Effects of acute and chronic administration of trace amine-associated receptor 1 (TAAR1) ligands on in vivo excitability of central monoamine-secreting neurons in rats.

Trace amine-associated receptor 1 (TAAR1) has been recently identified as a target for the future antidepressant, antipsychotic, and anti-addiction drugs. Full (e.g. RO5256390) and partial (e.g. RO5263397) TAAR1 agonists showed antidepressant-, antipsychotic- and anti-addiction-like behavioral effects in rodents and primates. Acute RO5256390 suppressed, and RO5263397 stimulated serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN) and dopamine neurons of the ventral tegmental area (VTA) in brain slices, suggesting that the behavioral effects of TAAR1 ligands involve 5-HT and dopamine. For more comprehensive testing of this hypothesis, we examined acute and chronic effects of RO5256390 and RO5263397 on monoamine neurons in in vivo conditions. Excitability of 5-HT neurons of the DRN, noradrenaline neurons of the locus coeruleus (LC), and dopamine neurons of the VTA was assessed using single-unit electrophysiology in anesthetized rats. For acute experiments, RO5256390 and RO5263397 were administered intravenously; neuronal excitability after RO5256390 and RO5263397 administration was compared to the basal activity of the same neuron. For chronic experiments, RO5256390 was administered orally for fourteen days prior to electrophysiological assessments. The neuronal excitability in RO5256390-treated rats was compared to vehicle-treated controls. We found that acute RO5256390 inhibited 5-HT and dopamine neurons. This effect of RO5256390 was reversed by the subsequent and prevented by the earlier administration of RO5263397. Acute RO5256390 and RO5263397 did not alter the excitability of LC noradrenaline neurons in a statistically significant way. Chronic RO5256390 increased excitability of 5-HT neurons of the DRN and dopamine neurons of the VTA. In conclusion, the putative antidepressant and antipsychotic effects of TAAR1 ligands might be mediated, at least in part, via the modulation of excitability of central 5-HT and dopamine neurons.

Trace Amines and Their Receptors.

Trace amines are endogenous compounds classically regarded as comprising ß-phenylethyalmine, p-tyramine, tryptamine, p-octopamine, and some of their metabolites. They are also abundant in common foodstuffs and can be produced and degraded by the constitutive microbiota. The ability to use trace amines has arisen at least twice during evolution, with distinct receptor families present in invertebrates and vertebrates. The term "trace amine" was coined to reflect the low tissue levels in mammals; however, invertebrates have relatively high levels where they function like mammalian adrenergic systems, involved in "fight-or-flight" responses. Vertebrates express a family of receptors termed trace amine-associated receptors (TAARs). Humans possess six functional isoforms (TAAR1, TAAR2, TAAR5, TAAR6, TAAR8, and TAAR9), whereas some fish species express over 100. With the exception of TAAR1, TAARs are expressed in olfactory epithelium neurons, where they detect diverse ethological signals including predators, spoiled food, migratory cues, and pheromones. Outside the olfactory system, TAAR1 is the most thoroughly studied and has both central and peripheral roles. In the brain, TAAR1 acts as a rheostat of dopaminergic, glutamatergic, and serotonergic neurotransmission and has been identified as a novel therapeutic target for schizophrenia, depression, and addiction. In the periphery, TAAR1 regulates nutrient-induced hormone secretion, suggesting its potential as a novel therapeutic target for diabetes and obesity. TAAR1 may also regulate immune responses by regulating leukocyte differentiation and activation. This article provides a comprehensive review of the current state of knowledge of the evolution, physiologic functions, pharmacology, molecular mechanisms, and therapeutic potential of trace amines and their receptors in vertebrates and invertebrates.

The Pharmacological Profile of Second Generation Pyrovalerone Cathinones and Related Cathinone Derivative.

Pyrovalerone cathinones are potent psychoactive substances that possess a pyrrolidine moiety. Pyrovalerone-type novel psychoactive substances (NPS) are continuously detected but their pharmacology and toxicology are largely unknown. We assessed several pyrovalerone and related cathinone derivatives at the human norepinephrine (NET), dopamine (DAT), and serotonin (SERT) uptake transporters using HEK293 cells overexpressing each respective transporter. We examined the transporter-mediated monoamine efflux in preloaded cells. The receptor binding and activation potency was also assessed at the 5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C receptors. All pyrovalerone cathinones were potent DAT (IC50 = 0.02-8.7 µM) and NET inhibitors (IC50 = 0.03-4.6 µM), and exhibited no SERT activity at concentrations < 10 µM. None of the compounds induced monoamine efflux. NEH was a potent DAT/NET inhibitor (IC50 = 0.17-0.18 µM). 4F-PBP and NEH exhibited a high selectivity for the DAT (DAT/SERT ratio = 264-356). Extension of the alkyl chain enhanced NET and DAT inhibition potency, while presence of a 3,4-methylenedioxy moiety increased SERT inhibition potency. Most compounds did not exhibit any relevant activity at other monoamine receptors. In conclusion, 4F-PBP and NEH were selective DAT/NET inhibitors indicating that these substances likely produce strong psychostimulant effects and have a high abuse liability.

TAAR1 Expression in Human Macrophages and Brain Tissue: A Potential Novel Facet of MS Neuroinflammation.

TAAR1 is a neuroregulator with emerging evidence suggesting a role in immunomodulation. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. Here, we investigate TAAR1 expression in human primary monocytes, peripherally-derived macrophages, and MS brain tissue. RT-qPCR was used to assess TAAR1 levels in MS monocytes. Using a previously validated anti-human TAAR1 antibody and fluorescence microscopy, TAAR1 protein was visualized in lipopolysaccharide-stimulated or basal human macrophages, as well as macrophage/microglia populations surrounding, bordering, and within a mixed active/inactive MS lesion. In vivo, TAAR1 mRNA expression was significantly lower in MS monocytes compared to age- and sex-matched healthy controls. In vitro, TAAR1 protein showed a predominant nuclear localization in quiescent/control macrophages with a shift to a diffuse intracellular distribution following lipopolysaccharide-induced activation. In brain tissue, TAAR1 protein was predominantly expressed in macrophages/microglia within the border region of mixed active/inactive MS lesions. Considering that TAAR1-mediated anti-inflammatory effects have been previously reported, decreased mRNA in MS patients suggests possible pathophysiologic relevance. A shift in TAAR1 localization following pro-inflammatory activation suggests its function is altered in pro-inflammatory states, while TAAR1-expressing macrophages/microglia bordering an MS lesion supports TAAR1 as a novel pharmacological target in cells directly implicated in MS neuroinflammation.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats.

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

TAAR1 levels and sub-cellular distribution are cell line but not breast cancer subtype specific.

Trace amine-associated receptors are G protein-coupled receptors of which TAAR1 is the most well-studied. Recently, Vattai et al. (J Cancer Res Clin Oncol 143:1637-1647 https://doi.org/10.1007/s00432-017-2420-8 , 2017) reported that expression of TAAR1 may be a marker of breast cancer (BC) survival, with a positive correlation also suggested between TAAR1 expression and HER2 positivity. Neither a role for TAAR1 in breast tissue, nor in cancer, had previously been suspected. We, therefore, sought to provide independent validation and to further examine these putative relationships. First, a bioinformatic analysis on 58 total samples including normal breast tissue, BC-related cell lines, and tumour samples representing different BC sub-types found no clear correlation between TAAR1 mRNA levels and any BC subtype, including HER2 + . We next confirmed the bioinformatics data correlated to protein expression using a well validated anti-human TAAR1 antibody. TAAR1 mRNA levels correlated with the relative intensity of immunofluorescence staining in six BC cell lines (MCF-7, T47D, MDA-MB-231, SKBR3, MDA-MB-468, BT-474), but not in the MCF-10A immortalized mammary gland line, which had high mRNA but low protein levels. As expected, TAAR1 protein was intracellular in all cell lines. Surprisingly MCF-7, SKBR3, and MDA-MB-468 showed pronounced nuclear localization. The relative protein expression in MCF-7, MDA-MB-231, and MCF-10A lines was further confirmed by semi-quantitative flow cytometry. Finally, we demonstrate that the commercially available anti-TAAR1 antibody has poor selectivity, which likely explains the lack of correlation with the previous study. Therefore, while we clearly demonstrate variable expression and sub-cellular localization of TAAR1 across BC cell lines, we find no evidence for association with BC subtype.

In Vitro Characterization of Psychoactive Substances at Rat, Mouse, and Human Trace Amine-Associated Receptor 1.

Trace amine-associated receptor 1 (TAAR1) has been implicated in the behavioral effects of amphetamine-type stimulant drugs in rodents. TAAR1 has also been suggested as a target for novel medications to treat psychostimulant addiction. We previously reported that binding affinities at TAAR1 can differ between structural analogs of psychostimulants, and species differences have been observed. In this study, we complement our previous findings with additional substances and the determination of functional activation potencies. In summary, we present here pharmacological in vitro profiles of 101 psychoactive substances at human, rat, and mouse TAAR1. p-Tyramine, ß-phenylethylamine, and tryptamine were included as endogenous comparator compounds. Functional cAMP measurements and radioligand displacement assays were conducted with human embryonic kidney 293 cells that expressed human, rat, or mouse TAAR1. Most amphetamines, phenethylamine, and aminoindanes exhibited potentially physiologically relevant rat and mouse TAAR1 activation (EC50 < 5 µM) and showed full or partial (Emax < 80%) agonist properties. Cathinone derivatives, including mephedrone and methylenedioxypyrovalerone, exhibited weak (EC50 = 5-10 µM) to negligible (EC50 > 10 µM) binding properties at TAAR1. Pipradrols, including methylphenidate, exhibited no affinity for TAAR1. We found considerable species differences in activity at TAAR1 among the highly active ligands, with a rank order of rat > mouse > human. This characterization provides information about the pharmacological profile of psychoactive substances. The species differences emphasize the relevance of clinical studies to translationally complement rodent studies on the role of TAAR1 activity for psychoactive substances.

A UGT2B10 splicing polymorphism common in african populations may greatly increase drug exposure.

RO5263397 [(S)-4-(3-fluoro-2-methyl-phenyl)-4,5-dihydro-oxazol-2-ylamine], a new compound that showed promising results in animal models of schizophrenia, is mainly metabolized in humans by N-glucuronidation. Enzyme studies, using the (then) available commercial uridine 5'-diphosphate-glucuronosyltransferases (UGTs), suggested that UGT1A4 is responsible for its conjugation. In the first clinical trial, in which RO5263397 was administered orally to healthy human volunteers, a 136-fold above-average systemic exposure to the parent compound was found in one of the participants. Further administration in this trial identified two more such poor metabolizers, all three of African origin. Additional in vitro studies with recombinant UGTs showed that the contribution of UGT2B10 to RO5263397 glucuronidation is much higher than UGT1A4 at clinically relevant concentrations. DNA sequencing in all of these poor metabolizers identified a previously uncharacterized splice site mutation that prevents assembly of full-length UGT2B10 mRNA and thus functional UGT2B10 protein expression. Further DNA database analyses revealed the UGT2B10 splice site mutation to be highly frequent in individuals of African origin (45%), moderately frequent in Asians (8%) and almost unrepresented in Caucasians (<1%). A prospective study using hepatocytes from 20 individual African donors demonstrated a >100-fold lower intrinsic clearance of RO5263397 in cells homozygous for the splice site variant allele. Our results highlight the need to include UGT2B10 when screening the human UGTs for the enzymes involved in the glucuronidation of a new compound, particularly when there is a possibility of N-glucuronidation. Moreover, this study demonstrates the importance of considering different ethnicities during drug development.

TAAR1-dependent effects of apomorphine in mice.

G protein-coupled trace amine-associated receptor 1 (TAAR1) is expressed in several brain regions and modulates dopaminergic activity partially by affecting D2 dopamine receptor function. In vitro, the nonselective dopamine agonist apomorphine can activate mouse and rat TAAR1. The aim of the present study was to evaluate whether apomorphine activity at the rodent TAAR1 observed in in vitro studies contributes to its behavioral manifestation in mice. For this purpose, we compared the behavioral effects of a wide range of apomorphine doses in wild type (WT) and TAAR1 knockout (TAAR1-KO) mice. Apomorphine-induced locomotor responses (0.01-4.0 mg/kg) were tested in locomotor activity boxes, and stereotypic behavior at 5 mg/kg was tested by ethological methods. A gnawing test was used to analyze the effects of the highest dose of apomorphine (10 mg/kg). No statistically significant differences were observed between TAAR1-KO and WT mice following inhibitory pre-synaptic low doses of apomorphine. At higher doses (2.0-5.0 mg/kg), apomorphine-induced climbing behavior was significantly reduced in TAAR1 mutants relative to WT controls. Moreover, the lack of TAAR1 receptors decreased certain types of stereotypies (as reflected in by measures of the global stereotypy score, licking but not sniffing or gnawing) that were induced by high doses of apomorphine. These data indicate that apomorphine activity at TAAR1 contributes to some behavioral manifestations, particularly climbing, in rodents following high doses of this drug. The contribution of TAAR1 to apomorphine-induced climbing in rodents should be considered when apomorphine is used as a screening tool in the search for potential antipsychotics.

TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity.

The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and ß-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT(1A) receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission. In WT but not Taar1(-/-) mice, RO5166017 prevented stress-induced hyperthermia and blocked dopamine-dependent hyperlocomotion in cocaine-treated and dopamine transporter knockout mice as well as hyperactivity induced by an NMDA antagonist. These results tie TAAR1 to the control of monoamine-driven behaviors and suggest anxiolytic- and antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.

Wakefulness Induced by TAAR1 Partial Agonism is Mediated Through Dopaminergic Neurotransmission.

Trace amine-associated receptor 1 (TAAR1) is known to negatively regulate dopamine (DA) release. The partial TAAR1 agonist RO5263397 promotes wakefulness and suppresses NREM and REM sleep in mice, rats, and non-human primates. We tested the hypothesis that the TAAR1-mediated effects on sleep/wake were due, at least in part, to DA release. Male C57BL6/J mice (n=8) were intraperitoneally administered the D1R antagonist SCH23390, the D2R antagonist eticlopride, a combination of D1R+D2R antagonists or saline at ZT5.5, followed 30 min later by RO5263397 or vehicle (10% DMSO in DI water) at ZT6 per os. EEG, EMG, subcutaneous temperature, and activity were recorded in each mouse across the 8 treatment conditions and sleep architecture was analyzed for 6 hours post-dosing. Consistent with our previous reports, RO5263397 increased wakefulness as well as the latency to NREM and REM sleep. D1, D2, and D1+D2 pretreatment reduced RO5263397-induced wakefulness during the first 1-2 hours after dosing, but only the D1+D2 combination attenuated the wake-promoting effect of RO5263397 from ZT6-8, mostly by increasing NREM sleep. Although D1+D2 antagonism blocked the wake-promoting effect of RO5263397, only the D1 antagonist significantly reduced the TAAR1-mediated increase in NREM latency. Neither the D1 nor the D2 antagonist affected TAAR1-mediated suppression of REM sleep. These results suggest that, whereas TAAR1 effects on wakefulness are mediated in part through the D2R, D1R activation plays a role in reversing the TAAR1-mediated increase in NREM sleep latency. By contrast, TAAR1-mediated suppression of REM sleep appears not to involve D1R or D2R mechanisms.

In vitro and in vivo metabolism of psilocybin's active metabolite psilocin.

In vivo, psilocybin is rapidly dephosphorylated to psilocin which induces psychedelic effects by interacting with the 5-HT2A receptor. Psilocin primarily undergoes glucuronidation or conversion to 4-hydroxyindole-3-acetic acid (4-HIAA). Herein, we investigated psilocybin's metabolic pathways in vitro and in vivo, conducting a thorough analysis of the enzymes involved. Metabolism studies were performed using human liver microsomes (HLM), cytochrome P450 (CYP) enzymes, monoamine oxidase (MAO), and UDP-glucuronosyltransferase (UGT). In vivo, metabolism was examined using male C57BL/6J mice and human plasma samples. Approximately 29% of psilocin was metabolized by HLM, while recombinant CYP2D6 and CYP3A4 enzymes metabolized nearly 100% and 40% of psilocin, respectively. Notably, 4-HIAA and 4-hydroxytryptophol (4-HTP) were detected with HLM but not with recombinant CYPs. MAO-A transformed psilocin into minimal amounts of 4-HIAA and 4-HTP. 4-HTP was only present in vitro. Neither 4-HIAA nor 4-HTP showed relevant interactions at assessed 5-HT receptors. In contrast to in vivo data, UGT1A10 did not extensively metabolize psilocin in vitro. Furthermore, two putative metabolites were observed. N-methyl-4-hydroxytryptamine (norpsilocin) was identified in vitro (CYP2D6) and in mice, while an oxidized metabolite was detected in vitro (CYP2D6) and in humans. However, the CYP2D6 genotype did not influence psilocin plasma concentrations in the investigated study population. In conclusion, MAO-A, CYP2D6, and CYP3A4 are involved in psilocin's metabolism. The discovery of putative norpsilocin in mice and oxidized psilocin in humans further unravels psilocin's metabolism. Despite limitations in replicating phase II metabolism in vitro, these findings hold significance for studying drug-drug interactions and advancing research on psilocybin as a therapeutic agent.

TAAR1 Regulates Purinergic-induced TNF Secretion from Peripheral, But Not CNS-resident, Macrophages.

Trace amine-associated receptor 1 (TAAR1) is an established neuroregulatory G protein-coupled receptor with recent studies suggesting additional functions related to immunomodulation. Our lab has previously investigated TAAR1 expression within cells of the innate immune system and herein we aim to further elucidate TAAR1 function in both peripherally-derived and CNS-resident macrophages. The selective TAAR1 agonist RO5256390 was used in combination with common damage associated molecular patterns (ATP and ADP) to observe the effect of TAAR1 agonism on modulating cytokine secretion and metabolic profiles. In mouse bone-marrow derived macrophages, TAAR1 agonism inhibited TNF secretion following ATP stimulation, which appeared to be downstream of an associated pro-inflammatory shift in metabolic profile and transcriptional regulation of TNF synthesis. In contrast, TAAR1 agonism had no effect on ADP-induced TNF and IL-6 secretion in mouse microglia in either the presence or absence of astrocytes. In summary, we report a novel interaction between TAAR1 and purinergic signaling in peripherally-derived, but not CNS-resident, macrophages. These findings provide the first evidence of trace aminergic and purinergic crosstalk, and support the potential for TAAR1 as a novel therapeutic target in inflammatory disorders.

Use of the head-twitch response to investigate the structure-activity relationships of 4-thio-substituted 2,5-dimethoxyphenylalkylamines.

RATIONALE: 4-Thio-substituted phenylalkylamines such as 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and 2,5-dimethoxy-4-n-propylthiophenethylamine (2C-T-7) produce psychedelic effects in humans and have been distributed as recreational drugs. OBJECTIVES: The present studies were conducted to examine the structure-activity relationships (SAR) of a series of 4-thio-substituted phenylalkylamines using the head twitch response (HTR), a 5-HT2A receptor-mediated behavior induced by psychedelic drugs in mice. The HTR is commonly used as a behavioral proxy in rodents for human psychedelic effects and can be used to discriminate hallucinogenic and non-hallucinogenic 5-HT2A agonists. METHODS: HTR dose-response studies with twelve different 4-thio-substituted phenylalkylamines were conducted in male C57BL/6 J mice. To detect the HTR, head movement was recorded electronically using a magnetometer coil and then head twitches were identified in the recordings using a validated method based on artificial intelligence. RESULTS: 2C-T, the parent compound of this series, had relatively low potency in the HTR paradigm, but adding an α-methyl group increased potency fivefold. Potency was also increased when the 4-methylthio group was extended by one to three methylene units. Fluorination of the 4-position alkylthio chain, however, was detrimental for activity, as was the presence of a 4-allylthio substituent versus a propylthio group. 2C-T analogs containing a 4-benzylthio group showed little or no effect in the HTR paradigm, which is consistent with evidence that bulky 4-substituents can dampen agonist efficacy at the 5-HT2A receptor. Binding and functional studies confirmed that the compounds have nanomolar affinity for 5-HT2 receptor subtypes and act as partial agonists at 5-HT2A. CONCLUSIONS: In general, there were close parallels between the HTR data and the known SAR governing activity of phenylalkylamines at the 5-HT2A receptor. These findings further support the classification of 2C-T compounds as psychedelic drugs.

Effects of the α2-adrenergic agonist clonidine on the pharmacodynamics and pharmacokinetics of 3,4-methylenedioxymethamphetamine in healthy volunteers.

The mechanism of action of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) involves the carrier-mediated and potentially vesicular release of monoamines. We assessed the effects of the sympatholytic α2-adrenergic receptor agonist clonidine (150 µg p.o.), which inhibits the neuronal vesicular release of norepinephrine, on the cardiovascular and psychotropic response to MDMA (125 mg p.o.) in 16 healthy subjects. The study used a randomized, double-blind, placebo-controlled crossover design with four experimental sessions. The administration of clonidine 1 h before MDMA reduced the MDMA-induced increases in plasma norepinephrine concentrations and blood pressure but only to the extent that clonidine lowered norepinephrine levels and blood pressure compared with placebo. Thus, no interaction was found between the cardiovascular effects of the two drugs. Clonidine did not affect the psychotropic effects or pharmacokinetics of MDMA. The lack of an interaction of the effects of clonidine and MDMA indicates that vesicular release of norepinephrine, which is inhibited by clonidine, does not critically contribute to the effects of MDMA in humans. Although clonidine may be used in the treatment of stimulant-induced hypertensive reactions, the present findings do not support a role for α2-adrenergic receptor agonists in the prevention of psychostimulant dependence.

Optimisation of imidazole compounds as selective TAAR1 agonists: discovery of RO5073012.

A series of imidazole compounds has been identified which affords potent and selective partial and full agonists of the TAAR1 receptor. Starting from 2-benzyl-imidazoline screening hits, a series of structurally related 2-benzyl- and 4-benzyl-imidazoles was investigated first, but it proved highly challenging to obtain compounds having sufficient selectivity against the adrenergic alpha 2 receptor. This issue could be successfully addressed by modification of the linker region and SAR exploration led to the discovery of highly selective isopropyl-substituted 4-aminomethyl-imidazole compounds. The work culminated in the identification of the selective TAAR1 partial agonist RO5073012 (4-chlorophenyl)-(1H-imidazol-4-ylmethyl)-isopropyl-amine, 24), which has a good pharmacokinetic profile after oral administration in rodents. RO5073012 has been found to be active in a behavioural rat model which is considered indicative for schizophrenia.

The selective antagonist EPPTB reveals TAAR1-mediated regulatory mechanisms in dopaminergic neurons of the mesolimbic system.

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) that is nonselectively activated by endogenous metabolites of amino acids. TAAR1 is considered a promising drug target for the treatment of psychiatric and neurodegenerative disorders. However, no selective ligand to identify TAAR1-specific signaling mechanisms is available yet. Here we report a selective TAAR1 antagonist, EPPTB, and characterize its physiological effects at dopamine (DA) neurons of the ventral tegmental area (VTA). We show that EPPTB prevents the reduction of the firing frequency of DA neurons induced by p-tyramine (p-tyr), a nonselective TAAR1 agonist. When applied alone, EPPTB increases the firing frequency of DA neurons, suggesting that TAAR1 either exhibits constitutive activity or is tonically activated by ambient levels of endogenous agonist(s). We further show that EPPTB blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current. When applied alone, EPPTB induces an apparent inward current, suggesting the closure of tonically activated K(+) channels. Importantly, these EPPTB effects were absent in Taar1 knockout mice, ruling out off-target effects. We additionally found that both the acute application of EPPTB and the constitutive genetic lack of TAAR1 increase the potency of DA at D2 receptors in DA neurons. In summary, our data support that TAAR1 tonically activates inwardly rectifying K(+) channels, which reduces the basal firing frequency of DA neurons in the VTA. We hypothesize that the EPPTB-induced increase in the potency of DA at D2 receptors is part of a homeostatic feedback mechanism compensating for the lack of inhibitory TAAR1 tone.

Pharmacological characterization of 3,4-methylenedioxyamphetamine (MDA) analogs and two amphetamine-based compounds: N,α-DEPEA and DPIA.

3,4-methylenedioxyamphetamine (MDA) is a psychoactive compound chemically related to the entactogen MDMA. MDA shares some of the entactogenic effects of MDMA but also exerts stimulant effects and psychedelic properties at higher doses. Here, we examined the pharmacological properties of MDA analogs and related amphetamine-based compounds detected in street drug samples or in sport supplements. We examined the key pharmacological mechanisms including monoamine uptake inhibition and release using human embryonic kidney 293 cells stably transfected with the respective human transporters. Additionally, we assessed monoamine transporter and receptor binding and activation properties. MDA, its fluorinated analogs, as well as the α-ethyl containing BDB and the dimeric amphetamine DPIA inhibited NET with the greatest potency and preferentially inhibited 5-HT vs. dopamine uptake. The ß­methoxy MDA analog 3C-BOH and the amphetamine-based N,α-DEPEA inhibited NET and preferentially inhibited dopamine vs. 5-HT uptake. The test drugs mediated efflux of at least one monoamine with the exception of DPIA. Most compounds bound to 5-HT2A and 5-HT2C receptors (Ki ≤ 10 µM) and several substances activated the 5-HT2A and 5-HT2B receptor as partial or full agonists. Furthermore, several compounds interacted with adrenergic receptors and the trace amine-associated receptor 1 (TAAR1) in the micromolar range. The pharmacological profiles of some fluorinated and nonfluorinated MDA analogs resemble the profile of MDMA. In contrast, 3C-BOH and N,α-DEPEA displayed more pronounced dopaminergic activity similar to amphetamine. Pharmacokinetics and pharmacodynamics studies are necessary to better establish the risks and therapeutic potential of the tested drugs.

Selective antagonists of mouse trace amine-associated receptor 1 (mTAAR1): discovery of EPPTB (RO5212773).

High throughput screening of the Roche compound library identified benzanilides such as 1 and 2 as antagonists of TAAR1. Optimisation of this hit series led to the first selective TAAR1 antagonist (N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB (RO5212773, 9f) having IC(50) of 28 nM at mouse TAAR1.

Receptor Interaction Profiles of 4-Alkoxy-3,5-Dimethoxy-Phenethylamines (Mescaline Derivatives) and Related Amphetamines.

3,4,5-Trimethoxyphenethylamine (mescaline) is a psychedelic alkaloid found in peyote cactus. Related 4-alkoxy-3,5-dimethoxy-substituted phenethylamines (scalines) and amphetamines (3C-scalines) are reported to induce similarly potent psychedelic effects and are therefore potential novel therapeutics for psychedelic-assisted therapy. Herein, several pharmacologically uninvestigated scalines and 3C-scalines were examined at key monoamine targets in vitro. Binding affinity at human serotonergic 5-HT1A, 5-HT2A, and 5-HT2C, adrenergic α1A and α2A, and dopaminergic D2 receptors, rat and mouse trace amine-associated receptor 1 (TAAR1), and human monoamine transporters were assessed using target specific transfected cells. Furthermore, activation of human 5-HT2A and 5-HT2B receptors, and TAAR1 was examined. Generally, scalines and 3C-scalines bound with weak to moderately high affinity to the 5-HT2A receptor (K i = 150-12,000 nM). 3C-scalines showed a marginal preference for the 5-HT2A vs the 5-HT2C and 5-HT1A receptors whereas no preference was observed for the scalines. Extending the 4-alkoxy substituent increased 5-HT2A and 5-HT2C receptors binding affinities, and enhanced activation potency and efficacy at the 5-HT2A but not at the 5-HT2B receptor. Introduction of fluorinated 4-alkoxy substituents generally increased 5-HT2A and 5-HT2C receptors binding affinities and increased the activation potency and efficacy at the 5-HT2A and 5-HT2B receptors. Overall, no potent affinity was observed at non-serotonergic targets. As observed for other psychedelics, scalines and 3C-scalines interacted with the 5-HT2A and 5-HT2C receptors and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline.