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Screening of 100 acylsulfonamides from the Bristol-Myers Squibb compound collection identified the C3-cyclohexyl indole 6 as a potent Nav1.7 inhibitor. Replacement of the C2 furanyl ring of 6 with a heteroaryl moiety or truncation of this group led to the identification of 4 analogs with hNav1.7 IC50 values under 50 nM. Fluorine substitution of the truncated compound 12 led to 34 with improved potency and isoform selectivity. The inverted indole 36 also maintained good activity. Both 34 and 36 exhibited favorable CYP inhibition profiles, good membrane permeability and a low efflux ratio and, therefore, represent new leads in the search for potent and selective Nav1.7 inhibitors to treat pain.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Nav1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and NaV1.7 potency associated with 29.

Replacement of the piperidine ring in the lead benzenesulfonamide Nav1.7 inhibitor 1 with a weakly basic morpholine core resulted in a significant reduction in Nav1.7 inhibitory activity, but the activity was restored by shortening the linkage from methyleneoxy to oxygen. These efforts led to a series of morpholine-based aryl sulfonamides as isoform-selective Nav1.7 inhibitors. This report describes the synthesis and SAR of these analogs.

Major depressive disorder is a leading cause of disability globally. Improvements in the efficacy of antidepressant therapy are needed as a high proportion (>40%) of individuals with major depressive disorder fail to respond adequately to current treatments. The non-selective N-methyl-D-aspartate receptor channel blocker, (±)-ketamine, has been reported to produce a rapid and long-lasting antidepressant response in treatment-resistant major depressive disorder patients, which provides a unique opportunity for investigation of mechanisms that mediate its therapeutic effect. Efforts have also focused on the development of selective N-methyl-D-aspartate receptor subtype 2B antagonists which may retain antidepressant activity but have lower potential for dissociative/psychotomimetic effects. In the present study, we examined the central nervous system effects of acute, intravenous administration of (±)-ketamine or the N-methyl-D-aspartate receptor subtype 2B antagonist, traxoprodil, in awake rats using pharmacological magnetic resonance imaging. The study contained five treatment groups: vehicle, 3 mg/kg (±)-ketamine, and three doses of traxoprodil (0.3 mg/kg, 5 mg/kg, and 15 mg/kg). Non-linear model fitting was performed on the temporal hemodynamic pharmacological magnetic resonance imaging data to generate brain activation maps as well as regional responses based on blood oxygen level dependent signal changes for group analysis. Traxoprodil at 5 mg/kg and 15 mg/kg produced a dose-dependent pharmacological magnetic resonance imaging signal in rat forebrain regions with both doses achieving >80% N-methyl-D-aspartate receptor subtype 2B occupancy determined by ex vivo [3H]Ro 25-6981 binding. The middle dose of traxoprodil (5 mg/kg) generated region-specific activations in medial prefrontal cortex, ventral orbital cortex, and anterior cingulate cortex whereas the high dose (15 mg/kg) produced a widespread pharmacological magnetic resonance imaging response in both cortical and subcortical brain regions which was similar to that produced by (±)-ketamine (3 mg/kg, intravenous).

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169) and the phosphate prodrug 4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl dihydrogen phosphate (BMS-986163) were identified from a drug discovery effort focused on the development of novel, intravenous glutamate N-methyl-d-aspartate 2B receptor (GluN2B) negative allosteric modulators (NAMs) for treatment-resistant depression (TRD). BMS-986169 showed high binding affinity for the GluN2B subunit allosteric modulatory site (Ki = 4.03-6.3 nM) and selectively inhibited GluN2B receptor function in Xenopus oocytes expressing human N-methyl-d-aspartate receptor subtypes (IC50 = 24.1 nM). BMS-986169 weakly inhibited human ether-a-go-go-related gene channel activity (IC50 = 28.4 μM) and had negligible activity in an assay panel containing 40 additional pharmacological targets. Intravenous administration of BMS-986169 or BMS-986163 dose-dependently increased GluN2B receptor occupancy and inhibited in vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding, confirming target engagement and effective cleavage of the prodrug. BMS-986169 reduced immobility in the mouse forced swim test, an effect similar to intravenous ketamine treatment. Decreased novelty suppressed feeding latency, and increased ex vivo hippocampal long-term potentiation was also seen 24 hours after acute BMS-986163 or BMS-986169 administration. BMS-986169 did not produce ketamine-like hyperlocomotion or abnormal behaviors in mice or cynomolgus monkeys but did produce a transient working memory impairment in monkeys that was closely related to plasma exposure. Finally, BMS-986163 produced robust changes in the quantitative electroencephalogram power band distribution, a translational measure that can be used to assess pharmacodynamic activity in healthy humans. Due to the poor aqueous solubility of BMS-986169, BMS-986163 was selected as the lead GluN2B NAM candidate for further evaluation as a novel intravenous agent for TRD.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

Adaptor-associated kinase 1 (AAK1), a member of the Ark1/Prk1 family of serine/threonine kinases, plays a role in modulating clatherin coated endocytosis of specific surface receptors. We have demonstrated that AAK1 inhibitors are efficacious in rodent models of neuropathic pain (Kostich et al., 2016). Here we have characterized the binding properties and distribution pattern of the tritiated AAK1 radioligand, [3H]BMT-046091, in rodents and cynomolgus monkeys, and used the radioligand to measure the brain target occupancy following systemic administration of AAK1 inhibitors. We have found that [3H]BMT-046091 is potent and selective AAK1 inhibitor. It inhibits AAK1 phosphorylation of a peptide derived from a physiologic substrate, the μ2 subunit of the adaptor protein complex, with an IC50 value of 2.8 nM, and is inactive at >5 μM in a panel of functional or binding assays for receptors, transporters and enzymes. [3H]BMT-046091 binding in the brain is absent in the AAK1 knockout mouse, and is displaceable with a high concentration of AAK1 inhibitors in wild type mice. Specific [3H]BMT-046091 binding is widespread in the brain and spinal cord with the highest density in the cortex, hippocampus, amygdala, striatum and thalamus. In the spinal cord, [3H]BMT-046091 binding appears enriched in the dorsal horn superficial layers. Oral administration of LP-935509, an AAK1 inhibitor, results in a dose-dependent occupation of AAK1 binding sites in the brain and spinal cord. The increase in AAK1 binding site occupancy by LP-935509 correlates with the decrease in antinociceptive responses in the rat chronic constriction injury model of neuropathic pain.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Background: A significant proportion of patients suffering from major depression fail to remit following treatment and develop treatment-resistant depression. Developing novel treatments requires animal models with good predictive validity. MRL/lpr mice, an established model of systemic lupus erythematosus, show depression–like behavior. Aims: We evaluated responses to classical antidepressants, and associated immunological and biochemical changes in MRL/lpr mice. Methods and results: MRL/lpr mice showed increased immobility in the forced swim test, decreased wheel running and sucrose preference when compared with the controls, MRL/MpJ mice. In MRL/lpr mice, acute fluoxetine (30 mg/kg, intraperitoneally (i.p.)), imipramine (10 mg/kg, i.p.) or duloxetine (10 mg/kg, i.p.) did not decrease the immobility time in the Forced Swim Test. Interestingly, acute administration of combinations of olanzapine (0.03 mg/kg, subcutaneously)+fluoxetine (30 mg/kg, i.p.) or bupropion (10 mg/kg, i.p.)+fluoxetine (30 mg/kg, i.p.) retained efficacy. A single dose of ketamine but not three weeks of imipramine (10 mg/kg, i.p.) or escitalopram (5 mg/kg, i.p.) treatment in MRL/lpr mice restored sucrose preference. Further, we evaluated inflammatory, immune-mediated and neuronal mechanisms. In MRL/lpr mice, there was an increase in autoantibodies’ titers, [3H]PK11195 binding and immune complex deposition. There was a significant infiltration of the brain by macrophages, neutrophils and T-lymphocytes. p11 mRNA expression was decreased in the prefrontal cortex. Further, there was an increase in the 5-HT2aR expression, plasma corticosterone and indoleamine 2,3-dioxygenase activity. Conclusion: In summary, the MRL/lpr mice could be a useful model for Treatment Resistant Depression associated with immune dysfunction with potential to expedite antidepressant drug discovery.

Screening of 100 acylsulfonamides from the Bristol-Myers Squibb compound collection identified the C3-cyclohexyl indole 6 as a potent Na

N-methyl-D-aspartate (NMDA) receptor antagonists, including open channel blockers and GluN2B receptor subtype selective antagonists, have been developed for the treatment of depression. The current study investigated effects of systemically administered NMDA channel blockers and GluN2B receptor antagonists on NMDA receptor activity in rodents using in vivo [(3)H]MK-801 binding. The receptor occupancy of GluN2B antagonists was measured using ex vivo [(3)H]Ro 25-6981 binding. Ketamine, a NMDA receptor channel blocker, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~100%. The complete inhibition of in vivo [(3)H]MK-801 binding was also observed with NMDA receptor channel blockers, AZD6765 (Lanicemine) and MK-801 (Dizocilpine). CP-101,606 (Traxoprodil), a GluN2B antagonist, produced a dose/exposure- and time-dependent inhibition of in vivo [(3)H]MK-801 binding that was maximal at ~60%. Partial inhibition was also observed with other GluN2B antagonists including MK-0657 (CERC-301), EVT-101, Ro 25-6981 and radiprodil. For all GluN2B antagonists tested, partial [(3)H]MK-801 binding inhibition was achieved at doses saturating GluN2B receptor occupancy. Combined treatment with ketamine (10mg/kg, i.p.) and Ro 25-6981(10mg/kg, i.p.) produced a level of inhibition of in vivo [(3)H]MK-801 binding that was similar to treatment with either agent alone. In conclusion, this in vivo [(3)H]MK-801 binding study shows that NMDA receptor activity in the rodent forebrain can be inhibited completely by channel blockers, but only partially (~60%) by GluN2B receptor antagonists. At doses effective in preclinical models of depression, ketamine may preferentially inhibit the same population of NMDA receptors as Ro 25-6981, namely those containing the GluN2B subunit.

Replacement of the piperidine ring in the lead benzenesulfonamide Na

The Na

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Na

Sibutramine was formerly marketed as an anti-obesity agent. The current study investigated the relationships between monoamine reuptake site occupancy for sibutramine and both its antidepressant-like efficacy and thermogenic effects. Sibutramine’s effects on locomotor activity (LMA) and food intake were also evaluated. Sibutramine occupied monoamine reuptake binding sites with the rank order of potency of NET>SERT>DAT; at 10 mg/kg, po, occupancy was 95% NET, 81% SERT and 73% DAT. Sibutramine produced antidepressant-like behavior in the forced swim test; at the lowest effective dose (3 mg/kg, po) occupancy was 61%, 90% and 23% at SERT, NET and DAT sites, respectively. Sibutramine also increased body core temperature in a dose- and time-dependent manner; at the lowest effective dose (30 mg/kg) SERT, NET and DAT occupancies were respectively 78%, 86% and 59%. A significant decrease in food consumption was observed at 3 and 10 mg/kg, po. LMA was increased at ≥10 mg/kg, sc. The relationship between efficacy in the FST and occupancy was also determined for citalopram, fluoxetine and reboxetine. Similarly, the relationship between thermogenesis and target occupancy for several single or double/triple reuptake inhibitors was measured and showed that >40–50% DAT binding was required for thermogenesis. Thermogenesis was blocked by the D1 antagonist SCH39166 (3 mg/kg, sc). Our findings indicate that the antidepressant-like effect of sibutramine may result from additive or synergistic actions on the three reuptake binding targets. At higher doses, sibutramine produces thermogenesis; DAT inhibition and activation of dopamine D1 receptors are required for this effect.

Introduction A radioligand for measuring the density of corticotropin-releasing factor subtype-1 receptors (CRF 1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF 1 receptor ligands based on a core 3-(phenylamino)‐pyrazin-2(1 H )-one scaffold. Methods CRF 1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF 1 receptors (displacement of [ 125 I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF 1 receptor density ( B max ) was assessed in rhesus brain cortical and cerebellum membranes with the CRF 1 receptor ligand, [ 3 H]BMS-728300. Results The three ligands selected for development showed high binding affinity ( IC 50 values, 0.3–8 nM) at CRF 1 receptors and moderate lipophilicity ( LogD , 2.8–4.4). [ 3 H]BMS-728300 and the two 18 F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF 1 receptor B max in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [ 18 F]BMS-721313, [ 18 F]BMS-732098 and [ 11 C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro . Conclusions Candidate CRF 1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF 1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF 1 receptors.

The α7 nicotinic acetylcholine receptor is thought to play an important role in human cognition. Here we describe the in vivo effects of BMS-902483, a selective potent α7 nicotinic acetylcholine receptor partial agonist, in relationship to α7 nicotinic acetylcholine receptor occupancy. BMS-902483 has low nanomolar affinity for rat and human α7 nicotinic acetylcholine receptors and elicits currents in cells expressing human or rat α7 nicotinic acetylcholine receptors that are about 60% of the maximal acetylcholine response. BMS-902483 improved 24h novel object recognition memory in mice with a minimal effective dose (MED) of 0.1mg/kg and reversed MK-801-induced deficits in a rat attentional set-shifting model of executive function with an MED of 3mg/kg. Enhancement of novel object recognition was blocked by the silent α7 nicotinic acetylcholine receptor agonist, NS6740, demonstrating that activity of BMS-902483 was mediated by α7 nicotinic acetylcholine receptors. BMS-902483 also reversed ketamine-induced deficits in auditory gating in rats, and enhanced ex vivo hippocampal long-term potentiation examined 24h after dosing in mice. Results from an ex vivo brain homogenate binding assay showed that α7 receptor occupancy ranged from 64% (novel object recognition) to ~90% (set shift and gating) at the MED for behavioral and sensory processing effects of BMS-902483.

The known interactions between the serotonergic and neurokinin systems suggest that serotonin reuptake inhibitor (SSRIs) efficacy may be improved by neurokinin-1 receptor (NK1R) antagonism. In the current studies combination of a subeffective dose of an SSRI (0.3 mg/kg fluoxetine or 0.03 mg/kg citalopram) with a subeffective dose of an NK1R antagonist (0.3 mg/kg aprepitant or 1 mg/kg CP-122,721) produced efficacy in the gerbil forced swim test (FST). Serotonin transporter (SERT) occupancy produced by 1 mg/kg fluoxetine (lowest efficacious dose) was 52 ± 5% and was reduced to 29 ± 4% at 0.3 mg/kg, a dose that was efficacious in combination with 0.3 mg/kg aprepitant or 1 mg/kg CP-122,721; the corresponding NK1R occupancies were 79 ± 4% and 61 ± 4% for aprepitant and CP-122,721, respectively. For citalopram, SERT occupancy at the lowest efficacious dose (0.1 mg/kg) was 50 ± 4% and was reduced to 20 ± 5% at 0.03 mg/kg, a dose that was efficacious when combined with aprepitant (0.3 mg/kg). Aprepitant (10 mg/kg) augmented the serotonin elevation produced by fluoxetine (1 or 10 mg/kg) in the gerbil prefrontal cortex; i.e. NK1R antagonism can modulate serotonin responses. A novel orally-available dual-acting NK1R antagonist/SERT inhibitor BMS-795176 is described; gerbil Ki = 1.4 and 1 nM at NK1R and SERT, respectively. BMS-795176 was efficacious in the gerbil FST; efficacy was observed with 35 ± 3% SERT occupancy and 73 ± 3% NK1R occupancy. The interaction between NK1R antagonism and SERT inhibition to lower the SERT occupancy required for antidepressant-like efficacy suggests that BMS-795176 has the potential to improve efficacy with a reduction in SSRI-associated side effects.

A series of substituted pyridines, ether linked to a phenylpiperidine core were optimized for dual NK(1)/SERT affinity. Optimization based on NK(1)/SERT binding affinities, and minimization of off-target ion channel activity lead to the discovery of compound 44. In vivo evaluation of 44 in the gerbil forced swim test (a depression model), and ex-vivo NK(1)/SERT receptor occupancy data support the potential of a dual acting compound for the treatment of depression.

The alpha7 (α7) nicotinic acetylcholine receptor is a therapeutic target for cognitive disorders. Here we describe 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide (B-973), a novel piperazine-containing molecule that acts as a positive allosteric modulator of the α7 receptor. We characterize the action of B-973 on the α7 receptor using electrophysiology and radioligand binding. At 0.1 mM acetylcholine, 1 μM B-973 potentiated peak acetylcholine-induced currents 6-fold relative to maximal acetylcholine (3 mM) and slowed channel desensitization, resulting in a 6900-fold increase in charge transfer. The EC 50 of B-973 was approximately 0.3 μM at acetylcholine concentrations ranging from 0.03 to 3 mM. At a concentration of 1 μM, B-973 shifted the acetylcholine EC 50 of peak currents from 0.30 mM in control to 0.007 mM. B-973 slowed channel deactivation upon acetylcholine removal (τ=50 s) and increased the affinity of the α7 agonist [ 3 H]A-585539. In the absence of exogenously added acetylcholine, application of B-973 at concentrations >1 μM induced large methyllycaconitine-sensitive currents, suggesting B-973 can function as an Ago-PAM at high concentrations. B-973 will be a useful probe for investigating the biological consequences of increasing α7 receptor activity through allosteric modulation.

The goal of this study was to develop and validate ex vivo binding assays for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, and to use these assays to evaluate the binding site occupancy of triple and double monoamine reuptake inhibitors in rat brains. This study demonstrated that while autoradiographic methods provided anatomic precision and regional resolution, the homogenate binding method for site occupancy assessment yielded comparable sensitivity with markedly improved throughput. For ex vivo binding assays, the reduction of temperature and time during the in vitro process (primarily incubation with a radioligand) markedly decreased the dissociation of test agents from binding sites in brain tissues. This reduction, in turn, minimized the potential for underestimation of site occupancy in vivo especially for test compounds with affinity >10nM. The ratios of measured occupancy ED(50) values (doses at which 50% occupancy occurs) among SERT, NET and DAT sites for duloxetine, venlafaxine, nomifensine, indatraline, DOV 21,947 and DOV 216,303 were consistent with the ratios of the in vitro affinities between these target binding sites. The biological relevance of the monoamine transporter occupancy for these compounds is discussed.

The long lasting antidepressant response seen following acute, i.v. ketamine administration in patients with treatment-resistant depression (TRD) is thought to result from enhanced synaptic plasticity in cortical and hippocampal circuits. Using extracellular field recordings in rat hippocampal slices, we show that a single dose of the non-selective NMDA receptor antagonist ketamine or CP-101,606, a selective antagonist of the NR2B subunit of the NMDA receptor, enhances hippocampal synaptic plasticity induced with high frequency stimulation (HFS) 24h after dosing - a time at which plasma concentrations of the drug are no longer detectable in the animal. These results indicate that acute inhibition of NMDA receptors containing the NR2B subunit can lead to long-lasting changes in hippocampal plasticity.

Bioisosteric replacement studies led to the identification of N -(1-benzo[1,3]dioxol-5-yl-ethyl)-3-(2-chloro-phenyl)-acrylamide (( S )- 3 ) as a highly potent KCNQ2 opener, and 3-(2,6-difluoro-phenyl)- N -[1-(2,3-dihydro-benzofuran-5-yl)-ethyl]-acrylamide (( S )- 4 ), and N -[1-(2,3-dihydro-1 H -indol-5-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide (( S )- 5 ) as highly efficacious KCNQ2 openers. In contrast, their respective R enantiomers showed significantly less or no appreciable KCNQ2 opener activity even at the highest concentration tested (10 μM). Because of its high potency and moderate efficacy as well as its convenient synthesis, (±)- 3 was selected as a reference compound for analyzing efficacies of KCNQ openers in electrophysiology studies. Compounds ( S )- 4 and ( S )- 5 demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices. The synthesis and the KCNQ2 opener activity of these acrylamides are described.

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.

The effect of γ-aminobutyric acid (GABA) on neuronal firing rate in rat suprachiasmatic nucleus (SCN) slices was examined using continuous recording methods. GABA inhibited neuronal discharge during both the subjective day and the subjective night in a concentration-dependent manner characterized by two apparent affinity states. The GABAA receptor agonist muscimol caused potent inhibition regardless of circadian time; repeated applications of the agonist did not reverse the direction of effect. The GABAA receptor antagonists bicuculline and picrotoxin increased excitability when applied during either subjective day or subjective night. A significant increase in GABAA receptor– mediated inhibition, as well as endogenous GABAergic tone, was observed on the second day after slice preparation. The GABAB receptor agonist baclofen inhibited cell firing during subjective day and night, but the GABAB antagonist phaclofen had no significant effect. These data provide additional strong support for a predominantly inhibitory role of GABA in the rat SCN, regardless of the time of application in relation to the circadian rhythm, and demonstrate an important level of plasticity of this system in vitro.

Serotonin 5-HT(1A) receptor antagonists potentiate the effects of serotonin reuptake inhibitors on extracellular serotonin levels in a variety of brain regions. These effects are quite variable, however, with reports indicating potentiations of anywhere from 100-1900%. One factor that might impact the magnitude of such potentiations is the timing of administration of the two agents; reports in which the reuptake inhibitor is given prior to the serotonin receptor antagonist consistently report larger potentiations than reports in which the antagonist is given first. To test this relationship directly, microdialysis and electrophysiology studies were performed to assess the magnitude of increase in extracellular serotonin and changes in cellular activity produced by the serotonin reuptake inhibitor fluoxetine and the 5-HT(1A) receptor antagonist WAY-100635 under various dosing regimens. In microdialysis studies, when WAY-100635 (0.5 mg/kg s.c.) was administered 80 min after fluoxetine (10 mg/kg i.p.) the increase in serotonin was more than twice that observed when the compounds were coadministered. In electrophysiology studies in vivo, WAY-100635 reversed the depression of cell firing produced by fluoxetine when administered 30 min after fluoxetine, but when the two compounds were coadministered, a depression in firing rate was observed comparable to that produced by fluoxetine alone. In contrast, slice recording studies showed that WAY-100635 blocked the effects of fluoxetine regardless of the order of administration. These results indicate that fluoxetine and WAY-100635 can interact in a fashion not predicted by the currently accepted model. It is likely that neuronal circuitry outside of the raphe nuclei underlies this relationship.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that [.gamma]-amino butyric acid (GABA)Areceptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath-or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.

The hormone, melatonin, is produced rhythmically in the vertebrate pineal gland (13). Rhythmic melatonin production plays a critical role in the regulation of reproduction in seasonally breeding mammals (1, 24). In nonmammalian vertebrates, melatonin also plays a major role in the regulation of circadian rhythms (4, 46). While endogenous melatonin appears to play only a subtle role in the regulation of circadian rhythms in mammals, exogenous melatonin influences circadian rhythms in several rodent species and in humans (2, 3, 9, 21, 22, 39, 46, 47). While the magnitude of this phase shifting effect is small for adult mammals, it is nevertheless critical in some situations; e.g., daily melatonin administration appears to be useful for entrainment of non-24-h circadian cycles to the 24-hour day for blind individuals (22). Two high-affinity receptors for melatonin have been identified in mammals (for reviews, see references 33 and 43). These receptors, the Mel1a and Mel1b receptor subtypes, are members of the G protein-coupled receptor superfamily (29, 30). A melatonin receptor-related receptor (also called H9 and GPR50) has sequence homology to the Mel1a and Mel1b receptors but does not bind melatonin (7, 16, 32). In addition to these three members of the gene family in mammals, nonmammalian species have a third high-affinity melatonin receptor subtype, the Mel1c receptor (31). The mammalian Mel1a and Mel1b receptors are also called the MT1 and MT2 receptors, respectively [10]. The inability of this MT nomenclature system to accommodate the nonmammalian Mel1c receptor has resulted in persistence of dual nomenclature systems; here, we use the original nomenclature. The relative importance of the mammalian melatonin receptor subtypes in mediating circadian responses to the hormone is unclear. The circadian clocks of neonatal hamsters are very efficiently set by even a single injection of melatonin, and several physiological and in vitro responses of the suprachiasmatic nucleus (SCN) to melatonin have been observed in hamsters (25, 36, 39, 47). Remarkably, however, the Mel1b receptor gene of several hamster species, including those with robust circadian and reproductive responses to the hormone, does not encode a functional melatonin receptor (47; see also GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY145849","term_id":"24251143","term_text":"AY145849"}}AY145849). Pharmacological studies with mice, however, suggest that the mouse Mel1b receptor mediates circadian responses (9, 19). Other data, derived from mice with targeted disruption of the Mel1a melatonin receptor, indicate that this subtype mediates an acute suppressive effect of melatonin on SCN neuronal firing, and they reveal apparent redundancy of receptor subtypes in mediating the phase shifting response to melatonin (23). To determine whether the mouse Mel1b receptor contributes to responses to the hormone, the mouse Mel1b receptor cDNA was isolated. Finding that the mouse Mel1b receptor cDNA encodes a high-affinity melatonin receptor, we then disrupted the Mel1b receptor gene. Analysis of the mutant mice indicates that the Mel1b receptor encodes a functionally relevant melatonin receptor. The Mel1b receptor appears to mediate a response to the hormone occurring at higher ligand concentrations in mice lacking the Mel1a melatonin receptor.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slicesin vitro(n= 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5–8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration–response relationships and very different phase–response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAAantagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13–36 and (Cys2,8-aminooctanoic acid5,24,d-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [d-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.