Fentanyl dose-sparing in polyarthritic rats requires full agonism at 5-HT1A receptors: Comparison between NLX-112, (±)8-OH-DPAT, and buspirone.

BACKGROUND: NLX-112 (a.k.a. F13640, befiradol) is a highly selective and fully efficacious agonist at 5-hydroxytryptamine (5-HT1A) receptors. It has been shown to be robustly and potently active in nociceptive, neuropathic and traumatic pain models in rats and mice. In particular, NLX-112 decreases oral fentanyl self-administration (FSA) in polyarthritic rats, ie, it has opioid dose-sparing effects. OBJECTIVE: To examine if the dose-sparing effects of NLX-112 in polyarthritic rats are shared by other 5-HT1A ligands: the prototypical 5-HT1A receptor agonist 8-HYDROXY-2-(DI-n-PROPYLAMINO)TETRALIN ((±)8-OH-DPAT), and the 5-HT1A receptor partial agonist and weak dopamine D2 receptor blocker, -buspirone. DESIGN: Polyarthritis was induced by inoculating rats with heat-killed Mycobacterium butyricum. They then had access to either a fentanyl (0.008 mg/mL) or a sweetened solution in their home cage. NLX-112, (±)8-OH-DPAT, or buspirone was administered via an osmotic minipump (5 µL/h) during a 2-week infusion period from day 14 to day 28 post-inoculation with Mycobacterium butyricum. Control infusions consisted of sterile 0.9 percent NaCl. RESULTS: NLX-112 (0.63 mg/day) significantly decreased FSA by 47 percent and increased total fluid consumption (TFC) by 7 percent (vehicle-loaded minipumps as controls). Both (±)8-OH-DPAT and buspirone (0.63 and 2.5 mg/day, respectively) failed to reduce FSA; (±)8-OH-DPAT did not modify TFC, while buspirone significantly decreased it by 17 percent. CONCLUSIONS: These results suggest that oral FSA dose-sparing effect, in this rat polyarthritis pain model, requires high efficacy activation of 5-HT1A receptors, such as that afforded by NLX-112. By contrast, the agonist efficacy of (±)8-OH-DPAT and buspirone seems insufficient for FSA dose-sparing.

Pharmacodynamic, pharmacokinetic and rat brain receptor occupancy profile of NLX-112, a highly selective 5-HT1A receptor biased agonist.

NLX-112 (i.e., F13640, befiradol) exhibits nanomolar affinity, exceptional selectivity and full agonist efficacy at serotonin 5-HT1A receptors. NLX-112 shows efficacy in rat, marmoset and macaque models of L-DOPA induced dyskinesia (LID) in Parkinson's disease and has shown clinical efficacy in a Phase 2a proof-of-concept study for this indication. Here we investigated, in rats, its pharmacodynamic, pharmacokinetic (PK) and brain 5-HT1A receptor occupancy profiles, and its PK properties in the absence and presence of L-DOPA. Total and free NLX-112 exposure in plasma, CSF and striatal ECF was dose-proportional over the range tested (0.04, 0.16 and 0.63 mg/kg i.p.). NLX-112 exposure increased rapidly (Tmax 0.25-0.5h) and exhibited approximately threefold longer half-life in brain than in plasma (1.1 and 3.6h, respectively). At a pharmacologically relevant dose of 0.16 mg/kg i.p., previously shown to elicit anti-LID activity in parkinsonian rats, brain concentration of NLX-112 was 51-63 ng/g from 0.15 to 1h. In microPET imaging experiments, NLX-112 showed dose-dependent reduction of 18F-F13640 (i.e., 18F-NLX-112) brain 5-HT1A receptor labeling in cingulate cortex and striatum, regions associated with motor control and mood, with almost complete inhibition of labeling at the dose of 0.63 mg/kg i.p.. Co-administration of L-DOPA (6 mg/kg s.c., a dose used to elicit LID in parkinsonian rats) together with NLX-112 (0.16 mg/kg i.p.) did not modify PK parameters in rat plasma and brain of either NLX-112 or L-DOPA. Here, we demonstrate that NLX-112's profile is compatible with 'druggable' parameters for CNS indications, and the results provide measures of brain concentrations and 5-HT1A receptor binding parameters relevant to the anti-dyskinetic activity of the compound.

Rapid reorganization of serotonin projections and antidepressant response to 5-HT1A-biased agonist NLX-101 in fluoxetine-resistant cF1ko mice.

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) like fluoxetine remain a first-line treatment for major depression, but are effective in less than half of patients and can take 4-8 weeks to show results. In this study, we examined cF1ko mice with genetically induced upregulation of 5-HT1A autoreceptors that reduces 5-HT neuronal activity. These mice display anxiety- and depression-related behaviors that did not respond to chronic fluoxetine treatment. We examined treatment with NLX-101, a biased agonist that preferentially targets 5-HT1A heteroreceptors. By testing different doses of NLX-101, we found that a dose of 0.2 mg/kg was effective in reducing depression-related behavior in cF1ko mice without causing hypothermia, a 5-HT1A autoreceptor-mediated response. After 1 h, this dose activated dorsal raphe 5-HT neurons and cells in the medial prefrontal cortex (mPFC), increasing nuclear c-fos labelling in cF1ko mice. In cF1ko mice but not wild-type littermates, 0.2 mg/kg NLX-101 administered 1 h prior to each behavioral test for two weeks reduced depressive behavior in the forced swim test, but increased anxiety-related behaviors in the open field, elevated plus maze, and novelty suppressed feeding tests. During this treatment, NLX-101 induced widespread increases in the density of 5-HT axons, varicosities, and especially synaptic and triadic structures, particularly in depression-related brain regions including mPFC, hippocampal CA1 and CA2/3, amygdala and nucleus accumbens of cF1ko mice. Overall, NLX-101 was rapid and effective in reducing depressive behavior in SSRI-resistant mice, but also induced anxiety-related behaviors. The increase in serotonin innervation induced by intermittent NLX-101 may contribute to its behavioral actions.

The 5-HT1A receptor biased agonists, NLX-204 and NLX-101, like ketamine, elicit rapid-acting antidepressant activity in the rat chronic mild stress model via cortical mechanisms.

BACKGROUND: The highly selective 5-HT1A serotonin receptor "biased" agonists NLX-101 and NLX-204 display, like ketamine, potent and efficacious rapid-acting antidepressant (RAAD) activity in the rat chronic mild stress (CMS) model with systemic (i.p.) administration. They rapidly (within 1 day) reverse anhedonia (i.e., CMS-induced sucrose consumption deficit), attenuate working memory deficit (novel object recognition: NOR), and decrease anxiety behavior in the elevated-plus maze (EPM). AIMS: Here, we sought to explore the contribution of prefrontal cortex (PFC) 5-HT1A receptor activation in the RAAD activity of NLX compounds. RESULTS/OUTCOMES: In male Wistar rats, unilateral PFC microinjections of NLX-204 and NLX-101 (16 µg), like ketamine (10 µg), reproduced the effects of their systemic administration: they reversed CMS-induced sucrose consumption deficit, attenuated anxiety (EPM), and reduced working memory deficits (NOR). In addition, unilateral PFC microinjections of the selective 5-HT1A antagonist, WAY-100,635 (2 µg), attenuated the beneficial effects of systemic NLX-204 and NLX-101 (0.16 mg/kg i.p.) in the sucrose intake and NOR models, indicating that these compounds exert their RAAD activity specifically through activation of PFC 5-HT1A receptors. CONCLUSIONS/INTERPRETATION: These data indicate that 5-HT1A receptor biased agonists share with ketamine a common neuroanatomical site for RAAD activity, which can be obtained not only by targeting glutamatergic/NMDA neurotransmission (ketamine's primary mechanism of action) but also by activating 5-HT1A receptors, as is the case for the NLX compounds. The present observations also reinforce the notion that biased agonism at 5-HT1A receptors constitutes a promising strategy to achieve RAAD effects, with additional benefits against cognitive deficits and anxiety in depressed patients, without ketamine's troublesome side effects.

Multimodal imaging study of the 5-HT1A receptor biased agonist, NLX-112, in a model of L-DOPA-induced dyskinesia.

INTRODUCTION: The leading treatment for motor signs of Parkinson's disease is L-DOPA, but, upon extended use, it can lead to levodopa-induced dyskinesia (LID). Serotonergic neurons are involved in LID etiology and previous pre-clinical studies have shown that NLX-112, a 5-HT1A biased agonist, has robust antidyskinetic effects. Here, we investigated its effects in hemiparkinsonian (HPK) rats with a unilateral nigrostriatal 6-OHDA lesion. METHODS: We compared HPK rats with LID (i.e., sensitized to the dyskinetic effects of chronic L-DOPA) and without LID (HPK-non-LID), using [18F]FDG PET imaging and fMRI functional connectivity following systemic treatment with saline, L-DOPA, NLX-112 or L-DOPA + NLX-112. RESULTS: In HPK-non-LID rats, [18F]FDG PET experiments showed that L-DOPA led to hypermetabolism in motor areas (cerebellum, brainstem, and mesencephalic locomotor region) and to hypometabolism in cortical regions. L-DOPA effects were also observed in HPK-LID rats, with the additional emergence of hypermetabolism in raphe nuclei and hypometabolism in hippocampus and striatum. NLX-112 attenuated L-DOPA-induced raphe hypermetabolism and cingulate cortex hypometabolism in HPK-LID rats. Moreover, in fMRI experiments NLX-112 partially corrected the altered neural circuit connectivity profile in HPK-LID rats, through activity in regions rich in 5-HT1A receptors. CONCLUSION: This neuroimaging study sheds light for the first time on the brain activation patterns of HPK-LID rats. The 5-HT1A receptor agonist, NLX-112, prevents occurrence of LID, likely by activating pre-synaptic autoreceptors in the raphe nuclei, resulting in a partial restoration of brain metabolic and connectivity profiles. In addition, NLX-112 also rescues L-DOPA-induced deficits in cortical activation, suggesting potential benefit against non-motor symptoms of Parkinson's disease.

The 5-HT1A receptor biased agonists, NLX-204 and NLX-101, display ketamine-like RAAD and anti-TRD activities in rat CMS models.

OBJECTIVES: NLX-101 and NLX-204 are highly selective serotonin 5-HT1A 'biased' agonists, displaying potent and efficacious antidepressant-like activity upon acute administration in models such as the forced swim test. METHODS: we compared the effects of repeated administration of NLX-101, NLX-204 and ketamine in the chronic mild stress (CMS) model of depression, considered to have high translational potential, on sucrose consumption (anhedonia measure), novel object recognition (NOR; working memory measure) and elevated plus maze (EPM; anxiety measure) in male Wistar and Wistar-Kyoto rats (the latter being resistant to classical antidepressants). RESULTS: in Wistar rats, NLX-204 and NLX-101 (0.08-0.16 mg/kg i.p.), like ketamine (10 mg/kg i.p.) dose-dependently reversed CMS-induced sucrose intake deficit from treatment Day 1, with nearly full reversal observed at the higher dose at Days 8 and 15. These effects persisted for 3 weeks following treatment cessation. In the NOR test, both doses of NLX-101/NLX-204, and ketamine, rescued the deficit in discrimination index caused by CMS on Days 3 and 17; all three compounds increased time spent in open arms (EPM) but only NLX-204 achieved statistical significance on Days 2 and 16. In Wistar-Kyoto rats, all 3 compounds were also active in the sucrose test and, to a lesser extent, in the NOR and EPM. In non-stressed rats (both strains), the three compounds produced no significant effects in all tests. CONCLUSIONS: these observations further strengthen the hypothesis that biased agonism at 5-HT1A receptors constitutes a promising strategy to achieve rapid-acting/sustained antidepressant effects combined with activity against TRD, in addition to providing beneficial effects against memory deficit and anxiety in depressed patients.

[18F]F13640: a selective agonist PET radiopharmaceutical for imaging functional 5-HT1A receptors in humans.

PURPOSE: F13640 (a.k.a. befiradol, NLX-112) is a highly selective 5-HT1A receptor ligand that was selected as a PET radiopharmaceutical-candidate based on animal studies. Due to its high efficacy agonist properties, [18F]F13640 binds preferentially to functional 5-HT1A receptors, which are coupled to intracellular G-proteins. Here, we characterize brain labeling of 5-HT1A receptors by [18F]F13640 in humans and describe a simplified model for its quantification. METHODS: PET/CT and PET-MRI scans were conducted in a total of 13 healthy male volunteers (29 ± 9 years old), with arterial input functions (AIF) (n = 9) and test-retest protocol (n = 8). Several kinetic models were compared (one tissue compartment model, two-tissue compartment model, and Logan); two models with reference region were also evaluated: simplified reference tissue model (SRTM) and the logan reference model (LREF). RESULTS: [18F]F13640 showed high uptake values in raphe nuclei and cortical regions. SRTM and LREF models showed a very high correlation with kinetic models using AIF. As concerns test-retest parameters and the prolonged binding kinetics of [18F]F13640, better reproducibility, and reliability were found with the LREF method. Cerebellum white matter and frontal lobe white matter stand out as suitable reference regions. CONCLUSION: The favorable brain labeling and kinetic profile of [18F]F13640, its high receptor specificity and its high efficacy agonist properties open new perspectives for studying functionally active 5-HT1A receptors, unlike previous radiopharmaceuticals that act as antagonists. [18F]F13640's kinetic properties allow injection outside of the PET scanner with delayed acquisitions, facilitating the design of innovative longitudinal protocols in neurology and psychiatry. TRIAL REGISTRATION: Trial Registration EudraCT 2017-002,722-21.

The 5-HT1A receptor biased agonist, NLX-204, shows rapid-acting antidepressant-like properties and neurochemical changes in two mouse models of depression.

Activation of cortical serotonin 5-HT1A receptors may be a promising strategy to achieve rapid-acting antidepressant (RAAD) activity. NLX-204 is a selective 5-HT1A receptor biased agonist that, in naïve mice, robustly decreases immobility in the forced swim test (FST), and preferentially phosphorylates extracellular signal-regulated kinase (ERK1/2), involved in antidepressant activity. Here, we evaluated the properties of NLX-204 in two mouse models of depression. Male CD-1 mice were subjected to unpredictable chronic mild stress (UCMS) for 4-weeks or to repeated corticosterone (CORT, 20 mg/kg s.c./day) for 3-weeks before receiving acute administration of NLX-204 (2 mg/kg, p.o.). Depressive-like behavior was assessed in the FST, anhedonia-like behavior in the sucrose preference test (SPT) and locomotor activity was also recorded. Phosphorylation of ERK1/2 (pERK1/2) and cAMP response binding element (pCREB) were measured ex vivo in hippocampus and prefrontal cortex (PFC). UCMS or CORT treatment increased immobility in the FST, elicited a sucrose preference deficit, and decreased pERK1/2 and pCREB levels in PFC and hippocampus. NLX-204 reduced depressive-like behavior in the FST in CORT and UCMS mice, and normalized sucrose preference in CORT mice, suggesting anti-anhedonic activity. NLX-204 increased pERK1/2 levels in PFC of UCMS mice. NLX-204 also increased pCREB levels in PFC of CORT mice. These data suggest that NLX-204 has RAAD-like properties not only in naïve mice, but also in mice in a "depressive-like" state, and that these involve changes in PFC and hippocampal pERK1/2 and pCREB levels. These data provide additional evidence that activation of 5-HT1A receptors by selective biased agonists, such as NLX-204, may constitute a promising RAAD strategy.

Neural serotonergic circuits for controlling long-term voluntary alcohol consumption in mice.

Alcohol-use-disorders are chronic relapsing illnesses, often co-morbid with anxiety. We have previously shown using the "drinking-in-the-dark" model in mice that the stimulation of the serotonin receptor 1A (5-HT1A) reduces ethanol binge-drinking behaviour and withdrawal-induced anxiety. The 5-HT1A receptor is located either on Raphe neurons as autoreceptors, or on target neurons as heteroreceptors. By combining a pharmacological approach with biased agonists targeting the 5-HT1A auto- or heteroreceptor and a chemogenetic approach (DREADDs), here we identified that ethanol-binge drinking behaviour is dependent on 5-HT1A autoreceptors and 5-HT neuronal function, with a transition from DRN-dependent regulation of short-term (6 weeks) ethanol intake, to MRN-dependent regulation after longer ethanol exposure (12 weeks). We further identified a serotonergic microcircuit (5-HTMRN→DG) originating from the MRN and projecting to the dentate gyrus (DG) of the hippocampus, that is specifically affected by, and modulates long-term ethanol consumption. The present study indicates that targeting Raphe nuclei 5-HT1A autoreceptors with agonists might represent an innovative pharmacotherapeutic strategy to combat alcohol abuse.

The selective 5-HT 1A receptor agonist, NLX-112, overcomes tetrabenazine-induced catalepsy and depression-like behavior in the rat.

Tetrabenazine, a preferential inhibitor of the vesicular monoamine transporter type 2, depletes the brain monoamines dopamine, serotonin and norepinephrine. Tetrabenazine and deutetrabenazine (Austedo ®) are used to treat chorea associated with Huntington's disease. However, both compounds are known to aggravate Parkinsonism and depression observed in Huntington's disease patients. NLX-112 (a.k.a. befiradol/F13640) is a highly selective, potent and efficacious serotonin 5-HT 1A agonist. In animal models, it has robust efficacy in combating other iatrogenic motor disorders such as L-DOPA-induced dyskinesia and has marked antidepressant-like activity in rodent tests. In the present study, we investigated, in rats, the efficacy of NLX-112 to counteract tetrabenazine-induced catalepsy (a model of Parkinsonism) and tetrabenazine-induced potentiation of immobility in the forced swim test (FST, a model to detect antidepressant-like activity). The prototypical 5-HT 1A agonist, (±)8-OH-DPAT, and the 5-HT 1A partial agonist/dopamine D2 receptor blocker, buspirone, were used as comparators. Both NLX-112 and (±)8-OH-DPAT (0.16-2.5 mg/kg p.o. or s.c., respectively) abolished catalepsy induced by tetrabenazine (2 mg/kg i.p.). In comparison, buspirone (0.63-5.0 mg/kg p.o.) was ineffective and even tended to potentiate tetrabenazine-induced catalepsy at 0.63 mg/kg. In the FST, NLX-112 and (±)8-OH-DPAT (0.63 mg/kg) strongly reduced immobility when administered alone but also significantly opposed potentiation of immobility induced by tetrabenazine (1.5 mg/kg i.p.). Buspirone (0.63 and 2.5 mg/kg p.o.) had no effect by itself or against tetrabenazine. These results strongly suggest that selective and highly efficacious 5-HT 1A agonists, such as NLX-112, may be useful in combating tetrabenazine-induced Parkinsonism and/or depression in Huntington's disease patients.

Molecular Signaling Mechanisms for the Antidepressant Effects of NLX-101, a Selective Cortical 5-HT1A Receptor Biased Agonist.

Depression is the most prevalent of the mental illnesses and serotonin (5-hydroxytryptamine, 5-HT) is considered to be the major neurotransmitter involved in its etiology and treatment. In this context, 5-HT1A receptors have attracted interest as targets for therapeutic intervention. Notably the activation of presynaptic 5-HT1A autoreceptors delays antidepressant effects whereas the stimulation of postsynaptic 5-HT1A heteroreceptors is needed for an antidepressant action. NLX-101 (also known as F15599) is a selective biased agonist which exhibits preferred activation of cortical over brain stem 5-HT1A receptors. Here, we used behavioral, neurochemical and molecular methods to examine the antidepressant-like effects in rats of a single dose of NLX-101 (0.16 mg/kg, i.p.). NLX-101 reduced immobility in the forced swim test when measured 30 min but not 24 h after drug administration. NLX-101 increased extracellular concentrations of glutamate and dopamine in the medial prefrontal cortex, but no changes were detected in the efflux of noradrenaline or 5-HT. NLX-101 also produced an increase in the activation of pmTOR, pERK1/2 and pAkt, and the expression of PSD95 and GluA1, which may contribute to its rapid antidepressant action.

Biased 5-HT1A receptor agonists F13714 and NLX-101 differentially affect pattern separation and neuronal plasticity in rats after acute and chronic treatment.

Pattern separation is a hippocampal process in which highly similar stimuli are recognized as separate representations, and deficits could lead to memory impairments in neuropsychiatric disorders such as schizophrenia. The 5-HT1A receptor (5-HT1AR) is believed to be involved in these hippocampal pattern separation processes. However, in the dorsal raphe nucleus (DRN), the 5-HT1AR is expressed as a somatodendritic autoreceptor, negatively regulates serotonergic signaling, and could thereby counteract the effects of hippocampal postsynaptic 5-HT1A receptors. Therefore, this study aims to identify how pre- and post-synaptic 5-HT1AR activity affects pattern separation. Object pattern separation (OPS) performance was measured in male Wistar rats after both acute and chronic treatment (i.p.) with 5-HT1AR biased agonists F13714 (0.0025 mg/kg acutely, 0.02 mg/kg/day chronically) or NLX-101 (0.08 mg/kg acutely, 0.32 mg/kg/day chronically), which preferentially activate autoreceptors or postsynaptic receptors respectively, for 14 days. Body temperature - a functional correlate of hypothalamic 5-HT1AR stimulation - was measured daily. Additionally, 5-HT1AR density (DRN) and plasticity markers (hippocampus) were assessed. Acute treatment with F13714 impaired OPS performance, whereas chronic treatment normalized this, and a drop in body temperature was found from day 4 onwards. NLX-101 enhanced OPS performance acutely and chronically, and caused an acute drop in body temperature. Chronic NLX-101 treatment increased doublecortin positive neurons in the dorsal hippocampus, while chronic treatment with F13714 resulted in a downregulation of 5-HT1A autoreceptors, which likely reversed the acute impairment in OPS performance. Chronic treatment with NLX-101 appears to have therapeutic potential to improve brain plasticity and OPS performance.

Dissecting the contribution of 5-HT1A auto- and heteroreceptors in sucrose overconsumption in mice.

The rise in obesity prevalence has been linked to overconsumption of high-sugar containing food and beverages. Recent evidence suggests that chronic sucrose consumption leads to changes in serotonergic neuroplasticity within the neural circuits involved in feeding control. Although there is a relationship between serotonin signalling in the brain and diet-induced obesity, the specific serotonin (5-HT) receptors or pathways involved remain unknown. The 5-HT1A receptor subtype plays a role in regulating mood, anxiety, and appetite, and has been associated with reversing addiction to substances of abuse. However, the respective role of 5-HT1A auto- vs heteroreceptors in sucrose consumption has not been examined. Mice were given controlled access to either 5%, 10% or 25% w/v sucrose, or water as a control, for 12 weeks using the well-established "drinking in the dark" protocol (n = 6-8 mice per group). Ligands selectively targeting 5-HT1A auto- and/or heteroreceptors (NLX-112, unbiased 5-HT1A receptor agonist; NLX-101, preferential heteroreceptor agonist; F13714, preferential autoreceptor agonist) were administered i.p. acutely after 6 and 12 weeks of sucrose consumption. The specific involvement of 5-HT1A receptors in these effects was verified by blockade with the selective 5-HT1A receptors antagonist WAY-100,635. The specific subpopulation of 5-HT1A receptors involved in sucrose consumption was dependent on the concentration of sucrose solution and the duration of exposure to sucrose (6 weeks vs 12 weeks). Long-term sucrose consumption leads to accentuated 5-HT1A autoreceptor function. Thus, targeting 5-HT1A autoreceptors might represent an effective therapeutic strategy to combat the rise in obesity resulting from the overconsumption of high-sugar diet.

The selective 5-HT1A receptor agonist NLX-112 displays anxiolytic-like activity in mice.

Anxiety is amongst the commonest neuropsychiatric disorders, and there is a large body of evidence to suggest that abnormalities in serotonergic function are involved in its pathogenesis. Several studies have implicated 5-HT1A receptor activation in mitigating anxiety disorders, so this study investigated the acute effects of a highly selective, potent and efficacious 5-HT1A receptor full agonist, NLX-112 (a.k.a. befiradol, F13640), in middle-aged C57bl/6 J male mice. Video tracking was used to measure several parameters including time spent in the open and closed arms of an elevated plus maze (EPM), distance travelled and thigmotaxis in an open field test (OFT). At 0.1 to 1.0 mg/kg s.c., NLX-112 markedly decreased thigmotaxis and increased exploratory behaviour in the OFT and EPM assays. Hence, at 0.3 mg/kg, NLX-112 augmented locomotor activity in the centre of an open field arena by 164% and increased the time spent in the open arms of the EPM by 119% of control. These results indicate that anxiety-like behaviours in mice are significantly diminished with low doses of NLX-112. NLX-112 may therefore possess anxiolytic properties which complement its known activity in models of movement disorders.

Perspectives for therapy of treatment-resistant depression.

A high proportion of depressed patients fail to respond to antidepressant drug treatment. Treatment-resistant depression (TRD) is a major challenge for the psychopharmacology of mood disorders. Only in the past decade have novel treatments, including deep brain stimulation (DBS) and ketamine, been discovered that provide rapid and sometimes prolonged relief to a high proportion of TRD sufferers. In this review, we consider the current status of TRD from four perspectives: the challenge of developing an appropriate regulatory framework for novel rapidly acting antidepressants; the efficacy of non-pharmacological somatic therapies; the development of an animal model of TRD and its use to understand the neural basis of antidepressant non-response; and the potential for rapid antidepressant action from targets (such as 5-HT1A receptors) beyond the glutamate receptor. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc.

Translating biased agonists from molecules to medications: Serotonin 5-HT1A receptor functional selectivity for CNS disorders.

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 ß-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.

Discriminative stimulus properties of the 5-HT1A receptor biased agonists NLX-101 and F13714, in rats trained to discriminate 8-OH-DPAT from saline.

NLX-101 and F13714 are selective, full efficacy, biased agonists of the serotonin (5-HT1A) receptor. NLX-101 preferentially activates cortical postsynaptic 5-HT1A receptors, whereas F13714 preferentially activates raphe nuclei presynaptic 5-HT1A receptors. We compared NLX-101 and F13714 for their efficacy and potency to substitute for the discriminative cue produced by the prototypical, nonbiased 5-HT1A 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 pretreatment) 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., whereas NLX-101 only achieved full substitution at 0.5 mg/kg i.p., a dose which is known to also activate presynaptic 5-HT1A receptors. The 5-HT1A 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-HT1A receptor antagonist WAY-100 635 (1 mg/kg s.c., 40 min pretreatment) 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 presynaptic 5-HT1A receptors. They also further demonstrate the distinct profiles in behavioral models of 5-HT1A receptor-biased agonists.

Different Alterations of Agonist and Antagonist Binding to 5-HT1A Receptor in a Rat Model of Parkinson's Disease and Levodopa-Induced Dyskinesia: A MicroPET Study.

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.

The selective 5-HT1A receptor biased agonists, F15599 and F13714, show antidepressant-like properties after a single administration in the mouse model of unpredictable chronic mild stress.

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.

NLX-101, a cortical 5-HT1A receptor biased agonist, reverses scopolamine-induced deficit in the delayed non-matching to position model of cognition.

NLX-101 is a selective, high efficacy, biased agonist at post-synaptic cortical 5-HT1A receptors. We have previously shown that it opposes deficits produced by blockade of NMDA receptors and has pro-cognitive activity of its own. Based on the strong interaction between 5-HT1A receptors and the central cholinergic system, we tested NLX-101 on scopolamine-induced impairment of cognition in a delayed non-matching to position (DNMTP) model. The cholinesterase inhibitor, tacrine, was used as a comparator. In operant chambers with two retractable levers, male rats were trained to press one randomly presented lever during a "sample" phase. Following a time delay of either 1, 5 or 10 s, both levers were then presented, the rat being required to press the correct lever (i.e. the one not previously presented) to receive a food pellet reward. Scopolamine (0.16 mg/kg i.p.) significantly impaired accuracy (i.e. choice of correct lever) at 5 and 10 s delays. In contrast, NLX-101 (0.04, 0.16, 0.63 mg/kg i.p.) did not worsen accuracy, except at 0.63 mg/kg. Moreover, NLX-101 (0.04 and 0.16 mg/kg) dose-dependently and significantly opposed scopolamine-induced impairment for 5 and 10 s delays, with near-total reversal at 10 s. The acetylcholinesterase inhibitor, tacrine, also opposed scopolamine-induced impairment but was less potent and efficacious, with a single significant effect at 2.5 mg/kg and 5 s delay only. The present data suggest that biased agonism at post-synaptic, cortical 5-HT1A receptors could prove useful in neurological or neuropsychiatric pathologies characterized by cognitive deficits consecutive to a reduced central cholinergic tone.