Functional mechanism of ASP5736, a selective serotonin 5-HT5A 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.

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.

4-Hydroxypyridazin-3(2H)-one derivatives as novel D-amino acid oxidase inhibitors.

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

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 Alzheimer's 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 Alzheimer's 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 Alzheimer's disease, suggesting that ASP2535 may satisfy currently unmet medical needs for the treatment of these diseases.

Chronic treatment with olanzapine via a novel infusion pump induces adiposity in male rats.

AIMS: Clinical use of olanzapine has been suggested to be associated with weight gain and adiposity in schizophrenic patients. While studies in experimental animals have noted weight gain in olanzapine-treated female rats, these findings have yet to be replicated in males. This study investigated the effect of chronic subcutaneous infusion of olanzapine in male rats via a recently developed electrical microinfusion pump. MAIN METHODS: An electrical microinfusion pump was subcutaneously implanted in male Sprague-Dawley rats who were then chronically administered olanzapine. Plasma olanzapine concentration and body weight were monitored, and fat pads were weighed after six weeks' olanzapine treatment. KEY FINDINGS: Plasma olanzapine concentration plateaued within 4h of commencement of chronic olanzapine 1.5mg/animal/day infusion and remained constant until day 21. Six-week infusion of olanzapine at 1.5 but not 1mg/animal/day induced significant adiposity in subcutaneous, epididymal, and retroperitoneal fat. Body weight and food intake values did not differ between olanzapine- and vehicle-treated rats throughout the experiment. SIGNIFICANCE: The present study demonstrated that chronic infusion of olanzapine induced adiposity in male rats without inducing weight gain or hyperphagia, even with sufficient plasma concentration. This report is the first to provide information about adiposity-inducible plasma concentration of olanzapine in male rats.

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.

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.

Anxiolytic activity of a novel potent serotonin 5-HT2C receptor antagonist FR260010: a comparison with diazepam and buspirone.

Hyperfunction of brain 5-hydroxytryptamine(2C) (5-HT(2C)) receptor is suggested to be involved in anxiety as evidenced by the fact that a putative 5-HT(2C) receptor agonist 1-(m-chlorophenyl)-piperazine (m-CPP) causes anxiety in humans. We have recently identified FR260010 (N-[3-(4-methyl-1H-imidazol-1-yl)phenyl]-5,6-dihydrobenzo[h]quinazolin-4-amine dimethanesulfonate) as novel 5-HT(2C) receptor antagonist from diaryl amine derivatives, and here characterized in vitro and in vivo profiles of the compound. FR260010 showed high affinity for human 5-HT(2C) receptor (K(i): 1.10 nM) and high selectivity over 5-hydroxytryptamine(2A) (5-HT(2A)) receptor (K(i): 386 nM) and many other transmitter receptors. FR260010 showed antagonist activity at human 5-HT(2C) receptor in an intracellular calcium assay and showed no detectable intrinsic activity. The compound dose-dependently inhibited the hypolocomotion (ID(50): 1.89 mg/kg, p.o.) and hypophagia (ID(50): 2.84 mg/kg, p.o.) in rats induced by m-CPP, putative indices of brain 5-HT(2C) receptor antagonist activity. We then compared the effects of FR260010 with those of two other anxiolytics belonging to different classes, diazepam and buspirone, in anxiety models in rats and mice and adverse effect tests in mice. FR260010 (0.1-3.2 mg/kg, p.o.) and diazepam (1-10 mg/kg, p.o.) decreased behavioral indices of anxiety in all models, whereas buspirone (0.32-10 mg/kg, p.o.) did not significantly affect them in any models. In adverse effect tests, FR260010 and buspirone showed modest effects, whereas diazepam showed significant effects in all tests. These results suggest that FR260010 is a novel, potent, orally active and brain penetrable antagonist of 5-HT(2C) receptor, and may have therapeutic potential for treatment of anxiety, with more desirable profiles than benzodiazepines or 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agonists.

Pharmacological characterization of FR194921, a new potent, selective, and orally active antagonist for central adenosine A1 receptors.

Adenosine A1 receptors in the brain are believed to play an important role in brain functioning. We have discovered a novel adenosine A1 receptor antagonist, FR194921 (2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone), and characterized the pharmacological activity in the present study. FR194921 showed potent and selective affinity for the adenosine A1 receptor without affinity for A2A and A3 receptors and did not show any species differences in binding affinity profile among human, rat, and mouse. Pharmacokinetic study in rats revealed that FR194921 was orally active and highly brain penetrable. Oral administration of FR194921 dose-dependently ameliorated the hypolocomotion induced by the A1 receptor agonist N6-cyclopentyladenosine in rats, indicating this compound exerts A1-antagonistic action in vivo. In the passive avoidance test, scopolamine (1 mg/kg)-induced memory deficits were significantly ameliorated by FR194921 (0.32, 1 mg/kg). In two animal models of anxiety, the social interaction test and elevated plus maze, FR194921 showed specific anxiolytic activity without significantly influencing general behavior. In contrast, FR194921 did not show antidepressant activity even at a dose of 32 mg/kg in the rat forced swimming test. These results indicate that the novel, potent, and selective adenosine A1 receptor antagonist FR194921 exerts both cognitive-enhancing and anxiolytic activity, suggesting the therapeutic potential of this compound for dementia and anxiety disorders.