[Discovery and development of donepezil hydrochloride for the treatment of Alzheimer's disease].

The most consistent change of neurotransmitter in the brain of Alzheimer's patients is the dramatic decrease of cholinergic innervation due to the loss of neurons in the basal forebrain. The most widely studied acetylcholinesterase inhibitors (AChEIs) have been physostigmine and tacrine. Physostigmine has very short duration, and tacrine has liability to hepatotoxicity. These are the defects of the inhibitors. Our objective was to find a new type of AChEIs that would overcome the disadvantages of physostigmine and tacrine. Through a random screening, we incidentally found an N-benzylpiperazine derivative which showed positive cholinergic behavior in rats. We replaced the N-benzylpiperazine moiety with N-benzylpiperidine moiety and found a dramatic increase in anti-AChE activity. Even after the replacement of an amide group with a ketone group the activity was held. Furthermore, the cyclic-amide derivative showed enhanced inhibitory activity. On the basis of these results, an indanone derivative was designed. Among these indanone derivatives, donepazil hydrochloride (E2020), brand name ARICEPT was found to be the most balanced compound. The clinical studies of donepezil hydrochloride demonstrated statistically significant effects on ADAS-cog (Alzheimer's Disease Assessment Scale cognitive sub.) and CIBIC Plus (Clinician's Interview-Based Impression of Change plus).

Synthesis and structure-activity relationships of acetylcholinesterase inhibitors: 1-benzyl-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine hydrochloride and related compounds.

Following the discovery of a new series of anti-acetylcholinesterase (anti-AChE) inhibitors such as 1-benzyl-4-[2-(N-benzoylamino)ethyl]piperidine (1), we reported that its rigid analogue, 1-benzyl-4-(2-isoindolin-2-ylethyl)piperidine (5), had more potent activity. We have extended the structure-activity relationship (SAR) study for the rigid analogue and found that the 2-isoindoline moiety in compound 5 can be replaced with a indanone moiety (8) without a major loss in potency. Among the indanone derivatives, 1-benzyl-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine (13e) (E2020) (IC50 = 5.7 nM) was found to be one of the most potent anti-AChE inhibitors. Compound 13e showed a selective affinity 1250 times greater for AChE than for butyrylcholinesterase. In vivo studies demonstrated that 13e has a longer duration of action than physostigmine at a dose of 5 mg/kg (po) and produced a marked and significant increase in acetylcholine content in rat cerebral cortex. We report the synthesis, SAR, and a proposed hypothetical binding site of 13e (E2020).

Effects of repeated doses of compound E for 4 and 9 weeks on the male reproductive organs.

Results of two toxicity studies of Compound E, which is an anticancer drug, on male reproductive organs and fertility by oral repeated dosing at dose levels of 12.5, 25 and 50 mg/kg/day for 4 and 9 weeks in rats were compared. After repeated dosing, the male fertility was studied by mating with untreated female animals. At the dose of 50 mg/kg/day in the 4-week study, Compound E significantly decreased testes weight and number of epididymal sperm, caused histopathological changes in the testis and epididymis characterized by decreased germ cells, but did not affect fertility. The dose of 25 mg/kg/day in the 9-week study caused reduction in epididymal weight and number of epididymal sperm and histopathological changes in the testis. The dose of 50 mg/kg/day in the 9-week study was lethal and caused more prominent toxic effects in the reproductive organs and loss of fertility. The present studies suggest that the order of sensitivity of parameters on male reproductive organs is as follows; histopathological examination = organ weight > number of sperm in epididymis > pregnancy index> copulation index. Further, 4-weeks repeated dosing is of sufficient duration to predict adverse effects of test compounds.

Mutagenicity of benzoquinones for Ames Salmonella tester strains.

The mutagenicity of 12 simple benzoquinone (BQ) derivatives was studied using five different Ames Salmonella mutagenicity tester strains in the presence and absence of S9 mix. Seven of the BQs used displayed mutagenicity with and/or without S9 mix, and most of them produced a marginal increase in revertants. p-Benzoquinone (p-BQ) showed the most potent mutagenic activity (17 induced revertants/nmol/plate for strain TA104 without S9 mix) among the BQs tested. TA104, which is sensitive to oxidative mutagens, was the most sensitive to the mutagenicity of the BQs of the five strains used, while the second most sensitive strain was TA2637, which detects bulky DNA adducts. Significant reductions in the mutagenicity of p-BQ, and 2,3-diCl-5,6-diCN-BQ without S9 mix were observed in the presence of catalase. These findings suggest that the mutagenicity of BQs for S. typhimurium is attributable to oxidative injury after BQ reduction and to DNA adducts that form with BQs that have electrophilic substituents.

Effects of a daily feeding-restriction on the reproductive and development toxicity parameters in female rats.

On the assumption of that the oral administration of an acid-unstable test compound into the empty stomach could enhance the systemic exposure to the test compound, the non-pregnant and pregnant rats had free access to the diet only for five hours per day. Female rats under the restricted feeding for a period of 21 days took diet at two thirds of the daily food-intake by the control animals, and lost their weight more than 10%. The vaginal smear test in these animals revealed a prolonged estrous cycle and diestrous period over four days. On the other hand, the restricted feeding from Day 0 to Day 17 of gestation suppressed the weight gain of pregnant animals but did not cause any significant influence upon the litter data, incidence of external anomalies or fetal skeletal development. The restricted feeding from Day 0 of gestation to Day 7 of lactation seriously disturbed the nursing behavior and the growth of offspring. These results suggested that the dosing method under the above-mentioned restricted feeding might be applicable to the teratology study but could not be applied to the fertility study nor to the peri- and post-natal study.

Effects of dietary restriction on induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) in rat liver.

The effects of dietary restriction on the induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in the hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl4. The animals were given food ad libitum, a restricted amount of food (4 g/rat/overnight) or no food. Hepatocytes were isolated 2 h after oral administration of DMN at a dose of 5 mg/kg body weight and 48 h after oral administration of CCl4 at a dose of 400 mg/kg body weight, and incubated for 4 h in Williams' medium E supplemented with either [3H]thymidine for UDS or 5-bromodeoxyuridine for RDS. UDS was determined by autoradiography and RDS was determined by the immunoenzymatic staining method. The background levels of UDS (net grains/nucleus) and RDS (cells in S phase) in control were -12.4 and 0.64% for ad libitum feeding, -6.8 and 0.04% for restricted feeding, and -8.1 and 0% for fasting. UDS induced by DMN and RDS induced by CCl4 were 19.4 and 3.3% for ad libitum feeding, 34.5 and 10.4% for restricted feeding, and 47.8 and 15.1% for fasting. DMN demethylase activity in rat liver was also found to increase with dietary restriction. These results indicate that dietary restriction modulates the responses of UDS and RDS in the liver of rats.

Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains.

The molecular mechanisms involved in quinone cytotoxicity, especially mutagenicity, are still largely unknown. In order to better understand the molecular aspects of the mechanisms of quinone mutagenicity and cytotoxicity, we examined them by using a series of 13 simple structural naphthoquinone (NQ) derivatives for 9 Ames Salmonella mutagenicity tester strains in the presence of absence of liver homogenate S9 mix from rats induced with phenobarbital and 5,6-benzoflavone. Most NQs used in this study showed mutagenicity with and/or without S9 mix. The most potent mutagenic NQ was 2,3-dichloro-1,4-NQ, with mutagenicity of 18 induced revertants/nmol/plate for strain TA104 without S9 mix. Among the strains used, TA104, which is sensitive to oxidative mutagens, was the most sensitive to the NQs, and the second most sensitive strain was TA2637, which detects bulky DNA adducts. The relationship of mutagenic potency to the one-electron reduction potential with TA104 suggested that the higher redox potential NQs were more mutagenic than the lower redox potential NQs. Significant reduction of the mutagenicity of 1,4-naphthoquinone without S9 mix was observed in the presence of catalase. Enhancement of the mutagenic potential of the NQs by the pKM101 plasmid implicated in error-prone repair was also observed. The most cytotoxic NQ was 2,3-dichloro-5,8-dihydroxy-1,4-NQ, and the least cytotoxic NQ was beta-NQ-4-sulfonic acid potassium salt, a 700-fold range in potency. The cytotoxic effect of the NQs was largely dependent on the structures of their substituents.(ABSTRACT TRUNCATED AT 250 WORDS)

Cumulative effects of chromosome aberrations and sister chromatid exchanges in rat liver induced in vivo by heterocyclic amines.

Cumulative effects of chromosome aberrations and sister chromatid exchanges (SCEs) were studied in hepatocytes of F344 rats exposed in vivo to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) at doses of 12.5, 25 or 50 mg/kg body wt/day or 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ) at doses of 12.5, 25 or 50 mg/kg body wt/day. Hepatocytes were isolated 24 h after 1, 7, 14 or 28 repeated doses (once a day) by gastric intubation and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Multiple treatment with IQ or nitro-IQ induced significant chromosome aberrations time- and dose-dependently, the maximum frequency of chromosome aberrations in metaphase cells being 39 and 33% respectively, while that in controls was 1.1%. Single treatment with IQ or nitro-IQ induced significant SCEs dose-dependently, the maximum frequency being 0.83 and 0.79 per chromosome respectively, while the control value was 0.51. Multiple treatment with nitro-IQ induced significant SCEs to a plateau level of 0.90 per chromosome. Cytogenetic damage in the liver by IQ was greater than that by nitro-IQ. These results show that this assay of chromosome aberrations and SCEs in rat liver in vivo without partial hepatectomy or mitogen treatment in vivo is a sensitive method for evaluating the cumulative tumor-initiating activities of carcinogenic heterocyclic amines at low doses and should be useful for the detection of unknown hepatocarcinogens.

Dimethyl sulfoxide (DMSO) is mutagenic for bacterial mutagenicity tester strains.

We used the Ames method with the modification of pre-incubation to evaluate the potential mutagenicity of DMSO. We performed the assays using nine different Ames Salmonella strains and Escherichia coli strains WP2 and WP2uvrA. DMSO was found to be mutagenic for Salmonella typhimurium TA1537 and TA2637 (the latter strain being isogenic to TA1537 but carrying plasmid pKM101) and for E. coli WP2uvrA. The mutagenic activity of DMSO observed at a concentration of 33% was about 10 times higher than the background level (65 revertants induced) for TA1537 after 20 min of incubation, where some lethal toxicity was observed. The mutagenicity of DMSO was observed in the presence and absence of rat liver S9 mix.

Synthesis and structure-activity relationships of acetylcholinesterase inhibitors: 1-benzyl-4-(2-phthalimidoethyl)piperidine and related derivatives.

Following the discovery of a new series of 1-benzyl-4-[2-(N-benzoyl-N-methylamino)ethyl]piperidine (2) derivatives with a potent anti-acetylcholinesterase (anti-AChE) activity, we extended the structure-activity relationships (SAR) to rigid analogues (4) and 1-benzyl-4-[2-(N-benzoyl-N-phenylamino)ethyl]piperidine derivatives (3). Introduction of a phenyl group on the nitrogen atom of the amide moieties resulted in enhanced activity. The rigid analogue containing isoindolone (9) was found to exhibit potent anti-AChE activity comparable to that of 2. Furthermore, replacement of the isoindolone with other heterobicyclic ring systems was examined. Among the compounds prepared in these series, 1-benzyl-4-[2-[4-(benzoylamino)phthalimido]ethyl]piperidine hydrochloride (19) (IC50 = 1.2 nM) is one of the most potent inhibitors of AChE. Compound 19 showed a definite selectivity to AChE over the BuChE (about 34700-fold) and, at dosages of 10-50 mg/kg, exerted a dose-dependent inhibitory effect on AChE in rat brain.

A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions.

A new mutant strain of inbred Sprague Dawley rats with autosomal recessive hyperbilirubinuria, were studied by biochemical, histologic, and ultrastructural methods. The plasma bilirubin concentration in the homozygote was significantly higher than that of the heterozygote, and about 80% of the bilirubin was conjugated. Plasma BSP and ICG clearance were both severely delayed in the homozygote. Plasma BSP elimination kinetics suggested that the pathophysiologic defect was not hepatic uptake or storage but rather in secretion into bile. Histopathology of the liver demonstrated brown pigment in the hepatocytes that appeared to be lipofuscin. The electron microscopic features of the hepatic pigment resembled those of the Dubin-Johnson syndrome. Homozygote histopathology also revealed glomerular lesions with mesangial expansion and proliferation in the kidneys. Immunohistologic studies disclosed mesangial granular deposition of IgG, IgA, and to a lesser degree, IgM and C3. These renal changes resembled those of IgA nephropathy. The spontaneous hyperbilirubinuric rat (EHBR) may be a useful animal model for studying constitutive conjugated hyperbilirubinemia, bilirubin metabolism, cholestasis, and glomerulonephropathy subsequent to hepatic dysfunction.

Ganglioside-induced inhibition of in vivo immune response in mice.

In vitro immunomodulatory properties of gangliosides have been well characterized such as the ganglioside-induced modulation of CD4 on T lymphocytes and inhibition of lectin-induced proliferative response of lymphocytes. These findings have led to an interesting suggestion that gangliosides play a role as in vivo immune modulators, although this possibility is not clearly defined yet. We then first confirmed in vitro effects of gangliosides on murine immunocytes and examined in vivo effects of gangliosides on immune response in mice. Murine spleen cells that were treated with a ganglioside mixture (GS) purified from bovine brain exhibited a marked decrease in CD4 expression, while CD8 expression was slightly suppressed. Transplantation of GS-untreated control immunocytes that were isolated from syngeneic mice into the immune suppressed mice by X-ray irradiation restored in vivo immune responses, while GS-treated cells could not. Immune response was assayed by the evaluation of footpad swelling which was induced by immunization with sheep erythrocytes as antigens. Moreover, intramuscular administration of gangliosides into mice suppressed both immediate (Arthus)-type and delayed-type allergic reactions. These results suggest that gangliosides would be potential in vivo immune modulators.

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines.

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.

Physical dependence liability of dynorphin A analogs in rodents.

To assess the physical dependence liability of dynorphin A analogs, mice were given repeated injections of various dynorphin A analogs twice daily for 5 days, and rats were given repeated administration of [N-methyl-Tyr1,N-methyl-Arg7,D-Leu8]dynorphin-A-(1-8) ethylamide (E-2078) twice daily for up to 7 weeks. Mice that had received repeated [D-Cys2,Cys5,N-methyl-Arg7,D-Leu8]dynorphin-A-(1-9) amide displayed jumping behavior after subcutaneous injection of naloxone, an opioid receptor antagonist. In contrast, the animals that had received repeated E-2078 or [N-methyl-Tyr1,Phe4(p-NO2),N-methyl-Arg7,D-Leu8]dynorphin-A-(1-8) ethylamide displayed very few jumps after naloxone administration. Rats that had received repeated E-2078 administration did not display withdrawal signs, such as weight loss, after either abrupt withdrawal or naloxone administration. These results indicate that E-2078 and [N-methyl-Tyr1,Phe4(p-NO2),N-methyl-Arg7,D-Leu8]dynorphin-A-(1-8) ethylamide may have little dependence liability and that [D-Cys2,Cys5,N-methyl-Arg7,D-Leu8]dynorphin-A-(1-9) amide can cause physical dependence.

Spinal kappa receptor-mediated analgesia of E-2078, a systemically active dynorphin analog, in mice.

E-2078 ([N-methyl-Tyr1, N-methyl-Arg7, D-Leu8]dynorphin A (1-8) ethylamide) is a systematically active dynorphin analog. We examined the sites of action of analgesia induced by systemic application of E-2078 compared with morphine in mice. When administered either intracerebroventricularly or intrathecally, E-2078 produced maximal dose-dependent analgesia in the tail-pinch, tail-flick and formalin tests. Its peak effect was observed 15 min after both injections, contrasted with a slow peak (120 min) by subcutaneous injection. The intrathecal site was relatively more sensitive than the intracerebroventricular site and many times more sensitive than the subcutaneous route. In contrast, morphine was equipotent when given intracerebroventricularly and intrathecally. When E-2078 was administered subcutaneously and naloxone or nor-binaltorphine were given either intracerebroventricularly or intrathecally, the analgesic action of E-2078 was most potently and totally reversed by intrathecal injection of nor-binaltorphimine. Intracerebroventricular and intrathecal injections of naloxone were equally effective for antagonism of morphine-analgesia. These data indicate that systemically administered E-2078 produces analgesia via central actions, in which the activation of the spinal kappa receptors is most important.

Protective effect of the novel anti-ischemic agent 2-(4-(p-fluorobenzoyl)-piperidin-1-yl)-2'-acetonaphthone hydrochloride against ischemia-induced impairment of the passive avoidance response in gerbils.

E2001 (2-(4-(p-fluorobenzoyl)-piperidin-1-yl)-2'-acetonaphthone hydrochloride, CAS 107025-80-9) is a novel anti-ischemic agent, which is reported to protect against delayed neuronal death in the CA1 subfield of the hippocampus. The effect of E2001 on ischemia-induced impairment of the passive avoidance response in gerbils was studied. The passive avoidance response was not disturbed by transient cerebral ischemia of 3 min duration, but was impaired by 5-min ischemia. E2001 at oral doses of 3 and 10 mg/kg significantly improved the impaired passive avoidance response induced by 5-min cerebral ischemia. It is speculated that the improvement by E2001 may be partly due to the inhibition of extracellular glutamate accumulation, and the suppression of lipid peroxides formation during cerebral ischemia.

Synthesis and biological activity of [MeTyr1,MeArg7,D-Leu8]-dynorphin A(1-9)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-dynorphin A(1-9)-NH2.

The opioid activities of [MeTyr1]-Dyn(1-7)-NH2, [MeTyr1,D-Leu8]-Dyn(1-8)-NH2, [MeTyr1,D-Leu8]-Dyn(1-9)-NH2, [MeTyr1,D-Leu8]-Dyn(1-10)-NH2, [MeTyr1,D-Leu8]-Dyn(1-11)-NH2, and [MeTyr1,D-Leu8,12]-Dyn(1-13)-NH2 were examined in the bioassays (guinea pig ileum, mouse vas deferens and rabbit vas deferens). Because [MeTyr1,D-Leu8]-Dyn(1-9)-NH2 showed the most potent opioid activity of the peptides tested, the biological activities of two kinds of Dyn(1-9) analogues, [MeTyr1,MeArg7,D-Leu8]-Dyn(1-9)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-Dyn(1-9)-NH2 were determined and compared with those of [MeTyr1,MeArg7,D-Leu8]-Dyn(1-8)-NHEt and [D-Cys2-Cys5,MeArg7,D-Leu8]-Dyn(1-8)-NHEt in the three bioassays, in the receptor binding assays, and in the mouse tail pinch test after subcutaneous administration. The results suggest that the extension of the C-terminal in the peptide chain of [MeArg7,D-Leu8]-Dyn(1-8)-NH2 analogues by Arg is ineffective for increasing the kappa-opioid activities, kappa-receptor selectivity and/or analgesic effects of the peptides.

Analgesia produced by E-2078, a systemically active dynorphin analog, in mice.

E-2078 is a very stable dynorphin analog that has the same affinity and selectivity for opioid receptors as dynorphin-A by in vitro bioassay. In the present study, we have characterized the receptor selectivity of E-2078 in mu-, delta- and kappa-representative binding assays, and have evaluated the analgesic effect of systemically administered E-2078 by the tail pinch, tail flick and formalin tests in mice. E-2078 possessed a higher affinity for kappa-receptors than for mu- or delta-receptors in the receptor-binding assay. Dose-related, long-lasting analgesia was produced by s.c. injection of E-2078, and its peak effect was observed 2 hr after s.c. administration in all the analgesic tests. This analgesic effect was produced at doses that did not affect rotarod latency. Post-treatment with naloxone dose-dependently reversed the analgesic effects of both E-2078 and morphine, but E-2078-induced analgesia was relatively resistant to naloxone antagonism. Pre-treatment with the kappa-antagonist, nor-binaltorphimine, antagonized the analgesic effect of E-2078 but had little effect on the analgesic action of morphine. These data indicate that E-2078 is a systemically active analgesic and suggest that the activation of kappa-opioid receptors contributes to analgesia.