Tadashi Shiotani

Improvement by nefiracetam of β‐amyloid‐(1‐42)‐induced learning and memory impairments in rats

We have previously demonstrated that continuous i.c.v. infusion of amyloid β‐peptide (Aβ), the major constituent of senile plaques in the brains of patients with Alzheimers disease, results in learning and memory deficits in rats. In the present study, we investigated the effects of nefiracetam [N‐(2,6‐dimethylphenyl)‐2‐(2‐oxo‐1‐pyrrolidinyl) acetamide, DM‐9384] on Aβ‐(1–42)‐induced learning and memory deficits in rats. In the Aβ‐(1–42)‐infused rats, spontaneous alternation behaviour in a Y‐maze task, spatial reference and working memory in a water maze task, and retention of passive avoidance learning were significantly impaired as compared with Aβ‐(40–1)‐infused control rats. Nefiracetam, at a dose range of 1–10 mg kg−1, improved learning and memory deficits in the Aβ‐(1–42)‐infused rats when it was administered p.o. 1 h before the behavioural tests. Nefiracetam at a dose of 3 mg kg−1 p.o. increased the activity of choline acetyltransferase in the hippocampus of Aβ‐(1–42)‐infused rats. Nefiracetam increased dopamine turnover in the cerebral cortex and striatum of Aβ‐(1–42)‐infused rats, but failed to affect the noradrenaline, serotonin and 5‐hydroxyindoleacetic acid content. These results suggest that nefiracetam may be useful for the treatment of patients with Alzheimers disease.

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors.

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimers disease.

Involvement of the cholinergic system in the effects of nefiracetam (DM-9384) on carbon monoxide (CO)-induced acute and delayed amnesia

The effects of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)-acetamide (DM-9384, nefiracetam), a cyclic derivative of GABA, were investigated in the carbon monoxide (CO)-induced amnesia model in mice using the passive avoidance task. Memory deficiency occurred when mice were exposed to CO before memory was completely consolidated after training (acute amnesia), at 7 days before training and 7 days after training (delayed amnesia). DM-9384 prolonged the step-down latency in mice with CO-induced amnesia. Scopolamine blocked the anti-amnesic effect of DM-9384 on delayed amnesia that had been induced by pre- or post-training exposure to CO. Bicuculline had a tendency to antagonize the anti-amnesic effect of DM-9384, but this tendency was not significant. Under these conditions, no significant change in the activity of choline acetyltransferase and glutamic acid decarboxylase was observed in the frontal cortex, striatum and hippocampus. These results suggest that DM-9384 potentiates cholinergic neuronal function and that it may modify acquisition and/or consolidation of memory.

Effects of nefiracetam on drug-induced impairment of latent learning in mice in a water finding task

We investigated the effects of nefiracetam (DM-9384), a pyrrolidone derivative, on chlordiazepoxide-, apomorphine-, and methamphetamine-induced impairment of latent learning in a water finding test in mice. Pretreatment with nefiracetam reversed the inhibitory effects of chlordiazepoxide and apomorphine, but not those of methamphetamine, on latent learning. The ameliorative effects of nefiracetam on apomorphine-induced, but not chlordiazepoxide-induced impairment of latent learning were antagonized by scopolamine. These results provide further evidence that nefiracetam has anti-amnesic effects. Further, it is suggested that the cholinergic neuronal system may be involved in the ameliorative effects exerted by nefiracetam on apomorphine-induced impairment of latent learning.

Attenuation of the development of morphine dependence/tolerance by nefiracetam: involvement of adenosine 3′:5′-cyclic monophosphate system

Biochemical changes such as intracellular cAMP and Ca(2+) underlying morphine dependence and tolerance have been suggested. Therefore, we investigated the effects of nefiracetam (N-(2, 6-dimethyl-phenyl)-2(2-oxo-1-pyrrolidinyl) acetamide), which increases intracellular cAMP and Ca(2+) levels, on the development of morphine dependence and tolerance. Mice administered morphine (6 or 20 mg kg(-1)) twice daily for 5 days, showed withdrawal symptoms (jumping, diarrhea and body weight loss) after naloxone challenge (5 mg kg(-1)), indicating morphine dependence. Furthermore, tolerance to the analgesic effect of morphine was observed in these mice. Co-administration of nefiracetam (5 or 10 mg kg(-1)) with morphine during the pretreatment period, significantly reduced the signs of withdrawal symptoms, moreover, the tolerance was significantly attenuated. Elevation of cAMP levels in the cortex was observed in morphine-dependent mice, but not in mice co-administered nefiracetam. Acute administration of nefiracetam shows no effect on the withdrawal symptoms and the analgesic effect in morphine-naive mice. Theophylline (3 or 10 mg kg(-1)) tended to attenuate and enprofylline (10 or 30 mg kg(-1)) significantly attenuated the development of morphine dependence and tolerance. These findings suggest that co-administration of nefiracetam or compounds, which increase the cAMP level, may be a useful strategy for attenuating the development of morphine dependence and tolerance in the clinic.

Effects of nefiracetam on amnesia animal models with neuronal dysfunctions

The effects of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide (nefiracetam; DM-9384), on learning and memory in several amnesia animal models with neuronal dysfunctions were investigated. Nefiracetam improved scopolamine-, bicuculline-, picrotoxin-, ethanol-, chlordiazepoxide- and cycloheximide-induced amnesia. Anti-amnesic action of nefiracetam on scopolamine model was antagonized by nifedipine and flunarizine, but not by diltiazem. Repeated administration of nefiracetam to AF64A-treated animals improved impairment of learning and memory as well as the alterations in cholinergic and monoaminergic neurotransmitters in the hippocampus. Basal forebrain (BF) lesioned rats induced by excitotoxin or by thermal coagulation showed impairment of learning accompanied by a marked reduction in choline acetyltransferase (ChAT) and acetylcholine esterase activities. Nefiracetam improved the learning deficit of the BF-lesioned rats. Nefiracetam also improved the carbon monoxide-induced delayed and acute amnesia. Nefiracetam stimulated acetylcholine release in the frontal cortex. Repeated administration of nefiracetam increased ChAT activity, gamma-aminobutyric acid (GABA) turnover and glutamic acid decarboxylase activity, and facilitated the Na(+)-dependent high-affinity GABA uptake. Nefiracetam activated the high voltage-activated (N/L-type) Ca2+ channel. The dose-response curves of nefiracetam were bell-shaped in both behavioral and biochemical studies. Therefore, it is suggested that nefiracetam improves the dysfunction of cholinergic, GABAergic and/or monoaminergic neuronal function by acting at Ca2+ channel and enhancing the release of neurotransmitters, and modifies impairment of memory processes induced by drugs and hypoxia.

Presynaptic nicotinic acetylcholine receptors as a functional target of nefiracetam in inducing a long-lasting facilitation of hippocampal neurotransmission.

Nefiracetam (1–10 &mgr;M), a nootropic (or cognition-enhancing) agent, persistently potentiated currents through Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes as a result of interacting with a protein kinase C pathway and the ensuing protein kinase C phosphorylation of the receptors. A similar effect was found in neuronal nicotinic ACh receptors (&agr;4&bgr;2 and &agr;7). In contrast, the other nootropic agents such as piracetam and aniracetam had no potentiating action on the receptors. A sustained enhancement in the activity of nicotinic ACh receptors induced by nefiracetam caused a marked increase in the glutamate release, leading to a long-term potentiation-like facilitation of hippocampal synaptic transmissions. One of the consistent neuropathologic features of the Alzheimer brain is a loss of nicotinic ACh receptors. This fact, together with the results of our study, raises the possibility that the loss of nicotinic ACh receptors may be a key factor in the decline of cognitive function observed in Alzheimer disease and that agents targeting neuronal nicotinic ACh receptors like nefiracetam could, therefore, be of great therapeutic importance.

Cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384)

We have studied cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384), a newly developed cognitive enhancer, by biochemical experiments on cholinergic and GABAergic transmissions as well as electrophysiological experiments on neuronal Ca2+ channels. In behavioral experiments in rats, nefiracetam (3 mg/kg) ameliorated amnesia induced by basal forebrain (BF) lesion or treatment of scopolamine. Biochemical experiments revealed that nefiracetam increased uptake and release of transmitters in both cholinergic and GABAergic systems in rat brain. In electrophysiological studies, nefiracetam (1 microM) increased long-lasting (N/L-type) Ca2+ channel currents in NG108-15 cells. The nefiracetam action on Ca2+ channels was blocked by pertussis toxin (PTX). The results suggest that nefiracetam improves impaired memory by facilitating cholinergic and GABAergic transmissions in the brain. It is further suggested that PTX-sensitive G-proteins and Ca2+ channels associated with these G-proteins are responsible for the action of nefiracetam on neurotransmission.

Peripheral-type benzodiazepine receptors in association with epileptic seizures in EL mice

Peripheral-type benzodiazepine receptors (PBR) in the brain were studied in association with epileptic seizures using EL mice, an animal model of epilepsy, and DDY mice as controls. Ro 5-4864 (i.p.), a specific agonist for PBR, elicited tonic-clonic convulsions in EL mice 2.6-times more potently than in DDY mice with CD50s of 11.9 and 31.2 mg/kg for EL and DDY mice, respectively. In contrast, pentylenetetrazole (i.p.) exerted convulsant actions on EL and DDY mice in a less differential way with CD50s of 29.2 and 48.1 mg/kg for EL and DDY mice, respectively. PK 11195 (i.v.), a specific antagonist for PBR, raised seizure thresholds of EL mice at a dose of 2 mg/kg. Binding assay revealed a 50% higher density of [3H]Ro 5-4864 binding sites in the mitochondrial fraction isolated from the cerebrum of EL mice in comparison with DDY mice. Similarly, a 40% higher density of [3H]flunitrazepam binding was observed in the mitochondrial fraction of EL mice. The results support the hypothesis that PBR, particularly those associated with mitochondria, are involved in the pathogenesis of epileptic seizures in EL mice.

Anticonvulsant actions of nefiracetam on epileptic EL mice and their relation to peripheral-type benzodiazepine receptors

Anticonvulsant actions of the nootropic drug nefiracetam were studied using EL mice, an animal model of epilepsy, in which peripheral-type benzodiazepine receptors (PBRs) might be involved in their epileptogenesis. Nefiracetam, when administered orally t o EL mice, inhibited convulsions induced by the PBR agonist, Ro 5-4864, with an ED(50) of 17.2 mg/kg, whereas it did not inhibit the drug-induced convulsions in control DDY mice. When administered intravenously (i.v.) to DDY mice, nefiracetam and other piracetam-like nootropics inhibited the Ro 5-4864-induced convulsions in the sequence of nefiracetam>aniracetam>>oxiracetam, piracetam. Spontaneous EL mouse seizures were also inhibited by these nootropics with a similar rank order of potencies. Binding studies for PBRs, performed on crude membranes of brain tissues of these mice, revealed that [3H]Ro 5-4864 and [3H]PK 11195 bindings were both inhibited by micromolar concentrations of nootropic agents in the sequence of nefiracetam> aniracetam>>oxiracetam, piracetam. The results suggest that nefiracetam may exert an anticonvulsant action through interacting with a low-affinity type of PBR in the brain, and could be developed as a promising therapeutic drug for neurological disorders including epilepsies.