Katsuya Harada

Activation of the serotonin 5-HT2C receptor is involved in the enhanced anxiety in rats after single-prolonged stress

We have recently confirmed that exposure of rats to the single-prolonged stress (SPS) paradigm induces enhanced hypothalamic-pituitary-adrenal (HPA) axis negative feedback and enhanced anxiety, and found that these changes develop time-dependently following stress exposure, suggesting that it could model the neuroendocrinological and behavioral abnormalities of the post-traumatic stress disorder (PTSD) patients. In the present study, microarray analysis was performed using RNA from the hippocampus, amygdala and anterior cingulate cortex of SPS rats and unstressed controls to unveil the molecular changes underlying SPS-induced behavioral changes. Thirty-one genes were found whose time course of expression corresponded to that of behavioral changes. One gene, 5-hydroxytryptamine2C (5-HT2C) receptor, was identified as a putative candidate. The overexpression of the gene in the amygdala of SPS rats was confirmed using real-time PCR 7 days after the SPS exposure. This molecule was then pharmacologically validated using FR260010 (N-[3-(4-methyl-1H-imidazol-1-yl)phenyl]-5,6-dihydrobenzo[h]quinazolin-4-amine dimethanesulfonate), a selective 5-HT2C receptor antagonist. FR260010 (1-10 mg/kg, s.c.) significantly inhibited the enhancement of anxiety in SPS rats. These results demonstrate for the first time that activation of the brain 5-HT2C receptor is involved in the development of behavioral abnormality in this model. This suggests that selective 5-HT2C receptor antagonists might provide novel therapeutic avenues for PTSD treatment.

A novel glycine transporter-1 (GlyT1) inhibitor, ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole), improves cognition in animal models of cognitive impairment in schizophrenia and Alzheimer's disease.

Hypofunction of brain N-methyl-d-aspartate (NMDA) receptors has been implicated in psychiatric disorders such as schizophrenia and Alzheimers disease. Inhibition of glycine transporter-1 (GlyT1) is expected to increase glycine, a co-agonist of the NMDA receptor and, consequently, to facilitate NMDA receptor function. We have identified ASP2535 (4-[3-isopropyl-5-(6-phenyl-3-pyridyl)-4H-1,2,4-triazol-4-yl]-2,1,3-benzoxadiazole) as a novel GlyT1 inhibitor, and here describe our in vitro and in vivo characterization of this compound. ASP2535 potently inhibited rat GlyT1 (IC(50)=92 nM) with 50-fold selectivity over rat glycine transporter-2 (GlyT2). It showed minimal affinity for many other receptors except for μ-opioid receptors (IC(50)=1.83 μM). Oral administration of ASP2535 dose-dependently inhibited ex vivo [(3)H]-glycine uptake in mouse cortical homogenate, suggesting good brain permeability. This profile was confirmed by pharmacokinetic analysis. We then evaluated the effect of ASP2535 on animal models of cognitive impairment in schizophrenia and Alzheimers disease. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both attenuated by ASP2535 (0.3-3mg/kg, p.o. and 0.3-1mg/kg, p.o., respectively). ASP2535 (1-3mg/kg, p.o.) also improved the PCP-induced deficit in prepulse inhibition in rats. Moreover, the working memory deficit in scopolamine-treated mice and the spatial learning deficit in aged rats were both attenuated by ASP2535 (0.1-3mg/kg, p.o. and 0.1mg/kg, p.o., respectively). These studies provide compelling evidence that ASP2535 is a novel and centrally-active GlyT1 inhibitor that can improve cognitive impairment in animal models of schizophrenia and Alzheimers disease, suggesting that ASP2535 may satisfy currently unmet medical needs for the treatment of these diseases.

Two models for weight gain and hyperphagia as side effects of atypical antipsychotics in male rats: Validation with olanzapine and ziprasidone

Body weight gain is one of the most serious side effects associated with clinical use of antipsychotics. However, the mechanisms by which antipsychotics induce body weight gain are unknown, and no reliable animal models of antipsychotics-induced weight gain have been established. The present studies were designed to establish male rat models of weight gain induced by chronic and acute treatment with antipsychotics. Six-week chronic treatment with olanzapine (5, 7.5, and 10mg/kg/day) in male Sprague-Dawley rats fed a daily diet resembling a human macronutrient diet, significantly increased body weight gain and weight of fatty tissues. In contrast, ziprasidone (1.25, 2.5, and 5mg/kg/day) administration caused no observable adverse effects. We then investigated feeding behavior with acute antipsychotic treatment in male rats using an automated food measurement apparatus. Rats were allowed restricted access to normal laboratory chow (4h/day). With acute olanzapine (0.5, 1, and 2mg/kg, i.p.) treatment in the light phase, food intake volume and duration were significantly increased, while treatment with ziprasidone (0.3, 1, and 3mg/kg, i.p.) did not increase food intake volume or meal time duration. Findings from the present studies showed that chronic treatment with olanzapine in male rats induced body weight gain, and acute injection induced hyperphagia, suggesting that hyperphagia may be involved in the weight gain and obesity-inducing properties of chronically administered olanzapine. These animal models may provide useful experimental platforms for analysis of the mechanism of hyperphagia and evaluating the potential risk of novel antipsychotics to induce weight gain in humans.

Novel 5-HT5A receptor antagonists ameliorate scopolamine-induced working memory deficit in mice and reference memory impairment in aged rats.

Despite the human 5-HT5A receptor being cloned in 1994, the biological function of this receptor has not been extensively characterized due to a lack of specific ligands. We recently reported that the selective 5-HT5A receptor antagonist ASP5736 ameliorated cognitive impairment in several animal models of schizophrenia. Given that areas of the brain with high levels of 5-HT5A receptor expression, such as the hippocampus and cerebral cortex, have important functions in cognition and memory, we evaluated the chemically diverse, potent and brain-penetrating 5-HT5A receptor antagonists ASP5736, AS2030680, and AS2674723 in rodent models of cognitive dysfunction associated with dementia. Each of these compounds exhibited a high affinity for recombinant 5-HT5A receptors that was comparable to that of the non-selective ligand of this receptor, lysergic acid diethylamide (LSD). Although each compound had a low affinity for other receptors, 5-HT5A was the only receptor for which all three compounds had a high affinity. Each of the three compounds ameliorated scopolamine-induced working memory deficit in mice and improved reference memory impairment in aged rats at similar doses. Further, ASP5736 decreased the binding of LSD to 5-HT5A receptors in the olfactory bulb of rats in a dose-dependent manner and occupied 15%-50% of brain 5-HT5A receptors at behaviorally effective doses. These results indicate that the 5-HT5A receptor is involved in learning and memory and that treatment with 5-HT5A receptor antagonists might be broadly effective for cognitive impairment associated with not only schizophrenia but also dementia.

ASP5736, a novel 5-HT5A receptor antagonist, ameliorates positive symptoms and cognitive impairment in animal models of schizophrenia

We recently identified ASP5736, (N-(diaminomethylene)-1-(3,5-difluoropyridin-4-yl)-4-fluoroisoquinoline-7-carboxamide (2E)-but-2-enedioate), a novel antagonist of 5-HT5A receptor, and here describe the in vitro and in vivo characterization of this compound. ASP5736 exhibited a high affinity for the human 5-HT5A receptor (Ki = 3.6 ± 0.66 nM) and antagonized 5-carboxamidotryptamine (5-CT)-induced Ca(2+) influx in human cells stably expressing the 5-HT5A receptor with approximately 200-fold selectivity over other receptors, including other 5-HT receptor subtypes, enzymes, and channels except human 5-HT2c receptor (Ki = 286.8 nM) and 5-HT7 receptor (Ki = 122.9 nM). Further, ASP5736 dose-dependently antagonized the 5-CT-induced decrease in cAMP levels in HEK293 cells stably expressing the 5-HT5A receptor. We then evaluated the effects of ASP5736 on cognitive impairments in several animal models of schizophrenia. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both ameliorated by ASP5736. In addition, ASP5736 also attenuated MK-801- and methamphetamine (MAP)-induced hyperactivity in mice without causing sedation, catalepsy, or plasma prolactin increase. The addition of olanzapine did not affect ASP5736-induced cognitive enhancement, and neither the sedative nor cataleptogenic effects of olanzapine were worsened by ASP5736. These results collectively suggest that ASP5736 is a novel and potent 5-HT5A receptor antagonist that not only ameliorates positive-like symptoms but also cognitive impairments in animal models of schizophrenia, without adverse effects. Present studies also indicate that ASP5736 holds potential to satisfy currently unmet medical needs for the treatment of schizophrenia by either mono-therapy or co-administered with commercially available antipsychotics.

Norzotepine, a major metabolite of zotepine, exerts atypical antipsychotic-like and antidepressant-like actions through its potent inhibition of norepinephrine reuptake

The antipsychotic drug zotepine [ZTP; 2-[(8-chlorodibenzo[b,f]thiepin-10-yl)oxy]-N,N-dimethylethan-1-amine] is known to have not only atypical antipsychotic effects but also antidepressive effects in schizophrenia patients. Norzotepine [norZTP; N-desmethylzotepine, 2-[(8-chlorodibenzo[b,f]thiepin-10-yl)oxy]-N-methylethan-1-amine] has been postulated to be a major metabolite of ZTP in humans. Here, we characterized norZTP through several in vitro studies and in animal models of psychosis, depression, and extrapyramidal symptoms (EPS) and compared the pharmacological profiles with those of ZTP. Although both compounds showed similar overall neurotransmitter receptor binding profiles, norZTP showed 7- to 16-fold more potent norepinephrine reuptake inhibition than ZTP. In a pharmacokinetic study, both ZTP and norZTP showed good brain permeability when administered individually in mice, although norZTP was not detected in either plasma or brain after intraperitoneal injection of ZTP. In the methamphetamine-induced hyperlocomotion test in mice, norZTP and ZTP showed similar antipsychotic-like effects at doses above 1 mg/kg i.p. In contrast, unlike ZTP, norZTP did not induce catalepsy up to 10 mg/kg i.p. norZTP significantly antagonized the hypothermia induced by reserpine [(3β,16β,17α,18β,20α)-11,17-dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester], suggesting in vivo inhibition of the norepinephrine transporter. In the forced-swim test, norZTP exerted an antidepressant-like effect at the effective doses for its antipsychotic action, whereas ZTP neither antagonized reserpine-induced hypothermia nor showed antidepressant-like effect. These results collectively demonstrate that norZTP exerts more potent inhibitory action than ZTP on norepinephrine transporters both in vitro and in vivo, presumably accounting for its antidepressant-like effect and low EPS propensity. Given that norZTP is the major metabolite observed in humans, norZTP may contribute to the unique clinical profiles of its mother compound, ZTP.

Functional mechanism of ASP5736, a selective serotonin 5-HT 5A receptor antagonist with potential utility for the treatment of cognitive dysfunction in schizophrenia

The 5-HT5A receptor is arguably the least understood 5-HT receptor. Despite widespread expression in human and rodent brains it lacks specific ligands. Our previous results suggest that 5-HT5A receptor antagonists may be effective against cognitive impairment in schizophrenia. In this study, using behavioral, immunohistochemical, electrophysiological and microdialysis techniques, we examined the mechanism by which ASP5736, a novel and selective 5-HT5A receptor antagonist, exerts a positive effect in animal models of cognitive impairment. We first confirmed the effect of ASP5736 on cognitive deficits in rats treated subchronically with phencyclidine hydrochloride (PCP) using an attentional set shifting task. Subsequently, we identified 5-HT5A receptors in dopaminergic (DAergic) neurons and parvalbumin (PV)-positive interneurons in the ventral tegmental area (VTA) and in PV-positive interneurons in the medial prefrontal cortex (mPFC). Burst firing of the DAergic cells in the parabrachial pigmental nucleus (PBP) in the VTA, which predominantly project to the mPFC, was significantly enhanced by treatment with ASP5736. In contrast, ASP5736 exerted no significant effect on either the firing rate or burst firing in the DA cells in the paranigral nucleus (PN), that project to the nucleus accumbens (N. Acc.). ASP5736 increased the release of DA and gamma-aminobutyric acid (GABA) in the mPFC of subchronically PCP-treated rats. These results support our hypothesis that ASP5736 might block the inhibitory 5-HT5A receptors on DAergic neurons in the VTA that project to the mPFC, and interneurons in the mPFC, and thereby improve cognitive impairment by preferentially enhancing DAergic and GABAergic neurons in the mPFC.