Central and peripheral activity of cholinesterase inhibitors as revealed by yawning and fasciculation in rats.

This study was designed to investigate the central and peripheral activity profile of cholinesterase inhibitors in rats. Intravenous injection of cholinesterase inhibitors caused fasciculation, a fine involuntary muscular movement. This peripheral cholinergic sign was tightly correlated with in vitro anti-acetylcholinesterase activity by cholinesterase inhibitors, suggesting that fasciculation is a valid index of peripheral cholinergic activation. Yawning, used as a marker of central cholinergic activation, was also monitored. E2030 (3-(2-(1-(1,3-dioxolan-2-ylmethyl)-4-piperidyl)ethyl)-2H-3,4-dihydro-1,3-benzoxazin-2,4-dione hydrochloride) elicited yawning at more than 4 mg/kg, while fasciculation was significantly intensified only at a dose of 16 mg/kg. Donepezil and tacrine induced both yawning and fasciculation at doses greater than 4 mg/kg, whereas physostigmine induced both behaviors at a dose of 8 mg/kg and above. Finally, ipidacrine elicited yawning at a dose of 16 mg/kg and fasciculation at doses greater than 8 mg/kg. Thus, all putative centrally acting cholinesterase inhibitors elicited yawning. TAK-147 (3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-benzazepin-8-yl)-1-propanone fumarate) did not significantly elicit yawning at doses under 16 mg/kg, but elicited fasciculation at a dose of more than 4 mg/kg. Distigmine, a peripherally acting cholinesterase inhibitor, evoked fasciculations, but not yawning. When mild to moderate fasciculation was evoked, donepezil and E2030 elicited more than nine yawns over 30 min, while the other cholinesterase inhibitors elicited approximately five yawns at most during this period. These results indicated that E2030 and donepezil exhibited the most marked preferential central cholinergic activity, relative to peripheral activity, among cholinesterase inhibitors tested. Scopolamine, a centrally acting antimuscarinic drug, completely inhibited E2030-induced yawning, while peripherally acting methylscopolamine did not. Haloperidol, a dopamine receptor antagonist, partially blocked E2030-induced yawning, but did not block donepezil-induced yawning. These results suggest that central cholinergic and, in part, dopaminergic mechanisms are involved in E2030-induced yawning.

Donepezil, a centrally acting acetylcholinesterase inhibitor, alleviates learning deficits in hypocholinergic models in rats.

Donepezil is a member of a new class of centrally acting cholinesterase inhibitors which preferentially inhibit acetylcholinesterase rather than butyrylcholinesterase. The effects of donepezil on learning impairments were investigated in some hypocholinergic models in rats. In nucleus basalis magnocellularis (NBM)-lesioned rats, donepezil alleviated deficits in passive avoidance response at a dose of 0.125 mg/kg and higher, while tacrine had only a tendency toward improved performance. Donepezil at 0.5 mg/kg effectively counteracted acquisition impairments in the water maze task induced by lesions of the medial septum; tacrine had no significant effects on impairments in this task. Scopolamine caused an increase of errors in the 8-arm radial maze. Donepezil significantly decreased scopolamine-induced errors in the radial maze at 0.5 mg/kg, whereas tacrine decreased errors at 2 mg/kg. These results suggest that donepezil can clearly minimize learning impairments induced by treatments that cause central cholinergic deficiencies in rats. These findings support the clinical efficacy of donepezil in Alzheimer's disease.

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 Alzheimer's 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.

Comparison of inhibitory activities of donepezil and other cholinesterase inhibitors on acetylcholinesterase and butyrylcholinesterase in vitro.

This study was designed to compare the in vitro inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of donepezil and some other cholinesterase (ChE) inhibitors which have been developed for the treatment of Alzheimer's disease. The carbamate derivatives physostigmine and rivastigmine needed preincubation to exhibit appropriate anti-ChE activity. The maximum ChE inhibition by physostigmine developed within 30-60 min, while the inhibitory effect of rivastigmine on AChE and BuChE activities reached its peak after 48 and 6 h, respectively. The order of inhibitory potency (IC50) towards AChE activity under optimal assay conditions for each ChE inhibitor was: physostigmine (0.67 nM) > rivastigmine (4.3 nM) > donepezil (6.7 nM) > TAK-147 (12 nM) > tacrine (77 nM) > ipidacrine (270 nM). The benzylpiperidine derivatives donepezil and TAK-147 showed high selectivity for AChE over BuChE. The carbamate derivatives showed moderate selectivity, while the 4-aminopyridine derivatives tacrine and ipidacrine showed no selectivity. The inhibitory potency of these ChE inhibitors towards AChE activity may illustrate their potential in vivo activity.

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.

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 Alzheimer's 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.

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.

Thoracoscopic en bloc total esophagectomy with radical mediastinal lymphadenectomy.

OBJECTIVE: Total esophagectomy with en bloc mediastinal lymphadenectomy for cancer carries a substantial morbidity and mortality rate. To investigate the feasibility of thoracoscopic technique, we carried out an extensive laboratory study. Encouraged by our excellent results, we conducted a clinical trial. METHODS: From September 1994 to September 1995, 39 patients thoracic esophageal cancer lesions not invading surrounding organs underwent total esophagectomy with mediastinal lymphadenectomy by means of thoracoscopy. Ages ranged from 47 to 86 years. The procedures were conventional except for the thoracic portion, which was performed as a thoracoscopic procedure with six trocar holes instead of thoracotomy. All harvested lymph nodes were counted for each station. Spirometric data and plethysmographically determined vital capacity were measured before and after operation for all patients. RESULTS: All procedures were accomplished as scheduled, and none was converted to open thoracotomy. The operating time was 200 +/- 41 minutes (mean +/- standard deviation). Estimated blood loss was 270 +/- 157 ml. The harvested lymph nodes numbered 19.7 +/- 11.1 per patient. Seventeen patients (45%) had positive lymph nodes. There were no in-hospital deaths within 30 days. Twenty-two patients did not require postoperative ventilatory support. Vital capacity decreased to 85% +/- 11% of the preoperative values, and forced expiratory volume in 1 second decreased to 82% +/- 16%. CONCLUSIONS: Thoracoscopic mediastinal lymphadenectomy is technically feasible, and its completeness is comparable to that of the open technique. The decline in pulmonary function is significantly less than that seen in our previous experience with the open technique.

[Cytophotometric DNA analysis of the early and superficial esophageal cancer].

Although it is widely known that early esophageal cancer (n-) patients have a favorable prognosis after surgery and the superficial esophageal cancer (n+) patients have a poor one, the differences between them have not been found out under the microscopic examination of the primary lesion. Cytophotometric DNA analysis of the primary lesion was done to search for the differences between early and superficial esophageal cancer. Significant differences were obtained in the mean DNA value (p less than 0.01), the percentage of nuclei beyond tetraploid (p less than 0.001) and the product of 2 indices above (p less than 0.001). The result suggests that cytophotometric DNA analysis is a useful means for differentiating the early esophageal cancer from the superficial esophageal cancer and that the most suitable perioperative combined therapy can be chosen through the Feulgen-stained biopsy specimen.

Postoperative cancer therapy for patients with carcinoma of the thoracic esophagus: an attempt of aggressive chemotherapy combined with proper nutritional support for patients with distant node metastasis.

The prognosis of patients with advanced thoracic esophageal carcinoma is still poor. In general, patients having distant lymph-node metastasis do not survive for more than 3 years. Therefore, a new therapeutic regimen employing aggressive chemotherapy for these patients has been attempted. In this regimen, three kinds of anticancer drugs Pepleomycin, and twice as much as Adriamycin and Mitomycin of the conventional level for a short period of time are used. Eleven cases were treated with this chemotherapy under active enteral and parenteral nutritional support of 45 to 50 kcal/kg daily. Two patients over 70 years of age died of pneumonitis. Four patients with recurrence of cancer in the lungs, liver and cervical nodes died after 10, 12, 13 and 14 months following surgery, respectively. Five patients have been alive for more than 2 years. These results indicate that this therapy is more effective than other conventional therapies. It has been shown that aggressive chemotherapy combined with proper nutritional support is effective for patients with node metastasis.

A new cell line (TE-3) derived from human squamous cell carcinoma of the esophagus.

A new epithelial cell line (TE-3) has been established from a metastatic lesion at the right chest wall which was originated from a well differentiated human squamous cell carcinoma of the esophagus. TE-3 has been subcultured more than 150 times for 3 years and 1 month. Cultured cells have grown as isolated colonies of epithelial cells. The average doubling time of the TE-3 cell line was 48 hr and the plating efficiency was 10 to 40% in MEM supplemented with 8% fetal calf serum. Distinctive marker chromosomes and a male karyotype were present in TE-3. Electron microscopic examination of the TE-3 cells disclosed the presence of desmosomes and microvilli in connection of the cells which were rich in cell organelles. In early passages of culture, the cytoplasm of cells was slightly positive with PAS stain but negative with Sudan III or mucicarmin stain. Heterotransplantation of the culture cells to BALB/c nude mice produced tumors, the histological appearance of which was similar to that of the original one. The carcinoembryonic antigen level of the medium in the confluent culture of TE-3 was 2 ng/10(4) cells.

Correlation between the mixed lymphocyte tumor cell culture reaction (MLTR) and some histological characteristics in esophageal cancer.

The mixed lymphocyte tumor cell culture reaction (MLTR) using autochthonous peripheral and lymph node lymphocytes was performed on 21 patients with esophageal cancer. Ten of 16 cases (62%) in peripheral lymphocytes and 9 of 16 cases (56%) in the lymph node lymphocytes showed positive reaction. There was no correlation between stimulation index (SI) and the degree of lymph node metastasis which is one of the determinants of clinical stage (Guide Lines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus, 1976). Many cases showed positive reaction in MLTR even if their tumors were at advanced stage. SI of MLTR on the patients who died of a relapse in a short time within one year after operation was found to be low levels. There was no remarkable correlation between MLTR and PPD skin reaction. The results of MLTR were compared with histologic differentiation grades of tumor tissues; intraepithelial spread, vascular invasion, sinus histiocytosis of the regional lymph nodes, and follicular hyperplasia of the regional lymph nodes. There was a minor correlation between the results of MLTR and sinus histiocytosis of the regional lymph nodes, and follicular hyperplasia of the regional lymph nodes. There was a minor correlation between the results of MLTR and sinus histioction. The results of MLTR were compared with histologic differentiation grades of tumor tissues; intraepithelial spread, vascular invasion, sinus histiocytosis of the regional lymph nodes, and follicular hyperplasia of the regional lymph nodes. There was a minor correlation between the results of MLTR and sinus histiocytosis of the regional lymph nodes.

Characteristics of two cell lines (TE-1 and TE-2) derived from human squamous cell carcinoma of the esophagus.

Two epithelial cell lines (TE-1 and TE-2) have been established from a well or poorly differentiated human squamous cell carcinoma of the esophagus. TE-1 has been subcultured 120 times during 2 years and 10 months, and TE-2, 50 times for almost 2 years. Cultured cells grew as isolated and piled-up colonies of epithelial cells. The average doubling time of the TE-1 cell line was 60 hr and that of TE-2, 72 hr. Distinctive marker chromosomes and a male karyotype were present in TE-1, but no marker chromosomes were seen in TE-2. Scanning electron microscopic examinations of both TE-1 and TE-2 confirmed the presence of desmosomes and interdigitated microvilli. Transmission electron micrographs of TE-1 showed the presence of abundant cell organelles, and a few organelles were found in the scanty cytoplasm of TE-2. There was a marked difference in the cell organelles between TE-1 and TE-2. Heterotransplantation of the cultured TE-1 and TE-2 cells produced tumors, the histological appearance of which was similar to that of the original ones. The carcinoembryonic antigen level of the medium in the confluent culture of TE-2 was 270 ng/10(6) cells. In the cytoplasm of TE-1 cells the number of paracrystals, which were produced by treatment with vinblastine sulfate, increased by the addition of cholera toxin to the medium.

The screening test of various chemotherapeutic drugs in primary malignant melanoma cells and human malignant melanoma cell line (TM-1).

The sensitivities to various chemotherapeutic drugs of the primary malignant melanoma cell of the esophagus and the malignant melanoma cells established from human skin were studied by determining the incoporation of 3H-thymidine into tumor cells using the tissue culture method. The incorporation of 3H-thymidine was depressed by a low concentration of Actinomycin D in malignant melanoma cells of the esophagus and by a low concentration of Adriamycin or Actinomycin D in the established malignant melanoma cells. When Actinomycin D was clinically used according to the experimental results, the subcutaneously metastasized tumor was remarkably reduced.

On a new continuous cell line (TM-1) derived from malignant melanoma.

A continuous cell line of malignant melanoma has been established. The tumor specimen was obtained from a left axillary lymph node of a malignant melanoma originating in the abdominal skin of a 67-year-old woman. The cell line has been designated TM-1. Explant specimens showed positive dopa staining, but after 2 years and 4 months, or 60 generations of cell passage, the dopa staining is now weakly positive. Treatment with DBcAMP that causes marked morphological changes induces melanin production and lowers the proliferative ability of the cells. These changes were reversible. Transplantation of the cells to nude mice resulted in the formation of large tumors consisting of epithelioid cells and spindle cells. Morphologically, the majority of the cell population in vitro was polygonal epithelioid cells, but the cell passage produced a large number of spindle cells. The shape of the TM-1 cells was examined by scanning electron microscopy. Depending upon the culture conditions, either of these cell types could be predominated, indicating that both cells are of the same origin.