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International audience; Background: The gold-standard treatment for Parkinson’s disease is L-DOPA, which in the long term often leads to levodopa-induced dyskinesia. Serotonergic neurons are partially responsible for this, by converting L-DOPA into dopamine leading to its uncontrolled release as a “false neurotransmitter”. The stimulation of 5-HT1A receptors can reduce involuntary movements but this mechanism is poorly understood. Objective: This study aimed to investigate the functionality of 5-HT1A receptors using positron emission tomography in hemiparkinsonian rats with or without dyskinesia induced by 3-weeks daily treatment with L-DOPA. Imaging sessions were performed “off” L-DOPA. Methods: Each rat underwent a positron emission tomography scan with [18F]F13640, a 5-HT1AR agonist which labels receptors in a high affinity state for agonists, or with [18F]MPPF, a 5-HT1AR antagonist which labels all the receptors. Results: There were decreases of [18F]MPPF binding in hemiparkinsonian rats in cortical areas. In dyskinetic animals, changes were slighter but also found in other regions. In hemiparkinsonian rats, [18F]F13640 uptake was decreased bilaterally in the globus pallidus and thalamus. On the non-lesioned side, binding was increased in the insula, the hippocampus and the amygdala. In dyskinetic animals, [18F]F13640 binding was strongly increased in cortical and limbic areas, especially in the non-lesioned side. Conclusion: These data suggest that agonist and antagonist 5-HT1A receptor-binding sites are differently modified in Parkinson’s disease and levodopa-induced dyskinesia. In particular, these observations suggest a substantial involvement of the functional state of 5-HT1AR in levodopa-induced dyskinesia and emphasize the need to characterize this state using agonist radiotracers in physiological and pathological conditions.

Rationale The prevalence of depression is ever-increasing throughout the population. However, available treatments are ineffective in around one-third of patients and there is a need for more effective and safer drugs. Objectives The antidepressant-like and procognitive effects of the “biased agonists” F15599 (also known as NLX-101) which preferentially targets postsynaptic 5-HT1A receptors and F13714, which targets 5-HT1A autoreceptors, were investigated in mice. Methods Antidepressant-like properties of the compounds and their effect on cognitive functions were assessed using the forced swim test (FST) and the novel object recognition (NOR), respectively. Next, we induced a depressive-like state by an unpredictable chronic mild stress (UCMS) procedure to test the compounds’ activity in the depression model, followed by measures of sucrose preference, FST, and locomotor activity. Levels of phosphorylated cyclic AMP response element-binding protein (p-CREB) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) were also determined. Results F15599 reduced immobility time in the FST over a wider dose-range (2 to 16 mg/kg po) than F13714 (2 and 4 mg/kg po), suggesting accentuated antidepressant-like properties in mice. F15599 did not disrupt long-term memory consolidation in the NOR at any dose tested, while F13714 impaired memory formation, notably at higher doses (4–16 mg/kg). In UCMS mice, a single administration of F15599 and F13714 was sufficient to robustly normalize depressive-like behavior in the FST but did not rescue disrupted sucrose preference. Both F15599 and F13714 rescued cortical and hippocampal deficits in p-ERK1/2 levels of UCMS mice but did not influence the p-CREB levels. Conclusions Our studies showed that 5-HT1A receptor biased agonists such as F13714 and especially F15599, due to its less pronounced side effects, might have potential as fast-acting antidepressants.

Obsessive compulsive disorder (OCD) is a psychiatric disorder characterized by excessive and repetitive thoughts and gestures, mainly treated pharmacologically with selective serotonin reuptake inhibitors (SSRIs). The marble burying test in mice is commonly used to model OCD and has been shown to be sensitive to SSRIs, which decrease burying behavior. The activity of SSRIs in this model is mediated through activation of 5-hydroxytryptamine (5-HT) 1A receptors, but the respective implication of pre- versus postsynaptic 5-HT1A receptors has not been elucidated. Here, we investigated marble burying behavior by male NMRI mice following acute administration of 3 biased agonists, which preferentially activate presynaptic 5-HT1A receptors (F13714) or postsynaptic receptors (NLX-101) or which exhibit balanced activation of both pre- and postsynaptic 5-HT1A receptors (NLX-112). When administered at the dose of 2.5 mg/kg i.p., all 3 biased agonists completely or nearly completely abolished marble burying behavior. However, they varied in their potency with minimal effective doses of 0.16, 0.63, and 2.5 mg/kg i.p., for F13714, NLX-112, and NLX-101, respectively. The selective 5-HT1A receptor antagonist, WAY100,635 was inactive up to 2.5 mg/kg. These results suggest that marble burying behavior in male NMRI mice is preferentially sensitive to activation of pre- versus postsynaptic 5-HT1A receptors. Moreover, they suggest that targeting 5-HT1A receptors with biased agonists could provide an innovative therapeutic approach to combat OCD.

Novel 1-(1-benzoylpiperidin-4-yl)methanamine derivatives with high affinity and selectivity for serotonin 5-HT1A receptors were obtained and tested in four functional assays: ERK1/2 phosphorylation, adenylyl cyclase inhibition, calcium mobilization, and β-arrestin recruitment. Compounds 44 and 56 (2-methylaminophenoxyethyl and 2-(1H-indol-4-yloxy)ethyl derivatives, respectively) were selected as biased agonists with highly differential "signaling fingerprints" that translated into distinct in vivo profiles. In vitro, 44 showed biased agonism for ERK1/2 phosphorylation and, in vivo, it preferentially exerted an antidepressant-like effect in the Porsolt forced swimming test in rats. In contrast, compound 56 exhibited a first-in-class profile: it preferentially and potently activated β-arrestin recruitment in vitro and potently elicited lower lip retraction in vivo, a component of "serotonergic syndrome". Both compounds showed promising developability properties. The presented 5-HT1A receptor-biased agonists, preferentially targeting various signaling pathways, have the potential to become drug candidates for distinct central nervous system pathologies and possessing accentuated therapeutic activity and reduced side effects.

NLX-101 and F13714 are selective, full efficacy, biased agonists of the 5-HT(1A) receptor. NLX-101 preferentially activates cortical post-synaptic 5-HT(1A) receptors whereas F13714 preferentially activates Raphe nuclei pre-synaptic 5-HT(1A) receptors. We compared NLX-101 and F13714 for their efficacy and potency to substitute for the discriminative cue produced by the prototypical, non-biased 5-HT(1A) receptor agonist, 8-OH-DPAT (racemate). Male and female Sprague-Dawley rats were trained to discriminate 8-OH-DPAT (0.1 mg/kg i.p., 20 min pre-treatment) from saline using a classical two-lever drug-discrimination procedure. 8-OH-DPAT (0.01 and 0.05 mg/kg i.p.) dose-dependently substituted for the training dose, with about 50 % responding on the 8-OH-DPAT-associated lever at 0.05 mg/kg. F13714 fully and very potently substituted for the training dose of 8-OH-DPAT from 0.018 mg/kg i.p., while NLX-101 only achieved full substitution at 0.5 mg/kg i.p., a dose which is known to also activate pre-synaptic 5-HT(1A) receptors. The 5-HT(1A) receptor partial agonist, buspirone, partially substituted (~80%) at 1 and 2 mg/kg i.p., doses which also decreased response rates. F13714 decreased response rates at 0.05 mg/kg. The selective 5-HT(1A) receptor antagonist WAY-100,635 (1 mg/kg s.c., 40 min pre-treatment) elicited almost no responding on the 8-OH-DPAT-associated lever by itself, but blocked the discriminative stimulus effects produced by administration (20 min pretreatment) of 8-OH-DPAT (0.1 mg/kg), F13714 (0.025 mg/kg), NLX-101 (0.5 mg/kg), or buspirone (1 mg/kg). These data suggest that the discriminative cue produced by 0.1 mg/kg i.p. 8-OH-DPAT results from activation of pre-synaptic 5-HT(1A) receptors. They also further demonstrate the distinct profiles in behavioral models of 5-HT(1A) receptor biased agonists.

Background:NLX-101 (also known as F15599) is a highly selective and efficacious ‘biased’ agonist at cortical 5-hydroxytryptamine 1A (5-HT1A) heteroreceptors. In rodents, it possesses marked antidepressant-like activity, potently and completely abolishing immobility in the forced swim test (FST) with extended duration of action.Methods:We investigated the antidepressant-like activity of NLX-101 using the rat chronic mild stress (CMS) model of depression, considered to have a higher translational potential than the FST, as it possesses construct, face and predictive validity. The effects of CMS and repeated NLX-101 treatment were tested using sucrose consumption (a measure of anhedonia), novel object recognition (NOR; a measure of working memory) and elevated plus maze (EPM; a measure of anxiety) tests.Results:NLX-101 reversed the CMS-induced decrease of sucrose intake on day 1 of testing, with full reversal observed at the dose of 0.16 mg/kg and a less pronounced but still significant effect at 0.04 mg/kg, both given twice a day intraperitoneally. The effects of NLX-101 were maintained over the 2 week treatment period and persisted for four weeks following cessation of treatment. In the NOR test, both doses of NLX-101 rescued the deficit in discrimination index caused by CMS, without any effect on locomotor activity. However, NLX-101 had no effect on the reduction of open-arms entries produced by CMS in the EPM model. In control, non-stressed rats, NLX-101 produced non-significant effects in all three models.Conclusions:NLX-101 displayed efficacious activity in the CMS test, with more rapid (1 day) antidepressant-like effects than pharmacological compounds tested previously under the same experimental conditions. These observations suggest that biased agonist targeting of cortical 5-HT1Areceptors constitutes a promising strategy to achieve rapid-acting and sustained antidepressant effects.

When placed in an unfamiliar and brightly lit open-field, two adult male rats that have not previously interacted display a low level of social interaction (SI) attributed to an anxiety-like state. The SI test has therefore been used to explore anxiolytic/antistress activity. Here, we investigated the effects of serotonin 5-HT1A receptor agonists displaying various activity profiles, i.e. partial vs full agonist efficacy and pre- versus postsynaptic 5-HT1A receptor preferential activation by "biased agonists". Adult male Sprague-Dawley rats were housed singly before starting the social interaction session. At 30 min before being placed in an open-field, both rats of the dyad were injected (i.p or s.c.) with either vehicle, diazepam (as a reference compound), or one of six 5-HT1A receptor agonists: NLX-101 (a.k.a. F15599), F13714, S15535, flesinoxan, 8-OH-DPAT, and buspirone. Time spent in SI (following, sniffing, playing) was recorded for 10 min. Time spent in SI was inversely correlated with light intensity, with values dropping nearly by half (212.6 ± 18.8 vs 113.7 ± 7.0 s) between 10 and 300 lx (measured at floor level). Under the high light intensity conditions (300 lx), diazepam showed a bell-shaped curve, significantly increasing SI (78% increase in interaction time above control) at 1 mg/kg i.p. only. In the case of 5-HT1A receptor ligands, full agonists, whether nonpreferential (flesinoxan, (±)8-OH-DPAT) or preferential for presynaptic receptors (F13714), showed the strongest activity in this model. The preferential presynaptic receptor partial agonist, S15535, was also active over a wide dose-range, although with lower efficacy than F13714. In contrast, NLX-101, a high-efficacy biased agonist that preferentially activates postsynaptic 5-HT1A receptors, exhibited little activity. The clinical anxiolytic, buspirone, showed a marked effect likely due to its partial agonist activity at 5-HT1A presynaptic receptors. These data support the hypothesis that enhancement of SI in this model is mediated by preferential agonist activation of presynaptic 5-HT1A receptors, and confirm previous studies using local microinjections of (±)8-OH-DPAT. They further support the utility of noninvasive administration of biased agonists for exploring the activity of 5-HT1A receptor subpopulations.

International audience; Serotonin 5-HT1A receptors constitute an attractive therapeutic target for various psychiatric or neurodegenerative disorders. These receptors are expressed in multiple brain regions on different neuronal populations and can be coupled with distinct G-protein subtypes; such functional diversity complicates the use of 5-HT1A ligands in several pathologies where it would be desirable to stimulate the receptors in a precise region. Therefore, using “biased agonists” able to target specifically certain subpopulations of 5-HT1A receptors would enable achievement of better therapeutic benefit. Several 5-HT1A receptor biased agonists are currently in development, including NLX-101 (aka F15599) and NLX-112 (aka F13640, befiradol), with preclinical data suggesting that they preferentially target different populations of 5-HT1A receptors. However, most previous studies used invasive and regionally limited approaches. In this context, [18F]-fluorodesoxyglucose (FDG)-positron emission tomography (PET) imaging constitutes an interesting technique as it enables noninvasive mapping of the regional brain activity changes following a pharmacological challenge in conscious animals. We report here the evaluation of cerebral glucose metabolism following intraperitoneal injection of different doses of NLX-112 or NLX-101 in conscious or isoflurane-anesthetized rats. The biased agonists produced different metabolic “fingerprints” with distinct regional preferences, consistent with previous studies. At equal doses, the effect of NLX-101 was less marked than NLX-112 in the piriform cortex, in the striatum (in terms of inhibition), and in the pontine nuclei and the cerebellum (in terms of activation); furthermore, only NLX-112 increased the glucose metabolism in the parietal cortex, whereas only NLX-101 induced a clear activation in the colliculi and the frontal cortex, which may be related to its distinctive procognitive profile. Both agonist effects were almost completely unapparent in anesthetized animals, underlining the importance of studying serotonergic neurotransmission in the conscious state. In this regard, [18F]FDG-PET imaging seems very complementary with other functional imaging techniques such as pharmacological MRI.

The emerging concept of "biased agonism" denotes the phenomenon whereby agonists can preferentially direct receptor signalling to specific intracellular responses among the different transduction pathways, thus potentially avoiding side effects and improving therapeutic effects. The aim of this study was to investigate biased agonism by using pharmacological magnetic resonance imaging (phMRI). The cerebral blood oxygen level dependent (BOLD) signal changes induced by increasing doses of two serotonin 5-HT1A receptor biased agonists, NLX-112 and NLX-101, were mapped in anaesthetized rats. Although both compounds display high affinity, selectivity and agonist efficacy for 5-HT1A receptors, NLX-101 is known to preferentially activate post-synaptic receptors, whereas NLX-112 targets both pre- and post-synaptic receptors. We used several doses of agonists in order to determine if the regional selectivity of NLX-101 was dose-dependent. NLX-112 and NLX-101 induced different positive and negative hemodynamic changes patterns at equal doses. Importantly, NLX-101 had no significant effect in regions expressing pre-synaptic receptors contrary to NLX-112. NLX-112 also produced higher BOLD changes than NLX-101 in the orbital cortex, the somatosensory cortex, and the magnocellular preoptic nuclei. In other regions such as the retrosplenial cortex and the dorsal thalamus, the drugs had similar effects. In terms of functional connectivity, NLX-112 induced more widespread changes than NLX-101. The present phMRI study demonstrates that two closely-related agonists display notable differences in their hemodynamic "fingerprints". These data support the concept of biased agonism at 5-HT1A receptors and raise the prospect of identifying novel therapeutics which exhibit improved targeting of brain regions implicated in neuropsychiatric disorders. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

l-DOPA is the gold-standard pharmacotherapy for treatment of Parkinson's disease (PD) but can lead to the appearance of troubling dyskinesia which are attributable to ‘false neurotransmitter’ release of dopamine by serotonergic neurons. Reducing the activity of these neurons diminishes l-DOPA-induced dyskinesia (LID), but there are currently no clinically approved selective, high efficacy 5-HT1A receptor agonists. Here we describe the effects of NLX-112, a highly selective and efficacious 5-HT1A receptor agonist, on LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated marmosets, a non-human primate model of PD. NLX-112 exhibited modest plasma half-life (~2h) and marked plasma protein binding (96%). When administered to parkinsonian marmosets with l-DOPA (7 mg/kg p.o.), NLX-112 (0.025, 0.1 and 0.4 mg/kg p.o.) reduced LID scores at early time-points after administration, whilst only minimally interfering with the l-DOPA-induced reversal of motor disability. In contrast, the prototypical 5-HT1A receptor agonist, (+)8-OH-DPAT (0.6 and 2 mg/kg p. o.), reduced LID but also abolished l-DOPA's anti-disability activity. Administered by itself, NLX-112 (0.1, 0.2 mg/kg p.o.) produced very little dyskinesia or locomotor activity, but reduced motor disability scores by about half the extent elicited by l-DOPA, suggesting that it may have motor facilitation effects of its own. Both NLX-112 and (+)8-OH-DPAT induced unusual and dose-limiting behaviors in marmoset that resembled ‘serotonin behavioral syndrome’ observed previously in rat. Overall, the present study showed that NLX-112 has anti-LID activity at the doses tested as well as reducing motor disability. The data suggest that additional investigation of NLX-112 is desirable to explore its potential as a treatment for PD and PD-LID., Highlights • l-DOPA-induced dyskinesia (LID) develops in many Parkinson's disease patients. • NLX-112 is a highly selective and high efficacy serotonin 5-HT1A receptor agonist. • In MPTP-treated marmosets NLX112 reduced LID without impairing therapeutic activity. • NLX-112 by itself exhibited antiparkinsonian activity (decreased motor disability). • NLX-112 is a promising drug candidate for treatment of Parkinson's disease.

International audience; The vast majority of PET radiopharmaceuticals used for receptor neuroimaging are antagonists. This is particularly true for 5-HT1A receptor radiopharmaceuticals such as 18F-MPPF or 11C-WAY100635. These radiolabeled antagonists allow PET neuroimaging of the total pool of receptors, regardless of their functional state. In contrast, agonists bind preferentially to the functionally active state of receptors, a pharmacological property that has not yet been exploited for PET imaging of functional 5-HT1A receptors [1]. To this end, we have developed a highly selective 5-HT1A receptor agonist, 18FF13640, and validated it in animal models [2]. A postmortem study in Alzheimer subjects showed that the in vitro binding of 18F-F13640 differs from that of 18F-MPPF, the prototypical radiopharmaceutical [3]. Here we show the first human brain image obtained in vivo with 18F-F13640 (study registration, EudraCT2017-002722-21). A summed brain PET image was obtained in a 32-year-old healthy man after IV injection of the radiopharmaceutical (90-min scan). 18F-F13640 brain distribution, expressed by the standardized uptake value with the body weight (SUV bw), showed a binding pattern different from that seen with conventional antagonist 5-HT1A radiopharmaceuticals. Although the binding regions are well correlated to the density of 5-HT1A receptors, the ROIs are mainly cortical, indicating that these brain regions are enriched in functionally active receptors. 18F-F13640 is the first radiopharmaceutical that enables in vivo investigation of functional 5-HT1A receptors that are likely to be altered in pathological conditions such as neurodegenerative diseases or psychiatric disorders. The development of this agonist PET radiopharmaceutical opens new opportunities for nuclear medicine.

Biased agonism (or “functional selectivity”) at G-protein-coupled receptors has attracted rapidly increasing interest as a means to improve discovery of more efficacious and safer pharmacotherapeutics. However, most studies are limited to in vitro tests of cellular signaling and few biased agonists have progressed to in vivo testing. As concerns 5-HT1A receptors, which exert a major control of serotonergic signaling in diverse CNS regions, study of biased agonism has previously been limited by the poor target selectivity and/or partial agonism of classically available ligands. However, a new generation of highly selective, efficacious and druggable agonists has advanced the study of biased agonism at this receptor and created new therapeutic opportunities. These novel agonists show differential properties for G-protein signaling, cellular signaling (particularly pERK), electrophysiological effects, neurotransmitter release, neuroimaging by PET and pharmacoMRI, and behavioral tests of mood, motor activity and side effects. Overall, NLX-101 (a.k.a. F15599) exhibits preferential activation of cortical and brain stem 5-HT1A receptors, whereas NLX-112 (a.k.a. befiradol or F13640) shows prominent activation of 5-HT1A autoreceptors in Raphe nuclei and in regions associated with motor control. Accordingly, NLX-101 is potently active in rodent models of depression and respiratory control, whereas NLX-112 shows promising activity in models of Parkinson's disease across several species – rat, marmoset and macaque. Moreover, NLX-112 has also been labeled with 18F to produce the first agonist PET radiopharmaceutical (known as [18F]-F13640) for investigation of the active state of 5-HT1A receptors in rodent, primate and human. The structure-functional activity relationships of biased agonists have been investigated by receptor modeling and novel compounds have been identified which exhibit increased affinity at 5-HT1A receptors and new profiles of cellular signaling bias, notably for β-arrestin recruitment versus pERK. Taken together, the data suggest that 5-HT1A receptor biased agonists constitute potentially superior pharmacological agents for treatment of CNS disorders involving serotonergic mechanisms.

International audience; In neuropharmacology, the recent concept of 'biased agonism' denotes the capacity of certain agonists to target-specific intracellular pathways of a given receptor in specific brain areas. In the context of serotonin pharmacotherapy, 5-HT1A receptor-biased agonists could be of great interest in several neuropsychiatric disorders. The aim of this study was to determine whether biased agonists could be differentiated in terms of regional targeting by use of simultaneous functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) brain imaging. We compared two 5-HT1A-biased agonists, NLX-112 and NLX-101, injected at three different doses in anaesthetised cats (n = 4). PET imaging was acquired for 90 min after bolus administration followed by constant infusion of the 5-HT1A radiotracer, [18F]MPPF. Drug occupancy was evaluated after injection at 50 min and BOLD fMRI was simultaneously acquired to evaluate subsequent brain activation patterns. 5-HT1A receptor occupancy was found to be dose-dependent for both agonists, but differed in magnitude and spatial distribution at equal doses with distinct BOLD patterns. Functional connectivity, as measured by BOLD signal temporal correlations between regions, was also differently modified by NLX-112 or NLX-101. Voxel-based correlation analyses between PET and fMRI suggested that NLX-112 stimulates both 5-HT1A autoreceptors and post-synaptic receptors, whereas NLX-101 preferentially stimulates post-synaptic cortical receptors. In cingulate cortex, the agonists induced opposite BOLD signal changes in response to receptor occupancy. These data constitute the first simultaneous exploration of 5-HT1A occupancy and its consequences in terms of brain activation, and demonstrates differential signalling by two 5-HT1A-biased agonists. Combined PET/fMRI represents a powerful tool in neuropharmacology, and opens new ways to address the concept of biased agonism by translational approaches.

NLX-112 (a.k.a. F13640 or befiradol), exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine 5-HT1A receptors. It possesses marked activity in a variety of animal models of depression, pain and L-DOPA-induced dyskinesia. However, its influence on translational biomarkers of central 5-HT1A receptor activation has not been previously described. Here, we report on the activity, in rats, of NLX-112 to increase plasma corticosterone levels and produce hypothermia, two responses which are also elicited by 5-HT1A receptor agonists in humans. NLX-112 elicited dose-dependent hypothermia (minimal effective dose, MED: 0.31 mg/kg p.o.) and also increased plasma corticosterone both by oral and intraperitoneal routes (MED: 0.63 mg/kg in both cases). The increase in corticosterone induced by NLX-112 (0.63 mg/kg p.o.) was abolished by co-administration of the selective 5-HT1A receptor antagonist, WAY100635. Additionally, NLX-112 also dose-dependently induced flat body posture, forepaw treading and lower lip retraction (MEDs 0.31–0.63 mg/kg p.o.). The doses of NLX-112 which induce hypothermia or corticosterone release were similar to those inducing serotonergic behaviors but greater than those reported previously in models of therapeutic-like activity (range 0.04 to 0.16 mg/kg). Overall, the present study provides information for clinical dose estimations of NLX-112 and suggests that therapeutic effects may occur at doses below those at which biomarker responses are observed.

Objectives NLX-112 (befiradol, F13640) is a selective serotonin 5-HT1A receptor agonist. Although it has been tested in vivo, little has been reported on its in vitro signal transduction profile. Methods NLX-112 was tested on G-protein activation, inhibition of adenylyl cyclase, ERK1/2 phosphorylation (pERK) and receptor internalization in recombinant cell lines. NLX-112 was also tested on G-protein activation in rat hippocampal membranes. Gα subunit mRNA expression in cell lines and rat brain tissue was quantified by quantitative PCR. Key findings For all signalling measures, NLX-112 exhibited agonist efficacy greater than for reference compounds ((±)8-OH-DPAT or buspirone), but similar to the endogenous agonist, serotonin, and was more potent for pERK than other responses. In rat hippocampal membranes, NLX-112 stimulated ‘total G-proteins' but, unlike (±)8-OH-DPAT and buspirone, was more potent for Gαo activation. Cell lines predominantly expressed Gαi1 and Gαi2 mRNA, with low levels of Gαo, whereas in rat brain Gαo subunits showed highest mRNA expression. Conclusions Unlike reference compounds, NLX-112 was a highly efficacious agonist in vitro, preferentially activating pERK in cell lines and Gαo proteins in rat hippocampal membranes. However, Gα subunit mRNA levels differ markedly between rat brain and cell lines, warranting caution when extrapolating from recombinant systems to native tissues.

Despite extensive efforts to design serotonin 5-HT1A receptor compounds, there are currently no clinically available selective agonists to explore the therapeutic potential of activating this receptor. Commonly used drugs targeting 5-HT1A receptors, such as buspirone or other azapirone compounds, possess only limited selectivity over cross-reacting sites, act as partial agonists for 5-HT1A receptor activation, and are metabolically labile, generating active metabolites. In addition, drug discovery has been hampered by the multiplicity of 5-HT1A receptor subpopulations, expressed in different brain regions, that are coupled to distinct molecular signaling mechanisms and mediate a wide variety of physiological responses, both desired and undesired. : In this context, advances in 5-HT1A receptor drug discovery have attracted attention of novel ‘biased agonists’ that are selective, efficacious and preferentially target the brain regions that mediate therapeutic activity without triggering side effects. The prototypical first-in-class compound NLX-101 (a.k.a. F15599; 3-chloro-4-fluorophenyl-[4-fluoro-4-[[(5-methylpyrimidin-2-ylmethyl)amino]methyl]piperidin- 1-yl]methanone), preferentially activates 5-HT1A receptors in cortical regions and exhibits potent, rapidacting and sustained antidepressant-like and procognitive properties in animal models. : Here the background has been reviewed that led to the discovery of the class of 1-(1-benzoylpiperidin-4- yl)methanamine derivatives, including NLX-101, as well as recent advances in discovery of novel 5-HT1A receptor biased agonists, notably aryloxyethyl derivatives of 1‑(1-benzoylpiperidin-4yl)methanamine which show promising pharmacological activity both in vitro and in vivo. : Overall, the results suggest that opportunities exist for innovative drug discovery of selective 5-HT1A receptor biased agonists that may open new avenues for the treatment of CNS disorders involving dysfunction of serotonergic neurotransmission.

Novel 1-(1-benzoylpiperidin-4-yl)methanamine derivatives were designed as “biased agonists” of serotonin 5-HT1A receptors. The compounds were tested in signal transduction assays (ERK1/2 phosphorylation, cAMP inhibition, Ca2+ mobilization, and β-arrestin recruitment) which identified ERK1/2 phosphorylation-preferring aryloxyethyl derivatives. The novel series showed high 5-HT1A receptor affinity, >1000-fold selectivity versus noradrenergic α1, dopamine D2, serotonin 5-HT2A, histamine H1, and muscarinic M1 receptors, and favorable druglike properties (CNS-MPO, Fsp3, LELP). The lead structure, (3-chloro-4-fluorophenyl)(4-fluoro-4-(((2-(pyridin-2-yloxy)ethyl)amino)methyl)piperidin-1-yl)methanone (17, NLX-204), displayed high selectivity in the SafetyScreen44 panel (including hERG channel), high solubility, metabolic stability, and Caco-2 penetration and did not block CYP3A4, CYP2D6 isoenzymes, or P-glycoprotein. Preliminary in vivo studies confirmed its promising pharmacokinetic profile. 17 also robustly sti...

NLX-101 (also known as F15599) exhibits nanomolar affinity, exceptional selectivity and biased agonist activation of serotonin 5-HT1A receptors. Given systemically, it displays antidepressant-like activity in the rat forced swim test (FST), and preferentially activates 5-HT1A post-synaptic heteroreceptors in the prefrontal cortex (PFC), a brain region involved in the control of mood. Here, we assessed the ability of NLX-101 to produce antidepressant-like activity in the FST following in-situ PFC unilateral microinjection. (+)8-OH-DPAT and F13714, two 5-HT1A receptor agonists that do not display cortical biased agonism, were tested as comparators. NLX-101 decreased time spent in immobility in a bi-modal manner, with a first MED of 0.25 μg (immobility reduced from 160 to 80 s) but immobility returned to control levels at the next dose (1 μg). At higher doses, immobility decreased monotonically, with a second MED of 16 μg and a maximal effect (36 s) at 32 μg. (+)8-OH-DPAT and F13714 also diminished immobility but, unlike NLX-101, they did so in a unimodal manner, with MEDs of 1 and 4 μg, and maximal responses of 31 and 4 s, for (+)8-OH-DPAT and F13714, respectively. The effects of (+)8-OH-DPAT (16 μg) and of both active doses of NLX-101 (0.25 and 16 μg) were prevented by the 5-HT1A receptor antagonist WAY-100,635 (0.63 mg/kg s.c.). In conclusion, activation of 5-HT1A receptors in the PFC by NLX-101 produces robust antidepressant-like effects in the rat FST, with a distinctive bimodal dose-response pattern. These data suggest that NLX-101 may target specific 5-HT1A receptor subpopulations in PFC, likely located on GABAergic and/or glutamatergic neurons.

Serotonin 1A receptors are known to play an important role in many psychiatric and neurodegenerative disorders. Currently, all available 5-HT1A receptor PET radiopharmaceuticals that are radiolabeled with fluorine-18 are antagonists. As agonists bind preferentially to the high-affinity state of receptors, it would be of great interest to develop agonist radioligands which could provide a measure of the functional 5-HT1A receptors in pathophysiological processes. The 5-HT1A receptor agonist candidates we recently proposed had promising in vitro properties but were not optimal in terms of PET imaging. F13640, a.k.a befiradol or NLX-112, is a 5-HT1A receptor agonist with a high affinity (Ki = 1 nM) and a high selectivity that would be suitable for a potential PET radiopharmaceutical. With propose here the first preclinical evaluation of 18F-F13640. 18F-F13640’s nitro-precursor was synthesized and radiolabeled via a fluoro-nucleophilic substitution. Its radiopharmacological characterization included autoradiographic studies, metabolic studies, and in vivo PET scans in rat, cat and non-human primate. Some of the results were compared with the radiotracer 18F-MPPF, a 5-HT1A receptor antagonist. The radiochemical purity of 18F-F13640 was > 98%. In vitro binding pattern was consistent with the 5-HT1A receptor distribution. Metabolic studies revealed that the radiotracer rapidly entered the brain and led to few brain radiometabolites. Although 18F-F13640 in vivo binding was blocked by the 5-HT1A antagonist WAY-100635 and the 5-HT1A agonist 8-OH-DPAT, the distribution pattern was markedly different from antagonist radiotracers in the three species, suggesting it provides novel information on 5-HT1A receptors. Preliminary studies also suggest a high sensitivity of 18F-F13640 to endogenous serotonin release. 18F-F13640 has suitable characteristics for probing in vitro and in vivo the 5-HT1A receptors in high-affinity state. Quantification analyses with kinetic modeling are in progress to prepare the first-in-man study of 18F-F13640.

Although serotonin 5-HT1A receptors constitute attractive therapeutic targets, there is a lack of potential clinical candidates that have a high degree of selectivity and full agonist efficacy. Recently, novel 5-HT1A receptor "biased agonists" F15599 (also known as NLX-101) and F13714 have been reported that exhibit distinctive properties for in vitro signaling, neurochemical, electrophysiological effects, and in brain imaging. The present study characterized their effects in rat models of anxiety (elevated plus-maze, EPM, and Vogel tests), in depressive-like behavior (forced swim test), and on the induction of the three serotonergic behaviors (forepaw treading, flat body posture, and lower lip retraction). The prototypical 5-HT1A receptor ligands (±)8-OH-DPAT and buspirone were tested as comparators. In the elevated plus-maze, F15599, F13714, and (±)8-OH-DPAT dose-dependently increased the amount and percentage of time spent in the open arms with minimal effective doses (MED) of 5 mg/kg p.o., 2.5 mg/kg p.o. and 1.25 mg/kg s.c., respectively. The effects of the three agonists were abolished by pretreatment with the selective 5-HT1A receptor antagonist, WAY100635 (0.63 mg/kg s.c.). Buspirone did not show significant activity in the EPM. In contrast, in the Vogel test only buspirone was active, significantly increasing the number of licks and shocks accepted (active dose: 1.25 mg/kg s.c.). However, WAY100635 failed to reverse the effects of buspirone in this test, suggesting that they were not 5-HT1A receptor-mediated. In the forced swim test, F15599, F13714, and (±)8-OH-DPAT were potently active, abolishing immobility (MED: 0.63 mg/kg p.o., 0.63 mg/kg p.o. and 0.16 mg/kg s.c., respectively). Buspirone was not active. In measures of serotonergic behavior, F13714 and (±)8-OH-DPAT robustly elicited all three signs of serotonergic behaviors, whereas F15599 and buspirone elicited only lower-lip retraction. Taken together, these observations highlight the distinct profiles of activity of 5-HT1A agonists and suggest that the novel biased agonist F15599 combines pronounced activity in a test of anxiety (elevated plus-maze) with potent antidepressant-like effects and low propensity to induce serotonergic behaviors. These data suggest that selective biased agonists could constitute promising pharmacotherapeutics for mood disorders.

L-3,4-dihydroxyphenylalanine (L-DOPA) is the most effective anti-parkinsonian agent available, but upon chronic administration, patients with Parkinson's disease (PD) experience abnormal involuntary movements, dyskinesia. Modulation of serotonin 1A (5-HT1A) receptors is regarded as an effective way to alleviate dyskinesia, yet this approach has been marred by a reduction of the therapeutic effectiveness of L-DOPA. We hypothesised that highly-selective 5-HT1A stimulation might be a way to alleviate dyskinesia without compromising L-DOPA anti-parkinsonian action. F15599 (also known as NLX-101) is a highly-selective 5-HT1A agonist that displays over 1000 × selectivity over off-target receptors. Seven cynomolgus macaques were administered MPTP and developed severe parkinsonism. Following chronic administration of L-DOPA, they developed severe and reproducible dyskinesia. F15599 (0.003, 0.01, 0.03 and 0.1 mg/kg) or vehicle was administered in combination with L-DOPA and its effect on dyskinesia and L-DOPA anti-parkinsonian was assessed. In combination with L-DOPA, F15599 (0.1 mg/kg) reduced the severity of peak-dose dyskinesia, by ≈45% (P < 0.001), compared to L-DOPA alone. F15599 (any dose) had no effect on duration of on-time or motor activity counts compared to L-DOPA alone. F15599 at 0.03 and 0.1 mg/kg significantly reduced duration of on-time with disabling dyskinesia (by ≈49% and ≈71%, P < 0.05 and P < 0.001, respectively). These results suggest that F15599, a highly-selective 5-HT1A receptor agonist, alleviates dyskinesia without exerting a deleterious effect on L-DOPA anti-parkinsonian action.

Schizophrenia is characterised by positive, negative, cognitive, depressive and anxiety symptoms. Over the last decades a number of novel treatment strategies with better clinical efficacy and scope, but with lower side-effect profiles have been developed. These have significantly improved the management and prognosis of the disease. Of these approaches, modulation of the serotonergic receptor system is a common, recurring, theme; particularly the use of 5-HT1A receptor agonism as part of or adjunct to existing therapies. Here we review data exploring the utility of 5-HT1A receptor agonists for extending the actions of antipsychotic agents, while limiting their side-effect profile. Notably, interest has grown concerning 5-HT1A receptor activation in schizophrenia because of the development of novel antipsychotics, such as lurasidone and cariprazine, the characterisation of 5-HT1A receptor polymorphisms in schizophrenia patients and the possible beneficial influence of 5-HT1A agonists on neurogenesis.

Serotonin 5-HT1A receptor agonists reduce L-DOPA-induced dyskinesia (LID) in animal models of Parkinson's disease (PD). Here, we compared the effects of novel 5-HT1A receptor 'biased agonists' on LID in hemiparkinsonian rats. F13714 preferentially activates pre-synaptic 5-HT1A autoreceptors. F15599 preferentially activates cortical postsynaptic 5-HT1A heteroreceptors. The partial agonist, tandospirone, does not differentiate these receptor subpopulations. The drugs were also tested on rotational behavior, rotarod and cylinder test for evaluation of locomotor activity, motor coordination and forelimb akinesia. Finally, the effects of F13714 and F15599 on 5-HT, DA, glutamate, and GABA release were investigated by microdialysis. F13714 abolished L-DOPA-induced AIMs even at very low doses (0.02-0.04 mg/kg). This effect was reversed by the selective 5-HT1A receptor antagonist, WAY100635. F13714 also elicited ipsilateral rotations (which were blocked by WAY100635) and potentiated the rotational activity of a sub-threshold dose of L-DOPA (2 mg/kg). F13714 profoundly inhibited striatal 5-HT release on both sides of the brain, and slightly increased DA release on the intact side. F15599 inhibited the L-DOPA-induced AIMs only at a dose (0.16 mg/kg) that reduced 5-HT release. Tandospirone produced a modest attenuation of peak AIMs severity and did not elicit rotations. F13714, F15599 and tandospirone did not modify the action of L-DOPA in the cylinder test but impaired rotarod performance at the highest doses tested. Targeting 5-HT1A receptors with selective biased agonists exerts distinct effects in the rat model of PD and LID. Preferential activation of 5-HT1A autoreceptors could potentially translate to superior antidyskinetic and L-DOPA dose-sparing effects in PD patients.

Summary Aims Positron emission tomography (PET) imaging using 5-HT1A receptor radioligands shows a decreased expression of this serotonin receptor in the hippocampus of patients with Alzheimer's disease (AD) at advanced stages. However, previous 5-HT1A receptor radioligands used in human imaging were antagonists, thought to bind to 5-HT1A receptors in different functional states (i.e., both the one which displays high affinity for agonists and is thought to mediate receptor activation, as well as the functional state which has low affinity for agonists). Comparing the PET imaging obtained using an agonist radioligand, which binds selectively to the functional state of the receptors, with the PET imaging obtained using an antagonist radioligand would therefore provide original information on 5-HT1A receptor impairment during AD. Methods Quantitative autoradiography using 18F-F15599 and 18F-MPPF, a 5-HT1A agonist and antagonist, respectively, was measured in hippocampi of 18 patients with AD. Results Functional 5-HT1A receptors, labeled by 18F-F15599, represented ~35% of total receptors, as estimated by 18F-MPPF labeling. 18F-F15599 binding decreased in dentate gyrus of patients with AD, as indicated by Braak's stages. In contrast, binding of 18F-MPPF was statistically unchanged. Conclusion These in vitro results support testing the concept of functional PET imaging using agonist radioligands in clinical studies.

A previous study observed bell-shaped concentration-response isotherms for activation of Gαi3 G-protein subunits by high efficacy 5-HT1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gαi3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT1A receptors in other cellular environments. Here, using an antibody capture-based [35S]GTPγS binding assay for Gαi3 activation, we investigated whether efficacious 5-HT1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F ) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gαi3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gαi3 activation. These data indicate that Gαi3 G-protein activation by 5-HT1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.

International audience; Serotonin 1A (5-HT 1A) receptors are involved in several physiological and pathological processes and constitute therefore an important therapeutic target. The recent pharmacological concept of biased agonism asserts that highly selective agonists can preferentially direct receptor signaling to specific intracellular responses, opening the possibility of drugs targeting a receptor subtype in specific brain regions. The present study brings additional support to this concept thanks to functional magnetic resonance imaging (7 Tesla-fMRI) in anaesthetized rats. Three 5-HT 1A receptor agonists (8-OH-DPAT, F13714 and F15599) and one 5-HT 1A receptor antagonist (MPPF) were compared in terms of influence on the brain blood oxygen level-dependent (BOLD) signal. Our study revealed for the first time contrasting BOLD signal patterns of biased agonists in comparison to a classical agonist and a silent antagonist. By providing functional information on the influence of pharmacological activation of 5-HT 1A receptors in specific brain regions, this neuroimaging approach, translatable to the clinic, promises to be useful in exploring the new concept of biased agonism in neuropsychopharmacology. 5-HT 1A receptors belong to the family of serotonin receptors, composed of 13 receptor subtypes. 5-HT 1A receptors are known to play a key role in serotonin neurotransmission due to their localization both as pre-synaptic receptors located on serotonin cell bodies in the raphe nuclei (somatodendritic receptors) and as post-synaptic heteroreceptors in forebrain areas that receive serotonergic projections. In such areas, 5-HT 1A receptors are located on pyramidal and GABAergic neurons of the neocortex and limbic system 1,2. Because of their distribution pattern and of their central role in the modulation of the serotoninergic neurotransmission, 5-HT 1A receptors are involved in several physiological and pathological processes and constitute therefore an important therapeutic target for psychiatric 3 and, more recently, for neurological disorders 4. Indeed, it is now well establish that 5-HT 1A receptors are one of the main targets for the treatment of mood disorders 5 , with different actions that depend strongly on their localization. For example, activation of soma-todendritic receptors by serotonin or 5-HT 1A receptor agonists decreases the firing of serotonin neurons in the raphe, and, consequently decreases its terminal release 6. This decrease is thought to be partially responsible for the delay in onset of the therapeutic action of selective serotonin reuptake inhibitors (SSRI) antidepressants 5. A recent study renewed this concept, showing that expression levels of 5-HT 1A somatodendritic receptors, are critically important for SSRI treatment response by controlling serotoninergic tone 7. On the other hand, the activation of postsynaptic 5-HT 1A receptors seems to be equally important for response to antidepressants 5,8. In a different therapeutic area, 5-HT 1A receptor agonism is also known as an important feature of some atypical antipsychotics including clozapine, aripiprazole, ziprasidone and quetiapine 9–12. Recently, 5-HT 1A receptors have attracted renewed interest as possible targets in neuropharmacology. For example, it was described that blockade of post-synaptic 5-HT 1A receptors, located on pyramidal cells, can improve cognition by enhancing glutamatergic transmission 13. This led to clinical trials using the 5-HT 1A antagonist lecozotan as a procognitive drug in Alzheimer's disease 14,15. Other 5-HT 1A receptor ligands such as sarizotan, buspirone and tandospirone were shown to alleviate dyskinesia in Parkinson's disease patients 16,17. More recently

Background In vivo mapping by positron emission tomography of the serotonin 1A receptors has been hindered by the lack of suitable agonist positron emission tomography probes. 18 F-labeled F13714 is a recently developed biased agonist positron emission tomography probe that preferentially targets subpopulations of serotonin 1A receptors in their " active state, " but its brain labeling pattern in nonhuman primate has not been described. In addition, a potential confound in the translatability of PET data between nonhuman animal and human arise from the use of anesthetics that may modify the binding profiles of target receptors. Methods Positron emission tomography scans were conducted in a cohort of common marmosets (n = 4) using the serotonin 1A receptor biased agonist radiotracer, 18 F-F13714, compared with a well-characterized 18 F-labeled antagonist radiotracer, 18 F-MPPF. Experiments on each animal were performed under both consciousness and isoflurane-anesthesia conditions. Results 18 F-F13714 binding distribution in marmosets by positron emission tomography differs markedly from that of the of the 18F-MPPF. Whereas 18F-MPPF showed highest binding in hippocampus and amygdala, 18F-F13714 showed highest labeling in other regions, including insular and cingulate cortex, thalamus, raphe, caudate nucleus, and putamen. The binding potential values of 18F-F13714 were about one-third of those observed with 18F-MPPF, with marked individual- and region-specific differences under isoflurane-anesthetized vs conscious conditions. Conclusions These findings highlight the importance of investigating the brain imaging of serotonin 1A receptors using agonist probes such as 18F-F13714, which may preferentially target subpopulations of serotonin 1A receptors in specific brain regions of nonhuman primate as a biased agonist.

Background and purpose: F15599, a novel 5-hydroxytryptamine (5-HT)1A receptor agonist with 1000-fold selectivity for 5-HT compared with other monoamine receptors, shows antidepressant and procognitive activity at very low doses in animal models. We examined the in vivo activity of F15599 at somatodendritic autoreceptors and postsynaptic 5-HT1A heteroreceptors. Experimental approach: In vivo single unit and local field potential recordings and microdialysis in the rat. Key results: F15599 increased the discharge rate of pyramidal neurones in medial prefrontal cortex (mPFC) from 0.2 µg·kg−1 i.v and reduced that of dorsal raphe 5-hydroxytryptaminergic neurones at doses >10-fold higher (minimal effective dose 8.2 µg·kg−1 i.v.). Both effects were reversed by the 5-HT1A antagonist (±)WAY100635. F15599 did not alter low frequency oscillations (∼1 Hz) in mPFC. In microdialysis studies, F15599 increased dopamine output in mPFC (an effect dependent on the activation of postsynaptic 5-HT1A receptors) with an ED50 of 30 µg·kg−1 i.p., whereas it reduced hippocampal 5-HT release (an effect dependent exclusively on 5-HT1A autoreceptor activation) with an ED50 of 240 µg·kg−1 i.p. Likewise, application of F15599 by reverse dialysis in mPFC increased dopamine output in a concentration-dependent manner. All neurochemical responses to F15599 were prevented by administration of (±)WAY100635. Conclusions and implications: These results indicate that systemic administration of F15599 preferentially activates postsynaptic 5-HT1A receptors in PFC rather than somatodendritic 5-HT1A autoreceptors. This regional selectivity distinguishes F15599 from previously developed 5-HT1A receptor agonists, which preferentially activate somatodendritic 5-HT1A autoreceptors, suggesting that F15599 may be particularly useful in the treatment of depression and of cognitive deficits in schizophrenia.

Typical antipsychotics such as haloperidol exert their therapeutic effects via blockade of dopamine (DA) D 2 receptors, leading to extrapyramidal symptoms (EPS) in humans and catalepsy in rodents. In contrast, atypical antipsychotics and new generation D 2 /5-HT 1A antipsychotics have low cataleptogenic potential. However, there has been no systematic comparative study on the effects of these different classes of antipsychotics in non-human primates, a species displaying a more sophisticated repertoire of behavioural/motor activity than rats. Once weekly, six young adult female non-haloperidol-sensitised cynomolgus monkeys were treated i.m. with a test compound and videotaped to score catalepsy-associated behaviour (CAB: static postures, unusual positions and crouching). Haloperidol, risperidone, olanzapine, nemonapride and remoxipride induced, to different extents, an increase in unusual positions (a response akin to dystonia), some crouching and static postures. In contrast, clozapine, quetiapine, ziprasidone and aripiprazole produced much lower or no unusual positions; clozapine also produced marked increases in static postures and crouching. Among novel D 2 /5-HT 1A antipsychotics, SLV313 and F15063 augmented the number of unusual positions, albeit at doses 16–63 times higher than those of haloperidol for approximately the same score. SSR181507 and bifeprunox produced moderate static postures, little crouching and negligible unusual positions. These data provide the first comparative analysis in cynomolgus monkeys of EPS liability of conventional, atypical and novel D 2 /5-HT 1A antipsychotics. They indicate that the latter are less prone than haloperidol to produce CAB, and provide a basis for comparison with rodent catalepsy studies.

Background and purpose: Activation of post-synaptic 5-HT1A receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT1A receptor agonist. Experimental approach: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. Key results: F15599 was highly selective for 5-HT1A receptors in binding experiments and in [35S]-GTPγS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT1A receptors. In cell lines expressing h5-HT1A receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT1A receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [35S]-GTPγS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated Gαi than Gαo activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT1A receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT1A receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT1A receptors in vivo almost as potently as F13714. Conclusions and implications: F15599 showed a distinctive activation profiles for 5-HT1A receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT1A receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition.

Small rodents (mice, rats) are the species of choice for evaluating the pharmacology of centrally acting compounds, such as antipsychotics, whereas toxicology data are routinely obtained from other species (rabbits, dogs, monkeys). Whilst there is a substantial number of "therapeutically relevant" pharmacological models for "antipsychotic-like" activity in small rodents, based on hyperdopaminergic or hypoglutamatergic/NMDA approaches, there is a remarkable paucity of such models in other species. Here, we compared the efficacy and potency of reference and new generation dopamine D2/5-HT(1A) putative antipsychotics, administered orally, against apomorphine-induced emesis in dogs, a model of central D2 receptor activation that can be implemented with relative ease. Risperidone potently and fully (10 microg/kg) prevented emesis/retching induced by 0.1 mg/kg s.c. apomorphine. SLV313 and F15063 (D2 receptor antagonists/5-HT(1A) receptor agonists) also abolished emesis/retching, albeit less potently than risperidone (minimal effective dose, MEDs: 10 and 40 microg/kg, respectively). The D2 receptor partial agonists/5-HT(1A) receptor agonists aripiprazole and bifeprunox, (up to 80 microg/kg) only partially attenuated emesis, as did the peripheral D2 receptor antagonist domperidone. Under the present experimental conditions, haloperidol was only efficacious at the highest dose tested (320 microg/kg). To summarize, dogs are very sensitive to the dopaminergic blocking effects of antipsychotics in this model of D2 receptor activation. This model can thus be advantageously used to investigate the pharmacological activity of novel D2 receptor antagonists/partial agonists in dogs.

The novel, potential antipsychotic, S33138 (N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide), displayed approximately 25-fold higher affinity at human (h) dopamine D(3) versus hD(2L) (long isoform) and hD(2S) (short isoform) receptors (pK(i) values, 8.7, 7.1, and 7.3, respectively). Conversely, haloperidol, clozapine, olanzapine, and risperidone displayed similar affinities for hD(3), hD(2L), and hD(2S) sites. In guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) filtration assays, S33138 showed potent, pure, and competitive antagonist properties at hD(3) receptors, displaying pK(B) and pA(2) values of 8.9 and 8.7, respectively. Higher concentrations were required to block hD(2L) and hD(2S) receptors. Preferential antagonist properties of S33138 at hD(3) versus hD(2L) receptors were underpinned in antibody capture/scintillation proximity assays (SPAs) of Galpha(i3) recruitment and in measures of extracellular-regulated kinase phosphorylation. In addition, in cells cotransfected with hD(3) and hD(2L) receptors that assemble into heterodimers, S33138 blocked (pK(B), 8.5) the inhibitory influence of quinpirole upon forskolin-stimulated cAMP formation. S33138 had low affinity for hD(4) receptors (

F15063 (N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine fumarate salt) is a novel potential antipsychotic with dopamine D(2)/D(3) blocking properties and agonist activity at 5-HT(1A) and D(4) receptors. The pertinent parameter for pharmacological activity of antipsychotics appears to be central D2-like receptor occupancy. However, its duration is not necessarily correlated with drug plasma levels, on which clinical dosing regimens are often based. Thus, we compared in mice the duration of actions of F15063 and haloperidol to (1) inhibit apomorphine-induced climbing and sniffing (behavioural measures of D2-like receptor antagonism) and (2) occupy D2-like receptors in vivo in the striatum and olfactory tubercles (inhibition of [(3)H]nemonapride binding). Finally, we measured plasma levels of F15063. D2-like receptor occupancy in the striatum remained elevated at 1, 4 and 8 h postadministration, with both F15063 (ID(50): 7.1, 3.6 and 16.5 mg/kg p.o., respectively) and the typical antipsychotic, haloperidol (ID(50): 1.4, 0.52 and 0.53 mg/kg p.o., respectively). This was paralleled by a protracted inhibition of apomorphine-induced climbing (ED(50): 0.9, 2.8 and 3.6 mg/kg p.o., and 0.21, 0.37 and 0.87 mg/kg p.o., respectively, for F15063 and haloperidol). In contrast, after administration of 10 mg/kg p.o. of F15063, its plasma levels decreased rapidly: 15.2, 2.1 and 0.6 ng/ml, 1, 4 and 8 h after administration, respectively. A similar pattern of results was observed when F15063 and haloperidol were administered i.p. and s.c., respectively. To summarise, the time-course of D2-like receptor occupancy and inhibition of apomorphine-climbing (and sniffing) behaviours was similarly long lasting with F15063 and haloperidol. In addition, the durations of action of F15063 and haloperidol in a behavioural model of antipsychotic-like activity were closely correlated to their occupancy of central D2-like receptors, and much longer than their presence in plasma.

Prepulse inhibition (PPI) of the startle reflex has been extensively studied because it is disrupted in several psychiatric diseases, most notably schizophrenia. In rats, and to a lesser extent, in humans, PPI can be diminished by dopamine (DA) D(2)/D(3) and serotonin 5-HT(1A) receptor agonists. A novel class of potential antipsychotics (SSR181507, bifeprunox, and SLV313) possess partial agonist/antagonist properties at D(2) receptors and various levels of 5-HT(1A) activation.It thus appeared warranted to assess, in Sprague-Dawley rats, the effects of these antipsychotics on basal PPI.SSR181507, sarizotan, and bifeprunox decreased PPI, with a near-complete abolition at 2.5-10 mg/kg; SLV313 had a significant effect at 0.16 mg/kg only. Co-treatment with the 5-HT(1A) receptor antagonist WAY100,635 (0.63 mg/kg) showed that the 5-HT(1A) agonist activity of SSR181507 was responsible for its effect. By contrast, antipsychotics with low affinity and/or efficacy at 5-HT(1A) receptors, such as aripiprazole (another DA D(2)/D(3) and 5-HT(1A) ligand), and established typical and atypical antipsychotics (haloperidol, clozapine, risperidone, olanzapine, quetiapine, and ziprasidone) had no effect on basal PPI (0.01-2.5 to 2.5-40 mg/kg).The present data demonstrate that some putative antipsychotics with pronounced 5-HT(1A) agonist activity, coupled with partial agonist activity at DA D(2) receptors, markedly diminish PPI of the startle reflex in rats.These data raise the issue of the influence of such compounds on sensorimotor gating in humans.

Combining antagonist/partial agonist activity at dopamine D2 and agonist activity at serotonin 5-HT1A receptors is one of the approaches that has recently been chosen to develop new generation antipsychotics, including bifeprunox, SSR181507 and SLV313. There have been, however, few comparative data on their pharmacological profiles. Here, we have directly compared a wide array of these novel dopamine D2/5-HT1A and conventional antipsychotics in rat models predictive of antipsychotic activity. Potency of antipsychotics to antagonize conditioned avoidance, methylphenidate-induced behaviour and D-amphetamine-induced hyperlocomotion correlated with their affinity at dopamine D2 receptors. Potency against ketamine-induced hyperlocomotion was independent of affinity at dopamine D2 or 5-HT1A receptors. Propensity to induce catalepsy, predictive of occurrence of extrapyramidal side effects, was inversely related to affinity at 5-HT1A receptors. As a result, preferential D2/5-HT1A antipsychotics displayed a large separation between doses producing 'antipsychotic-like' vs. cataleptogenic actions. These data support the contention that 5-HT1A receptor activation greatly reduces or prevents the cataleptogenic potential of novel antipsychotics. They also emphasize that interactions at 5-HT1A and D2 receptors, and the nature of effects (antagonism or partial agonism) at the latter has a profound influence on pharmacological activities, and is likely to affect therapeutic profiles.

The D 2 dopaminergic receptor represents a major target of antipsychotic drugs. Using the coupling of the human D 2long (hD 2L ) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus laevis oocytes, we examined the activity of antipsychotic agents of different classes – typical, atypical, and a “new generation” of compounds, exhibiting a preferential D 2 and 5-HT 1A receptor profile. When the hD 2L receptor was coexpressed with GIRK channels, a series of reference compounds exhibited full agonist (dopamine, and quinpirole), partial agonist (apomorphine, (−)3-PPP, and (+)-UH232) or inverse agonist (raclopride, and L741626) properties. Sarizotan exhibited only very weak partial agonist action. At higher levels of receptor cRNA injected per oocyte, both partial agonist activity and inverse agonist properties were generally more pronounced. The inverse agonist action of L741626 was reversed by interaction with sarizotan, thus confirming the constitutive activity of wild-type hD 2L receptors in the oocyte expression system. When antipsychotic agents were tested for their actions at the hD 2L receptor, typical (haloperidol) as well as atypical (nemonapride, ziprasidone, and clozapine) compounds acted as inverse agonists. In contrast, among D 2 /5-HT 1A antipsychotics, only SLV313 and F15063 behaved as inverse agonists, whilst the other members of this group (bifeprunox, SSR181507 and the recently marketed antipsychotic, aripiprazole) exhibited partial agonist properties. Thus, the X. laevis oocyte expression system highlights markedly different activity of antipsychotics at the hD 2L receptor. These differential properties may translate to distinct therapeutic potential of these compounds.

We report the discovery and the synthesis of novel, potential antipsychotic compounds combining potent dopamine D2 receptor antagonist and serotonin 5-HT1A receptor agonist properties in the same molecule. We describe the structure-activity relationship that lead us to the promising derivative: N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine 16. The latter has high affinity for D2 and 5-HT1A receptors, whereas it possesses only a weak affinity for 5-HT2A sites. In cellular models of signal transduction, 16 behaves as a silent antagonist at rD2 receptors while activating h5-HT1A receptors with an efficacy at least equivalent to that of the prototypical 5-HT1A agonist (+/-)-8-OH-DPAT. These dual actions confer a unique pharmacological profile to the product. In a behavioral model predictive of positive symptoms, 16 has an activity comparable to that of the typical antipsychotic haloperidol, while it is devoid of cataleptogenic effects. Although it produces behaviors characteristic of 5-HT1A receptor activation in rats, these occur at doses 100 times higher than those with (+/-)-8-OH-DPAT. We believe that the relative balance of D2 and 5-HT1A actions in 16 is appropriate, possibly optimal, to ensure superior efficacy and tolerability over existing antipychotic drugs.

Background and purpose: Desensitization of somatodendritic 5-HT1A receptors is involved in the mechanism of action of several antidepressants, but the rapidity of this effect and the amount of agonist stimulation needed are unclear. We evaluated the capacity of the high-efficacy 5-HT1A agonist, F13714 (3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methylaminopyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone) and of the partial agonist, flesinoxan, to desensitize somatodendritic 5-HT1A receptors involved in the control of 5-HT release. Experimental approach: Intracerebral microdialysis in the hippocampus of freely moving rats was used to examine the acute and chronic effects of the two compounds (administered by osmotic pumps for 3, 7 or 14 days) on extracellular 5-HT levels, measured by HPLC with electrochemical detection. Key results: When given acutely, F13714, flesinoxan and the low-efficacy 5-HT1A agonist, buspirone, dose-dependently decreased extracellular 5-HT concentrations (ED50 values: 0.04, 0.77 and 5.6 mg kg � 1 , respectively). The selective 5-HT1A antagonist WAY100635 inhibited the effects of the three compounds. F13714 (2.5 mg kg � 1 per day for 3, 7 or 14 days and 0.63 mg kg � 1 for 7 days) significantly attenuated the inhibition of 5-HT release induced by buspirone (10 mg kg � 1 ). In contrast, flesinoxan (10 mg kg � 1 per day) failed to alter the response to buspirone at any of the treatment durations. Conclusions and implications: Rat somatodendritic 5-HT1A receptors controlling hippocampal 5-HT release were rapidly desensitized by chronic activation with a high-efficacy 5-HT1A agonist, but not by chronic activation with a partial agonist. Thus, rapid 5-HT1A autoreceptor desensitization by high-efficacy agonists may accelerate the onset of the therapeutic effects of antidepressants.

The effects of antipsychotics targeting dopamine D2 and serotonin 5-HT1A receptors were compared with conventional antipsychotics on phosphorylation of Extracellular signal-Regulated Kinase 1/2 (ERK 1/2) in CHO cell lines stably expressing either the human serotonin 5-HT1A or human dopamine D2S receptor. All antipsychotics except haloperidol and olanzapine exhibited agonist properties at serotonin 5-HT1A receptors. Emax values (% effect of 10 microM 5-HT) were: bifeprunox (74), SSR181507 (73), SLV313 (72), aripiprazole (60), ziprasidone (56), clozapine (33). At dopamine D2S receptors, partial agonist activity (% effect of 10 microM dopamine) was observed for bifeprunox (76), SSR181507 (66) and aripiprazole (59). Other antipsychotics attenuated dopamine-induced ERK phosphorylation, with pK(B) values of : SLV313 (8.5), haloperidol (8.1), olanzapine (7.8), ziprasidone (7.7), and clozapine (6.4). Amongst the dopamine D2/serotonin 5-HT1A receptor compounds, aripiprazole acts as a partial dopamine D2S and serotonin 5-HT1A receptor agonist. SSR181507 and bifeprunox possess a profile of action similar to each other, efficaciously stimulating both serotonin 5-HT1A and dopamine D2S receptors. In contrast, SLV313, also an efficacious serotonin 5-HT1A receptor agonist, acted as a high potency dopamine D2 receptor antagonist. Thus, antipsychotics display varying efficacies at serotonin 5-HT1A and dopamine D2S receptors which may play a major role in their differential functional profiles in blocking the diverse symptoms of schizophrenia.

The dopamine D1/D2 agonist apomorphine (0.63 mg/kg) disrupted prepulse inhibition (PPI) of acoustic startle in rats, a model of sensorimotor gating deficits observed in schizophrenia. All current antipsychotics, which antagonize D2 receptors, prevent this apomorphine-induced deficit. A novel class of antipsychotics possesses, in addition to D2 antagonist property, various levels of 5-HT1A agonist activity. Considering that the latter itself produces PPI deficits, it appeared necessary to assess the potential of this novel class of antipsychotics to reverse apomorphine-PPI deficits. Potent D2 antagonists, like haloperidol (0.63-2.5 mg/kg), risperidone (0.63-10 mg/kg), and olanzapine (0.63-40 mg/kg) prevented apomorphine PPI disruption. The atypical antipsychotics, clozapine (40 mg/kg), nemonapride (0.01-2.5 mg/kg), ziprasidone (10 mg/kg), and aripiprazole (0.01 and 10 mg/kg), which all exhibit 5-HT1A agonist properties, reversed PPI deficits at some doses only, whereas the anti-dyskinetic agent sarizotan (0.16-10 mg/kg), an efficacious 5-HT1A agonist, did not. New generation antipsychotics with marked 5-HT1A agonist properties, such as SLV313 and SSR181507 (0.0025-10 mg/kg and 0.16-10 mg/kg, respectively) did not reverse these deficits whereas bifeprunox (0.04-2.5 mg/kg) did. To reveal the contribution of 5-HT1A agonist properties in the lack of effects of SLV313 and SSR181507, we pretreated rats with the 5-HT1A antagonist WAY100635 (0.63 mg/kg). Under these conditions, significant reversal of PPI deficit was observed, indicating that D2 antagonist properties of SLV313 and SSR181507 are now sufficient to overcome the disruptive effects of apomorphine. To summarize, antipsychotics possessing agonist efficacy at 5-HT1A receptors exhibit diverse profiles against apomorphine-induced PPI deficits, depending on the balance between D2 and 5-HT1A activities, suggesting that they may display distinct activity on some aspects of gating deficits in schizophrenic patients.

Compounds possessing 5-HT 1A agonist properties attenuate catalepsy induced by D 2 receptor blockade. Here we examined the role of 5-HT 1A receptor agonism in the reduced cataleptogenic potential of several novel antipsychotic agents in the crossed leg position (CLP) and the bar catalepsy tests in rats. When administered alone, ziprasidone produced marked catalepsy, whereas aripiprazole, bifeprunox, SLV313, SSR181507 and sarizotan did not. However, when 5-HT 1A receptors were blocked with the selective antagonist, WAY100635 (0.63 mg/kg, SC), robust cataleptogenic properties of SLV313, bifeprunox and sarizotan were unmasked and the catalepsy induced by ziprasidone was accentuated. In contrast, only modest catalepsy was induced by aripiprazole and SSR181507, even following a higher dose of WAY100635 (2.5 mg/kg). This suggests that these compounds possess other anti-cataleptic properties, such as partial agonism at dopamine D 2 receptors. The capacity to reverse neuroleptic-induced catalepsy was investigated in interaction studies with haloperidol (2.5 mg/kg, SC). Whereas ziprasidone and aripiprazole did not markedly reduce the effects of haloperidol, SLV313 and sarizotan attenuated CLP catalepsy. In contrast, SSR181507 and bifeprunox potently inhibited both CLP and bar catalepsy. Taken together, these data show that 5-HT 1A receptor activation reduces the cataleptogenic potential of novel antipsychotic agents but indicate marked diversity in the contribution of 5-HT 1A and/or other mechanisms to the profiles of the drugs.

Excessive activation of non-NMDA receptors, AMPA and kainate, contributes to neuronal degeneration in acute and progressive pathologies, possibly including schizophrenia. Because 5-HT(1A) receptor agonists have neuroprotective properties (e.g., against NMDA-induced neurotoxicity), we compared the effects of the antipsychotics, clozapine, ziprasidone and aripiprazole, that are partial agonists at 5-HT(1A) receptor, with those of haloperidol, which is devoid of 5-HT(1A) agonist properties, on kainic acid (KA)-induced striatal lesion volumes, in C57Bl/6N mice. The involvement of 5-HT(1A) receptors was determined by antagonist studies with WAY100635, and data were compared with those obtained using the potent and high efficacy 5-HT(1A) receptor agonist, F13714. Intra-striatal KA lesioning and measurement of lesion volumes using cresyl violet staining were carried out at 48 h after surgery. F13714, antipsychotics or vehicle were administered ip twice, 30 min before and 3 1/2 h after KA injection. WAY100635 (0.63 mg/kg) or vehicle were given sc 30 min before each drug injection. Clozapine (2 x 10 mg/kg), ziprasidone (2 x 20 mg/kg) and aripiprazole (2 x 10 mg/kg) decreased lesion volume by 61%, 59% and 73%, respectively. WAY100635 antagonized the effect of ziprasidone and of aripiprazole but only slightly attenuated that of clozapine. In contrast, haloperidol (2 x 0.16 mg/kg) did not affect KA-induced lesion volume. F13714 dose-dependently decreased lesion volume. The 61% decrease of lesion volume obtained with F13714 (2 x 0.63 mg/kg) was antagonized by WAY100635. WAY100635 alone did not affect lesion volume. These results show that 5-HT(1A) receptor activation protects against KA-induced striatal lesions and indicate that some atypical antipsychotic agents with 5-HT(1A) agonist properties may protect against excitotoxic injury, in vivo.

Dopamine D2 receptor blockade is thought to be mandatory for antipsychotic action because most of the currently used antipsychotics have high affinity at these receptors. Here, we examined the in vivo binding characteristics of the D2-like receptor antagonist [3H]nemonapride in rat brain areas including the striatum, olfactory lobes and frontal cortex and its inhibition by a series of D2 antagonist antipsychotics. In vivo affinity of [3H]nemonapride was similar (apparent Kd value: 0.05 µmol/kg) in all brain regions examined. The estimated number of binding sites was higher in the striatum (66 fmol/mg wet weight) than in the olfactory lobes (28 fmol/mg wet weight) and the frontal cortex (21 fmol/mg wet weight). In the striatum, [3H]nemonapride binding was inhibited in a dose-dependent manner with the following order of potency (ED50, mg/kg): nemonapride (0.04), raclopride (0.13), spiperone and risperidone (0.14), haloperidol (0.21), clozapine (7.2) and thioridazine (9.4); in the olfactory lobes: nemonapride (0.03), raclopride and spiperone (0.09), haloperidol (0.10), risperidone (0.15), thioridazine and clozapine (11); in the frontal cortex, only the high affinity dopamine D2 antagonist compounds nemonapride (0.05), haloperidol (0.09), and raclopride (0.12) significantly decreased the binding of [3H]nemonapride. The present data suggest that conventional and atypical antipsychotics may be distinguished by their differential occupancy of striatal versus frontocortical D2-like receptors in vivo.

S32504 [(+)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine] displayed marked affinity for cloned, human (h)D(3) receptors (pK(i), 8.1) at which, in total G-protein ([(35)S]GTPgammaS binding, guanosine-5'-O-(3-[(35)S]thio)-triphosphate), Galpha(i3) (antibody capture/scintillation proximity), and mitogen-activated protein kinase (immunoblot) activation procedures, it behaved as an agonist: pEC(50) values, 8.7, 8.6, and 8.5, respectively. These actions were blocked by haloperidol and the selective D(3) receptor antagonist S33084 [(3aR,9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl]-(4-phenyl) benzamide)]. S32504 showed lower potency at hD(2S) and hD(2L) receptors in [(35)S]GTPgammaS binding (pEC(50) values, 6.4 and 6.7) and antibody capture/scintillation proximity (hD(2L), pEC(50), 6.6) procedures. However, reflecting signal amplification, it potently stimulated hD(2L) receptor-coupled mitogen-activated protein kinase (pEC(50), 8.6). These actions were blocked by haloperidol and the selective D(2) receptor antagonist L741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol]. The affinity of S32504 for hD(4) receptors was low (5.3) and negligible for hD(1) and hD(5) receptors (pK(i),5.0). S32504 showed weak agonist properties at serotonin(1A) ([(35)S]GTPgammaS binding, pEC(50), 5.0) and serotonin(2A) (G(q), pEC(50), 5.2) receptors and low affinity for other (50) sites. In anesthetized rats, S32504 (0.0025-0.01 mg/kg, i.v.) suppressed electrical activity of ventrotegmental dopaminergic neurons. Correspondingly, S32504 (0.0025-0.63 mg/kg, s.c.) potently reduced dialysis levels (and synthesis) of dopamine in striatum, nucleus accumbens, and frontal cortex of freely moving rats, actions blocked by haloperidol and L741,626 but not by S33084. In contrast, S32504 only weakly inhibited serotonergic transmission and failed to affect noradrenergic transmission. Actions of S32504 were expressed stereospecifically versus its less active enantiomer S32601 [(-)-trans-3,4,4a,5,6,10b-hexahydro-9-carbomoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine]. Although the D(3)/D(2) agonist and antiparkinsonian agent ropinirole mimicked the profile of S32504, it was less potent. In conclusion, S32504 is a potent and selective agonist at dopamine D(3) and D(2) receptors.

This study employed [(35)S]guanosine 5'- O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT(1A) receptors with those of reference agonists at postsynaptic 5-HT(1A) receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E(max), values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC(50)) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT(1A) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT(1A) receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding, consistent with partial agonist properties. In [(35)S]GTPgammaS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT(1A) receptors, was more potent in the septum (pEC(50) approximately 6.5) than in the dentate gyrus of the hippocampus (pEC(50) approximately 5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 microM) did not enhance [(35)S]GTPgammaS labelling in any structure, S16924 (10 micro M) modestly increased [(35)S]GTPgammaS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 microM)-stimulated [(35)S]GTPgammaS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT(1A) receptors in the hippocampus. These data support a role of postsynaptic 5-HT(1A) receptors in the functional profiles of certain antipsychotic agents.

Agomelatine (S20098) displayed p K i values of 6.4 and 6.2 at native (porcine) and cloned, human (h)5-hydroxytryptamine (5-HT)2C receptors, respectively. It also interacted with h5-HT2B receptors (6.6), whereas it showed low affinity at native (rat)/cloned, human 5-HT2A (

Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. British Journal of Pharmacology (2003) 138, 1077–1084. doi:10.1038/sj.bjp.0705140