Estimation of plasma IC50 of donepezil hydrochloride for brain acetylcholinesterase inhibition in monkey using N-[11C]methylpiperidin-4-yl acetate ([11C]MP4A) and PET.

Donepezil hydrochloride is a potent and selective inhibitor for brain acetylcholinesterase (AChE) and is currently used worldwide for the treatment of Alzheimer's disease. Until now, there is no in vivo study on the relation between the plasma concentration and the brain AChE inhibition. The purpose of this study was to estimate in vivo plasma IC(50) of donepezil in living monkeys by measuring plasma donepezil concentration (LC/MS/MS) and brain AChE activity with positron emission tomography (PET) and N-[(11)C]methylpiperidin-4-yl acetate, which is an acetylcholine analog recently developed by us for quantifying in vivo brain AChE activity. PET scans with donepezil at two doses, 100 microg/kg (donepezil-1; N=5) or 250 microg/kg (donepezil-2; N=5), were performed using the same monkeys at 4-week intervals. Before each PET scan, baseline PET scans (N=10 in total) were performed without donepezil. The plasma donepezil concentrations 14 min after intravenous injection were proportional to the doses, 17.2+/-2.9 ng/ml (donepezil-1) and 44.0+/-5.0 ng/ml (donepezil-2), and the mean AChE inhibitions in four neocortical regions as evaluated by PET were also dose-dependent, 27% (donepezil-1) and 53% (donepezil-2). In IC(50) estimation, measured plasma donepezil concentrations were corrected for the change during PET scan. The IC(50) values (estimate+/-SE) were 42+/-9.0 (ng/ml; donepezil-1), 34+/-3.2 (donepezil-2), and 37+/-4.1 (combined data). The present method may be useful for in vivo evaluation of other AChE inhibitors and novel drugs.     

Cerebral acetylcholinesterase imaging: development of the radioprobes

Cerebral acetylcholinesterase (AChE) imaging is not only useful for diagnosis of dementia disorders but also for therapeutic monitoring of the effects of cholinesterase (ChE) inhibitors and for decision of the appropriate clinical dosage of newly developed ChE inhibitors. Several ChE inhibitors or the derivatives such as 1,2,3,4-tetrahydro-9-methylaminoacridine (MTHA), donepezil, physostigmine, CP126,998 and 2-fluoro-CP118,954 have been labeled with positron emitters for mapping cerebral AChE by positron emission tomography (PET). When [(11)C]MTHA or [(11)C]donepezil was injected in animals, the uptake poorly reflect the regional distribution of AChE in the brain because of high non-specific binding and/or less specific to AChE in vivo in the brain tissue. [(11)C]physostigmine, [(11)C]CP126,998 and 2-[(18)F]fluoro-CP118,954 were distributed corresponding well to the regional AChE activity in animals, and also former two probes were successfully applied to clinical PET trial. The other approach is measuring cerebral AChE activity with radiolabeled acetylcholine analogue substrates. We have developed the principle for measuring cerebral enzyme activity by PET and radiolabeled N-methylpiperidinyl esters for quantitative measurement of cerebral AChE activity. N-[(11)C]methylipiperidin-4-yl acetate (MP4A) and N-[(11)C]methylpiperidin-4-yl propionate (MP4P) have been used for clinical studies of other demented disorders including Alzheimer's disease (AD), and the probes have demonstrated not only the reduction of AChE activity in the cerebral cortex of patients with AD but also the inhibitory effects of donepezil and rivastigmine on AChE activity in the brain of AD patients. Following this succession, widely available [(18)F]-labeled derivatives of MP4A and MP4P have been developed based on the structure-activity relationships between AChE and piperidinol esters.     

Regional effects of donepezil and rivastigmine on cortical acetylcholinesterase activity in Alzheimer's disease

Donepezil and rivastigmine are acetylcholinesterase (AChE) inhibitors used to improve cholinergic neurotransmission and cognitive function in Alzheimer's disease (AD). This study examined direct effects of these drugs on AChE activity in the frontal, temporal, and parietal cortices in AD. Six AD patients were scanned with positron emission tomography before and after 3 months of treatment with donepezil (10 mg/day), and five AD patients were scanned before and after 3 to 5 months of treatment with rivastigmine (9 mg/day). Healthy unmedicated controls were imaged twice to evaluate the reproducibility of the method. A specific AChE tracer, [methyl-11C]N-methyl-piperidyl-4-acetate, and a 3D positron emission tomography system with MRI coregistration were used for imaging. Treatment with donepezil reduced the AChE activity (k3 values) in the AD brain by 39% in the frontal (p < 0.001, Bonferroni corrected), 29% in the temporal (p = 0.02, corrected) and 28% in the parietal cortex (p = 0.05, corrected). The corresponding levels of inhibition for rivastigmine were 37% (p = 0.003, corrected), 28% (p = 0.03, uncorrected) and 28% (p = 0.05, corrected). When the treatment groups were combined, the level of AChE inhibition was significantly greater in the frontal cortex compared to the temporal cortex (p = 0.03, corrected). The test-retest analysis with healthy subjects indicated good reproducibility for the method, with a nonsignificant 0% to 7% intrasubject variability between scans. The present study provides first evidence for the effect of rivastigmine on cortical AChE activity. Our results indicate that the pooled effects of donepezil and rivastigmine on brain AChE are greater in the frontal cortex compared to the temporal cortex in AD. This regional difference is probably related to the prominent temporoparietal reduction of AChE in AD. We hypothesize that the clinical improvement in behavioral and attentional symptoms of AD due to AChE inhibitors is associated with the frontal AChE inhibition.     

Acetylcholinesterase imaging: its use in therapy evaluation and drug design

Several cholinesterase (ChE) inhibitors have been labeled with carbon-11 for visualizing binding sites on acetylcholinesterase (AChE) by positron emission tomography (PET). Following intravenous injection of 1,2,3,4-tetrahydro-9-[(11)C]methylaminoacridine or [(11)C]donepezil, however, the radioactivity distribution does not reflect the regional distribution of AChE in the brain of animals, probably because these compounds have high non-specific binding and/or other specific binding sites in vivo in the brain. PET studies with [(11)C]physostigmine and [(11)C]CP-126,998 in the brain of healthy subjects have shown a radioactivity distribution corresponding to the regional distribution of AChE activity measured in postmortem human brains. These radiotracers may be useful for measuring the occupancy of binding sites on AChE by AChE inhibitors, and for investigating the cerebral pharmacokinetics of such therapeutic drugs. An alternative approach to map AChE is the use of acetylcholine analogue substrates. We have developed N-methylpiperidinyl esters labeled with carbon-11 for quantitative measurement of AChE activity. Currently, two N-[(11)C]methylpiperidine esters, N-[(11)C]methylipiperidin-4-ylacetate (MP4A) and N-[(11)C]methylpiperidin-4-yl propionate (MP4P or PMP), have been used for clinical studies of Alzheimer's disease and other neurodegenerative diseases. Both [(11)C]MP4A- and [(11)C]MP4P-PET have demonstrated not only the reduction of AChE activity in the cerebral cortex of patients with Alzheimer's disease (AD) but also the inhibitory effects of donepezil and rivastigmine on AChE activity in the brain of AD patients. AChE imaging should prove useful for therapeutic monitoring of the effects of ChE inhibitors, including determination of the appropriate clinical doses of newly developed compounds, and can thus prompt the development of novel drugs targeting AChE.     

Evaluation of the binding characteristics of [5-(11)C-methoxy]Donepezil in the rat brain for in vivo visualization of acetylcholinesterase

Donepezil, an acetylcholinesterase (AChE) inhibitor, has not been evaluated for its binding characteristics using a radioactive tracer, although its inhibitory action on AChE has been studied. The aim of this research is to examine whether AChE can be visualized in vivo and in vitro with [(11)C]donepezil. [5-(11)C-methoxy]Donepezil was synthesized by O-methylation using [(11)C]methyl triflate. The binding of [(11)C]donepezil to brain homogenates was higher in the brain stem and striatum, and it was lowest in the cerebellum. The in vitro autoradiographic study successfully demonstrated the specific binding of [(11)C]donepezil to AChE in the rat brain. The IC(50) value of binding was approximately 10 nM, which is comparable to the reported value for inhibiting enzyme activity (6 nM). Saturation experiments revealed that the B(max) and K(d) of [(11)C]donepezil binding in vitro are 65 fmol/mg tissue and 39.8 nM, respectively. In accordance with the in vitro bindings, the in vivo distribution of [(11)C]donepezil was heterogeneous in the rat brain. In the blocking experiments, the heterogeneous distribution disappeared in the presence of a large amount of unlabeled donepezil. These data suggest that [5-(11)C-methoxy]donepezil can be potentially useful to image AChE non-invasively in the human brain by positron emission tomography.     

Effect of donepezil hydrochloride (E2020) on basal concentration of extracellular acetylcholine in the hippocampus of rats

The effects of oral administration of the centrally acting acetylcholinesterase (AChE) inhibitors, donepezil hydrochloride (donepezil: E2020: (±)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride), tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) and ENA-713 (rivastigmine: (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate), which have been developed for the treatment of Alzheimer's disease, on the extracellular acetylcholine concentration in the hippocampus of rats were evaluated by using a microdialysis technique without adding cholinesterase inhibitor to the perfusion solution. We also compared the inhibition of brain AChE and the brain concentrations of these drugs. Donepezil at 2.5 mg/kg and tacrine at 5 mg/kg showed significant effects for more than 6 h. At these doses, the maximum increases were observed at about 1.5 h after administration of donepezil, and at about 2 h with tacrine, and were 499% and 422% of the pre-level, respectively. ENA-713 produced significant effects at doses of 0.625, 1.25 and 2.5 mg/kg, which lasted for about 1, 2 and 4 h, respectively. The maximum increases produced by these doses at about 0.5 h after administration were 190, 346 and 458% of the pre-level, respectively. The time courses of brain AChE inhibition with donepezil at 2.5 mg/kg, tacrine at 10 mg/kg and ENA-713 at 2.5 mg/kg were mirror images of the extracellular acetylcholine-increasing action at the same doses. The time courses of the brain concentrations of drugs after oral administration of donepezil at 2.5 mg/kg and tacrine at 10 mg/kg were consistent with those of brain AChE inhibition at the same doses, and there was a linear relation between these parameters. Brain concentration of ENA-713 at 2.5 mg/kg was below the limit of quantification at all time points measured. These results suggest that oral administration of donepezil, tacrine and ENA-713 increases acetylcholine concentration in the synaptic cleft of the hippocampus mostly through AChE inhibition, and that donepezil has a more potent activity than tacrine and a longer-lasting effect than ENA-713 on the central cholinergic system.     

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.

     

In vivo measurement of the serotonin transporter with (S)-([18F]fluoromethyl)-(+)-McN5652.

The radiolabeled serotonin transporter (SERT) ligand [(11)C](+)-McN5652 has recently been used in clinical positron emission tomography (PET) studies for SERT imaging. However, this radioligand offers disadvantages in routine clinical settings because of its short radioisotope half-life (eg PET facilities within hospitals without a cyclotron need to acquire such radioligands from distant cyclotron units for clinical use). S-([(18)F]fluoromethyl)-(+)-McN5652 ([(18)F](+)-FMe-McN5652) is an analogue which has been synthesized newly, and has a significantly longer radioisotope half-life. In the porcine brain, it demonstrates the same characteristic distribution pattern of serotonin-uptake sites like the (11)C-labeled congener with the highest binding in the midbrain and thalamus and the lowest in the cerebellum and occipital cortex. It shows a 30% higher blood-brain transfer and a slower peripheral metabolism than [(11)C](+)-McN5652. Rather uniform brain binding was observed after injection of the pharmacologically inactive radiolabeled enantiomer, or after pretreatment with the highly selective SERT inhibitor citalopram. The norepinephrine uptake inhibitor maprotiline did not show any inhibitory effect. Using a one-tissue compartment model (K(1), k"(2)) or a two-tissue compartment model (K(1) to k(4)) with or without constraints for calculation, the regional binding parameters of [(11)C](+)-McN5652 and [(18)F](+)-FMe-McN5652 are highly correlated among each other and with the SERT density, as determined by in vitro binding of [(3)H]citalopram. Using constraints to correct for the free fraction and nonspecific binding of the radiotracers, a considerable increase of the midbrain-occipital cortex ratios with higher values for [(18)F](+)-FMe-McN5652 compared to [(11)C](+)McN5652 was revealed. It is concluded that [(18)F](+)-FMe-McN5652 has better features than [(11)C](+)McN5652 for SERT imaging with PET.     

Okadaic acid induced neurotoxicity leads to central cholinergic dysfunction in rats

Central cholinergic system is involved in regulation of memory and disturbances in these results in memory loss. Previously, we examined the effect of okadaic acid, OKA (200ng, i.c.v.) on memory impairment and mitochondrial dysfunction in rats. In the present study, we investigated effect of OKA (i.c.v) on cholinergic function by observing acetylcholine level (ACh), acetylcholinestrase (AChE) activity, and mRNA expression of acetylcholinestrase and α7nicotinic receptor (α7-nAChR) as a cholinergic markers in brain areas (cerebellum, striatum cortex and hippocampus). In present work OKA, caused a significant decrease in acetylcholine level, acetylcholinestrase activity and mRNA expression of acetylcholinestrase and α7-nicotinic receptor in rat but these changes were mainly observed in cortex and hippocampus. Further, histopathological study by cresyl violet staining showed neuronal loss in cortex and hippocampus after OKA administration indicating neurotoxicity. Pretreatment with anti-dementic drugs donepezil (AChE inhibitor; 5mg/kg, p.o) and memantine (NMDA receptor antagonist; 10mg/kg, p.o) daily for 13 day prevented cholinergic dysfunction and neuronal loss in cortex and hippocampus of OKA treated rat. Daily per se treatment for 13 day with donepezil decreased acetylcholinestrase activity and increased mRNA expression of acetylcholinestrase and α7-nicotinic receptor. Whereas, per se treatment with memantine daily for 13 day did not affect acetylcholinestrase activity, mRNA expression of acetylcholinestrase and α7-nicotinic receptor. Findings of this work shows that OKA (i.c.v.), apart from memory impairment and mitochondrial dysfunction, as our previous study showed, also induced cholinergic dysfunction and neuronal loss, which can be addressed by antidementic drugs like donepezil and memantine.     

Nicotine normalizes increased prefrontal cortical dopamine D1 receptor binding and decreased working memory performance produced by repeated pretreatment with MK-801: a PET study in conscious monkeys.

The effects of acute nicotine were determined on dopamine (DA) D(1) (D(1)R) and D(2) (D(2)R) receptor binding in the neocortex of conscious monkeys under control conditions as well as after chronic pretreatment with MK-801 (dizocilpine), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Extrastriatal neocortical D(1)R and D(2)R binding was evaluated with [(11)C]NNC112 and [(11)C]FLB457 with high-specific radioactivity using positron emission tomography (PET). Acute administration of nicotine bitartrate, given as an intravenous (i.v.) bolus plus infusion for 30 min at doses of 32 microg/kg+0.8 microg/kg/min or 100 microg/kg+2.53 microg/kg/min as base, induced slight but significant dose-dependent increases of DA in the extracellular fluid of prefrontal cortex (PFC) as determined by microdialysis. However, acute nicotine did not affect either [(11)C]NNC112 or [(11)C]FLB457 binding to D(1)R or D(2)R, respectively, in any cortical region. Chronic MK-801 (0.03 mg/kg, intramuscularly (i.m.), twice daily for 13 days) increased [(11)C]NNC112 binding to D(1)R in PFC. No significant changes were detected in [(11)C]FLB457 binding to PFC D(2)R. Although chronic MK-801 lowered baseline DA and glutamate levels in PFC, acute nicotine normalized reduced DA to control levels. Acute nicotine dose-dependently normalized the increased binding of [(11)C]NNC112 to D(1)R produced by chronic MK-801 but [(11)C]FLB457 binding to PFC D(2)R did not change. Working memory performance, impaired after chronic MK-801, was partially improved by acute nicotine. These results demonstrate that acute nicotine normalizes MK-801-induced PFC abnormality of D(1)R in PFC.     

Effect of distigmine bromide on the central cholinergic system

AbstractDistigmine bromide (3, 3'-[hexamethylenebis (methyliminocarbonyloxy)] bis (1-methylpyridinium) dibromide), an acetylcholinesterase (AChE) inhibitor, produced a time-dependent and dose-dependent increase in acetylcholine (ACh) levels in the medial prefrontal cortex of rats. The overt cholinergic behaviours, such as tremor, fasciculation and lacrimation, were also elicited by distigmine bromide. The onset and duration of these behaviours were reflected in the microdialysis data showing that distigmine bromide enhances cholinergic neurotransmission in rat brain. The dose of distigmine bromide eliciting increase in ACh in the medial prefrontal cortex of rats correlated well with its dose for the induction of the cholinergic behaviours. Furthermore, distigmine bromide was an equipotent inhibitor of AChE and butyrylcholinesterase (BuChE) activities in the present study. From these findings, it is suggested that distigmine bromide may produce centrally mediated behavioural signs by increasing the ACh levels in the brain, resulting from its AChE and BuChE inhibitions.     

Comparative studies of huperzine A, donepezil, and rivastigmine on brain acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine levels in freely-moving rats

AIM: To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. METHODS: Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. RESULTS: Our results showed that huperzine A (0.25, 0.5, and 0.75 micromol/kg, po) dose-dependently elevated extracellular acetylcholine (ACh) levels in the medial prefrontal cortex (mPFC) and hippocampus. Oral administration of donepezil (5.4 micromol/kg) or rivastigmine (1 micromol/kg) also elicited significant increases in ACh in the mPFC and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of ACh at the same dose. The marked elevation of ACh after oral administration of huperzine A (0.5 micromol/kg) and donepezil (5.4 micromol/kg) was associated with a significantly increased release of dopamine (DA) in the mPFC or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the mPFC and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase ACh levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 micromol/kg, once per day for 30 d. CONCLUSION: The present findings showed that, in molar terms, huperzine A had similar potency on increasing mPFC ACh and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.     

Effect of donepezil hydrochloride (E2020) on extracellular acetylcholine concentration in the cerebral cortex of rats

Donepezil hydrochloride (donepezil), a potent and selective acetylcholinesterase inhibitor, has been developed for the treatment of Alzheimer's disease. We studied the effect of oral administration of this drug on the extracellular acetylcholine (ACh) concentration in the cerebral cortex of rats using microdialysis. We also observed fasciculation, a peripheral cholinergic sign induced by activation of neuromuscular transmission, after oral administration of the drug as an index of peripheral cholinergic activation. Other cholinesterase inhibitors, tacrine, ENA-713 and TAK-147, were used as reference drugs. Donepezil significantly and dose-dependently increased the extracellular ACh concentration in the rat cerebral cortex within the dose range of 2.5-10 mg/kg. Tacrine, ENA-713 and TAK-147 also elevated the extracellular concentration of ACh. The minimum effective doses of donepezil, tacrine, ENA-713 and TAK-147 were (< or = 2.5, 10, 10 and < or = 10 mg/kg, respectively. Donepezil produced fasciculation at doses of 2.5 mg/kg and above, with a dose-dependent increase in incidence and intensity. The reference compounds also induced fasciculation in a dose-dependent manner. The threshold doses of tacrine, ENA-713 and TAK-147 for fasciculation were 5, 2.5 and 2.5 mg/kg, respectively. The values of the ratio of the minimum effective dose for the ACh-increasing action to that for the fasciculation-producing action were: donepezil, < or = 1; tacrine, 2; ENA-713, 4; TAK-147, < or = 4. These results indicate that orally administered donepezil has a potent and selective activity on the central cholinergic system.     

Donepezil‐ or rivastigmine‐induced acetylcholinesterase inactivation is not modulated by neramexane in rat brain

In Alzheimer's disease (AD), the involvement of cholinergic deficit and overstimulation of N‐methyl‐D‐aspartate (NMDA) receptors by excessively released glutamate has been proposed. Since no existing drug currently addresses both pathologies, a combination therapy may be used clinically to target both mechanisms. The objective of the current study was to determine whether the NMDA receptor antagonist, neramexane affects the acetylcholinesterase (AChE) inhibition produced by donepezil and rivastigmine, two drugs used in the treatment of AD, or a prototype organophosphate compound diisopropylphosphorofluoridate (DFP) in rat cortex, hippocampus, striatum, and brainstem. Neramexane, at therapeutically relevant or higher doses (3.1, 6.2, or 12.4 mg/kg, ip), did not produce any apparent behavioral toxicity. Donepezil (0.75 mg/kg, ip), rivastigmine (0.35 mg/kg, ip) or DFP (1.5 mg/kg, ip) were administered at doses that produced approximately 50% inhibition of AChE and were well tolerated. Also, neramexane in combination with either of the AChE inhibitors did not produce any behavioral toxicity. This magnitude of AChE inhibition has been shown to increase extracellular acetylcholine (ACh) concentrations to therapeutically relevant levels. Neramexane (12.4 mg/kg) did not inhibit AChE activity in any of the brain regions tested when evaluated over a 24‐h period. Rats receiving neramexane alone or in combination with an AChE inhibitor (donepezil, rivastigmine, or DFP) were sacrificed 60 min after neramexane or DFP, 15 min after donepezil, and 30 min after rivastigmine administration. The times at which drug (donepezil, rivastigmine, or DFP) effects were assessed were based on maximal AChE inhibition established from previously reported time course studies. The current findings indicate that neramexane alone did not affect AChE activity at any dose tested nor did it influence the AChE inhibition produced by donepezil or rivastigmine in any brain region examined. However, a low dose of neramexane (3.1 mg/kg) significantly attenuated the inhibition of AChE produced by DFP in the hippocampus, striatum and brainstem, while higher doses (6.2 or 12.4 mg/kg) attenuated DFP‐induced AChE inhibition in all four of the brain regions evaluated. These results suggest that AChE inhibition produced by donepezil or rivastigmine is not altered by the co‐administration of neramexane. Drug Dev Res 68:253–260, 2007. © 2007 Wiley‐Liss, Inc.     

Pharmacological characterization of RS-1259, an orally active dual inhibitor of acetylcholinesterase and serotonin transporter, in rodents: possible treatment of Alzheimer's disease

A dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT), RS-1259 (4-[1S)-methylamino-3-(4-nitrophenoxy)]propylphenyl N,N-dimethylcarbamate (fumaric acid)(1/2)salt), was newly synthesized. RS-1259 simultaneously inhibited AChE and SERT in the brain following an oral administration in mice and rats. Actual simultaneous elevation of extracellular levels of 5-HT and ACh in the rat hippocampus was confirmed by microdialysis. The compound was as effective as SERT inhibitors such as fluoxetine and fluvoxamine in a 5-hydroxytryptophan-enhancing test in mice. Spatial memory deficits in the two-platform task of a water maze in aged rats were ameliorated by RS-1259 as well as donepezil. Both RS-1259 and donepezil increased the awake episodes in the daytime electroencephalogram of rats. Although RS-1259 was weaker than donepezil in enhancing central cholinergic transmission, as observed by ACh elevation in the hippocampus and memory enhancement in aged rats, the efficacy of RS-1259 on the consciousness level, which reflects the whole activity in the brain, was almost the same as that of donepezil. These results suggest that both cholinergic and serotonergic systems are involved in maintaining brain arousal and that a dual inhibitor of AChE and SERT may be useful for the treatment of cognitive disorders associated with reduced brain activity such as in Alzheimer's disease.     

Effect of subchronic galantamine treatment on neuronal nicotinic and muscarinic receptor subtypes in transgenic mice overexpressing human acetylcholinesterase

Overexpression of acetylcholinesterase (AChE) in mice causes cholinergic deficits with memory impairment. In this study, AChE overexpressing (hAChE-Tg) and control (FVB/N) mice were treated with the AChE inhibitor (AChEI) galantamine (4 mg/kg/day) for 10 days. The concentration of galantamine in plasma was 75-80 ng/ml. The inhibition of AChE was 20% in red blood cells (RBC) and 30% in brain cortical tissue. A significant increase in [(3)H]cytisine (alpha4 nicotinic receptor) binding was measured in the CA1 and CA3 area of the hippocampus of FVB/N mice following galantamine treatment. Similarly, a significant increase in [(125)I]alphabungarotoxin (alpha7 nicotinic receptor) binding was found in the frontal cortex, retrosplenial gr. cortex, motor cortex and thalamus in galantamine treated FVB/N compared to saline treated mice. No significant changes in nicotinic receptor binding sites were observed in galantamine treated hAChE-Tg mice. Significant decreases in the muscarinic receptors measured by [(3)H]AF-DX-384 (M2 muscarinic receptor) and [(3)H]pirenzepine (M1 muscarinic receptor) were observed in several brain regions of galantamine treated FVB/N and hAChE-Tg mice. This study shows regional and receptor subtype specific changes in the nicotinic receptor subtypes compared to the muscarinic receptors following galantamine treatment in FVB/N and hAChE-Tg mice.     

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes.     

Effects of donepezil on cortical metabolic response to activation during 18FDG-PET in Alzheimer’s disease: a double-blind cross-over trial

Rationale Cholinergic enhancement is among the best established treatments of Alzheimer’s disease (AD). The cognitive effects of treatment are thought to be mediated by improvement of neuronal transmission. Positron emission tomography using (18)F-fluorodeoxyglucose (FDG-PET) by measuring cortical metabolic response to activation assesses integrity of neuronal transmission in vivo.Objective To determine the effects of treatment with donepezil, a centrally selective acetylcholinesterase inhibitor, on cortical metabolism in AD using (18)FDG-PET.Methods We enrolled 23 patients, 18 of which completed the study, with mild to moderate probable AD (mini-mental status exam scores of 15–28, inclusive) in a double-blind cross over trial of 8 weeks donepezil compared to 8 weeks placebo with repeated double (18)FDG-PET examinations during passive audio-visual stimulation. Effects of treatment on cortical metabolic response to stimulation were determined with a linear model on a voxel level using Statistical Parametric Mapping (SPM 99, Wellcome Department of Imaging Neuroscience, London).Results Effects of treatment on cognitive measures were not different between donepezil and placebo. During passive audio-visual stimulation, patients showed activation in posterior visual and auditory areas and decreased activation in frontal cortex and basal ganglia. Resting state metabolism was increased with donepezil in left prefrontal cortex and decreased in right hippocampus. Cortical response to activation was increased in right hippocampus with donepezil compared to placebo.Conclusion Donepezil treatment shows a spatially limited functional effect on right hippocampus and left prefrontal cortical metabolism, independently of clinical response to treatment.     

Discovery and labeling of high-affinity 3,4-diarylpyrazolines as candidate radioligands for in vivo imaging of cannabinoid subtype-1 (CB1) receptors

Imaging of cannabinoid subtype-1 (CB1) receptors in vivo with positron emission tomography (PET) is likely to be important for understanding their role in neuropsychiatric disorders and for drug development. Radioligands for imaging with PET are required for this purpose. We synthesized new ligands from a 3,4- diarylpyrazoline platform of which (-)-12a ((-)-3-(4-chlorophenyl)-N'-[(4-cyanophenyl)sulfonyl]-4-phenyl- 4,5-dihydro-1H-pyrazole-1-carboxamidine) was found to have high-affinity and selectivity for binding to CB1 receptors. (-)-12a and its lower affinity enantiomer ((+)-12a) were labeled with carbon-11 (t1/2 ) 20.4 min) using [11C]cyanide ion as labeling agent and evaluated as PET radioligands in cynomolgus monkeys. After injection of [11C](-)-12a, there was high uptake and retention of radioactivity across brain according to the rank order of CB1 receptor densities. The distomer, [11C](+)-12a, failed to give a sustained CB1 receptor-specific distribution. Polar radiometabolites of [11C](-)-12a appeared moderately slowly in plasma. Radioligand [11C](-)-12a is promising for the study of brain CB1 receptors and merits further investigation in human subjects.     

Mechanisms of neuroprotective effects of therapeutic acetylcholinesterase inhibitors used in treatment of Alzheimer's disease

Donepezil, galanthamine, and tacrine are therapeutic acetylcholinesterase (AChE) inhibitors used for the treatment of Alzheimer's disease. The aim of this paper is to review recent findings on their neuroprotective properties and the mechanisms of neuroprotection against glutamate neurotoxicity in rat cortical neurons. First, the hallmark of neurotoxicity induced by two different glutamate treatment conditions was examined, revealing that acute glutamate treatment (1 mM, 10 min) induces necrotic neuronal death and that moderate glutamate treatment (100 microM, 24 hr) induces apoptotic neuronal death. Next, we showed that therapeutic AChE inhibitors protect cortical neurons from glutamate neurotoxicity in a time- and concentration-dependent manner. We examined the mechanism of this neuroprotective effect and found that the neuroprotective effects against both acute and moderate glutamate treatments are mediated through nicotinic acetylcholine receptors (nAChRs), or more specifically, the effects of donepezil and galanthamine are mediated through alpha4- and alpha7-nAChR. We also showed that donepezil and galanthamine protect cortical neurons against acute glutamate treatment-induced neurotoxicity at steps before, and that tacrine protects at steps after, nitric oxide radical formation. On the other hand, the neuroprotective effects of donepezil and galanthamine, but not of tacrine, against neurotoxicity induced by moderate glutamate treatment were mediated through the phosphatidylinositol 3-kinase-Akt pathway. These findings unveiled the hitherto unknown neuroprotective effects of therapeutic AChE inhibitors and provided valuable insights into its neuroprotective mechanisms. They may very likely form the basis for a novel treatment strategy against Alzheimer's disease.