Safety and pharmacokinetic profiles of MGS0274 besylate (TS-134), a novel metabotropic glutamate 2/3 receptor agonist prodrug, in healthy subjects.

AIMS: The safety and pharmacokinetics of single and multiple doses of a novel mGlu2/3 receptor agonist prodrug, MGS0274 besylate (TS-134), were investigated in healthy subjects. METHODS: Phase 1 single-ascending dose (5-20 mg) and multiple-ascending dose titration (5-80 mg) studies were conducted in healthy male and female subjects. Both studies were randomized, double-blinded and placebo-controlled. In one cohort of single-ascending dose study (10 mg), concentrations of MGS0008, the active compound, in the cerebrospinal fluid (CSF) were measured for up to 24 hours postdose. RESULTS: Following single and multiple oral administrations, MGS0274 was rapidly absorbed and extensively converted into MGS0008, which reached a maximum concentration (Cmax ) in plasma within 4 hours postdose and declined with a terminal half-life (t1/2 ) of around 10 hours. Plasma exposure to MGS0274 was minimal, accounting for approximately 3% of the area under the concentration-time curve (AUC) of MGS0008. Plasma Cmax and AUC of MGS0008 at steady state increased dose proportionally (5-80 mg). MGS0008 penetrated into CSF, with a CSF-to-plasma Cmax ratio of 3.66%, and was eliminated with a t1/2 of approximately 16 hours. The most frequent treatment-emergent adverse events observed following single and multiple oral administration included headache, nausea, somnolence, dizziness and vomiting. CONCLUSION: TS-134 is orally bioavailable in humans and converts rapidly and extensively to MGS0008, which exhibits good CSF penetration. Orally administered TS-134 was safe and generally well-tolerated; hence, TS-134 is a promising candidate for further clinical development for the treatment of disorders in which glutamatergic abnormalities are involved, such as schizophrenia.

Clarithromycin prevents smoke-induced emphysema in mice.

RATIONALE: Modulating the low-grade chronic inflammation in chronic obstructive pulmonary disease remains challenging. Clarithromycin (CAM), a macrolide antibiotic, reportedly ameliorates chronic inflammation via mechanisms independent of its antibacterial activity. OBJECTIVES: The aim of this study was to examine whether CAM can prevent or reduce emphysema induced by chronic cigarette smoke exposure. METHODS: Mice were exposed to cigarette smoke daily for 6 months and treated with orally administered CAM at doses of 25 to 100 mg/kg twice a day throughout the course of the experiment to test the preventive effects. The administration of CAM at 50 or 100 mg/kg was performed during the second half of a 6-month exposure period to assess the therapeutic effects. Histologic analysis was performed to evaluate the effect of CAM. MEASUREMENTS AND MAIN RESULTS: CAM treatment for 6 months decreased airspace enlargement and the destruction of the alveolar walls and impaired the accumulation of macrophages in bronchoalveolar lavage fluid in a dose-related fashion. The administration of clarithromycin at 100 mg/kg in the therapeutic protocol reduced emphysema compared with the smoke-exposed group without treatment. An immunohistologic analysis revealed that CAM reduced the number of F4/80-positive macrophages in the lung parenchyma. In an in vitro test, CAM at 5 to 20 microM directly suppressed the activation of macrophages stimulated with tumor necrosis factor-alpha. CONCLUSIONS: Our data demonstrated that CAM at a clinically achievable dose prevented cigarette smoke-induced emphysema by modulating lung inflammation. This study supports the possibility that low-dose CAM treatment might provide a new therapeutic strategy for chronic obstructive pulmonary diseases.

[Targeting metabotropic glutamate receptors to develop novel antipsychotics].

Based on the glutamate hypothesis of schizophrenia, extensive studies to develop drugs acting on glutamate receptors have been conducted. Among glutamate receptors, metabotropic glutamate (mGlu) receptors, all of which are GPCRs, have 8 subtypes, and are involved in regulation of glutamate transmissions. Of these, much attention has been paid to mGlu2/3 receptors and mGlu5 receptor. mGlu2/3 receptor agonists improve behavioral abnormalities such as locomotor hyperactivity and cognitive deficits induced by NMDA receptor antagonists. In addition, mGlu2/3 receptor agonists attenuate glutamate overflow in the prefrontal cortex, and regulate dopamine release and 5-HT2A receptor activity, all of which have been presumed to be involved in antipsychotic actions of mGlu2/3 receptor agonists. Recently, LY2140023, an mGlu2/3 receptor agonists developed by Eli Lilly, has been demonstrated to be effective for the treatment of positive and negative symptoms of schizophrenic patients in a phase II study, while it did not cause unwanted side effects often observed with current antipsychotic medications. Moreover, a series of experiments has demonstrated that mGlu5 receptor potentiators exert antipsychotic effects in animal models of schizophrenia. Therefore, mGlu2/3 receptor and mGlu5 receptor may provide exciting targets for the development of novel medications for schizophrenia.

Intense correlation between brain infarction and protein-conjugated acrolein.

BACKGROUND AND PURPOSE: We recently found that increases in plasma levels of protein-conjugated acrolein and polyamine oxidases, enzymes that produce acrolein, are good markers for stroke. The aim of this study was to determine whether the level of protein-conjugated acrolein is increased and levels of spermine and spermidine, the substrates of acrolein production, are decreased at the locus of infarction. METHODS: A unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. The level of protein-conjugated acrolein at the locus of infarction and in plasma was measured by Western blotting and enzyme-linked immunosorbent assay, respectively. The levels of polyamines at the locus of infarction and in plasma were measured by high-performance liquid chromatography. RESULTS: The level of protein-conjugated acrolein was greatly increased, and levels of spermine and spermidine were decreased at the locus of infarction at 24 hours after the induction of stroke. The size of infarction was significantly decreased by N-acetylcysteine, a scavenger of acrolein. It was also found that the increases in the protein-conjugated acrolein, polyamines, and polyamine oxidases in plasma were observed after the induction of stroke. CONCLUSIONS: The results indicate that the induction of infarction is well correlated with the increase in protein-conjugated acrolein at the locus of infarction and in plasma.

Effect of a new inhibitor of the synthesis of 20-HETE on cerebral ischemia reperfusion injury.

BACKGROUND AND PURPOSE: Arachidonic acid that is released following cerebral ischemia can be metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent vasoconstrictor that may contribute to ischemic injury. This study examined the effects of blockading the synthesis of 20-HETE with TS-011 on infarct size after transient occlusion of the middle cerebral artery (MCAO) of rats and after thromboembolic stroke in monkeys. METHODS: Rats were treated with TS-011 or vehicle at various times after MCAO. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining and plasma levels of 20-HETE were determined by liquid chromatography mass spectrometry (LC/MS). The effect of TS-011 on infarct size was also studied in monkeys after introduction of a clot into the internal carotid artery. RESULTS: Plasma levels of 20-HETE increased after MCAO in rats. TS-011 (0.01 to 1.0 mg/kg per hour) reduced infarct volume by 40%. Chronic administration of TS-011 for 7 days reduced neurological deficits after MCAO in rats. TS-011 given in combination with tissue plasminogen activator also improved neurological outcome in the stroke model in monkeys. CONCLUSIONS: These results suggest that blockade of the formation of 20-HETE with TS-011 may be useful for the treatment of ischemic stroke.

Group II metabotropic glutamate receptor-mediated regulation of dopamine release from slices of rat nucleus accumbens.

The role of metabotropic glutamate receptors (mGluRs) in the regulation of dopamine release in the rat nucleus accumbens was investigated. Fifteen millimolar of KCl stimulated the release of [(3)H]dopamine from the slices of the rat nucleus accumbens. Both an mGluR agonist 1S,3R-1-amino-cyclopentane-1,3-dicarboxylate (ACPD) and a preferential group II mGluR agonist, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1), significantly inhibited the KCl-evoked [(3)H]dopamine release in the nucleus accumbens. This inhibitory effect of L-CCG-1 on the KCl-evoked dopamine release was significantly attenuated by preferential group II mGluR antagonists such as (2S,3S,4S)-2-methyl-2-(carboxypropyl)glycine (MCCG) and (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG); in contrast, the preferential group III mGluR agonist L-2-amino-4-phosphonobutylate (L-AP4), failed to show any effect on the KCl-evoked [(3)H]dopamine release in the nucleus accumbens. Moreover, the inhibitory effect of L-CCG-1 on the KCl-evoked [(3)H]dopamine release from the slices of the rat nucleus accumbens was preserved in the presence of tetrodotoxin. These results show that group II mGluRs may play a more significant role in regulating dopamine release than group III mGluRs, and that the group II mGluRs may negatively regulate dopamine release, presumably through those expressed at the dopaminergic nerve terminals or through those expressed at glutamatergic nerve terminals in the nucleus accumbens.

Involvement of melanocortin-4 receptor in anxiety and depression.

The melanocortins, which are derived from proopiomelanocortin, have a variety of physiological functions mediated membrane surface receptors. To date, five subtypes have been cloned. With the cloning of melanocortin receptors, studies with genetic models, and development of selective compounds, the physiological roles of the five melanocortin receptors have begun to be understood. The melanocortin-4 receptor (MC4R), which is predominantly expressed in the central nervous system, has in particular become the focus of much attention in recent years because of the critical roles it plays in a wide range of functions, including feeding, sexual behavior, and stress. Recent development of selective antagonists for the MC4R has provided pharmacological evidence that blockade of MC4R could be a useful way of alleviating numerous conditions such as anxiety/depression, pain, and addiction to drugs of abuse.

Neuropharmacological profiles of antagonists of group II metabotropic glutamate receptors.

Glutamatergic abnormalities play roles in several psychiatric disorders. Glutamate acts at two classes of receptors, ionotropic and metabotropic glutamate receptors (mGluR), the latter is classified into three group, based on receptor homology and signaling mechanisms. Among them, recent pharmacological and histochemical studies suggest that the group II mGluR (mGluR2 and mGluR3) plays crucial roles in the control of emotional states. We previously reported that MGS0039, a selective group II mGluR antagonist, exhibited dose-dependent antidepressant-like effects in some animal models. However, the mechanism by which group II mGluR antagonists exhibit such effects is still unclear. In the present two studies, we examined neuropharmacological effects of group II mGluR antagonists on monoaminergic neurons. In an electrophysiological study, MGS0039 dose-dependently and significantly increased the firing rate of dorsal raphe nucleus (DRN) serotonergic neurons. LY341495, another group II mGluR antagonist, also increased DRN serotonergic neural activity significantly. Consistent with the findings of this electrophysiological study, MGS0039 significantly increased extracellular level of serotonin in rat medial prefrontal cortex in a microdialysis study. In contrast, MGS0039 had no effect on the activity of locus coeruleus noradrenergic neurons. These findings suggest that modulation of serotonergic neuron might be, at least in part, responsible for the antidepressant-like effects of group II mGluR antagonists.

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.

Efficacy of a glycine transporter 1 inhibitor TASP0315003 in animal models of cognitive dysfunction and negative symptoms of schizophrenia.

RATIONALE: Since the hypofunction of the N-methyl-D-aspartate (NMDA) receptor is known to be involved in the pathophysiology of schizophrenia, the enhancement of NMDA receptor function through glycine modulatory sites is expected to be a useful approach for the treatment of schizophrenia. OBJECTIVES: We investigated the efficacy of a glycine transporter 1 (GlyT1) inhibitor that potentiates NMDA receptor function by increasing synaptic glycine levels in animal models for cognitive dysfunction and negative symptoms, both of which are poorly managed by current antipsychotics. RESULTS: A newly synthesized GlyT1 inhibitor, 3-chloro-N-{(S)-[3-(1-ethyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide (TASP0315003) significantly improved cognitive deficit induced by MK-801 in the object recognition test in rats. Likewise, TASP0315003 significantly improved MK-801 impaired cognition in the social recognition test in rats and also enhanced social memory in treatment-naïve rats. In addition, repeated phencyclidine (PCP) treatment reduced the social interaction of paired mice, which may reflect negative symptoms such as social withdrawal, and both acute and sub-chronic treatment with TASP0315003 reversed the reduction in social interaction induced by PCP. Moreover, TASP0315003 additionally exhibited an antidepressant effect in the forced swimming test in rats. In contrast, TASP0315003 did not affect spontaneous locomotor activity or rotarod performance and did not induce catalepsy, indicating that TASP0315003 does not cause sedation or motor dysfunction, which is sometimes observed with the use of current antipsychotics. CONCLUSIONS: These results suggest that GlyT1 inhibitors including TASP0315003 may be useful for the treatment of cognitive dysfunction and the negative symptoms of schizophrenia without having undesirable central nervous system side effects.

Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture.

The non-essential amino acid L-serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L-serine biosynthesis enzyme 3-phosphoglycerate dehydrogenase in them. In the present study, we investigated L-[(3)H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L-[(3)H]serine uptake was dependent on temperature and Na(+) ions, and exhibited a single component of high-affinity uptake sites (K(m)=15.0 and 17.2 micro M for neurons and astrocytes, respectively). Kinetic analysis of L-[(3)H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L-[(3)H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L-alanine, L-serine, L-cysteine, and L-threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L-valine and L-proline also inhibited L-[(3)H]serine uptake. Neither alpha-methyl aminoisobutyric acid (a system A-specific substrate) nor 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (a system L-specific substrate) inhibited the uptake of L-[(3)H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L-serine by using a Na(+)-dependent, high-affinity transport system, operating predominantly through the ASCT1 transporter subtype.

MGS0039: a potent and selective group II metabotropic glutamate receptor antagonist with antidepressant-like activity.

The present study describes the pharmacological profile of (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039), a novel group II mGluR antagonist. MGS0039 showed high affinity for both mGluR2 (Ki = 2.2 nM) and mGluR3 (Ki = 4.5 nM), which are comparable to LY341495, another group II mGluR antagonist. MGS0039 attenuated both glutamate-induced inhibition of forskolin-evoked cyclic AMP formation in CHO cells expressing mGluR2 (IC50 = 20 nM) or mGluR3 (IC50 = 24 nM) and glutamate-increased [35S]GTPgammaS binding to mGluR2 (pA2 = 8.2), which means that MGS0039 acts as an antagonist. MGS0039 shifted the dose-response curve of glutamate-increased [35S]GTPgammaS binding rightward without altering the maximal response, and thereby indicating competitive antagonism. MGS0039 showed no significant effects on other mGluRs as well as the other receptors and transporters we studied. MGS0039 (0.3-3 mg/kg, i.p.) as well as LY341495 (0.1-3 mg/kg, i.p.) had dose-dependent antidepressant-like effects in the rat forced swim test and in the mouse tail suspension test. In contrast, MGS0039 (0.3-3 mg/kg, i.p.) had no apparent effect in the rat social interaction test and in the rat elevated plus-maze. These results indicate that MGS0039 is a potent and selective antagonist of group II mGluR, and that group II mGluR antagonists, like MGS0039, have an antidepressant-like potential in experimental animal models.

Possible involvement of induction of brain-derived neurotrophic factor in the neuroprotective effect of a 5-phenylpyrimidine derivative.

When primary cortical neurons prepared from the brains of rat embryos (E18) were cultured in the absence of serum, most of the neurons died after 3 days in vitro. We used this model to discover compounds which support neuronal survival, and found that a new 5-phenylpyrimidine derivative named FU248 (2-amino-5-(2,4-dichlorophenyl) pyrimidine) inhibited the neuronal cell death in a dose-dependent manner up to 1 microg/mL. Semiquantitative RT-PCR analysis revealed that an exposure of the primary cortical neurons to 1 microg/mL of FU248 transiently and significantly enhanced the expression of genes including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). The enhancement of the gene expression was maximal 6 hr after the addition of FU248, and the expression returned to the basal level after 24 hr. Expression of neurotrophin-4 was not detectable throughout the experimental period. The amount of the transcript for BDNF was approximately nine times and sixteen times more abundant than those for NT-3 and NGF, respectively (t=6 hr). Moreover, an anti-BDNF antibody suppressed the effect of FU248, whereas the control antibody did not show any effects on the neuronal survival. These findings strongly suggest that FU248 exerts its neuroprotective effect, at least in part, through induction of BDNF.

XIB4035, a novel nonpeptidyl small molecule agonist for GFRalpha-1.

We investigated the agonistic activities of N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035), at the glial cell line-derived neurotrophic factor (GDNF) family receptoralpha-1(GFRalpha-1) in Neuro-2A cells, a mouse neuroblastoma cell line which is a suitable model for investigating functions mediated through GFRalpha-1. XIB4035 concentration-dependently inhibited [(125)I]GDNF binding in Neuro-2A cells with an IC(50) of 10.4 microM. GDNF induced autophosphorylation of Ret protein, and promoted neurite outgrowth in Neuro-2A cells. XIB4035, like GDNF, induced Ret autophosphorylation in the Neuro-2A cells. Moreover, XIB4035 promoted neurite outgrowth in a concentration-dependent manner. These results show that XIB4035 may act as an agonist at GFRalpha-1 receptor complex, and mimic neurotrophic effects of GDNF in Neuro-2A cells. This is an interesting finding showing that a nonpeptidyl small molecule is capable of inducing activation of a receptor that normally bind a relatively large protein ligand such as GDNF.

Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase.

Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease. In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells. The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells. ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media. In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines. Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner. The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity. These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells. The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.

Cocaine- and amphetamine-regulated transcript peptide produces anxiety-like behavior in rodents.

Cocaine- and amphetamine-regulated transcript (CART) peptide (CART-(55-102)) is involved in the suppression of food intake. We now report that CART-(55-102) is involved in anxiety in rodents. Intracerebroventricularly administered CART-(55-102) as well as intraperitoneal administration of N-methyl-beta-carboline-3-carboxamide (FG-7142), a selective GABA(A)/benzodiazepine receptor inverse agonist, reduced time spent in the open arms in the elevated plus-maze task in mice. CART-(55-102)-induced anxiogenic-like behavior in this task was attenuated by widely prescribed anxiolytics such as diazepam and buspirone. Likewise, CART-(55-102) and FG-7142 significantly reduced social interaction in mice. Both diazepam and buspirone significantly reversed CART-(55-102)-induced anxiogenic-like behavior in social interaction tests. By contrast, another biologically active CART peptide, CART-(62-102), was without effect in the elevated plus-maze task in mice. Moreover, intracerebroventricular administration of CART-(55-102) markedly increased the firing rate of locus coeruleus neurons in single unit recording in anesthetized rats. As CART-(55-102) produced anxiety-like effects in rodents, this peptide may possibly be involved in anxiety and stress-related behavior.

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.

Electrophysiological effects of melanocortin receptor ligands on neuronal activities of monoaminergic neurons in rats.

The melanocortin-4 (MC4) receptor may possibly be involved in stress and stress-related behavior. In the present study, we examined effects of an intracerebroventricular injection of the MC4 receptor agonist, Ac-[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH 4-10-NH2 (MT II), and the MC4 receptor inverse agonist, Agouti-related protein (AGRP), on dorsal raphe nucleus (DRN) serotonergic neurons and the locus coeruleus (LC) noradrenergic neurons, both of which are brain neuronal systems related to responses to stress. The firing rate of DRN serotonergic neurons was increased by MTII, while AGRP had a lack of effect on the firing rate of DRN serotonergic neurons. In comparison with the DRN, MTII significantly reduced the firing rate of LC noradrenergic neurons, while AGRP increased LC neuronal activity. These findings suggest that MC4 receptor ligands differently regulate serotonergic and noradrenergic neuronal systems. The MC4 receptor mediating multiple regulation on the monoaminergic neuronal system may, in part, relate to stress responses (anxiety and/or depressive behavior).

Monkey corticotropin-releasing factor1 receptor: Complementary DNA cloning and pharmacological characterization.

The full-length complementary DNA (cDNA) of monkey corticotropin-releasing factor type 1 (CRF1) receptor was isolated from a rhesus monkey (Macaca mulatta) amygdala cDNA library. The cloned monkey CRF1 receptor cDNA has 2,374 bp with an open reading frame encoding a 415-amino acid protein. The sequence of the monkey CRF1 receptor cDNA showed a high degree of sequence identity with other species of CRF1 receptors, and being 99.5% identical to human CRF1 receptors. When monkey CRF1 was expressed into COS-7 cells, high specific binding of [125I]-ovine CRF was observed. CRF and CRF-related peptides inhibited [125I]-ovine CRF binding in a concentration-dependent manner. IC50 values of ovine CRF, human/rat CRF, sauvagine and urotensin I were 23.5 +/- 7.4, 22.7 +/- 10.8, 27.5 +/- 12.3 and 14.2 +/- 7.0 nM, respectively. CRF1 receptor specific antagonists, such as CP-154,526, SC241 and CRA1000, also inhibited the [125I]-ovine CRF binding, with IC50 values of 3.9 +/- 0.4, 43.5 +/- 8.0 and 19.8 +/- 2.0 nM, respectively. GTP and its nonhydrolyzed analogue, GTPgammaS, reduced [125I]-ovine CRF binding, while ATP had a negligible effect, thereby indicating that the monkey CRF1 receptor belongs to a family of G-protein coupled receptors. CRF and its related peptides increased cyclic AMP formation concentration-dependently in COS-7 cells transiently expressing the monkey CRF1 receptor. Monkey CRF1 was expressed abundantly in the pituitary, cerebral cortex, hippocampus, amygdala and cerebellum. Thus the monkey CRF1 receptor and the human CRF1 receptor have similar molecular and pharmacological characteristics.

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.