Yoshiharu Yamanishi

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 Alzheimers 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.

E2020 - THE PHARMACOLOGY OF A PIPERIDINE CHOLINESTERASE INHIBITOR

Alzheimer’s disease is associated with a relative decrease in the activity of the cholinergic system in the cerebral cortex and other areas of the brain (Bowen, 1981). This discovery has led to the development of agents designed to increase cholinergic function in the CNS. One such group of agents, the cholinesterase (ChE) inhibitors, increase the concentration of acetylcholine (ACh) by inhibiting one or more of the enzymes which hydrolyze it, i.e, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

Inhibitory effect of orally administered donepezil hydrochloride (E2020), a novel treatment for Alzheimer's disease, on cholinesterase activity in rats.

Donepezil hydrochloride ((+/-)-2-[(1-benzylpiperidin-4-yl)methyl]-5, 6-dimethoxy-indan-1-one monohydrochloride: E2020: donepezil) is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimers disease. The present experiments were designed to compare the inhibitory effects of orally administered donepezil and other cholinesterase inhibitors, tacrine (9-amino-1,2, 3,4-tetrahydroacridine hydrochloride), (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713, rivastigmine) and 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4, 5-tetrahydro-1H-1-benzazepin-8-yl)-1-propanone fumarate (TAK-147), on the cholinesterase activity in the brain and plasma of rats. Moreover, in order to validate the cholinesterase inhibition data, we measured the brain and plasma concentrations of these drugs. Oral administration of donepezil, tacrine, ENA-713 or TAK-147, caused a dose-dependent inhibition of brain and plasma cholinesterase activities. The ID(50) values of these compounds for brain cholinesterase activity were 6.3, 40.5, 7.2 and 26.8 micromol/kg, respectively. On the other hand, the ID(50)170, 9.7 and 51.2 micromol/kg, respectively. Thus, the ratios of the ID(50)4.2, 1.3 and 1.9, respectively. Brain and plasma concentrations of donepezil, tacrine and TAK-147 increased dose-dependently. The ratios of the concentrations (brain/plasma) of these compounds were 6.1-8.4 for donepezil, 14.5-54.6 for tacrine and 7.0-20.6 for TAK-147. The values of 50% inhibitory concentration of these drugs in the brain were 0.42, 3.5 and 1.1 nmol/g, respectively. In contrast, the brain and plasma concentrations of ENA-713 at all doses, except the two highest doses, were below the quantification limit. These results suggest that orally administered donepezil satisfactorily penetrates into the brain and inhibits cholinesterase there, and that donepezil is a potent and selective inhibitor of brain cholinesterase in comparison with plasma cholinesterase in vivo.

Inhibitory effects of donepezil hydrochloride (E2020) on cholinesterase activity in brain and peripheral tissues of young and aged rats

Donepezil hydrochloride (donepezil: E2020: (+/-)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride)) is a centrally acting acetylcholinesterase inhibitor developed for the treatment of Alzheimers disease. In the present study, its inhibitory effect on the activity of cholinesterase ex vivo was evaluated in the brain, plasma, erythrocytes, heart, small intestine, liver and pectoral muscle of young adult as well as aged rats, in comparison with that of tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride). In aged animals, cholinesterase activity in heart, small intestine and pectoral muscle was lower, whereas that in plasma and liver was higher than in young rats. Both groups showed the highest levels in the brain. Donepezil, at doses of 1.25, 2.5 and 5 mg/kg, p.o., inhibited brain, plasma, erythrocyte, liver and pectoral muscle cholinesterase activity in young rats in a dose-dependent manner but had less effect on cholinesterase activity in heart and small intestine. In aged animals, inhibition of cholinesterase activity in the brain, erythrocytes and pectoral muscle by donepezil was more potent than that in young animals. Tacrine, at doses of 5, 10 and 20 mg/kg, p.o., dose-dependently inhibited cholinesterase activity in all tissues of both young and aged animals, but most potently in heart, small intestine and liver. The inhibition of cholinesterase activity by tacrine in the brain, plasma, erythrocytes, heart and liver was more potent in aged rats than in tissues of young rats. Brain and plasma concentrations of unchanged donepezil and tacrine were measured in the same animals as used for the cholinesterase inhibition study. Brain and plasma concentrations of donepezil and tacrine were higher in aged than in young animals. It is concluded that the inhibitory effects of donepezil and tacrine on cholinesterase activity are greater in aged than in young rats, owing to differences in the tissue concentrations of these compounds between young and aged animals. It is also suggested that the effect of donepezil on cholinesterase activity is more tissue-selective than that of tacrine.

Differential Effects of Donepezil on Methamphetamine and Cocaine Dependencies

Abstract:  Donepezil, a choline esterase inhibitor, has been widely used as a medicine for Alzheimers disease. Recently, a study showed that donepezil inhibited addictive behaviors induced by cocaine, including cocaine‐conditioned place preference (CPP) and locomotor sensitization to cocaine. In the present study, we investigated the effects of donepezil on methamphetamine (METH)‐induced behavioral changes in mice. In counterbalanced CPP tests, the intraperitoneal (i.p.) administration of 3 mg/kg donepezil prior to 2 mg/kg METH i.p. failed to inhibit METH CPP, whereas pretreatment with 3 mg/kg donepezil abolished the CPP for cocaine (10 mg/kg, i.p.). Similarly, in locomotor sensitization experiments, i.p. administration of 1 mg/kg donepezil prior to 2 mg/kg METH i.p. failed to inhibit locomotor sensitivity to METH, whereas pretreatment with 1 mg/kg donepezil significantly inhibited locomotor sensitivity to cocaine (10 mg/kg, i.p.). These results suggest that donepezil may be a useful tool for treating cocaine dependence but not for treating METH dependence. The differences in the donepezil effects on addictive behaviors induced by METH and cocaine might be due to differences in the involvement of acetylcholine in the mechanisms of METH and cocaine dependencies.

Reduction in excessive muscle tone by selective depletion of serotonin in intercollicularly decerebrated rats.

Intercollicular decerebration in animals induces sustained facilitation of muscle tone of the limbs and this animal model has been used to assess centrally acting muscle relaxants. We have examined the involvement of central and spinal cord serotonergic pathways in the onset of excessive muscle tone in an intercollicularly decerebrated rat. Descending serotonergic pathways are known to modulate, directly or indirectly, the excitability of spinal cord motoneurons and it is inferred that serotonin (5-HT) plays an important role in locomotion. Alteration of muscle tone has been investigated in 5-HT-depleted rats with a neurotoxin, 5, 7-dihydroxytryptamine (5,7-DHT) after pretreatment with desipramine. Intracerebroventricular (i.c.v.) administration of 5,7-DHT reduced 5-HT content in the forebrain to 50.5% and that in the spinal cord to 10.5%, while intrathecal (i.t.) administration of 5,7-DHT decreased 5-HT content in the spinal cord to 8.9% without causing any change in the forebrain. In contrast, noradrenaline or dopamine content was not affected by the neurotoxin in both tissues. These treatments significantly attenuated the muscle tone in the animal models. Moreover, the measurement of 5-HT and 5-hydroxyindoleacetic acid content in intact rats after decerebration showed that facilitation of the 5-HT turnover in the spinal cord, but not in the forebrain, was enhanced compared with sham-operated rats. These findings suggest that the descending serotonergic pathways are essential to induce excessive muscle tone in the intercollicular decerebrated rats and that 5-HT antagonists might be candidates for centrally acting muscle relaxants.

Peptide N- and P/Q-type Ca2 blockers inhibit stimulant-induced hyperactivity in mice

omega-Conotoxin GVIA and omega-agatoxin IVA are specific peptide blockers of N- and P/Q-type calcium channel, respectively. Effects of their intracerebroventricular injection (1-3 pmol/mouse) on psychostimulant-induced hyperactivity were investigated in mice. omega-Conotoxin GVIA antagonized methylphenidate-, methamphetamine- and phencyclidine-induced hyperactivity in a dose-dependent manner. omega-Agatoxin IVA blocked methylphenidate-induced but not methamphetamine- or phencyclidine-induced hyperactivity. Neither peptides showed any effect on apomorphine-induced hyperactivity or spontaneous activity, suggesting that the inhibitory effects on psychostimulant-induced hyperactivity are not due to dopamine receptor blockage or nonspecific behavioral depression. Antagonism of calcium channels, particularly N-type, may ameliorate activation of the dopaminergic system induced by increased dopamine release.

Quantitative reverse transcription-PCR-HPLC for nerve growth factor mRNA using a deletion RNA as an internal standard.

We have developed a convenient method for the routine measurement of the absolute amount of nerve growth factor (NGF) mRNA in tissue samples. The method consists of RNA extraction, amplification by reverse transcription-PCR and detection by high-performance liquid chromatography. The addition of a deletion mutant RNA to tissue samples as an internal standard enabled correction for RNA recovery during extraction, and the target mRNA and the internal standard were both amplified with the same PCR primers. The conditions were optimized so that the procedure was conducted in the region where the calibration curve was linear, thereby allowing high reproducibility and reliability. The method was applied to the measurement of NGF mRNA in tissues such as skin and skeletal muscle, where the levels are too low to be easily detected by Northern blotting analysis: skin, 14.1 +/- 4.6 fg/mg tissue and skeletal muscle, 11.0 +/- 2.2 fg/mg tissue (mean +/- SD, n = 10). The coefficient of variation of this method was less than 2.8%. This approach should also be applicable to the routine assay of the absolute amount of other mRNAs present at low levels in tissues.

Effect of a platelet-activating factor antagonist, E5880, on cerebrovasospasm following subarachnoid hemorrhage in a canine double-hemorrhage model

We investigated the effects of a platelet-activating factor (PAF) antagonist, E5880 (1-ethyl-2-[N-(2-methoxy)benzoyl-N-[(2)-2-methoxy-3-(4-octadecycarbamoylox)piperidinocarbonyloxy-propyloxy]carbonyl]aminomethyl-pyridiniumchloride), on subarachnoid hemorrhage-induced prolongation of cerebral circulation time and decrease in the basilar artery diameter in a canine double-hemorrhage model. Animals were assigned to three groups, control (saline), E5880 1.2 mg/kg and E5880 2.4 mg/kg. For measurement of cerebral circulation time, regions of interest were chosen at the basilar artery and the straight sinus in order to obtain time-density curves. Cerebral circulation time was defined as the difference between the arterial and venous peaks. Cerebral circulation time and basilar artery diameter were assessed by intra-arterial digital subtraction angiography (IA-DSA) on Days 0, 2 and 7. The prolongation of cerebral circulation time following subarachnoid hemorrhage was significantly inhibited by intravenous administration of 2.4 mg/kg of E5880. Basilar artery constriction was also reduced by E5880. Thus, E5880 had preventive effects on the prolongation of cerebral circulation time and the vasoconstriction of basilar artery in this model. These results suggest that E5880 may have a preventive effect on neurological symptoms aggravated by cerebrovascular lesions following subarachnoid hemorrhage.

Basic research for Alzheimer's disease drugs.

アルツハイマー病（AD）はかつてnon-treatableと言われてきた．しかし近年，コリン仮説に基づいた創薬研究からコリンエステラーゼ阻害薬が開発され，薬物治療の対象となった．現在ではタクリン，ドネペジル，リバスチグミン，ガランタミンが上市されている．さらにNMDA受容体拮抗薬のメマンチンが承認されている．一方，ADの根本治療の研究開発も進められており，ADの原因および発症に密接に関与しているとされるアミロイド・βタンパク（Aβ）に関する研究から，Aβの産生・代謝に関与する酵素阻害薬および免疫療法などが開発されてきている．将来これらの研究成果が実を結び実際の治療に寄与していくことを期待したい．