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3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

A series of N -heterocyclic pyridinone catechol- O -methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and c log P , were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Objective: Preclinical models, receptor localization, and genetic linkage data support the role of D4 receptors in the etiology of ADHD. This proof-of-concept study was designed to evaluate MK-0929, a selective D4 receptor antagonist as treatment for adult ADHD. Method: A randomized, double-blind, placebo-controlled, crossover study was conducted in adults with primary ADHD. The primary end point was changed from baseline in total score on the Adult ADHD Investigator Symptom Rating Scale following a 4-week treatment regimen. Additional measures included Clinical Global Impression–Severity Scale, Hospital Anxiety and Depression Scale, and Brown Attention Deficit Disorder Scale and D4 genotype analysis. Results: No statistically significant treatment differences were found between MK-0929 and placebo in any of the primary or secondary assessments. Conclusion: Results from this study suggest that blockade of the D4 receptor alone is not efficacious in the treatment of adult ADHD.

Background Recent evidence suggests an involvement of T-type calcium channels in the effects of drugs of abuse. Methods We examined the influence of the novel, potent, and selective T-type calcium channel antagonist [2-(4-Cyclopropylphenyl)-N-((1R)-1-{5-[2,2,2-trifluoroethyl]oxo}pyridine-2-yl)ethyl]acetamide] (TTA-A2) (.3, 1, or 3 mg/kg) on motivation for nicotine, as measured by nicotine self-administration on a progressive ratio (PR) schedule, and nicotine- and cue-induced reinstatement for a response previously reinforced with nicotine delivery ( n = 11 or 12 Long Evans rats/group). Furthermore, we examined the specificity of the TTA-A2 effects by characterizing its influence on PR responding for food (in the absence or presence of nicotine-potentiated responding), food- versus nicotine-induced cue-potentiated reinstatement for a response previously reinforced by food administration ( n = 11 or 12 Wistar Hannover rats/group), and its ability to induce a conditioned place aversion. Results TTA-A2 dose-dependently decreased self-administration of nicotine on a PR schedule and the ability of both nicotine and a cue paired with nicotine to reinstate responding. The effects were specific for nicotine's incentive motivational properties, as TTA-A2 did not influence responding for food on a PR schedule but did attenuate the ability of nicotine to potentiate responding for food. Likewise, TTA-A2 did not alter food-induced cue-potentiated reinstatement for a response previously reinforced by food but did decrease nicotine-induced cue-potentiated reinstatement. Finally, TTA-A2 did not produce an aversive state, as indicated by a lack of ability to induce conditioned place aversion. Conclusions These data suggest that T-type calcium channel antagonists have potential for alleviating nicotine addiction by selectively decreasing the incentive motivational properties of nicotine.

Novel oxazolobenzimidazoles are described as potent and selective positive allosteric modulators of the metabotropic glutamate receptor 2. The discovery of this class and optimization of its physical and pharmacokinetic properties led to the identification of potent and orally bioavailable compounds (20 and 21) as advanced leads. Compound 20 (TBPCOB) was shown to have robust activity in a PCP-induced hyperlocomotion model in rat, an assay responsive to clinical antipsychotic treatments for schizophrenia.

Hit to lead optimization of (5R)-5-hexyl-3-phenyl-1,3-oxazolidin-2-one as a positive allosteric modulator of mGluR2 is described. Improvements in potency and metabolic stability were achieved through SAR on both ends of the oxazolidinone. An optimized lead compound was found to be brain penetrant and active in a rat ketamine-induced hyperlocomotion model for antipsychotic activity.

Rationale Compounds which decrease NMDA receptor functioning, such as PCP and ketamine have abuse liability, whereas co-agonists of the NMDA receptor attenuate some of the behavioral and neurochemical effects of stimulant drugs. Here we examined the effects of a glycine transporter (GlyT1) inhibitor, which elevates glycine and hence NMDA signaling, on the behavioral effects of nicotine. Objectives To examine the influence of a novel potent, selective, and brain penetrant GlyT1 inhibitor, compound 5 {(2-chloro-N-[1-(ethylsulfonyl)-4-isobutylpiperidin-4-yl]methyl)}-4-(trifluoromethyl)benzamide; human IC50 = 22 nM; rat = 30 nM), on nicotine-induced potentiation of progressive ratio responding for a food reward and nicotine- and food-induced cue-potentiated reinstatement for a response previously paired with sucrose. Results Compound 5 (33 mg/kg; p.o.; achieving ∼62% GlyT1 blockade) significantly attenuated nicotine-, but not food-induced cue-potentiated reinstatement for a response previously paired with sucrose whereas a lower dose (11 mg/kg, which achieved ∼34% GlyT1 blockade) did not. The effect of the higher dose was similar to that observed for mecamylamine (1 mg/kg i.p.), a non-selective nicotinic receptor antagonist. Conclusions These results suggest that compound 5 influences the ability of nicotine to promote reinstatement in the presence of a cue embedded with incentive motivation. Given the hypothesized contribution of reinstatement and conditioned stimuli to drug abuse and relapse, these findings suggest that GlyT1 inhibitors could have utility for treating nicotine addiction.

In the search for strategies to treat schizophrenia, attention has focused on enhancing NMDA receptor function. In vitro experiments show that metabotropic glutamate 5 receptor (mGluR5) activation enhances NMDA receptor activity, and in vivo experiments indicate that mGluR5 positive allosteric modulators (PAMs) are effective in preclinical assays measuring antipsychotic potential and cognition. Here we characterized the dose-effect function of CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide), an mGluR5 PAM, on novel object recognition memory in unimpaired Wistar Hannover rats (0, 10 or 30 mg/kg CDPPB) and animals with an MK-801-induced deficit (0, 3, 10, or 30 mg/kg CDPPB). In each experiment compound was given 30 min prior to the first exposure in order to affect acquisition/consolidation of the memory. In both cases, an inverted-U-shaped dose-effect function was observed, with lower doses improving recognition but higher doses having no effect. We then examined the effects of CDPPB (0, 3, 10, or 30 mg/kg) on markers of synaptic plasticity in prefrontal cortex and hippocampus, focusing on the expression and phosphorylation status of proteins involved in NMDA related signaling, including the NMDA receptor subunits NR1 and NR2B, the AMPA receptor subunit GluR1, alphaCa((2+))/CaM dependent Ser-Thr kinases II (alphaCaMKII), and the transcription factor CREB. Expression and phosphorylation of many of these proteins, particularly in the prefrontal cortex, were also characterized by an inverted-U-shaped dose-effect function. Taken together, these findings show that mGluR5 activation enhances NMDA receptor function and markers of neuronal plasticity commensurate with improvements in recognition memory. However, the effects of CDPPB are heavily dependent on dose, with higher doses being ineffective in improving recognition memory and producing downstream effects consistent with heightened NMDA receptor activation. These findings may have important implications for the development of mGluR5 PAMs to treat schizophrenia.

It was recently reported that administration of the metabotropic glutamate 2 and 3 (mGlu2/3) receptor agonist prodrug LY2140023 to schizophrenic patients decreased positive symptoms. However, at the single, potentially suboptimal, dose that was tested, LY2140023 trended towards being inferior to olanzapine on several indices of efficacy within the Positive and Negative Syndrome Scale.In this study, we examined whether the antipsychotic potential of mGlu2/3 receptor agonism can be enhanced with 5-HT(2A) receptor antagonism.Specifically, we characterized the effects of co-administering submaximally effective doses of the 5-HT(2A) receptor antagonist M100907 (0.2 mg/kg) and the mGlu2/3 receptor agonist LY379268 (1 mg/kg) on amphetamine-induced and MK-801-induced psychomotor activity in rats, an assay sensitive to antipsychotics. We also determined the effects of co-administering these two compounds on MK-801-induced dopamine and norepinephrine efflux in the nucleus accumbens (NAc).At the submaximally effective doses tested, the effects of M100907 and LY379268 on amphetamine-induced and MK-801-induced psychomotor activity were significantly greater when given together than when given separately. Furthermore, coadministration of these doses of M100907 and LY379268 reduced MK-801-induced dopamine efflux in the NAc. This effect on dopamine release was not observed with the administration of either compound alone, even at higher doses that attenuated MK-801-induced psychomotor activity.Our results suggest that a single compound having both mGlu2/3 receptor agonist and 5-HT(2A) receptor antagonist activity, or coadministration of two compounds selective for these receptors, could be superior in terms of efficacy and/or reduced side-effect liability relative to an mGlu2/3 receptor agonist alone.

Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.

Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 µm. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 µm, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.

The orexinergic system has been implicated in a number of behaviors, including reward and incentive motivation. Orexin 1 receptor antagonism has been reported to reduce drug self-administration, conditioned place preference, and reinstatement in rodents, but the role of the orexin 2 receptor is unclear. Here we evaluated the impact of the novel and selective orexin 2 receptor antagonist, 2-SORA 18, on motivation for nicotine as measured by responding on a progressive ratio schedule, as well as cue-induced reinstatement of a response previously associated with nicotine reward, and nicotine-induced reinstatement. 2-SORA 18 demonstrated selective effects on these behaviors. Specifically, doses up to 60 mg/kg 2-SORA 18 were without significant effect on nicotine self-administration or nicotine-induced reinstatement, but doses as low as 15 mg/kg 2-SORA 18 completely blocked cue-induced reinstatement. These findings indicate that orexin 2 receptor antagonism might have utility for attenuating relapse, particularly for patients sensitive to environmental stimuli associated with drug taking.

T-type calcium channels are important in burst firing and expressed in brain regions implicated in schizophrenia. Therefore, we examined the effects of novel selective T-type calcium channel antagonists in preclinical assays predictive of antipsychotic-like activity. TTA-A2 blocked the psychostimulant effects of amphetamine and MK-801 and decreased conditioned avoidance responding. These effects appeared mechanism based, rather than compound specific, as two structurally dissimilar T-type antagonists also reduced amphetamine-induced psychomotor activity. Importantly, the ability to reduce amphetamine's effects was maintained following 20 days pre-treatment with TTA-A2. To explore the neural substrates mediating the observed behavioral effects, we examined the influence of TTA-A2 on amphetamine-induced c-fos expression as well as basal and stimulant-evoked dopamine and glutamate release in the nucleus accumbens. TTA-A2 decreased amphetamine-induced c-fos expression as well as MK-801-induced, but not basal, glutamate levels in the nucleus accumbens. Basal, amphetamine- and MK-801-induced dopamine efflux was altered. These findings suggest that T-type calcium channel antagonism could represent a novel mechanism for treating schizophrenia.

The 'NMDA hypofunction hypothesis of schizophrenia' can be tested in a number of ways. DAO is the enzyme primarily responsible for the metabolism of d-serine, a co-agonist for the NMDA receptor. We identified novel DAO inhibitors, in particular, acid 1, which demonstrated moderate potency for DAO in vitro and ex vivo, and raised plasma d-serine levels after dosing ip to rats. In parallel, analogues were prepared to survey the SARs of 1.

The postnatal development of the binding of the GABA(A) receptor agonist [(3)H]gaboxadol in rat brain was investigated. Using brain tissue from rats obtained at postnatal days 1, 10, 25, and >25 (adult), the binding of [(3)H]gaboxadol and the benzodiazepine [(3)H]flunitrazepam to GABA(A) receptors was compared in homogenate binding assays and quantitative receptor autoradiography. Kinetic and equilibrium data obtained in homogenate binding studies revealed two different [(3)H]gaboxadol affinities. A kinetically derived K(D) of 3.7 nM in adult cerebellum, calculated from the association and dissociation rate constants k(on) (1.45 x 10(8) M(-1) min(-1)) and k(off) (0.54 min(-1)) was contrasted by an equilibrium K(D) of 38.6 nM, obtained by homologous competition experiments. Quantitative analysis of autoradiographic data revealed an increase in specific [(3)H]gaboxadol binding sites during brain development, which resembles the anatomical and temporal pattern of the postnatal expression of the extrasynaptic delta subunit of GABA(A) receptors. In conclusion, by the radioligand binding data obtained on native tissue, binding of gaboxadol to GABA(A) receptors located outside the synaptic junctions could be postulated.

Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 micro m. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 micro m, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.