5-HT1A receptor stimulation in the medial prefrontal cortex mediates the antidepressant effects of mGlu2/3 receptor antagonist in mice.

We previously reported that serotonergic transmission is involved in the antidepressant effects of metabotropic glutamate 2/3 (mGlu2/3) receptor antagonists. However, the detailed underlying mechanisms had not yet been explored. In the present study, we investigated the role of the 5-HT1A receptor and its signaling cascade in the medial prefrontal cortex (mPFC) in the antidepressant effects of LY341495, an mGlu2/3 receptor antagonist. LY341495 significantly reduced the immobility time in the forced swimming test which sustained for 24 h after administration. The antidepressant effect of LY341495 was attenuated by either intraperitoneal or intra-mPFC injection of WAY100635, a 5-HT1A receptor antagonist. Among the signaling cascades mediated by the 5-HT1A receptor, the role of phosphoinositide-3 kinase (PI3K)/Akt signaling, which has a critical role in neuroplasticity, was investigated. The sustained antidepressant effect of LY341495 was blocked by intra-mPFC injection of LY294002, a PI3K inhibitor. LY341495 increased the phosphorylation of Akt in the mPFC, which was blocked by both intra-mPFC injection of WAY100635 and LY294002. Thus, LY341495 activates PI3K/Akt signaling, presumably via stimulation of the 5-HT1A receptor in the mPFC, to exert its sustained antidepressant effect. Involvement of mechanistic target of rapamycin complex-1 (mTORC1) signaling was also demonstrated, as the sustained antidepressant effects of LY341495 was attenuated by intra-mPFC injection of rapamycin, an mTORC1 inhibitor. Finally, the sustained antidepressant effect of LY341495 was also attenuated by silencing of the dorsal raphe nucleus (DRN) neurons. These results suggest that stimulation of the 5-HT1A receptor in the mPFC and its signaling cascade, PI3K/Akt/mTORC1 signaling mediate the sustained antidepressant effect of LY341495, and that activation of the DRN neurons is also involved in these processes.

Role of 5-HT1A Receptor Stimulation in the Medial Prefrontal Cortex in the Sustained Antidepressant Effects of Ketamine.

Background: We previously reported that serotonergic transmission plays an important role in antidepressant effects of ketamine. However, detailed mechanisms have not been elucidated. Among the serotonin receptor subtypes, the serotonin1A receptor in the medial prefrontal cortex has an important role in depression. Here, we investigated the role of the medial prefrontal cortex serotonin1A receptor and its signaling mechanism in the antidepressant effects of ketamine. Methods: The role of serotonin1A receptor-mediated signaling mechanism (phosphoinositide-3 kinase/Akt) in the medial prefrontal cortex was examined in the mouse forced swimming test and western blotting. Results: Ketamine exerted antidepressant effects that lasted for 24 hours, and the sustained antidepressant effects were attenuated by intra-medial prefrontal cortex injection of a serotonin1A receptor antagonist, WAY100635. The sustained antidepressant effects were mimicked by intra- medial prefrontal cortex, but not systemic, administration of a serotonin1A receptor agonist, (±)-8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT). The sustained antidepressant effects of ketamine and 8-OH-DPAT were abrogated by intra- medial prefrontal cortex injection of a phosphoinositide-3 kinase inhibitor. Ketamine increased the phosphorylation of Akt in the medial prefrontal cortex at 60 minutes after administration, which was blocked by a serotonin1A receptor antagonist and a phosphoinositide-3 kinase inhibitor. Furthermore, the sustained antidepressant effects of ketamine and 8-OH-DPAT were attenuated by pretreatment of intra-medial prefrontal cortex injection of a mechanistic target of rapamycin complex-1 inhibitor. Conclusions: These results indicate that selective stimulation of the medial prefrontal cortex serotonin1A receptor and subsequent activation of the phosphoinositide-3 kinase/Akt/mechanistic target of rapamycin complex-1 pathway may be necessary for ketamine to exert the sustained antidepressant effects, and that this mechanism could be targeted to develop a novel and effective approach for treating depression.

Antidepressant/anxiolytic potential and adverse effect liabilities of melanin-concentrating hormone receptor 1 antagonists in animal models.

Melanin-concentrating hormone receptor 1 (MCH1 receptor) is known to be involved in the control of mood and stress, in addition to the regulation of feeding. Here, we report further evidence that the blockade of the MCH1 receptor exhibits antidepressant and anxiolytic-like effects in a variety of animal models using TASP0382650 and TASP0489838, newly synthesized MCH1 receptor antagonists, with different scaffolds. Both TASP0382650 and TASP0489838 exhibited high affinities for human MCH1 receptor with IC50 values of 7.13 and 3.80nM, respectively. Both compounds showed potent antagonist activities at the MCH1 receptor, as assessed using MCH-increased [(35)S]GTPγS binding to human MCH1 receptor and an MCH-induced [Ca(2+)]i assay in rat MCH1 receptor expressing cells. In contrast, neither TASP0382650 nor TASP0489838 showed an affinity for the MCH2 receptor, another MCH receptor subtype. The oral administration of TASP0382650 or TASP0489838 significantly reduced the immobility time during the forced swimming test in rats, and reduced hyperemotionality induced by an olfactory bulbectomy, both of which are indicative of an antidepressant-like potential. In the olfactory bulbectomy model, the antidepressant effect of TASP0382650 appeared following a single administration, suggesting a faster onset of action, compared with current medications. Moreover, both TASP0382650 and TASP0489838 exhibited anxiolytic effects in several animal models of anxiety. In contrast, both TASP0382650 and TASP0489838 did not affect spontaneous locomotor activity, motor function, spatial memory during the Morris water maze task, or the convulsion threshold to pentylenetetrazole. These findings provide additional evidence that the blockade of the MCH1 receptor exhibits antidepressant- and anxiolytic activities with no adverse effects in experimental animal models.

Dopamine receptor agonists reverse behavioral abnormalities of alpha-synuclein transgenic mouse, a new model of Parkinson's disease.

Parkinson's disease (PD) is characterized by loss of nigral dopaminergic (DAergic) neurons and presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residues 1-130) in DAergic neurons. Syn130m mice showed significant loss of DAergic neurons in the substantia nigra pars compacta. Subsequently, the striatal DA level and spontaneous locomotor activity of the mice were decreased significantly. In the present study, we investigated behavioral responses of Syn130m mice to L-DOPA and DA receptor agonists. Administration of L-DOPA dose dependently ameliorated the reduction of spontaneous locomotor activity of Syn130m mice. Similarly, D(2) agonists, quinpirole and talipexole, and a D1/D2 agonist, pergolide, were effective against the reduction. Syn130m mice also showed significant reduction in exploratory behavior compared with non-Tg littermates when they were placed in a novel environment, but this abnormality was ameliorated by treatment with pergolide. These results strongly suggest that the behavioral abnormalities of Syn130m mice were caused by low striatal DA content. On the other hand, the expression of postsynaptic D(2)-like receptors (DRD2) in the striatum was not increased in Syn130m mice, although the low striatal DA level is known to induce compensatory expression of DRD2. Because the abnormalities could be rectified by treatment with DA receptor agonists, it is likely that Syn130m mice provide a useful tool to explore therapeutic possibilities for PD as a new animal model of the disease.

Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists.

The optimization of the distance between two key pharmacophore features within our first hit compounds 1a and 2a led to the identification of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylaminoquinazolines. In particular, ATC0065 (2c), N2-[cis-4-([2-[4-Bromo-2-(trifluoromethoxy)phenyl]ethyl]amino)cyclohexyl]-N4,N4-dimethylquinazoline-2,4-diamine dihydrochloride, bound with high affinity to the MCH-R1 (IC50 value of 16 nM) and showed good metabolic stability in liver microsomes from human and rat.

MCL0042: a nonpeptidic MC4 receptor antagonist and serotonin reuptake inhibitor with anxiolytic- and antidepressant-like activity.

In the present study, we examined the anxiolytic and antidepressant effects of MCL0042, a novel compound showing activity in both MC4 receptor antagonism and serotonin transporter inhibition. MCL0042 showed relatively high affinity for the MC4 receptor and serotonin reuptake site, as determined by receptor binding assays. MCL0042 attenuated [Nle(4),d-Phe(7)]alpha-MSH-increased cAMP formation in MC4 receptor expressing cells, and it inhibited [(3)H]serotonin uptake by rat brain synaptosomes; thus, MCL0042 is an MC4 receptor antagonist and serotonin transporter inhibitor. Subcutaneous administration of MCL0042 significantly increased the number of licks in a Vogel punished drinking test in rats, and it also significantly attenuated swim stress-induced reduction in time spent in open arms in an elevated plus-maze task in rats, showing the anxiolytic-like potential of MCL0042. Moreover, repeated administration of MCL0042 for 14 days attenuated olfactory bulbectomy-induced locomotor hyperactivity in rats, indicating antidepressant-like potential. These data show that MCL0042 has unique properties of both the MC4 receptor antagonist and serotonin transporter inhibitor, and produces anxiolytic and antidepressant activity in rats. Moreover, blockade of both the MC4 receptor and serotonin reuptake sites might represent a useful approach in the treatment of anxiety and depression.

Lead optimization of 4-(dimethylamino)quinazolines, potent and selective antagonists for the melanin-concentrating hormone receptor 1.

The optimization of a series of 4-(dimethylamino)quinazoline antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. The combination of the elaboration of both the linker portion and the terminal phenyl ring provided N-(cis-4-{[4-(dimethylamino)quinazolin-2-yl]amino}cyclohexyl)-3,4-difluorobenzamide hydrochloride 28 (ATC0175), which showed excellent antagonist activity at the MCH-R1 (IC50 = 3.4 nM) as well as good selectivity over the Y5 and the alpha2A receptors.

Discovery of 4-(dimethylamino)quinazolines as potent and selective antagonists for the melanin-concentrating hormone receptor 1.

A series of 4-(dimethylamino)quinazoline based antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. This series was derived from a lead compound, AR129330, identified by HTS of a GPCR-directed library using a functional assay with a constitutively activated (CART) form of the receptor. The preliminary optimization resulted in the identification of compounds 20, 21, and 23.

Anxiolytic- and antidepressant-like profile of ATC0065 and ATC0175: nonpeptidic and orally active melanin-concentrating hormone receptor 1 antagonists.

Melanin-concentrating hormone (MCH) is a cyclic peptide produced in the lateral hypothalamus. It has been implicated in a number of physiological processes including feeding behavior, energy balance, and the regulation of emotional states. Here, we report in vitro and in vivo profiles of ATC0065 [N(2)-[cis-4-({2-[4-bromo-2-(trifluoromethoxy)phenyl]ethyl}amino)cyclohexyl]-N(4), N(4)-dimethylquinazoline-2,4-diamine dihydrochloride] and ATC0175 [N-(cis-4-{[4-(dimethylamino)quinazolin-2-yl]amino}cyclohexyl)-3,4-difluorobenzamide hydrochloride], newly synthesized MCH receptor 1 (MCHR1) antagonists. Both ATC0065 and ATC0175 had high affinities for human MCHR1 with IC(50) values of 15.7 +/- 1.95 and 7.23 +/- 0.59 nM, respectively. Both ATC0065 (IC(50) = 21.4 +/- 1.57 nM) and ATC0175 (IC(50) = 13.5 +/- 0.78 nM) showed potent antagonist activities at MCHR1, as assessed by MCH-increased guanosine 5'-O-(3-[(35)S]thio)phosphate ([(35)S]GTPgammaS) binding to human MCHR1. Oral administration of ATC0065 (3-30 mg/kg) or ATC0175 (1-10 mg/kg) significantly reduced immobility time in the forced swimming test in rats, indicating antidepressant-like effects. Both ATC0065 and ATC0175 significantly reversed swim stress-induced anxiety in the elevated plus-maze test in rats and stress-induced hyperthermia in mice. ATC0175 significantly increased social interaction between unfamiliar rats and reduced separation-induced vocalizations in guinea pig pups, indicating anxiolytic potential. In contrast, ATC0065 and ATC0175 did not affect spontaneous locomotor activity or rotarod performance in rats. These findings indicate that ATC0065 and ATC0175 are potent and orally active MCHR1 antagonists with anxiolytic and antidepressant activity in rodents.

Involvement of the melanocortin MC4 receptor in stress-related behavior in rodents.

The melanocortin subtype 4 (MC4) receptor has been postulated to be involved in stress and stress-related behavior. We made use of melanocortin MC4 receptor agonists and antagonist to investigate the relationship between the melanocortin MC4 receptor and stress related disorders. The nonspecific melanocortin receptor agonist alpha-melanocyte stimulating hormone (alpha-MSH) and the melanocortin MC4 receptor agonist, Ac-[Nle4,Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 (MT II) dose-dependently and significantly reduced the number of licking periods in the rat Vogel conflict test, suggesting that stimulation of the melanocortin MC4 receptor causes anxiogenic-like activity in rats. We synthesized a peptidemimetic melanocortin MC4 receptor selective antagonist, Ac-D-2Nal-Arg-2Nal-NH2 (MCL0020), which has high affinity for the melanocortin MC4 receptor with IC50 values of 11.63 +/- 1.48 nM, in contrast, the affinities for melanocortin MC1 and MC3 receptors were negligible. In addition, MCL0020 significantly attenuated the cAMP formation induced by alpha-MSH in COS-1 cells expressing the melanocortin MC4 receptor without affecting basal cAMP contents. Thus, we considered MCL0020 to be a selective melanocrotin MC4 receptor antagonist among melanocortin receptors. Restraint stress significantly reduced food intake in rats, and i.c.v. administration of MCL0020 dose-dependently and significantly attenuated restraint stress-induced anorexia without affecting food intake. Swim stress induced reduction in the time spent in the light area in the mouse light/dark exploration test, and MCL0020 significantly prevented it. Taken together our findings suggest that the melanocortin MC4 receptor might be related to stress-induced changes in behavior, and blockade of the melanocortin MC4 receptor may prevent stress-induced disorders such as anxiety.

Anxiolytic-like and antidepressant-like activities of MCL0129 (1-[(S)-2-(4-fluorophenyl)-2-(4-isopropylpiperadin-1-yl)ethyl]-4-[4-(2-methoxynaphthalen-1-yl)butyl]piperazine), a novel and potent nonpeptide antagonist of the melanocortin-4 receptor.

We investigated the effects of a novel melanocortin-4 (MC4) receptor antagonist,1-[(S)-2-(4-fluorophenyl)-2-(4-isopropylpiperadin-1-yl)ethyl]-4-[4-(2-methoxynaphthalen-1-yl)butyl]piperazine (MCL0129) on anxiety and depression in various rodent models. MCL0129 inhibited [(125)I][Nle(4)-D-Phe(7)]-alpha-melanocyte-stimulating hormone (alpha-MSH) binding to MC4 receptor with a K(i) value of 7.9 nM, without showing affinity for MC1 and MC3 receptors. MCL0129 at 1 microM had no apparent affinity for other receptors, transporters, and ion channels related to anxiety and depression except for a moderate affinity for the sigma(1) receptor, serotonin transporter, and alpha(1)-adrenoceptor, which means that MCL0129 is selective for the MC4 receptor. MCL0129 attenuated the alpha-MSH-increased cAMP formation in COS-1 cells expressing the MC4 receptor, whereas MCL0129 did not affect basal cAMP levels, thereby indicating that MCL0129 acts as an antagonist at the MC4 receptor. Swim stress markedly induced anxiogenic-like effects in both the light/dark exploration task in mice and the elevated plus-maze task in rats, and MCL0129 reversed the stress-induced anxiogenic-like effects. Under nonstress conditions, MCL0129 prolonged time spent in the light area in the light/dark exploration task and suppressed marble-burying behavior. MCL0129 shortened immobility time in the forced swim test and reduced the number of escape failures in inescapable shocks in the learned helplessness test, thus indicating an antidepressant potential. In contrast, MCL0129 had negligible effects on spontaneous locomotor activity, Rotarod performance, and hexobarbital-induced anesthesia. These observations indicate that MCL0129 is a potent and selective MC4 antagonist with anxiolytic- and antidepressant-like activities in various rodent models. MC4 receptor antagonists may prove effective for treating subjects with stress-related disorders such as depression and/or anxiety.

In vitro and in vivo pharmacological profile of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl] ethyl] piperidine (NRA0161).

Atypical antipsychotic properties of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl] piperidine (NRA0161) were investigated by in vitro receptor affinities, in vivo receptor occupancies and findings were compared with those of risperidone and haloperidol in rodent behavioral studies. In in vitro receptor binding studies, NRA0161 has a high affinity for human cloned dopamine D(4) and 5-HT(2A) receptor with Ki values of 1.00 and 2.52 nM, respectively. NRA0161 had a relatively high affinity for the alpha(1) adrenoceptor (Ki; 10.44 nM) and a low affinity for the dopamine D(2) receptor (Ki; 95.80 nM). In in vivo receptor binding studies, NRA0161 highly occupied the 5-HT(2A) receptor in rat frontal cortex. In contrast, NRA0161 did not occupy the striatal D(2) receptor. In behavioral studies, NRA0161, risperidone and haloperidol antagonized the locomotor hyperactivity in mice, as induced by methamphetamine (MAP). At a higher dosage, NRA0161, risperidone and haloperidol dose-dependently antagonized the MAP-induced stereotyped behavior in mice and NRA0161 dose-dependently and significantly induced catalepsy in rats. The ED(50) value in inhibiting the MAP-induced locomotor hyperactivity was 30 times lower than that inhibiting the MAP-induced stereotyped behavior and 50 times lower than that which induced catalepsy. These findings suggest that NRA0161 may have atypical antipsychotic activities yet without producing extrapyramidal side effects.

In vitro and in vivo pharmacological profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel and putative atypical antipsychotic.

In vitro and in vivo pharmacological properties of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel atypical antipsychotic, were investigated. NRA0562 showed high affinities for human cloned dopamine D(1), D(2), D(3) and D(4) receptors with Ki values of 7.09, 2.49, 3.48 and 1.79 nM. In addition, NRA0562 had high affinities for the 5-HT(2A) receptor and the alpha(1) adrenoceptor with Ki values of 1.5 and 0.56 nM, and moderate affinity for the histamine H(1) receptor. Using in vivo and ex vivo receptor binding studies in rats, we showed NRA0562 occupied frontal cortical 5-HT(2A) receptors and alpha(1) adrenoceptor potently, while occupancy of striatal dopamine D(2) receptor was moderate as were other atypical antipsychotics. NRA0562 dose-dependently inhibited methamphetamine (MAP)-induced locomotor hyperactivity in rats. At higher dosage, NRA0562 dose-dependently antagonized MAP-induced stereotyped behavior and induced catalepsy dose-dependently and significantly in rats. But, the ED(50) value in inhibiting MAP-induced locomotion hyperactivity was 10 times lower than that in inhibiting MAP-induced stereotyped behavior, and 30 times lower than that in inducing catalepsy. In addition, the potency of NRA0562 in antagonizing MAP-induced hyperactivity in rats was higher than that of other antipsychotics, clozapine, risperidone and olanzapine. NRA0562 had favorable properties in view of prediction of extrapyramidal side effects. As this antipsychotic has a unique profile with affinity and occupancy for receptors, we propose that NRA0652 may have unique atypical antipsychotic activities, and a moderate liability of extrapyramidal motor side effects seen in the treatment with classical antipsychotics.

Antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) in rats.

The antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) was investigated using the conditioned avoidance test in rats. NRA0562 is a putative "atypical" antipsychotic agent with moderate to high affinities for dopamine D(1), D(2), D(4), 5-hydroxytryptamine(2A) receptors and alpha(1) adrenoceptor. NRA0562 (1 and 3 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response. Likewise other atypical antipsychotics such as risperidone (1 and 3 mg/kg, p.o.) and clozapine (100 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response in rats. In addition, typical antipsychotics, haloperidol (1 and 3 mg/kg, p.o.) potently impaired the conditioned avoidance response.These results suggest that antipsychotic profile of NRA0562 is consistent with profiles of clozapine or risperidone and may be considered an atypical antipsychotic agent.