BACKGROUND AND PURPOSEDesigner b-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized. EXPERIMENTAL APPROACHWe determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity. KEY RESULTSMephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-b-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability. CONCLUSIONS AND IMPLICATIONSCathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction. AbbreviationsBBB, blood-brain barrier; DA, dopamine; DAT, dopamine transporter; MBDB, 3,4-methlyenedioxyphenyl-N-methyl-2-

The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and β-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 anxiolyticand antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.drug discovery | serotonin | depression | schizophrenia | anxiety

Schizophrenia is a chronic, severe and highly complex mental illness. Current treatments manage the positive symptoms, yet have minimal effects on the negative and cognitive symptoms, two prominent features of the disease with critical impact on the long-term morbidity. In addition, antipsychotic treatments trigger serious side effects that precipitate treatment discontinuation. Here, we show that activation of the trace amine-associated receptor 1 (TAAR1), a modulator of monoaminergic neurotransmission, represents a novel therapeutic option. In rodents, activation of TAAR1 by two novel and pharmacologically distinct compounds, the full agonist RO5256390 and the partial agonist RO5263397, blocks psychostimulant-induced hyperactivity and produces a brain activation pattern reminiscent of the antipsychotic drug olanzapine, suggesting antipsychotic-like properties. TAAR1 agonists do not induce catalepsy or weight gain; RO5263397 even reduced haloperidol-induced catalepsy and prevented olanzapine from increasing body weight and fat accumulation. Finally, TAAR1 activation promotes vigilance in rats and shows pro-cognitive and antidepressant-like properties in rodent and primate models. These data suggest that TAAR1 agonists may provide a novel and differentiated treatment of schizophrenia as compared with current medication standards: TAAR1 agonists may improve not only the positive symptoms but also the negative symptoms and cognitive deficits, without causing adverse effects such as motor impairments or weight gain.

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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, b-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 (EC 50 , 5 mM) and showed full or partial (E max , 80%) agonist properties. Cathinone derivatives, including mephedrone and methylenedioxypyrovalerone, exhibited weak (EC 50 5 5-10 mM) to negligible (EC 50 . 10 mM) 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.

The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]‐(2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine ([3H]‐DCG IV), was characterized in rat mGlu2 receptor‐transfected CHO cell membranes. [3H]‐DCG IV binding was pH‐dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg−1 protein. Binding was not sensitive to Na+‐dependent glutamate uptake blockers or Cl−‐dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N‐methyl‐D‐aspartic acid (NMDA), (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]‐DCG IV binding. Of the compounds observed to inhibit [3H]‐DCG IV binding, the most potent were the recently described selective group II agonist, (+)‐2‐aminobicyclo‐[3.1.0]hexane‐2,6‐dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2‐amino‐2‐(2‐carboxycyclopropan‐1‐yl)‐3‐(dibenzopyran‐4‐yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G‐protein‐coupled receptor, guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) inhibited [3H]‐DCG IV binding in a concentration‐dependent manner, with an IC50 value of 12 nM. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]‐DCG IV binding and potencies obtained for agonist activity in a GTPγ35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)‐2‐amino‐3‐phosphonopropionic acid (L‐AP3), L(+)‐2‐amino‐5‐phosphonopentanoic acid (L‐AP5), 3‐((RS)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (R‐CPP), N‐acetyl‐L‐aspartyl‐L‐glutamic acid (NAAG) and (RS)‐α‐methylserine‐O‐phosphate (MSOP). British Journal of Pharmacology (1998) 123, 497–504; doi:

Abstract:We have investigated the binding properties of [ 3 H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K D values of 27 Ϯ 4 and 81 Ϯ 22 nM for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S)-4-Carboxyphenylglycine, (S)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [ 3 H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM [ 3 H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 M kainate, revealing the distribution of mGlu1ϩ5 receptors. The latter were almost completely inhibited by the group I agonist (S)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively. Key Words: [ 3 H]Quisqualate binding-Metabotropic glutamate 1a and 5a receptors-Radioautography. J. Neurochem. 75, 2590 -2601 (2000).Eight G protein-coupled metabotropic glutamate (mGlu) receptors have been cloned to date (for review, see Pin et al., 1999). On the basis of their sequence similarities, signal transduction, and agonist rank order of potency, these receptors have been subdivided into three groups. Group I mGlu receptors, for which several splice variants have been identified , are activated by the weak group I-selective agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-3-hydroxyphenylglycine (3HPG) and the potent nonselective agonist quisqualate (Aramori and Nakanishi, 1992). This group comprises mGlu1, which is selectively blocked noncompetitively by 7-(hydroxyimino)cyclopropa [b]chromen1a-carboxylate ethyl ester (CPCCOEt) (Litschig et al., 1999), and mGlu5, which is selectively blocked, noncompetitively, with high potency by methylphenylethynylpyridine . Although precise physiological roles cannot yet be assigned to mGlu1 and mGlu5 receptors (potent, selective agonists/antagonists with good brain penetration are not yet widely available), group I receptors have, nevertheless, been implied in psychiatric and neurological diseases (see Bordi and Ugolini, 1999).

acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC 50 of 0.2~tMfor the high-affinity component. The binding was also affected by GTPyS, reducing agents, and Odd2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre-and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPyS show that [ 3H]DCGIV binding is probably to group II metabotropic glutamate receptors in rat brain. Key Words: (25,2'R,3'R)-2-(2 ',3 '-[3H] Dicarboxycyclopropyl) glycine-mGIu2/3 receptor-Binding -Radioautography.