Behavioral Characterization of the Novel GABA B Receptor- Positive Modulator GS39783 (N,NЈ-Dicyclopentyl-2- methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): Anxiolytic-Like Activity without Side Effects Associated with Baclofen or Benzodiazepines

ABSTRACT The role of GABA B receptors in various behavioral processes has been largely defined using the prototypical GABA B receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABA B receptors in these disorders is largely unclear. We recently identified GS39783 (N,NЈ-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABA B receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital-and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABA B receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines

Gastrin-Releasing Peptide Signaling Plays a Limited and Subtle Role in Amygdala Physiology and Aversive Memory

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe6, Leu-NHEt13, des-Met14)-Bombesin (6–14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders

JN403, in vitro characterization of a novel nicotinic acetylcholine receptor alpha7 selective agonist.

This report describes the in vitro features of a novel selective nicotinic acetylcholine receptor (nAChR) alpha7 agonist, JN403, (S)-(1-Aza-bicyclo[2.2.2]oct-3-yl)-carbamic acid (S)-1-(2-fluoro-phenyl)-ethyl ester. JN403 was evaluated in a number of in vitro systems of different species, at recombinant receptors using radioligand binding, signal transduction and electrophysiological studies. When using [(125)I] alpha-bungarotoxin (alpha-BTX) as a radioligand, JN403 has high affinity for human recombinant nAChR alpha7 (pK(D)=6.7). Functionally, JN403 is a partial and potent agonist at human nAChR alpha7. The compound stimulates calcium influx in GH3 cells recombinantly expressing the human nAChR with an pEC(50) of 7.0 and an E(max) of 85% (compared to the full agonist epibatidine). In Xenopus oocytes expressing human nAChR alpha7 JN403 induces inward currents with an pEC(50) of 5.7 and an E(max) of 55%. In both recombinant systems JN403 is a partial agonist and the agonistic effects are blocked after pre-administration of methyllycaconitine (MLA, 100nM), a nAChR alpha7 antagonist. In functional calcium influx assays, JN403 displays a significantly lower potency for other subtypes of human nAChRs like alpha4beta2, alpha3beta4, alpha1beta1gammadelta as well as 5HT(3) receptors when tested functionally as an antagonist (pIC(50)<4.8) and is devoid of agonistic activity (pEC(50)<4). Similarly, JN403 shows low binding activity at a wide panel of neurotransmitter receptors. Thus, JN403 is a potent and selective nAChR alpha7 agonist and will be a useful tool for the characterization of nAChR alpha7 mediated effects both in vitro and in vivo

Coupling of human nicotinic acetylcholine receptors alpha 7 to calcium channels in GH3 cells.

The neuronal nicotinic acetylcholine receptor alpha7 (nAChR alpha7) may be involved in cognitive deficits in Schizophrenia and Alzheimer's disease. A fast pharmacological characterization of homomeric alpha7 receptors is mostly hampered by their low functional expression levels in heterologous expression systems. In the present study expression of homomeric nAChR alpha7 was achieved in GH3 rat pituitary cells. Alpha7 subunits were heterologously expressed as components of [125I]-labeled alpha-bungarotoxin binding nAChRs (Bmax: 1.2 pmol/mg protein). Function of the expressed alpha7 ion channels was assessed by patch-clamp recording and calcium imaging. While acetylcholine-induced currents desensitized within much less than 1 s, calcium-sensitive fluorescence transients peaked after 5-10 s and returned to background levels within 30 s only. The fluorescence signal was blocked by isradipine and removal of extracellular sodium indicated that in these cells opening of rapidly desensitizing alpha7 nAChR triggers calcium influx via voltage-gated, DHP-sensitive calcium channels. In this cellular system, agonists revealed the following rank order of potency: epibatidine>anatoxin A>AAR17779>ABT-594>DMPP>nicotine>GTS-21>cytisine>ABT-418>acetylcholine>choline>ABT-089. All of the signals were inhibited by the alpha7 antagonists alpha-bungarotoxin (pIC50: 7.4) and methyllycaconitine (pIC50: 7.8). Further, marketed antidepressants showed antagonistic activity with the following rank order of potency: fluoxetine>imipramine>paroxetine>sertraline. These data illustrate that coupling to voltage-gated calcium channels allows a rapid and reliable functional examination of nAChR alpha7

Fear-reducing effects of intra-amygdala neuropeptide Y infusion in animal models of conditioned fear: an NPY Y1 receptor independent effect

RATIONALE: Neuropeptide Y (NPY) and its receptors are densely localized in brain regions involved in the mediation and modulation of fear, including the amygdala. Several studies showed that central NPY is involved in the modulation of fear and anxiety. OBJECTIVES: In the present study, we investigated (1) whether intra-amygdala injections of NPY affect the expression of conditioned fear and (2) whether NPY Y1 receptors (Y1R) mediates the effects of these intra-amygdaloid NPY injections. RESULTS: Intra-amygdala NPY injections robustly decreased the expression of conditioned fear measured by conditioned freezing and fear-potentiated startle. These NPY effects were not mimicked by intra-amygdala injections of the Y1R agonists Y-28 or Y-36, and co-infusion of the Y1R antagonist BIBO 3304 did not block the NPY effects. Furthermore, we tested Y1R-deficient mice in conditioned freezing and found no differences between wild type and mutant littermates. Finally, we injected NPY into the amygdala of Y1R-deficient mice. Y1R deficiency had no effect on the fear-reducing effects of intra-amygdala NPY. CONCLUSIONS: These data show an important role of the transmitter NPY within the amygdala for the expression of conditioned fear. Y1R do not appear to be involved in the mediation of the observed intra-amygdala NPY effects suggesting that these effects are mediated via other NPY receptors

AMPA receptor antagonists: potential therapeutic applications.

The current review will focus on the recent patents for AMPA receptor antagonists and their claims, evidence for their therapeutic effectiveness in the treatment of epilepsy and their potential role in psychiatric and neurodegenerative disorders. It will also highlight the proposed mechanisms of action and the implications thereof for our current understanding of the biomolecular basis of these pathologies. It will conclude with a summary of what we know, but also point out the remaining uncertainties, especially as this relates to the claims in the patent under discussion

Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021–1024

Activation of N-methyl-D-aspartate subtype glutamate receptors (NMDARs) is required for long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission at hippocampal CA1 synapses, the proposed cellular substrates of learning and memory. However, little is known about how activation of NMDARs leads to these two opposing forms of synaptic plasticity. Using hippocampal slice preparations, we showed that selectively blocking NMDARs that contain the NR2B subunit abolishes the induction of LTD but not LTP. In contrast, preferential inhibition of NR2A-containing NMDARs prevents the induction of LTP without affecting LTD production. These results demonstrate that distinct NMDAR subunits are critical factors that determine the polarity of synaptic plasticity. The molecular mechanisms underlying activity-dependent modification of synaptic strength have been under intensive investigation because of their fundamental importance in brain function and dysfunction (1, 2). Homosynaptic long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission mediated by ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid subtype glutamate receptors (AMPARs) at Schaffer collateral-CA1 synapses of the hippocampus are by far the best-characterized cellular models of synaptic plasticity. Both LTP and LTD require N-methyl-D-aspartate subtype glutamate receptor (NMDAR) activation (1, 3). However, the detailed mechanisms by which the activation of the same class of receptor can produce two opposing forms of synaptic modification remain unclear. A long-held belief has been that the degree of NMDAR activation, and hence the level of postsynaptic calcium elevation during the induction period, dictates the direction of NMDAR-dependent synaptic modification. The strongest evidence for this hypothesis comes from the conversion of LTP to LTD by a partial blockade of NMDARs with low concentrations of the NMDAR antagonist D,L-2-amino-5-phosphophonovaleric acid (APV) (4, 5 ). NMDARs are assembled from NMDAR subunit 1 (NR1) and at least one type of NR2 subunit (6). In the adult rat hippocampus, NR2A and NR2B are the predominant NR2 subunits We first investigated the effect of blocking NR2B-containing NMDARs on LTP and LTD of CA1 field excitatory postsynaptic potentials (fEPSPs) induced by high-and low-frequency stimulation (HFS and LFS), respectively (15). The NR2B subunitselective antagonist ifenprodil (3 M) (16) completely abolished the induction of LTD by LFS, suggesting that LTD requires the activation of NR2B-containing NMDARs It is unlikely that the selective blockade of LTD by NR2B-specific antagonists is due merely to a partial inhibition of NMDARs, because LTP rather than LTD is more sensitive to the partial NMDAR blockade produced by low concentrations of APV (4, 5). To rule out such a possibility, we tested the effect of low-dose APV that generated a partial blockade of NMDAR-mediated excitatory postsynaptic currents (EPSCs) similar to that produced by NR2B antagonist

Quinazolinedione sulfonamides: A Novel Class of Competitive AMPA Receptor Antagonists with Oral Activity

Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models

Design and synthesis of Selurampanel, a novel orally active and competitive AMPA receptor antagonist.

A series of potent quinazolinedione sulfonamide AMPA receptor antagonists were designed and synthesized. The SAR and in vivo activity of the series were investigated. In particular, compound 1S (Selurampanel) has shown excellent oral potency against MES-induced generalized tonic-clonic seizures in rodents. The X-ray structure of Selurampanel bound to AMPA was also obtained