Acute and Repeated Administration of NLX-101, a Selective Serotonin-1A Receptor Biased Agonist, Reduces Audiogenic Seizures in Developing Fmr1 Knockout Mice.

Fragile XSyndrome (FXS) is a leading known genetic cause of Autism Spectrum Disorders (ASD) and intellectual disability. A consistent and debilitating phenotype of FXS is sensory hypersensitivity that manifests strongly in the auditory domain and may lead to delayed language and high anxiety. The mouse model of FXS, the Fmr1 KO mouse, also shows auditory hypersensitivity, an extreme form of which is seen as audiogenic seizures (AGS). The midbrain inferior colliculus (IC) is critically involved in generating audiogenic seizures and IC neurons are hyper-responsive to sounds in developing Fmr1 KO mice. Serotonin-1A receptor (5-HT1A) activation reduces IC activity. Therefore, we tested whether 5-HT1A activation is sufficient to reduce audiogenic seizures in Fmr1 KO mice. A selective and post-synaptic 5-HT1A receptor biased agonist, 3-Chloro-4-fluorophenyl-[4-fluoro-4-[[(5-methylpyrimidin-2-ylmethyl)amino]methyl]piperidin-1-yl] methanone (NLX-101, 0.6, 1.2, 1.8 or 2.4 mg/kg, i.p.) was administered to Fmr1 KO mice 15 min before seizure induction. Whereas the 0.6 mg/kg dose was ineffective in reducing seizures, the 1.2, 1.8 and 2.4 mg/kg doses of NLX-101 dramatically reduced seizures and increased mouse survival. Treatment with a combination of NLX-101 and 5-HT1A receptor antagonists prevented the protective effects of NLX-101, indicating that NLX-101 acts selectively through 5-HT1A receptors to reduce audiogenic seizures. NLX-101 (1.8 mg/kg) was still strongly effective in reducing seizures even after repeated administration over 5 days, suggesting an absence of tachyphylaxis to the effects of the compound. Together, these studies point to a promising treatment option targeting post-synaptic 5-HT1A receptors to reduce auditory hypersensitivity in FXS, and potentially across autism spectrum disorders.

NLX-101, a 5-HT1A receptor-biased agonist, improves pattern separation and stimulates neuroplasticity in aged rats.

5-HT1A serotonin receptors may play a role in cognitive function changes related to advanced age. Here, we investigated the effects of acute and repeated treatment with NLX-101 (F15599), a postsynaptic 5-HT1A receptor-biased agonist, and F13714, a presynaptic 5-HT1A receptor-biased agonist on spatial object pattern separation (OPS) in aged (22-24 months) rats. Neuroplasticity markers including brain-derived neurotrophic factor, PSD95, synaptophysin, and doublecortin were evaluated in the hippocampus. Unlike younger rats, aged rats were incapable of discriminating any new position of the objects in the arena, reflecting the detrimental effect of aging on pattern separation. However, aged animals treated with NLX-101 showed a significant cognitive improvement in the OPS test, accompanied by increases in hippocampal brain-derived neurotrophic factor and PSD95 protein levels. In contrast, no improvement in OPS performance was observed when aged rats received F13714. Both F13714 and NLX-101 increased the number of newborn neurons in the hippocampi of aged rats. These findings provide a rationale for targeting post-synaptic 5-HT1A as a treatment for cognitive deficits related to aging.

NLX-112, a highly selective 5-HT1A receptor biased agonist, does not exhibit misuse potential in male rats or macaques.

NLX-112 (a.k.a. F13640 or befiradol) exhibits nanomolar affinity, exceptional selectivity and biased agonism at serotonin 5-HT1A receptors. NLX-112 displays robust analgesic activity in a number of rodent models of pain, and is currently developed as a treatment for l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD) patients. Noteworthy, PD patients can suffer from comorbid chronic pain, thus necessitating the use of analgesic drugs, such as opioids, which have potential for misuse. Additionally, dopamine agonists used to treat PD can produce cocaine-like effects in preclinical assays of misuse potential. The present study investigated whether NLX-112 possesses misuse potential of its own using two behavioural assays routinely used for this purpose: intracranial self-stimulation (ICSS) in rats, and cocaine discrimination in macaque monkeys. In rats, low doses of NLX-112 (0.03 and 0.1 mg/kg p.o.) did not alter ICSS frequency-rate curves, while higher doses (0.3 and 1.0 mg/kg) shifted the curve to the right and flattened it, i.e., reduced ICSS. As expected, cocaine (10 mg/kg i.p.) shifted the curve to the left, i.e., facilitated ICSS, but NLX-112 (0.03 and 0.1 mg/kg p.o.) did not further enhance cocaine-induced facilitation of ICSS. In monkeys trained to discriminate cocaine (0.4 mg/kg i.m.) from saline, NLX-112 (0.01-0.1 mg/kg p.o.) did not substitute for cocaine. Taken together, these results suggest that NLX-112, at doses displaying anti-dyskinetic activity in rat, marmoset and macaque models of LID, is free from misuse potential. From a translational perspective, this is a desirable property for a compound destined to be used in PD patients, who can suffer from comorbid chronic pain necessitating the use of potentially misused analgesic drugs.

NLX-101, a highly selective 5-HT1A receptor biased agonist, mediates antidepressant-like activity in rats via prefrontal cortex 5-HT1A receptors.

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.

The selective 5-HT1A receptor agonist, NLX-112, exerts anti-dyskinetic effects in MPTP-treated macaques.

BACKGROUND: Long-term treatment of Parkinson's disease (PD) with l-DOPA typically leads to development of l-DOPA induced dyskinesia (LID). Amantadine, an NMDA antagonist, attenuates LID, but with limited efficacy and considerable side-effects. NLX-112 (also known as befiradol or F13640), a highly selective and efficacious 5-HT1A receptor agonist, reduced LID when tested in rodent and marmoset models of PD. METHODS: The effects of NLX-112 (0.03, 0.1 and 0.3 mg/kg PO) on established LID evoked by acute challenge with l-DOPA (27.5 ± 3.8 mg/kg PO) were assessed in MPTP-treated cynomolgus macaques. Amantadine (10 mg/kg PO) was tested as a positive control. Plasma exposure of NLX-112 (0.1 mg/kg PO) was determined. RESULTS: NLX-112 significantly and dose-dependently reduced median LID levels by up to 96% during the first hour post-administration (0.3 mg/kg). Moreover, NLX-112 reduced the duration of 'bad on-time' associated with disabling LID by up to 48% (0.3 mg/kg). In contrast, NLX-112 had negligible impact on the anti-parkinsonian benefit of l-DOPA. NLX-112 exposure peaked at ~50 ng/ml at 30 min post-administration but decreased to ~15 ng/ml at 2h. Amantadine reduced by 42% 'bad on-time' associated with l-DOPA, thereby validating the model. CONCLUSION: These data show that, in MPTP-lesioned cynomolgus macaques, NLX-112 exerts robust anti-dyskinetic effects, without reducing the anti-parkinsonian benefit of l-DOPA. These observations complement previous findings and suggest that selective and high efficacy activation of 5-HT1A receptors by NLX-112 may constitute a promising approach to combat LID in PD, providing an alternative for patients in whom amantadine is poorly tolerated or without useful effect.

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling.

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.

NLX-112, a highly selective 5-HT1A receptor agonist, mediates analgesia and antidepressant-like activity in rats via spinal cord and prefrontal cortex 5-HT1A receptors, respectively.

NLX-112 (a.k.a. F13640 or befiradol) possesses marked activity in a variety of animal models of pain and of neuropsychiatric disorders; it exhibits nanomolar affinity, exceptional selectivity and high agonist efficacy at 5-hydroxytryptamine1A (5-HT1A) receptors. Although NLX-112 has been shown to activate 5-HT1A postsynaptic heteroreceptors in the prefrontal cortex (PFC), a brain region involved in the control of depressive states, the influence of NLX-112 on spinal cord 5-HT1A receptors (implicated in the control of pain) has not been described. Here we report on the ability, in rats, of NLX-112 to elicit analgesia in the intraplantar formalin model of nociceptive pain following intrathecal (i.t.) administration, and its ability to produce antidepressant-like activity in the forced swim test (FST) following in situ PFC microinjection. NLX-112, injected i.t. (L5-L6 region) induced analgesic effects in the formalin model of tonic nociceptive pain. At 20 µg, it almost abolished the effect of formalin on both the paw licking and paw elevation measures, and in both the early (0-5 min after formalin administration, reflecting acute pain) and the late (22.5-27.5 min, reflecting inflammatory pain) phases. The effects of NLX-112 (20 µg i.t.) were reversed by co-administration of 20 µg i.t. of the 5-HT1A receptor antagonist, WAY100635. Furthermore, the analgesic effects of systemically administered NLX-112 (0.63 mg/kg i.p.) were reversed by i.t. administration of WAY100635 (20 µg), most notably on paw licking. Finally, microinjection of NLX-112, bilaterally in the PFC, dose-dependently (MED 4 µg) and markedly reduced immobility in the FST (circa 90% reduction at 32 µg). In conclusion, the present data demonstrate that activation of spinal cord-located 5-HT1A receptors is sufficient for NLX-112 to mediate its analgesic effects in a rat model of tonic nociceptive pain. The data also highlight the involvement of PFC 5-HT1A receptors in the antidepressant-like activity of NLX-112 in the FST. Overall, the study suggests that highly selective and high efficacy 5-HT1A receptors agonists, such as NLX-112, could be useful to treat painful conditions associated with depressive states, through activation of different sub-populations of 5-HT1A receptors.

NLX-112, a highly selective 5-HT1A receptor agonist: Effects on body temperature and plasma corticosterone levels in rats.

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: 0.31mg/kg p.o.) and also increased plasma corticosterone both by oral and intraperitoneal routes (MED: 0.63mg/kg in both cases). The increase in corticosterone induced by NLX-112 (0.63mg/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 0.31-0.63mg/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 0.16mg/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.

F15063, a compound with D2/D3 antagonist, 5-HT 1A agonist and D4 partial agonist properties. III. Activity in models of cognition and negative symptoms.

BACKGROUND AND PURPOSE: The D(2)/D(3) receptor antagonist, D(4) receptor partial agonist, and high efficacy 5-HT(1A) receptor agonist F15063 was shown to be highly efficacious and potent in rodent models of activity against positive symptoms of schizophrenia. However F15063 induced neither catalepsy nor the 'serotonin syndrome'. Here, we evaluated its profile in rat models predictive of efficacy against negative symptoms/cognitive deficits of schizophrenia. EXPERIMENTAL APPROACH: F15063, given i.p., was assessed in models of behavioural deficits induced by interference with the NMDA/glutamatergic (phencyclidine: PCP) or cholinergic (scopolamine) systems. KEY RESULTS: Through 5-HT(1A) activation, F15063 partially alleviated (MED: 0.04 mg kg(-1)) PCP-induced social interaction deficit between two adult rats, without effect by itself, underlining its potential to combat negative symptoms. At doses above 0.16 mg kg(-1), F15063 reduced interaction by itself. F15063 (0.16 mg kg(-1)) selectively re-established PCP-impaired 'cognitive flexibility' in a reversal learning task, suggesting potential against adaptability deficits. F15063 (0.04-0.63 mg kg(-1)) also reversed scopolamine-induced amnesia in a juvenile-adult rat social recognition test, indicative of a pro-cholinergic influence. Activity in this latter test is consistent with its D(4) partial agonism, as it was blocked by the D(4) antagonist L745,870. Finally, F15063 up to 40 mg kg(-1) did not disrupt basal prepulse inhibition of startle reflex in rats, a marker of sensorimotor gating. CONCLUSIONS AND IMPLICATIONS: The balance of D(2)/D(3), D(4) and 5-HT(1A) receptor interactions of F15063 yields a promising profile of activity in models of cognitive deficits and negative symptoms of schizophrenia.

F15063, a compound with D2/D3 antagonist, 5-HT 1A agonist and D4 partial agonist properties. II. Activity in models of positive symptoms of schizophrenia.

BACKGROUND AND PURPOSE: F15063 is a high affinity D(2)/D(3) antagonist, D(4) partial agonist, and high efficacy 5-HT(1A) agonist, with little affinity (40-fold lower than for D(2) receptors) at other central targets. Here, the profile of F15063 was evaluated in models of positive symptoms of schizophrenia and motor side-effects. EXPERIMENTAL APPROACH: Rodent behavioural tests were based on reversal of hyperactivity induced by psychostimulants and on measures of induction of catalepsy and 'serotonin syndrome'. KEY RESULTS: F15063 potently (ED(50)s: 0.23 to 1.10 mg kg(-1) i.p.) reversed methylphenidate-induced stereotyped behaviors, blocked d-amphetamine and ketamine hyperlocomotion, attenuated apomorphine-induced prepulse inhibition (PPI) deficits, and was active in the conditioned avoidance test. In mice, it reversed apomorphine-induced climbing (ED(50)=0.30 mg kg(-1) i.p.). F15063, owing to its 5-HT(1A) agonism, did not produce (ED(50)>40 mg kg(-1) i.p.) catalepsy in rats and mice, a behavior predictive of occurrence of extra-pyramidal syndrome (EPS) in man. This absence of cataleptogenic activity was maintained upon sub-chronic treatment of rats for 5 days at 40 mg kg(-1) p.o. Furthermore, F15063 did not induce the 'serotonin syndrome' in rats (flat body posture and forepaw treading: ED(50) >32 mg kg(-1) i.p.). CONCLUSIONS AND IMPLICATIONS: F15063 conformed to the profile of an atypical antipsychotic, with potent actions in models of hyperdopaminergic activity but without inducing catalepsy. These data suggest that F15063 may display potent antipsychotic actions with low EPS liability. This profile is complemented by a favourable profile in rodent models of negative symptoms and cognitive deficits of schizophrenia (companion paper).

F15063, a potential antipsychotic with D2/D3 antagonist, 5-HT 1A agonist and D4 partial agonist properties. I. In vitro receptor affinity and efficacy profile.

BACKGROUND AND PURPOSE: Combining 5-HT(1A) receptor activation with dopamine D(2)/D(3) receptor blockade should improve negative symptoms and cognitive deficits in schizophrenia. We describe the in vitro profile of F15063 (N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine). EXPERIMENTAL APPROACH: F15063 was characterised in tests of binding affinity and in cellular models of signal transduction at monoamine receptors. KEY RESULTS: Affinities (receptor and pK(i) values) of F15063 were: rD(2) 9.38; hD(2L) 9.44; hD(2S) 9.25; hD(3) 8.95; hD(4) 8.81; h5-HT(1A) 8.37. F15063 had little affinity (40-fold lower than D(2)) at other targets. F15063 antagonised dopamine-activated G-protein activation at hD(2), rD(2) and hD(3) receptors with potency (pK (b) values 9.19, 8.29 and 8.74 in [(35)S]GTP gamma S binding experiments) similar to haloperidol. F15063 did not exhibit any hD(2) receptor agonism, even in tests of ERK1/2 phosphorylation and G-protein activation in cells with high receptor expression. In contrast, like (+/-)8-OH-DPAT, F15063 efficaciously activated h5-HT(1A) (E(max) 70%, pEC(50) 7.57) and r5-HT(1A) receptors (52%, 7.95) in tests of [(35)S]GTP gamma S binding, cAMP accumulation (90%, 7.12) and ERK1/2 phosphorylation (93%, 7.13). F15063 acted as a partial agonist for [(35)S]GTP gamma S binding at hD(4) (29%, 8.15) and h5-HT(1D) receptors (35%, 7.68). In [(35)S]GTP gamma S autoradiography, F15063 activated G-proteins in hippocampus, cortex and septum (regions enriched in 5-HT(1A) receptors), but antagonised quinelorane-induced activation of D(2)/D(3) receptors in striatum. CONCLUSIONS AND IMPLICATIONS: F15063 antagonised dopamine D(2)/D(3) receptors, a property underlying its antipsychotic-like activity, whereas activation of 5-HT(1A) and D(4) receptors mediated its actions in models of negative symptoms and cognitive deficits of schizophrenia (see companion papers).

Rapid desensitization of somatodendritic 5-HT1A receptors by chronic administration of the high-efficacy 5-HT1A agonist, F13714: a microdialysis study in the rat.

BACKGROUND AND PURPOSE: Desensitization of somatodendritic 5-HT(1A) 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-HT(1A) agonist, F13714 (3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone) and of the partial agonist, flesinoxan, to desensitize somatodendritic 5-HT(1A) 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-HT(1A) agonist, buspirone, dose-dependently decreased extracellular 5-HT concentrations (ED(50) values: 0.04, 0.77 and 5.6 mg kg(-1), respectively). The selective 5-HT(1A) 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-HT(1A) receptors controlling hippocampal 5-HT release were rapidly desensitized by chronic activation with a high-efficacy 5-HT(1A) agonist, but not by chronic activation with a partial agonist. Thus, rapid 5-HT(1A) autoreceptor desensitization by high-efficacy agonists may accelerate the onset of the therapeutic effects of antidepressants.

Selective serotonin 5-HT1A receptor biased agonists elicitdistinct brain activation patterns: a pharmacoMRI study.

Serotonin 1A (5-HT1A) 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-HT1A receptor agonists (8-OH-DPAT, F13714 and F15599) and one 5-HT1A 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-HT1A 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.

Divergent effects of the 'biased' 5-HT1 A receptor agonists F15599 and F13714 in a novel object pattern separation task.

BACKGROUND AND PURPOSE: Pattern separation, that is, the formation of distinct representations from similar inputs, is an important hippocampal process implicated in cognitive domains like episodic memory. A deficit in pattern separation could lead to memory impairments in several psychiatric and neurological disorders. Hence, mechanisms by which pattern separation can be increased are of potential therapeutic interest. EXPERIMENTAL APPROACH: 5-HT1A receptors are involved in spatial memory. Herein we tested the 'biased' 5-HT1A receptor agonists F15599, which preferentially activates post-synaptic heteroreceptors, and F13714, which preferentially activates raphe-located autoreceptors, in rats in a novel spatial task assessing pattern separation, the object pattern separation (OPS) task. KEY RESULTS: The acetylcholinesterase inhibitor donepezil, which served as a positive control, significantly improved spatial pattern separation at a dose of 1 mg·kg(-1) , p.o. F15599 increased pattern separation at 0.04 mg·kg(-1) , i.p., while F13714 decreased pattern separation at 0.0025 mg·kg(-1) , i.p. The selective 5-HT1A receptor antagonist WAY-100635 (0.63 mg·kg(-1) , s.c.) counteracted the effects of both agonists. These data suggest that acute preferential activation of post-synaptic 5-HT1A heteroreceptors improves spatial pattern separation, whereas acute preferential activation of raphe-located 5-HT1A autoreceptors impairs performance. CONCLUSIONS AND IMPLICATIONS: We successfully established and validated a novel, simple and robust OPS task and observed a diverging profile of response with 'biased' 5-HT1A receptor agonists based on their targeting of receptors in distinct brain regions. Our data suggest that the post-synaptic 5-HT1A receptor consists of a potential novel molecular target to improve pattern separation performance.

Activity of serotonin 5-HT(1A) receptor 'biased agonists' in rat models of Parkinson's disease and L-DOPA-induced dyskinesia.

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.

NLX-112, a novel 5-HT1A receptor agonist for the treatment of L-DOPA-induced dyskinesia: Behavioral and neurochemical profile in rat.

L-DOPA is the gold-standard treatment for Parkinson's disease (PD), but induces troublesome dyskinesia after prolonged treatment. This is associated with the 'false neurotransmitter' conversion of L-DOPA to dopamine by serotonin neurons projecting from the raphe to the dorsal striatum. Reducing their activity by targeting pre-synaptic 5-HT1A receptors should thus be an attractive therapeutic strategy, but previous 5-HT1A agonists have yielded disappointing results. Here, we describe the activity of a novel, highly selective and potent 5-HT1A agonist, NLX-112 (also known as befiradol or F13640) in rat models relevant to PD and its associated affective disorders. NLX-112 (0.16 mg/kg, i.p.) potently and completely reversed haloperidol-induced catalepsy in intact rats and abolished L-DOPA-induced Abnormal Involuntary Movements (AIMs) in hemiparkinsonian rats, an effect that was reversed by the selective 5-HT1A antagonist, WAY100635. In microdialysis experiments, NLX-112 profoundly decreased striatal 5-HT extracellular levels, indicative of inhibition of serotonergic function. NLX-112 also blunted the L-DOPA-induced surge in dopamine levels on the lesioned side of the brain, an action that likely underlies its anti-dyskinetic effects. NLX-112 (0.16 mg/kg, i.p.) robustly induced rotations in hemiparkinsonian rats, suggesting that it has a motor facilitatory effect. Rotations were abolished by WAY100635 and were ipsilateral to the lesioned side, suggesting a predominant stimulation of the dopamine system on the non-lesioned side of the brain. NLX-112 also efficaciously reduced immobility time in the forced swim test (75% reduction at 0.16 mg/kg, i.p.) and eliminated stress-induced ultrasonic vocalization at 0.08 mg/kg, i.p., effects consistent with potential antidepressant- and anxiolytic-like properties. In other tests, NLX-112 (0.01-0.16 mg/kg, i.p.) did not impair the ability of L-DOPA to rescue forepaw akinesia in the cylinder test but decreased rotarod performance, probably due to induction of flat body posture and forepaw treading which are typical of 5-HT1A agonists upon acute administration. However, upon repeated administration of NLX-112 (0.63 mg/kg, i.p., twice a day), flat body posture and forepaw treading subsided within 4 days of treatment. Taken together, these observations suggest that NLX-112 could exhibit a novel therapeutic profile, combining robust anti-dyskinetic properties without impairing the therapeutic properties of L-DOPA, and with additional beneficial effects on non-motor (affective) symptoms.

Inverse agonist properties of antipsychotic agents at cloned, human (h) serotonin (5-HT)(1B) and h5-HT(1D) receptors.

The actions of diverse antipsychotics at cloned h5-HT(1B) and h5-HT(1D) receptors were examined employing [3H]-GR125,743 and [35S]-GTPgammaS for determination of affinities and efficacies, respectively. Compared with hD(2) receptors, haloperidol, chlorpromazine and olanzapine showed markedly (>100-fold) lower affinity for h5-HT(1D) and h5-HT(1B) receptors at which they expressed inverse agonist properties. Clozapine, risperidone and ocaperidone likewise behaved as inverse agonists at h5-HT(1B) and h5-HT(1D) receptors but their affinities were only approximately 10-fold lower than at hD(2) receptors. Moreover, ziprasidone, S16924 and ORG5222 interacted at h5-HT(1B) and h5-HT(1D) receptors with affinities similar to hD(2) sites. While S16924 and ORG5222 were inverse agonists at h5-HT(1B) and h5-HT(1D) sites, ziprasidone was an inverse agonist at h5-HT(1D) receptors yet a partial agonist at h5-HT(1B) receptors. These actions of antipsychotics were abolished by the selective, neutral antagonist, S18127. In conclusion, with the exception of ziprasidone, all antipsychotics were inverse agonists at h5-HT(1B) and h5-HT(1D) receptors, although they differed markedly in their potency at these sites as compared to hD(2) receptors.

Inverse agonists and serotonergic transmission: from recombinant, human serotonin (5-HT)1B receptors to G-protein coupling and function in corticolimbic structures in vivo.

The concept of inverse agonism, whereby "antagonists" exert actions opposite to those of agonists at constitutively active receptors, has been documented both at receptor-modulated ion channels as well as at G-protein-coupled receptors (GPCR) in recombinant expression systems. However, it remains unclear whether physiologically or therapeutically relevant inverse agonists actions at GPCRs occur in the CNS in vivo. The present overview discusses our recent observations concerning 5-HT1B receptors, and focuses on the relationship between actions at heterologous Chinese hamster ovary (CHO) expression systems compared with native CNS populations of receptors. To this end, we have exploited several novel and selective ligands, notably the inverse agonist and neutral antagonist at 5-HT1B receptors, SB224,289 and S18127, respectively. Like 5-HT itself, the agonist, GR46611, markedly increases the binding of [35S]-GTP gamma S binding to h5-HT1B receptors expressed in CHO cells, while the "antagonist", GR127,935, modestly stimulates binding suggesting partial agonist properties. However, SB224,289 markedly suppresses binding at these sites. S18127, which does not alter [35S]GTP gamma S binding alone, abolishes the actions of both GR46611 and SB224,289. Nevertheless, in quantitative autoradiographical studies, S18127 and SB224,289 cannot be distinguished as concerns modulation of [35S]-GTP gamma S binding at substantia nigra and caudate nucleus-localized 5-HT1B receptors, inasmuch as they each block the action of the 5-HT1B agonist, CP93129, yet fail to modify binding alone. Further, S18217 and SB224,289, as well as GR127,935, all abolish the inhibitory influence of GR46611 upon dialysis levels of 5-HT in the frontal cortex of freely moving rats without themselves modifying release. Moreover, they all block the hypothermic actions of GR46611 without themselves modifying core temperature. Thus, differences in intrinsic activity of S18127, SB224,289 and GR127,935 seen at cloned, h5-HT1B receptors cannot be detected in vivo. Most notably, no evidence for opposite actions of the inverse agonist, SB224,289, as compared to 5-HT1B agonists is apparent. These data suggest that in vitro observations of inverse agonist actions cannot necessarily be extrapolated to intact systems in vivo.

Agonist and antagonist actions of (-)pindolol at recombinant, human serotonin1A (5-HT1A) receptors.

It has been proposed that the arylalkylamine, (-)pindolol, potentiates the therapeutic action of antidepressant drugs in humans by blockade of 5-HT1A autoreceptors. Its interactions at human 5-HT1A receptors have not, however, been directly characterized. Herein, we demonstrate that (-)pindolol exhibits nanomolar affinity at human 5-HT1A receptors expressed in Chinese Hamster Ovary cells (CHO-h5-HT1A; Ki = 6.4 nmol/L). In a functional test of receptor-mediated G-protein activation (stimulation of [35S]-GTP gamma S binding) (-)pindolol displays an efficacy of 20.3% relative to the endogenous agonist, 5-HT (= 100%). (-)Pindolol also antagonizes 5-HT (100 nmol/L)-stimulated [35S]-GTP gamma S binding, reducing it to 19.8% of control binding. These data indicate that (-)pindolol acts as a (weak) partial agonist at CHO-h5-HT1A receptors and that it blocks the action of 5-HT at these sites.

Constitutive activity at serotonin 5-HT(1D) receptors: detection by homologous GTPgammaS versus [(35)S]-GTPgammaS binding isotherms.

Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT(1D) receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) binding to CHO-h5-HT(1D) membranes whereas methiothepin and the 5-HT(1B/1D)-selective ligand, SB224,289, exerted robust inhibition of basal [(35)S]-GTPgammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT(1B/1D) ligand, S18127. Constitutive activity was investigated by homologous inhibition of [(35)S]-GTPgammaS binding to CHO-h5-HT(1D) membranes with unlabelled GTPgammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [(35)S]-GTPgammaS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT(1D) receptor-G-protein coupling. Thus, at 5-HT(1D) receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPgammaS versus [(35)S]-GTPgammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT(1D) receptors and, possibly, other GPCRs, inverse agonists will be detectable by [(35)S]-GTPgammaS binding if a HA component is present under basal conditions.