Effects of 5-HT1 and 5-HT 2 Receptor Agonists on Electromagnetic Field-Induced Analgesia in Rats.

Much evidence demonstrates the antinociceptive effect of magnetic fields (MFs). However, the analgesic action mechanism of the electromagnetic field (EMF) is not exactly understood. The aim of the present study was to investigate the effects of 5-HT1 and 5-HT2 receptor agonists (serotonin HCl and 2,5-dimethoxy-4-iodoamphetamine [DOI] hydrochloride) on EMF-induced analgesia. In total, 66 adult male Wistar albino rats with an average body mass of 225 ± 13 g were used in this study. The animals were subjected to repeated exposures of alternating 50 Hz and 5 mT EMF for 2 h a day for 15 days. Prior to analgesia tests, serotonin HCl (5-HT1 agonist) 4 mg/kg, WAY 100635 (5-HT1 antagonist) 0.04 mg/kg, DOI hydrochloride (5-HT2 receptor agonist) 4 mg/kg, and SB 204741 (5-HT2 antagonist) 0.5 mg/kg doses were injected into rats. For statistical analysis of the data, analysis of variance was used and multiple comparisons were determined by Tukey's test. Administration of serotonin HCl MF (5 mT)-exposed rats produced a significant increase in percent maximal possible effect (% MPE) as compared with EMF group (P < 0.05). On the contrary, injection of WAY 100635 to MF-exposed rats produced a significant decrease in analgesic activity (P < 0.05). Similarly, the administration of DOI hydrochloride significantly increased % MPE values as compared with the EMF group while SB 204741 reduced it (P < 0.05). In conclusion, our results suggested that serotonin 5-HT1 and 5-HT2 receptors play an important role in EMF-induced analgesia; however, further research studies are necessary to understand the mechanism. Bioelectromagnetics. 2019;40:319-330. © 2019 Bioelectromagnetics Society.

Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: synthesis, biological characterization, and behavioral studies.

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

Selective agonists for serotonin 7 (5-HT7) receptor and their applications in preclinical models: an overview.

The serotonin 7 (5-HT7) receptor was the last serotonin receptor subtype to be discovered in 1993. This receptor system has been implicated in several central nervous system (CNS) functions, including circadian rhythm, rapid eye movement sleep, thermoregulation, nociception, memory and neuropsychiatric symptoms and pathologies, such as anxiety, depression and schizophrenia. In 1999, medicinal chemistry efforts led to the identification of SB-269970, which became the gold standard selective 5-HT7 receptor antagonist, and later of various selective agonists such as AS-19, LP-44, LP-12, LP-211 and E-55888. In this review, we summarize the preclinical pharmacological studies performed using these agonists, highlighting their strengths and weaknesses. The data indicate that 5-HT7 receptor agonists can have neuroprotective effects against N-methyl-d-aspartate-induced toxicity, modulate neuronal plasticity in rats, enhance morphine-induced antinociception and alleviate hyperalgesia consecutive to nerve lesion in neuropathic animals.

Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin 1A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan.

Yokukansan (YKS), a traditional Japanese medicine, is composed of seven kinds of dried herbs. It is widely prescribed in clinical situation for treating psychiatric disorders such as aggressiveness in patients with dementia. We previously demonstrated that YKS and Uncaria hook (UH), which is a constituent herb of YKS, had a partial agonistic effect to 5-HT(1A) receptors in vitro. However, it has still been unclear whether this in vitro effect is reflected in in vivo, and what the active ingredients are. The purpose of the present study is to find the active ingredient in YKS and to demonstrate the effect in in vivo. In the present study, we first studied the effect of YKS and UH on aggressiveness and sociality in socially isolated mice. YKS and UH ameliorated the isolation-induced increased aggressiveness and decreased sociality, and these ameliorative effects were counteracted by coadministration of 5-HT(1A) receptor antagonist WAY-100635, or disappeared by eliminating UH from YKS. These results suggest that the effect of YKS is mainly attributed to UH, and the active ingredient is contained in UH. To find the candidate ingredients, we examined competitive binding assay and [(35)S] guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding assay of seven major alkaloids in UH using Chinese hamster ovary cells expressing 5-HT(1A) receptors artificially. Only geissoschizine methyl ether (GM) among seven alkaloids potently bound to 5-HT(1A) receptors and acted as a partial agonist. This in vitro result on GM was further demonstrated in the socially isolated mice. As did YKS and UH, GM ameliorated the isolation-induced increased aggressiveness and decreased sociality, and the effect was counteracted by coadministration of WAY-100635. These lines of results suggest that GM in UH is potent 5-HT(1A) receptor agonist and a candidate for pharmacological effect of YKS on aggressiveness and sociality in socially isolated mice.

Anti-amnesic effect of Ficus religiosa in scopolamine-induced anterograde and retrograde amnesia.

CONTEXT: Ficus religiosa Linn (Moraceae) is a variety of fig tree. Its figs are known to contain a high serotonergic content, and modulation of serotonergic neurotransmission plays a crucial role in the pathogenesis of amnesia. Thus, the present study was envisaged. OBJECTIVE: To investigate the effect of the methanol extract of figs of Ficus religiosa (FRFE) on scopolamine-induced anterograde and retrograde amnesia in mice. MATERIALS AND METHODS: Transfer latency (TL) to the preferred niche in the elevated plus-maze (EPM) and learning avoidance of passive behavior to avoid punishment in the modified passive avoidance paradigm (MPA) served as behavioral models for the assessment of memory. Scopolamine (1 mg/kg, i.p.) was administered before training for induction of anterograde amnesia and before retrieval for induction of retrograde amnesia in both models. TL in the EPM, step down latency (SDL), number of trials, and number of mistakes in the MPA were determined in vehicle control, FRFE treated (10, 50, and 100 mg/kg, i.p.), and standard groups (piracetam 200 mg/kg, i.p.). Cyproheptadine, a non-selective 5-HT(1/2) blocker (4 mg/kg, i.p.), was administered along with the FRFE to investigate the involvement of serotonergic pathways in the anti-amnesic effect of FRFE. RESULTS AND DISCUSSION: FRFE resulted in a significant improvement of memory, as its treatment attenuated the scopolamine-induced anterograde and retrograde amnesia dose-dependently. Further, cyproheptadine pretreatment significantly reversed the anti-amnesic effect of FRFE. CONCLUSION: FRFE has anti-amnesic activity against scopolamine-induced amnesia, in a dose-dependent manner. Inhibition of the anti-amnesic effect of FRFE by cyproheptadine substantiates the involvement of serotonergic pathways for its activity.

Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.

Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.

Agonists of the serotonin 5-HT2C receptor: preclinical and clinical progression in multiple diseases.

The serotonin 5-HT2C receptor is a G-protein-coupled receptor and is one of the 14 subtypes that constitutes the serotonin receptor family. Agonists of 5-HT2C have been implicated as potential treatments for diseases of significant unmet medical need, including obesity and schizophrenia. Despite approximately 10 years of discovery efforts, 5-HT2C agonists have only recently advanced into the clinic, likely because many of the early drug discovery efforts experienced significant difficulties with attaining receptor selectivity. Several of these issues related to receptor selectivity have now been overcome, resulting in the entry of compounds into advanced clinical trials. This review summarizes the progress in 5-HT2C agonist discovery and clinical development over the last 3 years. [sw1]what are the several issues - several issues relating to receptor selectivity?

Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.

1-Aminocyclopentane-1,2,4-tricarboxylic acids screening on glutamatergic and serotonergic systems.

Enantiopure constrained 1-aminocyclopentane-1,2,4-tricarboxylic acids containing the glutamic acid skeleton were prepared as two diastereomers characterized by having the carboxylic groups in position two and four cis-oriented to each other and trans with respect to 1-carboxylic group and all cis-oriented carboxylic groups, respectively. A biochemical screening of activity of the above amino acids was investigated on glutamate and 5-HT receptors to find a possible metabotropic agonist, acting on the serotoninergic system.

Screening of 5-HT1A receptor antagonists using molecularly imprinted polymers.

Molecular imprinting produces network polymers with recognition sites for imprint molecules. The high binding affinity and selectivity in conjunction with the polymers' physical robustness positions molecular imprinted polymers (MIPs) as candidates for use as preliminary screens in drug discovery. As such, MIPs can serve as crude mimics of native receptors. In an effort to evaluate the relationship between MIPs and native receptors, imprinted polymers for WAY-100635, an antagonist of the serotonin (5-HT) receptor subtype 5-HT1A were prepared. The resulting MIP P(WAY) was evaluated as an affinity matrix in the screening of serotonin receptor antagonists with known affinities for the native receptor. Rough correlations in affinity between the synthetic P(WAY) and native receptor 5-HT1A were found. These findings provide some support for the analogy between MIPs and native receptors and their possible use as surrogates.

Conformational restriction in novel NAN-190 and MP3022 analogs and their 5-HT(1A) receptor activity.

The newly synthesized analogs of NAN-190 containing m-Cl and m-CF(3) substituents in the arylpiperazine moiety and their conformationally restricted counterparts showed a very high 5-HT(1A )receptor affinity. In the LLR test, the flexible compounds 4a and 5a displayed features of a partial agonist and agonist, respectively. The conformational restriction in the tested structures caused alternations in the observed in vivo effects; compounds 4b and 5b were classified as an inactive agent and an antagonist of postsynaptic 5-HT(1A )receptors, respectively. Rigidification of MP3022 and its 5,6-dimethyl analog structures resulted in cis and trans stereoisomers 6b-9b with a 1- and 2-substituted benzotriazole moiety. In both series, in vitro experiments showed that the cis configurations of the compounds were better tolerated by 5-HT(1A) receptor sites than the trans ones. The conformational analysis revealed various spatial regions that can be explored by terminal benzotriazole fragments in those structures. Like the previously described cis-6b, the new ligand cis-7b, displayed features of a postsynaptic 5-HT(1A) receptor agonist, whereas cis-8b was characterized as a partial agonist of those receptor sites. It was suggested that the nonlinear geometry of the above agents has significant influence on the postsynaptic 5-HT(1A )receptor stimulation.

Synthesis, SAR studies, and evaluation of 1,4-benzoxazepine derivatives as selective 5-HT1A receptor agonists with neuroprotective effect: Discovery of Piclozotan.

A new series of 1,4-benzoxazepine derivatives was designed, synthesized, and evaluated for binding to 5-HT1A receptor and cerebral anti-ischemic effect. A lot of compounds exhibited nanomolar affinity for 5-HT1A receptor with good selectivity over both dopamine D2 and alpha1-adrenergic receptors. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1, 4-benzoxazepin-5(4H)-one (50: SUN N4057 (Piclozotan) as 2HCl salt) showed remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.

Synthesis and 5-HT(1A), 5-HT(2A) receptor activity of new beta-tetralonohydantoins.

A series of new 3-[4-(4-arylpiperazinyl)-butyl]-beta-tetralonohydantoins (8a-13a) were synthesized. The compounds exhibited high affinity for 5-HT(1A) receptors (K(i)=6 to 55 nM) combined with moderate-to-high 5-HT(2A) receptor affinities (K(i)=45 to 213 nM). The results of in vivo studies indicated that of the compounds tested, 3-[4-(4-phenylpiperazinyl)-butyl-beta-tetralonohydantoin (8a) showed features of full (pre- and postsynaptic) 5-HT(1A) receptor agonists, whereas compounds 9a-13a behaved like antagonists of postsynaptic 5-HT(1A) receptors; additionally, compound 13a produced an effect characteristic of presynaptic 5-HT(1A) receptor agonists. Moreover, compounds 8a and 10a-13a exhibited properties of 5-HT(2A) receptor antagonists. Due to the most interesting 5-HT(1A)/5-HT(2A) functional profile compounds 8a and 13a were further tested for their potential psychotropic activity. In fact, compound 8a (but not 13a) showed diazepam-like anxiolytic activity and behaved like a weak antidepressant.

Preclinical studies characterizing the anti-migraine and cardiovascular effects of the selective 5-HT1D receptor agonist PNU-142633.

The present study describes the preclinical pharmacology of a highly selective 5-HT1D receptor agonist PNU-142633. PNU-142633 binds with a Ki of 6 nm at the human 5-HT1D receptor and a Ki of> 18 000 nm at the human 5-HT1B receptor. The intrinsic activity of PNU-142633 at the human 5-HT1D receptor was determined to be 70% that of 5-HT in a cytosensor cell-based assay compared with 84% for that of sumatriptan. PNU-142633 was equally effective as sumatriptan and a half-log more potent than sumatriptan in preventing plasma protein extravasation induced by electrical stimulation of the trigeminal ganglion. Like sumatriptan, PNU-142633 reduced the increase in cat nucleus trigeminal caudalis blood flow elicited by electrical stimulation of the trigeminal ganglion compared with the vehicle control. The direct vasoconstrictor potential of PNU-142633 was evaluated in vascular beds. Sumatriptan increased vascular resistance in carotid, meningeal and coronary arteries while PNU-142633 failed to alter resistance in these vascular beds. These data are discussed in relation to the clinical findings of PNU-142633 in a phase II acute migraine study.

4-(Phenylsulfonyl)piperidines: novel, selective, and bioavailable 5-HT(2A) receptor antagonists.

On the basis of a spirocyclic ether screening lead, a series of acyclic sulfones have been identified as high-affinity, selective 5-HT(2A) receptor antagonists. Bioavailability lacking in the parent, 1-(2-(2,4-difluorophenyl)ethyl)-4-(phenylsulfonyl)piperidine (12), was introduced by using stability toward rat liver microsomes as a predictor of bioavailability. By this means, the 4-cyano- and 4-carboxamidophenylsulfonyl derivatives 26 and 31 were identified as orally bioavailable, brain-penetrant analogues suitable for evaluation in animal models. Bioavailability was also attainable by N substitution leading to the N-phenacyl derivative 35. IKr activity detected through counterscreening was reduced to insignificant levels in vivo with the latter compound.

Donitriptan (Pierre Fabre).

Pierre Fabre is developing donitriptan, a piperazide 5-HT1D agonist, as a potential treatment for migraine [175854]. In January 2001, donitriptan had completed phase I trials for migraine and was scheduled to enter phase II development [396027]. This compound has an increased potency and, importantly, markedly higher intrinsic activity in comparison to the well described tryptamine derivatives, naratriptan, zolmitriptan and sumatriptan [295769].

Effect of ring fluorination on the pharmacology of hallucinogenic tryptamines.

A series of fluorinated analogues of the hallucinogenic tryptamines N,N-diethyltryptamine (DET), 4-hydroxy-N,N-dimethyltryptamine (4-OH-DMT, psilocin), and 5-methoxy-DMT was synthesized to investigate possible explanations for the inactivity of 6-fluoro-DET as a hallucinogen and to determine the effects of fluorination on the molecular recognition and activation of these compounds at serotonin receptor subtypes. The target compounds were evaluated using in vivo behavioral assays for hallucinogen-like and 5-HT(1A) agonist activity and in vitro radioligand competition assays for their affinity at 5-HT(2A), 5-HT(2C), and 5-HT(1A) receptor sites. Functional activity at the 5-HT(2A) receptor was determined for all compounds. In addition, for some compounds functional activity was determined at the 5-HT(1A) receptor. Hallucinogen-like activity, evaluated in the two-lever drug discrimination paradigm using LSD-trained rats, was attenuated or abolished for all of the fluorinated analogues. One of the tryptamines, 4-fluoro-5-methoxy-DMT (6), displayed high 5-HT(1A) agonist activity, with potency greater than that of the 5-HT(1A) agonist 8-OH-DPAT. The ED(50) of 6 in the two-lever drug discrimination paradigm using rats trained to discriminate the 5-HT(1A) agonist LY293284 was 0.17 micromol/kg, and the K(i) at [(3)H]8-OH-DPAT-labeled 5-HT(1A) receptors was 0.23 nM. The results indicate that fluorination of hallucinogenic tryptamines generally has little effect on 5-HT(2A/2C) receptor affinity or intrinsic activity. Affinity at the 5-HT(1A) receptor was reduced, however, in all but one example, and all of the compounds tested were full agonists but with reduced functional potency at this serotonin receptor subtype. The one notable exception was 4-fluoro-5-methoxy-DMT (6), which had markedly enhanced 5-HT(1A) receptor affinity and functional potency. Although it is generally considered that hallucinogenic activity results from 5-HT(2A) receptor activation, the present results suggest a possible role for involvement of the 5-HT(1A) receptor with tryptamines.

N-Substituted (2,3-dihydro-1,4-benzodioxin-2-yl)methylamine derivatives as D(2) antagonists/5-HT(1A) partial agonists with potential as atypical antipsychotic agents.

A series of N-substituted 1-(2,3-dihydro-1, 4-benzodioxin-2-yl)methylamine derivatives with D(2) antagonist/5-HT(1A) partial agonist activity has been prepared as potential atypical antipsychotic agents. Optimization of in vitro receptor binding activity and in vivo activity in rodent models of psychosis has led to compound 24, which showed good affinities for human D(2), D(3), and 5-HT(1A) receptors but significantly less affinity for human alpha(1) adrenoceptors and rat H(1) and muscarinic receptors. In rodents, 24 showed functional D(2)-like antagonism and 5-HT(1A) partial agonism. After oral dosing, 24 showed good activity in rodent antipsychotic tests and very little potential to cause extrapyramidal side effects (EPS), as measured by its ability to induce catalepsy in rats only at very high doses. In the light of this promising profile of activity, 24 has been selected for clinical investigation as a novel antipsychotic agent with a predicted low propensity to cause EPS.

Novel derivatives of 2-pyridinemethylamine as selective, potent, and orally active agonists at 5-HT1A receptors.

The aim of this work was to improve the oral bioavailability of a recently discovered, novel structural class of 5-HT1A receptor agonists: aryl-{[4-(6-R-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1 -yl-metha none. Incorporation of a fluorine atom in the beta-position to the amino function in the side chain led to analogues that exhibited, in general, enhanced and long-lasting 5-HT1A agonist activity in rats after oral administration. Location of the fluorine atom at the C-4 position of the piperidine ring was the most favorable, and among the various substituents tested, the ability of the fluorine was unique in improving the oral activity of this family of ligands. Thus, the derivatives 39, 46, and 61 bound with higher affinity and selectivity to 5-HT1A receptors (versus dopaminergic D2 and adrenergic alpha1 receptors) and displayed more potent 5-HT1A agonist activity in vitro and in vivo than their C-4 desfluoro analogues. To examine the relationship between the conformation of the pharmacophore and the level of agonistic activity of this type of ligand, we synthesized a series of 3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-(H or CH3)-6-R-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-+ ++methanone derivatives and found that the combination of a 5-methyl and a 6-methylamino substituent on the pyridine ring synergistically affected their 5-HT1A agonist properties. Thus, the 3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-methyl-6-methylamino-pyridin- 2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone 40 behaved as a more potent 5-HT1A receptor agonist in vitro and in vivo than its 5-unsubstituted analogue 38. The antidepressant potential of the lead compounds 40, 45, and 54 was examined by means of the forced swimming test (FST) in rats. The results indicated that, after a single oral administration, these compounds inhibited immobility in the FST more potently and more extensively than the clinically used antidepressant imipramine. Thus, 40, 45, and 54 are potent, orally active 5-HT1A receptor agonists with marked antidepressant potential.

Definition of a pharmacophore for partial agonists of serotonin 5-HT3 receptors.

A definition of a partial agonists serotonin 5-HT3 pharmacophore was carried out by considering a three-dimensional model which correlates the chemical structures of series of piperazinopyrrolothienopyrazines, piperazinopyridopyrrolopyrazines, piperazinopyrroloquinolaxines, piperazinopyridopyrroloquinoxalines, aminoalkyloximinopyrroloindoles, aminoalkyloximinothienopyrrolizines, and aminoalkyloximinopyrrolizines with the biological affinities. The model is formed by five features corresponding to two hydrogen bond acceptors, one aromatic ring, one hydrophobic group, and one positive ionizable site (quaternary ammonium ions). The nature of the features and the distances between them explain the partial agonist activities of these compounds.