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Supplementary Table from Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor

Supplementary Data from Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis

The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K–mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E–mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti–PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses.Significance:A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

Purpose:Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi.Experimental Design:The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi.Results:Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples.Conclusions:The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.

Supplementary Data from Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor

Purpose: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. Experimental Design: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. Results: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. Conclusions: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.

The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K–mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E–mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti–PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. Significance: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

Glioblastoma is an invariably deadly disease. A subpopulation of glioma stem-like cells (GSCs) drives tumor progression and treatment resistance. Two recent studies demonstrated that neurons form oncogenic glutamatergic electrochemical synapses with post-synaptic GSCs. This led us to explore whether glutamate signaling through G protein-coupled metabotropic receptors would also contribute to the malignancy of glioblastoma. We found that glutamate metabotropic receptor (Grm)3 is the predominantly expressed Grm in glioblastoma. Associations of GRM3 gene expression levels with survival are confined to the proneural gene expression subtype, which is associated with enrichment of GSCs. Using multiplexed single-cell qRT-PCR, GSC marker-based cell sorting, database interrogations, and functional assays in GSCs derived from patients’ tumors, we establish Grm3 as a novel marker and potential therapeutic target in GSCs. We confirm that Grm3 inhibits adenylyl cyclase and regulates extracellular signal-regulated kinase. Targeting Grm3 disrupts self-renewal and promotes differentiation of GSCs. Thus, we hypothesize that Grm3 signaling may complement oncogenic functions of glutamatergic ionotropic receptor activity in neuroglial synapses, supporting a link between neuronal activity and the GSC phenotype. The novel class of highly specific Grm3 inhibitors that we characterize herein have been clinically tested as cognitive enhancers in humans with a favorable safety profile., Graphical Abstract, Wirsching et al. establish glutamate metabotropic receptor (Grm)3 as the most abundantly expressed Grm in glioblastoma. Single-cell analyses establish preferential expression of Grm3 in glioma stem-like cells (GSCs). A novel class of clinically well-tolerated negative allosteric modulators of Grm3 compromised the GSC phenotype, warranting further exploration as anti-glioblastoma agents.

The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

Nociceptin/orphanin FQ receptor (NOP) agonists have been reported to produce antinociceptive effects in rhesus monkeys with comparable efficacy to μ-opioid receptor (MOP) agonists, but without their limiting side effects. There are also known to be species differences between rodents and nonhuman primates (NHPs) in the behavioral effects of NOP agonists. The aims of this study were the following: 1) to determine if the NOP agonist Ro 64-6198 could be trained as a discriminative stimulus; 2) to evaluate its pharmacological selectivity as a discriminative stimulus; and 3) to establish the order of potency with which Ro 64-6198 produces discriminative stimulus effects compared with analgesic effects in NHPs. Two groups of rhesus monkeys were trained to discriminate either fentanyl or Ro 64-6198 from vehicle. Four monkeys were trained in the warm-water tail-withdrawal procedure to measure antinociception. Ro 64-6198 produced discriminative stimulus effects that were blocked by the NOP antagonist J-113397 and not by naltrexone. The discriminative stimulus effects of Ro 64-6198 partially generalized to diazepam, but not to fentanyl, SNC 80, ketocyclazocine, buprenorphine, phencyclidine, or chlorpromazine. Fentanyl produced stimulus effects that were blocked by naltrexone and not by J-113397, and Ro 64-6198 did not produce fentanyl-appropriate responding in fentanyl-trained animals. In measures of antinociception, fentanyl, but not Ro 64-6198, produced dose-dependent increases in tail-withdrawal latency. Together, these results demonstrate that Ro 64-6198 produced stimulus effects in monkeys that are distinct from other opioid receptor agonists, but may be somewhat similar to diazepam. In contrast to previous findings, Ro 64-6198 did not produce antinociception in the majority of animals tested even at doses considerably greater than those that produced discriminative stimulus effects.

Psychopharmacology, 179 (1), ISSN:0033-3158, ISSN:1432-2072

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinson's disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.

We previously reported that the selective nociceptin orphanin peptide (NOP) receptor agonist, Ro64-6198, impairs mnemonic function through glutamatergic-dependent mechanisms. The aim of the current study was to determine whether the amnesic effects of Ro64-6198 involve a cholinergic component. The effects of systemic administration of Ro64-6198 (0.3 and 1 mg/kg, i.p.), the cholinergic nicotinic receptor antagonist, mecamylamine (0.1 and 1 mg/kg, s.c.), the cholinergic muscarinic receptor antagonist, scopolamine (0.1 and 0.3 mg/kg, s.c.), and the glutamatergic NMDA receptor antagonist, MK-801 (0.03 and 0.1 mg/kg, s.c.), were studied in the mouse object recognition task. All compounds tested were effective in disrupting formation of long-term (24-h delay) recognition memory. Drug interaction studies were then conducted to reveal the existence of functional interactions between NOP receptors and cholinergic and/or NMDA receptors. Co-administration of silent doses of Ro64-6198 (0.3 mg/kg) and MK-801 (0.01 mg/kg) produced clear-cut memory impairment. Similar synergistic effects were observed with the combination of mecamylamine (0.03 mg/kg) and scopolamine (0.1 mg/kg). In contrast, co-administration of Ro64-6198 (0.3 mg/kg) with either mecamylamine (0.03 and 0.1 mg/kg) or scopolamine (0.1 mg/kg) was without any effect on recognition memory. These findings suggest that NOP receptor may modulate memory formation through a functional interaction with glutamatergic but not cholinergic receptors.

Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor (NOP receptor) have been implicated in a host of brain functions and diseases, but the contribution of this neuropeptide system to behavioral processes of relevance to psychosis has not been investigated. We examined the effect of the NOP receptor antagonists, Compound 24 and J-113397, and the synthetic agonist, Ro64-6198, on time function (2-2000 ms prepulse-pulse intervals) of acoustic (80 dB/10 ms prepulse) and visual (1000 Lux/20 ms prepulse) prepulse inhibition of startle reflex (PPI), a preattentive sensory filtering mechanism that is central to perceptual and mental integration. The effects of the dopamine D1-like receptor agonist, SKF-81297, the D2-like receptor agonist, quinelorane, and the mixed D1/D2 agonist, apomorphine, were studied for comparison. When acoustic stimulus was used as prepulse, BALB/cByJ mice displayed a monotonic time function of PPI, and consistent with previous studies, apomorphine and SKF-81279 induced PPI impairment, whereas quinelorane had no effect. None of the NOP receptor ligands was effective on acoustic PPI. When flash light was used as prepulse, BALB/cByJ mice displayed a bell-shaped time function of PPI and all dopamine agonists were active. Ro64-6198 was also effective in reducing visual PPI. NOP receptor antagonists showed no activity but blocked disruptive effect of Ro64-6198. Finally, coadministration of the typical antipsychotic, haloperidol, attenuated PPI impairment induced by Ro64-6198, revealing involvement of a dopaminergic component. These findings show that pharmacological stimulation of NOP or dopamine D2-like receptors is more potent in disrupting visual than acoustic PPI in mice, whereas D1-like receptor activation disrupts both. They further suggest that dysfunction of N/OFQ transmission may be implicated in the pathogenesis of psychotic manifestations.

Altered glutamatergic synaptic transmission is among the key events defining the course of Alzheimer's disease (AD). mGlu2 receptors, a subtype of group II metabotropic glutamate receptors, regulate (as autoreceptors) fast synaptic transmission in the CNS via the controlled release of the excitatory amino acid glutamate. Since their pharmacological manipulation in rodents has been reported to affect cognition, they are potential drug targets for AD therapy. We examined the fate of these receptors in cases of AD as well as in aging PS2APP mice—a proposed model of the disease. In vitro binding of [3H]LY354740, a selective group II agonist (with selective affinity for mGlu2 receptors, under the assay conditions used) and quantitative radioautography revealed a partial, but highly significant, loss of receptors in amyloid-affected discrete brain regions of AD cases and PS2APP mice. Among the mouse brain regions affected were, above all, the subiculum but also frontolateral cortex, dentate gyrus, lacunosum moleculare and caudate putamen. In AD, significant receptor losses were registered in entorhinal cortex and lacunosum moleculare (40% and 35%, respectively). These findings have implications for the development of selective ligands for symptomatic therapy in AD and for its diagnosis.

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA(A) alpha5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.

Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides with improved pharmacokinetic properties have been designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.

The asymmetric synthesis and receptor pharmacology of (1S,2R,3R,5R,6S)-2-amino-3-Hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (+)-9 (HYDIA) and a few of its O-alkylated derivatives are described. The key step of the synthesis utilizes Sharpless' asymmetric dihydroxylation (AD-beta) for the kinetic resolution of a bicyclic racemic precursor olefin. In contrast to the bicyclic glutamate analogue LY354740, which is a potent and selective agonist for the group II metabotropic glutamate receptors (mGluRs), these new conformationally restricted and also hydroxylated or alkoxylated glutamate analogues are potent and selective antagonists for the group II mGluRs.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] is an atypical anxiolytic agent with unknown molecular target that has previously been demonstrated both in rodents and human to exert anxiolytic activity. Here, we report that fenobam is a selective and potent metabotropic glutamate (mGlu)5 receptor antagonist acting at an allosteric modulatory site shared with 2-methyl-6-phenylethynyl-pyridine (MPEP), the protypical selective mGlu5 receptor antagonist. Fenobam inhibited quisqualate-evoked intracellular calcium response mediated by human mGlu5 receptor with IC(50) = 58 +/- 2 nM. It acted in a noncompetitive manner, similar to MPEP and demonstrated inverse agonist properties, blocking 66% of the mGlu5 receptor basal activity (in an over expressed cell line) with an IC(50) = 84 +/- 13 nM. [(3)H]Fenobam bound to rat and human recombinant receptors with K(d) values of 54 +/- 6 and 31 +/- 4 nM, respectively. MPEP inhibited [(3)H]fenobam binding to human mGlu5 receptors with a K(i) value of 6.7 +/- 0.7 nM, indicating a common binding site shared by both allosteric antagonists. Fenobam exhibits anxiolytic activity in the stress-induced hyperthermia model, Vogel conflict test, Geller-Seifter conflict test, and conditioned emotional response with a minimum effective dose of 10 to 30 mg/kg p.o. Furthermore, fenobam is devoid of GABAergic activity, confirming previous reports that fenobam acts by a mechanism distinct from benzodiazepines. The non-GABAergic activity of fenobam, coupled with its robust anxiolytic activity and reported efficacy in human in a double blind placebo-controlled trial, supports the potential of developing mGlu5 receptor antagonists with an improved therapeutic window over benzodiazepines as novel anxiolytic agents.

Mit dem Ziel der Demokratisierung, Humanisierung und Pluralisierung erlebt das russische Bildungswesen seit Beginn der 1990er-Jahre eine tief greifende und komplexe Transformation. In zahlreichen Analysen wurden bisher vor allem die programmatischen, strukturellen, institutionellen sowie die schulischen und curricularen Veranderungen zu charakterisieren versucht. Die vorliegende Untersuchung greift einen bisher unberucksichtigt gebliebenen Bereich auf, dessen Veranderung aber in ganz starkem Mase diese Transformation mitbestimmt hat und zugleich deren Spiegelbild ist: den bildungspolitisch-padagogischen Diskurs der Zeitschrift Pedagogika. Mit Hilfe sowohl empirischer Daten als auch texthermeneutisch gewonnener Aussagen soll dargestellt werden, wie vor dem Hintergrund-einer ganz besonderen russischen Variante der geopolitischen und kulturellen Fragmentierung der Welt bestimmte internationale Impulse selektiv in den Diskurs zu integrieren und fur die eigene Reform des Bildungswesens nutzbar zu machen versucht werden. Die zentrale Frage ist, ob und inwieweit sich die russische Bildungspolitik und Padagogik entweder vorbehaltlos bestimmten internationalen Bildungsmodellen und globalen Prozessen offnet oder ob sie verstarkt auf eine Revitalisierung der russischen Bildungstradition setzt.

We have previously reported that the 5-HT uptake blocker and releaser, dexfenfluramine, attenuates ethanol intake, and that this may be mediated via a 5-HT(2C) receptor mechanism. Our goals were to further determine the contribution made by this receptor subtype in mediating the reduction in ethanol self-administration induced by dexfenfluramine using the selective 5-HT(2C) antagonist, SB242,084. Additionally, we wanted to compare dexfenfluramine's effects on ethanol motivated responding with those elicited by the 5-HT(2C) receptor agonist Ro60-0175. In male Wistar rats trained to self-administer a 12% w/v ethanol solution on an FR-4 schedule, both dexfenfluramine (0.05--2.5 mg/kg ip) and Ro60-0175 (0.1--1 mg/kg sc) produced a significant dose-dependent reduction in ethanol self-administration, which was reversed by SB242,084 (0.5 mg/kg ip). Interestingly, SB242,084 alone (0.1--1 mg/kg ip) significantly increased ethanol motivated responding in both high and low ethanol drinking animals. While dexfenfluramine had no effect on ethanol's kinetic profile, the selective 5-HT(2C) agents used had opposing effects, with the agonist Ro60-0175 decreasing and the antagonist SB242,084 increasing blood ethanol levels. Since there were incongruent drug effects on ethanol self-administration and blood ethanol levels, these data support a role for 5-HT(2C) receptors in modifying ethanol intake independent of their effects on blood ethanol kinetics. Furthermore, 5-HT(2C) receptors may exert a tonic control over ethanol self-administration behaviour, since agonist and antagonist administration had opposing effects on this behaviour.

Using a combination of the selective opioid receptor-like 1 (ORL 1 ) receptor agonist, Ro 64-6198, and orphanin FQ/nociceptin (OFQ/N) peptide knockout (KO) mice, the influence of OFQ/N on cognition has been studied in therodent. In wild type, C57BL/ 6J mice, Ro 64-6198 (0.3-1 mg/kg i.p.) impaired the acquisition of spatial learning in the Morris water maze, although a mild neurological impairment was evident which complicated precise interpretation. In Lister hooded rats, Ro 64-6198 (6 mg/kg i.p.) produced delay dependent impairments in rats performing either a delayed matching or a delayed nonmatching to position task with only a modest (< 20%) effect on omissions - an effect consistent with a short-term memory impairment. Electrophysiological studies demonstrated an inhibitory effect of OFQ/N on LTP recorded from the CA1 region of wild type mice, but not in ORL 1 receptor knockout mice. In contrast to the ORL 1 agonist, mice deficient in the OFQ/N peptide showed some evidence of improved spatial learning, fear conditioning and passive avoidance retention. However, CA1 LTP was similar between OFQ/N peptide KO mice and wild type controls. Subsequent receptor radioautography studies demonstrated the presence of ORL 1 receptors within various regions of the medial temporal lobe system: i.e. CA1, dentate gyrus molecular layer, subiculum, perirhinal cortex. Taken together, these results suggest a bi-directional effect of OFQ/N containing systems on aspects of cognitive behaviour, particularly those elements associated with hippocampal function. This is consistent with a likely modulatory role of OFQ/N on hippocampal and associated cortical circuitry.

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

Identification of synthetic agonists and antagonists at orphan receptors represents an important step for understanding their physiological function and therapeutic potential. Accordingly, we have recently described a non-peptide agonist at the opioid receptor like (ORL1) receptor (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro64-6198; Jenck et al., PNAS 94 (2000) 4938; Wichmann et al., Eur. J. Med. Chem. 35 (2000) 839). We have investigated the effects of this compound in various tests of rodent neurological function, utilising ORL1 knockout mice to examine the pharmacological specificity of Ro64-6198. In male C57BL/6J mice, effects on balance and motor co-ordination were detected following low doses (0.3-1mg/kg IP) of Ro64-6198. At higher doses (1-3mg/kg IP), effects on swim behaviour and hypothermia was observed. At 10mg/kg, each effect became more profound and a severe neurological disturbance appeared, including loss of righting reflex. These effects of Ro64-6198 (10mg/kg IP) were absent in ORL1 receptor knockout mice. In male, hooded Lister rats, Ro64-6198 (6-10mg/kg IP), produced some disturbance of neurological function, including hypoactivity, rotarod performance, grip strength and mild hypothermia. An impairment of food responding under a variable interval (VI) 20s schedule of reinforcement was noted at 3mg/kg. These results confirm Ro64-6198 to be a highly selective pharmacological tool to investigate ORL1 receptor function in vivo and, furthermore, that activation of this receptor is accompanied by a variety of effects on neurological function.

The development of 8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3, 8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (micro, kappa, delta) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPgamma(35)S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.

We have investigated 8-cycloalkyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o nes as ligands for the ORL1 receptor. These unsophisticated, achiral compounds show remarkable affinity for the ORL1 receptor. Optimizing for selectivity we show that the maximum of affinity and selectivity versus the other opioid receptors is achieved for 8-cyclodecyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o ne 2e and 8-(cis-4-isopropyl-cyclohexyl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one 2q. The identified compounds (2e, 2q) are more or less equipotent to the natural ligand itself, both in the binding assay and in the functional GTPgammaS assay.

Im Mittelpunkt dieser Analyse steht das in den USA und in einigen anderen Landern gegenwartig an Interesse gewinnende Modell der Service University. Im Gegensatz zu den Selbstdarstellungen seiner Verfechter wird dieses Modell nunmehr vor dem Hintergrund seiner spezifischen Entstehungsbedingungen kritisch im Hinblick auf seine grundlegenden Ideen und Forderungen sowie auf deren Neuigkeitsgehalt analysiert. Vor allem durch die Kontrastierung mit signifikanten Momenten der deutschen Universitatsgeschichte wird deutlich, dass in den USA der Service-Gedanke die Hochschulentwicklung von den ersten Grundungen bis heute begleitet hat. Daruber hinaus wird am Beispiel Russlands dargestellt, wie sich hier die Rezeption des Modells der Service University mit unterschiedlichen Ergebnissen und ambivalenten Reaktionen vollzieht. Generell will dieser Beitrag deutlich machen, dass unter den heutigen Bedingungen zunehmender internationaler Verflechtung notwendige Bildungsreformen auf nationaler Ebene ohne die Rezeption internationaler bildungspolitisch-padagogischer Modelle nicht mehr moglich sind. Gleichwohl wird aber auch deutlich, dass jede Rezeption nur auf der Grundlage kritischer Analysen und nicht in Gestalt euphoriegeleiteten Kopierens geschehen kann. Die Strukturen und Institutionen eines jeden Bildungssystems sind unter jeweils spezifischen kultur- und bildungsgeschichtlichen Bedingungen entstanden. Das Wissen uber diese Bedingungen versetzt uns in die Lage, die Moglichkeiten und Grenzen der Ubernahme internationaler Bildungsmodelle zu erkennen.

The conformationally restricted analog of L-glutamic acid (L-Glu, 1) (2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)-glycine (DCG-IV, 2) is a potent group II mGluR agonist. In order to study the distribution of group II mGluRs in the brain and to establish a radioligand binding assay we have developed a synthesis of [3H]-DCG-IV (2a). The key intermediate, α-bromo aldehyde 7, was prepared in four steps starting from (−)-Fiest's acid (3). The incorporation of tritium was performed by reaction of 7 with tri-n-butyltin tritide to give 8, which was transformed in two steps into 2a. Copyright © 2000 John Wiley & Sons, Ltd.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.

Fenobam is a clinically efficacious anxiolytic that acts as metabotropic glutamate receptor 5 (mGlu5) antagonist by binding to an allosteric site. Other mGlu5 receptor antagonists such as MPEP and MTEP bind to the same allosteric site and are efficacious in preclinical models of anxiety and depression. Consequently, the allosteric-binding site of the mGlu5 receptor is an attractive target for the discovery of novel psychiatric therapies. Radioligands of this binding site can be used for in vitro and in vivo pharmacodynamic studies. We report here a short synthesis of such a radioligand for the allosteric mGlu5 receptor-binding site, [3H]fenobam. Copyright © 2008 John Wiley & Sons, Ltd.

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).

Based on O-methylasparvenone (1), a N-free 5HT2C antagonist with moderate affinity (pKi = 6.7), derivatives bearing dimethylamino (7), (dimethylamino)methyl (17, 18, 21, and 22), and aminomethyl substituents (26) in place of the benzylic OH group of 1 as well as pyrrolidine- (33) and piperidine-fused derivatives (29, 43, and 45) were synthesized. In contrast to the lead structure 1, these new ligands were active in vivo in the rat. The tricycles 33 and 45 display high affinities for the 5HT2C receptor (pKi = 8).

The syntheses of a series of substituted 2-(indol-1-yl)-1-methylethylamines and 2-(indeno[1,2-b]pyrrol-1-yl)-1-methylethylamines are reported. The binding affinities of the compounds at 5HT 2C and 5HT 2A receptors (79% homology in the transmembrane domain) were determined. The ligands displayed selectivity for 5HT 2C receptors relative to 5HT 2A receptors. Compounds were functionally characterized both in vitro and in vivo as 5HT 2C receptor agonists. 5f, 5l, 5n, 5o, 5q, 14c, 14f, 14k, and 14m exhibited anticompulsive activity in an animal model of obsessive compulsive disorder.

1. The effects of selective agonists at group I, II and III metabotropic glutamate receptors (mGluRs) on adenosine A2 receptor-mediated cyclic AMP formation were compared in cross-chopped slices of adult and neonatal (8 days old) rat striatum, in the presence of 1 u ml(-1) adenosine deaminase. 2. The group II selective agonist, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), elicited a potentiation of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated cyclic AMP production with similar potencies in adult (EC50 value 122 +/- 35 nM) and neonatal (EC50 value 285 +/-6 nM) brain. In contrast, the group I selective agonist (S)-dihydroxyphenylglycine ((S)-DHPG) augmented the NECA cyclic AMP response in neonatal striatum (EC50 value 9 +/- 1 microM), but at a concentration of 100 microM, (S)-DHPG failed to affect the NECA response in adult striatal slices. 3. The potentiation evoked by (S)-DHPG was specific for group I mGluRs as (2S,3S,4S,)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a group II antagonist, was ineffective on the (S)-DHPG (100 microM) response at a concentration (500 microM) which reversed a similar augmentation elicited by DCG-IV (300 nM). Furthermore, a protein kinase C inhibitor (Ro 31-8220, 10 microM) markedly reversed the effect of (S)-DHPG without affecting the response to DCG-IV. 4. The mGluR agonist (2S,3S,4S,)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited a greater potentiation of NECA-stimulated cyclic AMP production in neonatal striatum in comparison with that observed in adult rat brain. Moreover, EC50 values obtained from adult and neonatal striatum were 2 +/-1 microM and 9 +/-1 microM, respectively. These differences in potency might reflect co-activation of both group I and group II mGluRs by L-CCG-I in neonatal striatum. 5. Distinct patterns of mGluR expression in various brain areas might account for previous conflicting data on the nature of the mGluR able to evoke such potentiated responses.

Potential antidepressant properties of preferential 5HT2C receptor agonists were investigated in stress-induced anhedonia, a validated simulation of depression. This simulation evaluates the hedonic state of stressed rats by recording variations in self-stimulation threshold measured before, during, and after exposure to intermittent, unpredictable, mild stressors. This stress regimen gradually elevates self-stimulation threshold, suggesting the development of an anhedonic state. In stressed animals, chronic treatment with the preferential 5HT2C receptor agonists Ro 60-0175 and Ro 60-0332 (3 mg/kg i.p. b.i.d.) prevented the loss of sensitivity to reward. Similarly, when stressed anhedonic animals were curatively treated with Ro 60-0175 (3 mg/kg i.p. b.i.d.), the stress-induced anhedonia was gradually reversed. These results suggest a role for 5HT2C receptors in some aspects of depression, and potential antidepressant properties for selective 5HT2C receptor agonists. Such compounds may offer an innovative approach to the treatment of mood disorders.

BACKGROUND AND PURPOSE Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu2) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu2/3 receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu2/3 NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu2 is characterized. EXPERIMENTAL APPROACH Site directed mutagenesis, functional measurements and β2-adrenoceptor-based modelling of mGlu2 were employed to identify important molecular determinants of two new potent mGlu2/3 NAMs. KEY RESULTS RO4988546 and RO5488608 affected both [3H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (3H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu2 TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu2 and directly interact with the NAMs: Arg3.28, Arg3.29, Phe3.36, HisE2.52, Leu5.43, Trp6.48, Phe6.55 and Val7.43. The mGlu2 specific residue HisE2.52 is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [3H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp6.48 residue. CONCLUSIONS AND IMPLICATIONS This is the first complete molecular investigation of the allosteric binding pocket of mGlu2 and Group II mGluRs and provides new information on what determines mGlu2 NAMs selective interactions and effects.

Ro 64-6198, the prototypical non-peptide nociceptin/orphanin FQ peptide (NOP) receptor agonist, has potent anxiolytic-like effects in several preclinical models and species. However the effects of Ro 64-6198 on distinctive anxiety-provoking conditions related to unconditioned conflict behavior as well as its role in despair-like behavior remain to be addressed.Here we examined the effects of Ro 64-6198 on unconditioned conflict anxiety using stimuli with different salience and on regulation of autonomic reactivity and compared these to the effects of benzodiazepine receptor agonists. We also addressed the potential effects of Ro 64-6198 on despair-like behavior.Ro 64-6198 (0.1 to 10 mg/kg i.p.) and either diazepam or chlordiazepoxide were tested in the Vogel conflict punished drinking test (VCT) in Sprague Dawley rats, in the social approach-avoidance (SAA) test in Lewis rats, in the novelty-induced hypophagia (NIH) in C57BL/6J mice, and in stress-induced hyperthermia in NMRI mice, as well as in the forced swim test (FST) in Sprague Dawley rats and the tail suspension test (TST) in C57BL/6J mice.Ro 64-6198 (0.3 to 3 mg/kg) dose-dependently produced anxiolytic-like effects in the VCT, SAA, NIH, and SIH, similar to benzodiazepine receptor agonists. Ro 64-6198 did not alter immobility time in the FST and TST.Ro 64-6198 produced marked anxiolytic-like effects in response to a variety of mild to strong anxiogenic stimuli, whereas it did not facilitate depression-related behaviors. This data extend previous literature suggesting that NOP receptors are a viable target for the treatment of anxiety disorders.

The reaction of the N-acylated enamino ketones 4a–d with trifluoroacetic acid, TMS triflate, or Lewis acids leads to the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines 5a–d in 84 to 94% yield. The enamino ketones 4f–i can only be cyclized with trifluoromethanesulfonic acid to the 1,2,3,4-tetrahydroisoquinolines 5f–i in 75–84% yield. For the synthesis of 5e from the enamino ketone 4e carrying the benzyloxycarbonyl group TMS triflate is the best mediator (93%). The trichloromethylcarbonyl group in 5 can easily be transformed into a methoxycarbonyl group by treatment with K2CO3 in methanol to give 6 in 83–90% yield.

An efficient three-step biomimetic synthesis of the four diastereomeric 18,19-dihydroantirhines 4a–d starting from the tetrahydrocarboline aldehydes 5a and 5b is described. Domino reaction of the aldehydes 5a and 5b, respectively, with Meldrum's acid (6) and the enol ethers 8a and 8b in the presence of catalytic amounts of ethylenediammonium diacetate leads to the strictosidine analogues 10a–d and 11a–h, respectively, in 74–86% yield, hydrogenation of which gives mainly 15 and 16 with the skeleton of the vallesiachotamine indole alkaloids (55–86%). In addition, small amounts of 17 and 18 with the corynanthe skeleton are also formed (10–12%). Reduction of both 15 and 16 with LiAlH4 yields the diastereomeric rac-18,19-dihydroantirhines 4a–d in 78–86% yield.