ellman’s assay. Antioxidant assays were performed using 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays.
Results
In the GC-MS analysis 141 and 122 compounds were indentified in Ph.LO and Ph.FO respectively. Caryophylene oxide (41.42 %) was the major component in Ph.FO while decahydronaphthalene (38.29 %) was prominent in Ph.LO. In AChE inhibition, Ph.LO and Ph.FO exhibited 87.00** and 79.66***% inhibitions at 1000 μg/ml with IC50 of 120 and 220 μg/ml respectively. The IC50 value for galanthamine was 15 μg/ml. In BChE inhibitory assay, Ph.LO and Ph.FO caused 82.66*** (IC50 130 μg/ml) and 77.50***% (IC50 225 μg/ml) inhibitions respectively at 1000 μg/ml concentration. In DPPH free radical scavenging assay, Ph.LO and Ph.FO exhibited IC50 of 20 and 200 μg/ml respectively. The calculated IC50s were 180 & 60 μg/ml for Ph.LO, and 45 & 50 μg/ml for Ph.FO in scavenging of ABTS and H2O2 free radicals respectively.
Conclusions
In the current study, essential oils from leaves and flowers of P. hydropiper exhibited dose dependent anticholinesterase and antioxidant activities. Leaves essential oil were more effective and can be subjected to further in-vitro and in-vivo anti-Alzheimer’s studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12944-015-0145-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12944-015-0145-8
PMCID: PMC4632677  PMID: 26530857
Polygonum hydropiper; Essential oils; GC-MS; Anticholinesterase and antioxidant
15.  Pharmacological characterization of nicotine-induced acetylcholine release in the rat hippocampus in vivo: evidence for a permissive dopamine synapse 
British Journal of Pharmacology  1999;127(6):1486-1494.
In this study, the mechanism of nicotine-induced hippocampal acetylcholine (ACh) release in awake, freely moving rats was examined using in vivo microdialysis.Systemic administration of nicotine (0.4 mg kg−1, s.c.) increased the levels of ACh in hippocampal dialysates.The nicotine-induced hippocampal ACh release was sensitive to the pretreatment of neuronal nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine (3.0 mg kg−1, s.c.) and dihydro-β-erythrodine (DHβE; 4.0 mg kg−1, s.c.) as well as systemic administration of the dopamine (DA) D1 receptor antagonist SCH-23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzazepine; 0.3 mg kg−1, s.c.).Local perfusion of mecamylamine (100 μM), DHβE (100 μM) or SCH-23390 (10 μM) through microdialysis probe did not increase basal hippocampal ACh release.Hippocampal ACh release elicited by systemic administration of nicotine (0.4 mg kg−1, s.c.) was antagonized by local perfusion of SCH-23390 (10 μM), but not by MEC (100 μM) or DHβE (100 μM).Direct perfusion of nicotine (1 mM, but not 0.1 mM) increased hippocampal ACh levels; however, this effect was relatively insensitive to blockade by co-perfusion of either mecamylamine (100 μM) or SCH-23390 (10 μM).These results suggest that nicotine-induced hippocampal ACh release occurs by two distinct mechanisms: (1) activation of nAChRs outside the hippocampus leading to DA release and subsequent ACh release involving a permissive DA synapse, and (2) direct action of nicotine within the hippocampus leading to ACh release via non-DA-ergic mechanism.
doi:10.1038/sj.bjp.0702683
PMCID: PMC1760670  PMID: 10455300
Nicotine; hippocampal ACh release; microdialysis; SCH-23390; mecamylamine; neuronal nicotinic acetylcholine receptors
16.  Conformational analysis and parallel QM/MM X-ray refinement of protein bound anti-Alzheimer drug donepezil 
The recognition and association of donepezil with acetylcholinesterase (AChE) has been extensively studied in the past several decades because of the former’s use as a palliative treatment for mild Alzheimer disease. Herein we examine the conformational properties of donepezil and we re-examine the donepezil-AChE crystal structure using combined quantum mechanical/molecular mechanical (QM/MM) X-ray refinement tools. Donepezil’s conformational energy surface was explored using the M06 suite of density functionals and with the MP2/complete basis set (CBS) method using the aug-cc-pVXZ (X = D and T) basis sets. The donepezil-AChE complex (PDB 1EVE) was also re-refined through a parallel QM/MM X-ray refinement approach based on an in-house ab initio code QUICK, which uses the message passing interface (MPI) in a distributed SCF algorithm to accelerate the calculation via parallelization. In the QM/MM re-refined donepezil structure, coordinate errors that previously existed in the PDB deposited geometry were improved leading to an improvement of the modeling of the interaction between donepezil and the aromatic side chains located in the AChE active site gorge. As a result of the re-refinement there was a 93% reduction in the donepezil conformational strain energy versus the original PDB structure. The results of the present effort offer further detailed structural and biochemical inhibitor-AChE information for the continued development of more effective and palliative treatments of Alzheimer disease.
doi:10.1021/ct300957x
PMCID: PMC3601759  PMID: 23526889
17.  Different Cholinesterase Inhibitor Effects on CSF Cholinesterases in Alzheimer Patients 
Current Alzheimer research  2009;6(1):4-14.
Background
The current study aimed to compare the effects of different cholinesterase inhibitors on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and protein levels, in the cerebrospinal fluid (CSF) of Alzheimer disease (AD) patients.
Methods and Findings
AD patients aged 50–85 years were randomized to open-label treatment with oral rivastigmine, donepezil or galantamine for 13 weeks. AChE and BuChE activities were assayed by Ellman’s colorimetric method. Protein levels were assessed by enzyme-linked immunosorbent assay (ELISA). Primary analyses were based on the Completer population (randomized patients who completed Week 13 assessments). 63 patients were randomized to treatment. Rivastigmine was associated with decreased AChE activity by 42.6% and decreased AChE protein levels by 9.3%, and decreased BuChE activity by 45.6% and decreased BuChE protein levels by 21.8%. Galantamine decreased AChE activity by 2.1% and BuChE activity by 0.5%, but increased AChE protein levels by 51.2% and BuChE protein levels by10.5%. Donepezil increased AChE and BuChE activities by 11.8% and 2.8%, respectively. Donepezil caused a 215.2%increase in AChE and 0.4% increase in BuChE protein levels. Changes in mean AChE-Readthrough/Synaptic ratios, which might reflect underlying neurodegenerative processes, were 1.4, 0.6, and 0.4 for rivastigmine, donepezil and galantamine, respectively.
Conclusion
The findings suggest pharmacologically-induced differences between rivastigmine, donepezil and galantamine. Rivastigmine provides sustained inhibition of AChE and BuChE, while donepezil and galantamine do not inhibit BuChE and are associated with increases in CSF AChE protein levels. The clinical implications require evaluation.
PMCID: PMC4046577  PMID: 19199870
Acetylcholinesterase; Alzheimer’s disease; Butyrylcholinesterase; Cerebrospinal fluid (CSF); Cholinesterase inhibitors
18.  Effects of thyroxine and donepezil on hippocampal acetylcholine content, acetylcholinesterase activity, synaptotagmin-1 and SNAP-25 expression in hypothyroid adult rats 
Molecular Medicine Reports  2014;11(2):775-782.
A growing number of studies have revealed that neurocognitive impairment, induced by adult-onset hypothyroidism, may not be fully restored by traditional hormone substitution therapies, including thyroxine (T4). The present study has investigated the effect of T4 and donepezil (DON; an acetylcholinesterase (AChE) inhibitor) treatment on the hypothyroidism-induced alterations of acetylcholine (ACh) content and AChE activity. Furthermore, we examined synaptotagmin-1 (syt-1) and SNAP-25 expression in the hippocampus of adult rats. Adding 0.05% propylthiouracil to their drinking water for five weeks induced hypothyroidism in the rat models. From the fourth week, the rats were treated with T4, DON or a combination of both. Concentration of ACh and the activity of AChE was determined colorimetrically. The results demonstrated that hypothyroidism induced a significant decrease of Ach content and AChE activity (by 17 and 34%, respectively), which were restored to control values by T4 administration. DON treatment also restored Ach to the normal level. Protein levels of syt-1 and SNAP-25 were determined by immunohistochemistry. The results demonstrated that syt-1 was expressed at significantly lower levels in hypothyroid rats, while SNAP-25 levels were notably higher compared with the controls. Two-week treatment with T4 alone failed to normalize the expression levels of these two proteins, while co-administration of T4 and DON was able to induce this effect. These data suggested that the thyroid hormone, T4, may have a direct effect on the metabolism of hippocampal ACh in adult rats, and that the DON treatment may facilitate the recovery of synaptic protein impairments induced by hypothyroidism.
doi:10.3892/mmr.2014.2825
PMCID: PMC4262484  PMID: 25371181
hypothyroidism; hippocampus; thyroxine; donepezil; acetylcholine; acetylcholinesterase; synaptotagmin-1; SNAP-25
19.  Donepezil Is Ineffective in Promoting Motor and Cognitive Benefits after Controlled Cortical Impact Injury in Male Rats 
Journal of Neurotrauma  2013;30(7):557-564.
Abstract
The acetylcholinesterase (AChE) inhibitor donepezil is used as a therapy for Alzheimer's disease and has been recommended as a treatment for enhancing attention and memory after traumatic brain injury (TBI). Although select clinical case studies support the use of donepezil for enhancing cognition, there is a paucity of experimental TBI studies assessing the potential efficacy of this pharmacotherapy. Hence, the aim of this pre-clinical study was to evaluate several doses of donepezil to determine its effect on functional outcome after TBI. Ninety anesthetized adult male rats received a controlled cortical impact (CCI; 2.8 mm cortical depth at 4 m/sec) or sham injury, and then were randomly assigned to six TBI and six sham groups (donepezil 0.25, 0.5, 1.0, 2.0, or 3.0 mg/kg, and saline vehicle 1.0 mL/kg). Treatments began 24 h after surgery and were administered i.p. once daily for 19 days. Function was assessed by motor (beam balance/walk) and cognitive (Morris water maze) tests on days 1–5 and 14–19, respectively. No significant differences were observed among the sham control groups in any evaluation, regardless of dose, and therefore the data were pooled. Furthermore, no significant differences were revealed among the TBI groups in acute neurological assessments (e.g., righting reflex), suggesting that all groups received the same level of injury severity. None of the five doses of donepezil improved motor or cognitive function relative to vehicle-treated controls. Moreover, the two highest doses significantly impaired beam-balance (3.0 mg/kg), beam-walk (2.0 mg/kg and 3.0 mg/kg), and cognitive performance (3.0 mg/kg) versus vehicle. These data indicate that chronic administration of donepezil is not only ineffective in promoting functional improvement after moderate CCI injury, but depending on the dose is actually detrimental to the recovery process. Further work is necessary to determine if other AChE inhibitors exert similar effects after TBI.
doi:10.1089/neu.2012.2782
PMCID: PMC3636588  PMID: 23227953
AChE inhibitor; behavior; CCI; functional recovery; learning and memory; Morris water maze; TBI
20.  Pharmacokinetic and pharmacodynamic profile of donepezil HCl following single oral doses 
Aim
The aim of this study was to characterize the pharmacokinetic and pharmacodynamic profile of donepezil HCl, a chemically distinct and specific acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer’s disease, following administration of single oral doses.
Methods
This was a double-blind, randomized, single-dose, placebo-controlled, sequential-group, ascending-dose study in healthy male volunteers (n = 48). Six dose levels were investigated, ranging from 0.3 to 6.0 mg. Donepezil concentrations in plasma were determined by HPLC with UV detection. Pharmacodynamic activity was determined by the radioenzymatic measurement of erythrocyte membrane acetylcholinesterase (rbc-AChE) inhibition.
Results
The pharmacokinetic disposition of donepezil was observed to be both linear and dose proportional following single-dose administration. The mean peak plasma concentration (Cmax) of donepezil was observed at 4.1±1.5 h. The mean terminal disposition half-life was 81.5±22.0 h. The post-absorption phase of the plasma concentration–time curves for the 4.0 mg and 6.0 mg doses appeared to be biphasic, but the rate of donepezil clearance was independent of dose. Plasma concentrations for the 0.3, 0.6 and 0.9 mg dose groups were generally below the level of HPLC detection (2.0 ng ml−1), preventing accurate characterization of these doses. A direct correlation was observed between plasma donepezil concentrations and extent of AChE inhibition. For the 4.0 and 6.0 mg donepezil dose groups, maximal AChE inhibition (Emax) ranged from 33% to 35% and there was significant correlation between AChE inhibition and donepezil plasma concentration (P<0.005).
Conclusions
The pharmacokinetics of donepezil were found to be linear and dose proportional following the administration of single doses to healthy volunteers. A direct correlation was also observed between plasma donepezil concentrations and AChE inhibition. The extended half-life of donepezil makes it suitable for once-daily dosing.
doi:10.1046/j.1365-2125.1998.0460s1001.x
PMCID: PMC1873812  PMID: 9839758
donepezil; selective acetylcholinesterase inhibitor; Alzheimer’s disease; pharmacokinetics; pharmacodynamics
21.  Effects of thyroxin and donepezil on hippocampal acetylcholine content and syntaxin-1 and munc-18 expression in adult rats with hypothyroidism 
Adult-onset hypothyroidism induces various impairments in hippocampus-dependent cognitive function, in which numerous synaptic proteins and neurotransmitters are involved. Donepezil (DON), an acetylcholinesterase inhibitor, has been shown to be efficient in improving cognitive function. The aim of the present study was to investigate the effects of adult-onset hypothyroidism on the expression levels of the synaptic proteins syntaxin-1 and munc-18, as well as the content of the neurotransmitter acetylcholine (ACh) in the hippocampus. In addition, the study explored the effects of thyroxin (T4) and DON treatment on the altered parameters. The study involved 55 Sprague-Dawley rats that were randomly divided into five groups: Control, hypothyroid (0.05% 6-n-propyl-2-thiouracil; added to the drinking water), hypothyroid treated with T4 (6 μg/100 g body weight once daily; intraperitoneal injection), hypothyroid treated with DON (0.005%; added to the drinking water) and hypothyroid treated with a combination of the two drugs (6 μg/100 g T4 and 0.005% DON). The concentration of ACh was determined in the homogenized hippocampus of each animal by alkaline hydroxylamine colorimetry. The protein levels of syntaxin-1 and munc-18 were determined by immunohistochemistry. The results showed that the content of ACh in the hippocampi of the hypothyroid rats was significantly decreased compared with that in the controls and that T4 monotherapy and DON administration restored the ACh content to normal values. In the hippocampi of the hypothyroid group, munc-18 was expressed at significantly lower levels, while the expression levels of syntaxin-1 were increased compared with the levels in the control group. Treatment with T4 alone restored the expression of syntaxin-1 but failed to normalize munc-18 expression levels. The co-administration of T4 and DON returned the munc-18 levels to normal values. These observations indicate that adult-onset hypothyroidism induces alterations in the levels of munc-18, syntaxin-1 and ACh in the hippocampus. Syntaxin-1 and ACh levels were restored by T4 monotherapy while munc-18 levels were not. In addition, the co-administration of T4 and DON resulted in more effective restoration than either alone. The thyroid hormone has a direct effect on metabolism of hippocampal ACh in adult rats and DON is helpful for treatment of synaptic protein impairment induced by hypothyroidism.
doi:10.3892/etm.2014.1487
PMCID: PMC3919934  PMID: 24520241
hypothyroidism; hippocampus; thyroxin; donepezil; acetylcholine; syntaxin-1; munc-18
22.  Synthesis and in silico evaluation of 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives against Alzheimer disease: to understand their interacting mechanism with acetylcholinesterase 
Journal of Chemical Biology  2012;5(4):151-166.
Anomalous action of human acetylcholinesterase (hAChE) in Alzheimer’s disease (AD) was restrained by various AChE inhibitors, of which the specific and potent lead candidate Donepezil is used for treating the disease AD. Besides the specificity, the observed undesirable side effects caused by Donepezil invoked the quest for new lead molecules with the increased potency and specificity for AChE. The present study elucidates the potency of six 1N-methyl-1S-methyl-2-nitroethylene (NMSM) derivatives to form a specific interaction with the peripheral anionic site and catalytic anionic subsite residues of hAChE. The NMSMs were prepared in good yield from 1,1-di(methylsulfanyl)-2-nitroethylene and primary amine (or) amino acid esters. In silico interaction analysis reveals specific and potent interactions between hAChE and selected ligand molecules. The site-specific interactions formed between these molecules also results in a conformational change in the orientation of active site residues of hAChE, which prevents them from being accessed by beta-amyloid protein (Aβ), which is a causative agent for amyloid plaque formation and acetylcholine (ACh). In silico interaction analysis between the ligand-bounded hAChE with Aß and ACh confirms this observation. The variation in the conformation of hAChE associated with the decreased ability of Aβ and ACh to access the respective functional residues of hAChE induced by the novel NMSMs favors their selection for in vivo analysis to present themselves as new members of hAChE inhibitors.
doi:10.1007/s12154-012-0084-z
PMCID: PMC3461197  PMID: 24052808
Alzheimer’s disease; Human acetylcholinesterase (hAChE); Amyloid beta (Aβ); 1N-methyl-1S-methyl-2-nitroethylene (NMSM); Donepezil and molecular docking calculation
23.  Description of 3,180 Courses of Chelation with Dimercaptosuccinic Acid in Children ≤5 y with Severe Lead Poisoning in Zamfara, Northern Nigeria: A Retrospective Analysis of Programme Data 
PLoS Medicine  2014;11(10):e1001739.
Jane Greig and colleagues from the medical humanitarian organization Médecins Sans Frontières describe the use of the oral chelating agent dimercaptosuccinic acid (DMSA) in several thousand young children with severe lead poisoning as a result of an environmental disaster in Zamfara, northern Nigeria.
Please see later in the article for the Editors' Summary
Background
In 2010, Médecins Sans Frontières (MSF) discovered extensive lead poisoning impacting several thousand children in rural northern Nigeria. An estimated 400 fatalities had occurred over 3 mo. The US Centers for Disease Control and Prevention (CDC) confirmed widespread contamination from lead-rich ore being processed for gold, and environmental management was begun. MSF commenced a medical management programme that included treatment with the oral chelating agent 2,3-dimercaptosuccinic acid (DMSA, succimer). Here we describe and evaluate the changes in venous blood lead level (VBLL) associated with DMSA treatment in the largest cohort of children ≤5 y of age with severe paediatric lead intoxication reported to date to our knowledge.
Methods and Findings
In a retrospective analysis of programme data, we describe change in VBLL after DMSA treatment courses in a cohort of 1,156 children ≤5 y of age who underwent between one and 15 courses of chelation treatment. Courses of DMSA of 19 or 28 d duration administered to children with VBLL ≥ 45 µg/dl were included. Impact of DMSA was calculated as end-course VBLL as a percentage of pre-course VBLL (ECP). Mixed model regression with nested random effects was used to evaluate the relative associations of covariates with ECP. Of 3,180 treatment courses administered, 36% and 6% of courses commenced with VBLL ≥ 80 µg/dl and ≥ 120 µg/dl, respectively. Overall mean ECP was 74.5% (95% CI 69.7%–79.7%); among 159 inpatient courses, ECP was 47.7% (95% CI 39.7%–57.3%). ECP after 19-d courses (n = 2,262) was lower in older children, first-ever courses, courses with a longer interval since a previous course, courses with more directly observed doses, and courses with higher pre-course VBLLs. Low haemoglobin was associated with higher ECP. Twenty children aged ≤5 y who commenced chelation died during the period studied, with lead poisoning a primary factor in six deaths. Monitoring of alanine transaminase (ALT), creatinine, and full blood count revealed moderate ALT elevation in <2.5% of courses. No clinically severe adverse drug effects were observed, and no laboratory findings required discontinuation of treatment. Limitations include that this was a retrospective analysis of clinical data, and unmeasured variables related to environmental exposures could not be accounted for.
Conclusions
Oral DMSA was a pharmacodynamically effective chelating agent for the treatment of severe childhood lead poisoning in a resource-limited setting. Re-exposure to lead, despite efforts to remediate the environment, and non-adherence may have influenced the impact of outpatient treatment.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lead, a toxic metal that occurs naturally in the earth's crust, is now present throughout the environment because of human activities. For many years, lead was added to paint and gasoline and used in solder for water pipes. In addition, the mining, smelting, and refining of some metallic ores releases lead into the environment. Inhalation of contaminated air, consumption of contaminated food and water, and contact with dust that contains lead raises venous blood lead levels (VBLLs) and causes many health problems, particularly in children. Children who ingest large amounts of lead can develop anemia, muscle weakness, kidney damage, and life-threatening encephalopathy (brain swelling). Although fatal lead poisoning is now rare in resource-rich countries, it nevertheless remains a major global health problem. Over a three-month period in early 2010, for example, about 400 young children died in Zamfara State, Nigeria, from unexplained, intractable fits. By May 2010, it was clear that recently expanded gold mining had caused widespread environmental lead contamination in the region, and an environmental management program was begun to reduce lead levels in the surface soils.
Why Was This Study Done?
In response to the lead poisoning outbreak, the not-for-profit organization Médecins Sans Frontières (MSF) began a medical management program to reduce VBLLs that included treatment with the oral chelation agent dimercaptosuccinic acid (DMSA). Chelation agents bind metal ions and facilitate their removal from the body, thereby reducing the likelihood of lead moving from the blood to the brain. Lead encephalopathy has been commonly treated by injecting another chelator called CaNa2EDTA, but the discovery of more than 1,000 cases of childhood lead poisoning in rural villages in Nigeria meant that MSF needed a chelation approach that could be applied rapidly in a remote resource-limited setting. Additionally, although CaNa2EDTA has been in common use for severe lead poisoning for longer than DMSA, and is commonly recommended in guidelines, the evidence base does not support one treatment as superior. Here, in a retrospective analysis of MSF program data, the researchers evaluate the changes in VBLLs before and after courses of oral DMSA treatment in children aged five years and below living in Zamfara to gain new insights into this understudied treatment for severe childhood lead poisoning.
What Did the Researchers Do and Find?
The researchers measured VBLLs before and after treatment with DMSA in 1,156 children (inpatient and outpatient) with high amounts of lead in their blood who underwent one or more courses of chelation treatment lasting 19 or 28 days by calculating each child's end-course VBLL as a percentage of the child's pre-course VBLL (ECP). Considering all the treatment courses given between June 2010 and June 2011, the mean (average) ECP was 74.5%. That is, on average, VBLLs measured at the end of treatment courses were reduced by a quarter compared to VBLLs at the start of treatment courses. Among 159 inpatient courses of DMSA, the ECP was 47.7% (a halving of pre-course VBLLs). The ECP after 19-day courses was lower in older children, after first-ever courses, after courses with a longer interval since a previous course, after courses that included more directly observed doses (DMSA given in the presence of a health-care worker), and in children with higher pre-course VBLLs. Nine of the children included in this analysis died during the study period; lead poisoning was probably involved in three of these deaths. Importantly, no clinically severe adverse effects related to DMSA were seen during the study period, and no laboratory findings were recorded that required treatment discontinuation.
What Do These Findings Mean?
Because many changes were made to the treatment given to the affected children in Zamfara during the study period and because no information is presented here on clinical outcomes, these findings cannot be used to reach any definitive conclusions about the effectiveness or safety of oral DMSA as a treatment for lead poisoning in young children. However, these findings show that chelation was associated with a large reduction in the death rate among probable or suspected cases of childhood lead poisoning in Zamfara and provide new information about oral chelation that may help agencies such as MSF provide urgent treatment for lead poisoning in resource-limited settings where intravenous chelation is not feasible. Moreover, the finding of a lower ECP after inpatient treatment courses compared to after outpatient courses suggests that re-exposure to lead and non-adherence to treatment may have influenced the impact of outpatient treatments. Thus, it is essential that medical management of lead poisoning in resource-limited settings be accompanied by environmental remediation and that efforts are made to support adherence to treatment in the community by implementing directly observed treatment wherever possible.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001739.
A related PLOS ONE Research Article by Greig et al. provides information about the association between VBLLs and neurological features in children affected by the acute lead poisoning outbreak in Zamfara
MSF provides information about the lead poisoning crisis in Zamfara State
Human Rights Watch, an international organization that works to uphold human dignity and advance the cause of human rights for all, also provides information about lead poisoning in Zamfara State, including photographs and a video
Tox Town, an interactive site about environmental health concerns from the US National Library of Medicine, provides information on exposure to lead (in English and Spanish)
The US Environmental Protection Agency provides information on lead and lead poisoning (in English and Spanish)
The US Centers for Disease Control and Prevention provides information about lead in the environment and about its lead poisoning prevention program
MedlinePlus provides a list of links to further information about lead poisoning (in English and Spanish)
doi:10.1371/journal.pmed.1001739
PMCID: PMC4188566  PMID: 25291378
24.  Pharmacokinetic and pharmacodynamic profile of donepezil HCl following multiple oral doses 
Aim
The aim of this study was to characterize the pharmacokinetics and pharmacodynamics of donepezil HCl, a new, chemically distinct and specific acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer’s disease, following multiple-dose administration.
Methods
This was a double-blind, randomized, placebo-controlled, multiple-dose study in healthy male volunteers (n =27). Three dose levels were investigated in sequential order: 1, 3 and 5 mg. Each dose was administered orally, once a day, for 21 consecutive days. Donepezil concentrations in plasma were quantified by HPLC. Pharmacodynamic activity was determined by the radioenzymatic measurement of erythrocyte membrane acetylcholinesterase (rbc-AChE) inhibition.
Results
The pharmacokinetic disposition of donepezil was observed to be dose proportional. The mean terminal disposition half-life was 79.5±19.0 h which resulted in a slow approach to steady state (14–21 days). A four- to sixfold increase in donepezil plasma concentration was observed during this time; however, no further increase was evident after achievement of steady state. The mean donepezil plasma concentration at steady state (Css) was 14.2 ng ml−1. Neither the rate of accumulation nor the rate of clearance was dose dependent. Inhibition of rbc-AChE was directly correlated with donepezil concentration over a wide concentration range, with the higher concentrations showing the expected hyperbolic relationship. Donepezil was well tolerated by all subjects with no clinically significant changes in laboratory or physical parameters observed at any dose.
Conclusions
The pharmacokinetics of donepezil were found to be dose proportional following the administration of multiple doses to healthy volunteers. A predictable relationship was also observed between plasma donepezil concentrations and rbc-AChE inhibition. The half-life of donepezil makes it suitable for once-daily dosing.
doi:10.1046/j.1365-2125.1998.0460s1007.x
PMCID: PMC1873811  PMID: 9839759
donepezil; selective acetylcholinesterase inhibitor; Alzheimer’s disease; pharmacokinetics; pharmacodynamics
25.  Effect of thyrotropin releasing hormone (TRH) on acetylcholine release from different brain areas investigated by microdialysis. 
British Journal of Pharmacology  1991;102(2):363-368.
1. The effect of thyrotropin releasing hormone (TRH) administration upon acetylcholine (ACh) release in freely moving rats was investigated by means of transversal microdialysis coupled to h.p.l.c. TRH administered either s.c. or via local perfusion increased the ACh release from cortex and hippocampus but not from the striatum. The increase in ACh release was maintained after 7 days of s.c. administration of TRH. 2. After s.c. injection of the neuropeptide, the increase in ACh release was dose-dependent and reached a maximum at 40 min after administration. The maximal percentage increases were 18, 52, 66 and 89% at doses of 1, 2.5, 5 and 10 mg kg-1 and 35, 48 and 54% at doses of 2.5, 5 and 10 mg kg-1 in the cortex and hippocampus, respectively. The effect of TRH was dependent on neuronal activity since it was completely inhibited by perfusion with tetrodotoxin (TTX), 5 X 10(-7) M. 3. Perfusion with TRH, 2.5 micrograms microliters-1, caused 198% and 150% increase in ACh release 60 and 80 min after the beginning of the perfusion in the cortex and hippocampus, respectively. After this initial peak, a 100% increase in ACh release persisted throughout the perfusion. 4. Systemic TRH administration was followed by marked hyperactivity and stereotyped behaviour that showed a time course shorter than that of the increase in ACh release. 5. These findings demonstrate that TRH exerts a strong stimulant action on cortical and hippocampal cholinergic pathways.
PMCID: PMC1918036  PMID: 1901747

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