TAAR1 (EC50 = 17~35 nM)

RO5263397 Increases cAMP Mediated BRET Signal in a Dose-Dependent Manner in HEK-293 Cells [3]
To study the pharmacology of RO5263397, we first used an in vitro approach. Since TAAR1 is a GPCR coupled to stimulatory G protein, its activation evokes the production of cAMP. To monitor in real time TAAR1 activation, we transfected, in addition of mouse and human TAAR1, a cAMP BRET biosensor that changes the emission of the light according to the cAMP fluctuation (see section “Materials and Methods”). We then tested a range of concentrations of RO5263397 both in cells transfected with mouse or human TAAR1. As shown in Figure 1, RO5263397 increases cAMP-mediated BRET signal in a concentration dependent manner (Figures 1A,B), with no desensitization over the observation period. RO5263397 did not provoke a BRET signal in cells not transfected with TAAR1 (data not shown). Comparison with a maximal concentration (10 μM) of β-phenylethylamine (PEA), a known TAAR1 full agonist indicated, RO5263397 behaves as a full agonist at the mTAAR1 (EC50: 0.12 nM and Emax: 100%) and partial agonist at the hTAAR1 (EC50: 47 nM and Emax: 82%) as shown in Figures 1C,D, respectively. The data also indicate that RO5263397 shows species related difference with 392-fold higher potency at the mTAAR1 compared to hTAAR1.

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The TAAR1 Antagonist EPPTB Inhibits RO5263397 Effect [3]
We further verified that the cAMP-mediated BRET signal seen with RO5263397 was due to activation of TAAR1 by pretreating the cells with the selective TAAR1 antagonist EPPTB. For these experiments, we used cells with mTAAR1 since EPPTB shows species selectivity mouse over human and rat. Pretreatment with EPPTB (1 μM) partially blocked the response to a maximal concentration (0.1 μM) of RO5263397 (Figure 1E). EPPTB alone did not affect BRET signal in mTAAR1 transfected cells. Furthermore, it did not affect the cAMP-BRET signal provoked by the adenylyl cyclase activator, forskolin (20μM) as shown in Figure 1F. These data indicate that mTAAR1 activation is necessary to observe the antagonist properties of EPPTB.

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RO5263397 Mediated ERK and CREB Phosphorylation in HEK293 Cells [3]
Apart from cAMP induction, TAAR1 signaling could involve other proteins, such as β-arrestin2 and AKT/GSK3 pathways. However, cAMP and also β-arrestin2 are linked to downstream effectors such as CREB and ERK, and TAAR1 seems to modulate the activity of these two proteins. In order to verify this hypothesis, we used HEK293 cells expressing mTAAR1 or hTAAR1. First, we treated the cells expressing mTAAR1 with RO5263397 and we performed a time course at a concentration of the compound that was the first to reach the efficacy of 100%, according to our previous BRET experiments (10 nM for mTAAR1 and 100 nM for hTAAR1). As shown in Figures 2A,B, RO5263397 was able to induce the phosphorylation of ERK2 and CREB, with the maximum effect at 5 min for pERK and at 15 min for pCREB. After measuring the optimal time for the induction of pERK and pCREB, we performed a concentration response curve using different concentrations of RO5263397 (Figures 2C,D). We then repeated the same time course experiment in cells expressing hTAAR1, and obtained similar results (Figure 3).

At 0.3 and 1 mg/kg, RO5263397 (0.1-1.0 mg/kg; side wall; during the mesophotopic phase (ZT6) medication) prolongs wake time [2]. RO5263397 (0.3 and 1.0 mg/kg; po) considerably lengthens the light period in the OE model by 5–6 hours while reducing WT mice RO5263397 (0.3 and 1.0 mg/kg; sidewall). NREM sleep is reduced for four to six hours in all models, but REM sleep.

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Trace amine-associated receptor 1 (TAAR1) agonists have been shown to have procognitive, antipsychotic-like, anxiolytic, weight-reducing, glucose-lowering, and wake-promoting activities. We used Taar1 knockout (KO) and overexpressing (OE) mice and TAAR1 agonists to elucidate the role of TAAR1 in sleep/wake. EEG, EMG, body temperature (Tb), and locomotor activity (LMA) were recorded in Taar1 KO, OE, and WT mice. Following a 24 h recording to characterize basal sleep/wake parameters, mice were sleep deprived (SD) for 6 h. In another experiment, mice were given three doses of the TAAR1 partial agonist RO5263397, caffeine, or vehicle p.o. Baseline wakefulness was modestly increased in OE compared with WT mice. Baseline theta (4.5-9 Hz) and low gamma (30-60 Hz) activity was elevated in KO compared with OE mice in NREM and REM sleep. Following SD, both KO and OE mice exhibited a homeostatic sleep rebound. In WT mice, RO5263397 increased waking and reduced NREM and REM sleep, decreased gamma power during wake and NREM, and decreased Tb without affecting LMA; these effects were absent in KO mice and potentiated in OE mice. In contrast, caffeine increased wake time, NREM gamma power, and LMA in all strains compared with vehicle; this effect was attenuated in KO and potentiated in OE mice. TAAR1 overexpression modestly increases wakefulness, whereas TAAR1 partial agonism increases wakefulness and also reduces NREM and also REM sleep. These results indicate a modulatory role for TAAR1 in sleep/wake and cortical activity and suggest TAAR1 as a novel target for wake-promoting therapeutics.[2]

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RO5263397 Inhibits Spontaneous Hyperactivity in DAT-KO Mice [3]
Trace amine-associated receptor 1 agonists can reduce dopaminergic hyperactivation, for example after cocaine treatment (Revel et al., 2011). We tested the ability of RO5263397 to reduce the hyperactivity spontaneously present in DAT-KO mice. RO5263397 dose-dependently suppressed the locomotor activity in DAT-KO mice at all doses tested (0.03, 0.1, and 0.3 mg/kg, i.p.) with the most pronounced action noted at the dose 0.1 mg/kg (Figure 4A). In WT mice, RO5263397 also demonstrated significant locomotor-inhibiting action (Figure 4B).

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Antidepressant-Like Effect of RO5263397 Involves AMPA and D1 Receptors [3]
Trace amine-associated receptor 1 selective agonists have multiple in vivo actions, affecting both the locomotor and emotional behaviors. However, while locomotor behavior is mostly mediated by the dopamine system, less is known about the neurobiological basis underpinning the TAAR1 effect on emotional behaviors. Thus, we tested RO5263397 in the FST to particularly examine the role of 5HT1A, AMPA or D1 receptors in its antidepressant-like activity in rats. RO5263397 significantly reduced immobility of rats at 1 and 10 mg/kg (p.o.), by 40 and 47%, respectively, compared to the vehicle group (Figure 5A). The lower dose of RO5263397 (0.1 mg/kg p.o.) had no significant effect on immobility time. RO5263397 significantly increased climbing behavior only at 10 mg/kg p.o., by 144%. At the lower doses 0.1 and 1 mg/kg RO5263397 increased climbing 26 and 62%, respectively although these effects were not significant. There was no effect on swimming activity at the doses examined. The RO5263397 provoked decrease in immobility time was similar in magnitude to fluoxetine (10 mg/kg, p.o.), although the latter drug also significantly increased swimming time (Figure 5B).
Then, to understand whether other neurotransmitters could be involved in the anti-depressant action of RO5263397, we treated rats with RO5263397 alone or in combination of antagonists of D1 receptor, 5-HT1A serotonin receptor and AMPA receptor. RO5263397 alone significantly reduced immobility time of rats at 1 mg/kg (p.o.), by 40% compared to the vehicle group (Figure 5C). When RO5263397 1 mg/kg was combined with D1 receptor antagonist SCH23390 (0.1 mg/kg, s.c.), immobility time was significantly increased, by 88% as compared to the effect of RO5263397 alone, 14% above the level of vehicle group. In combination with AMPA antagonist NBQX (10 mg/kg, s.c.), immobility time was also significantly increased, by 47% as compared to the effect of RO5263397 alone. 5-HT1A antagonist WAY100635 (1 mg/kg, s.c.) had no significant effect on RO5263397-induced decrease in immobility time. RO5263397 alone at the dose 1 mg/kg (p.o.) or in combination with D1, 5-HT1A or AMPA receptor antagonists did not have significant effect on climbing behavior. In this experiment, however, RO5263397 significantly increased swimming behavior which was attenuated by all three antagonists investigated.

Bioluminescence Resonance Energy Transfer (BRET) Measurement [3]
Bioluminescence resonance energy transfer experiments were performed as described previously (Espinoza et al., 2013). RO5263397 and EPPTB powder was dissolved in DMSO at the concentration of 10 mM and then diluted in PBS to the desired concentration. For time course experiments, the plate was read immediately after the addition of RO5263397 and for approximately 20 min. In order to calculate the EC50 values, a concentration response curve was performed using different concentration of the agonist. To evaluate the antagonistic effect of EPPTB, the antagonist was added 5 min before RO5263397. All the experiments were conducted in presence of the phosphodiesterase inhibitor 3-Isobutyl-1-methylxanthine at the final concentration of 200 μM. Readings were collected using a Tecan Infinite instrument that allows the sequential integration of the signals detected in the 465 to 505 nm and 515 to 555 nm windows using filters with the appropriate band pass and by using iControl software. The acceptor/donor ratio was calculated as previously described (Espinoza et al., 2013). We used an EPAC BRET biosensor to monitor cAMP levels. With this sensor an increase in cAMP is reflected in a decrease in the BRET ratio. Curve was fitted using a non-linear regression and one site specific binding with GraphPad Prism 5. Data are representative of four independent experiments and are expressed as means ± SEM.

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Assay for determining functional activity at the human adrenergic α2A receptor: [3]
Membrane Preparation: CHL cells stably expressing the adrenergic α2A receptor were maintained at 37 °C and 5% CO2 in DMEM high glucose medium containing fetal calf serum (5%, heat inactivated for 30 min at 56 °C) and 250 µg/ml geneticin. Cells were released from culture flasks using trypsin/ EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1’000 rpm for 5 min at 4 °C, frozen and stored at -80 °C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron at 14’000 rpm for 20 s. The homogenate was centrifuged at 48’000 x g for 30 min at 4 °C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14’000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80 °C.

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Wheatgerm agglutinin SPA beads assay: [3]
The radioligand [35S] GTPγS was used at a concentration of 0.5 nM final concentration. Nonspecific binding was defined as the amount of GTPγS bound in the presence of 10 µM final concentration of GTP (unlabeled ligand). Compounds were tested at a broad range of concentrations (30 pM to 30 µM) in duplicates. Norepinephrine was used as a reference for agonistic activity and RX821002 as an antagonist reference. A mix M was prepared containing GDP at 1.5 µM final concentration, membranes at 5 µg protein/well and wheatgerm agglutinin SPA beads at 1 mg/well. The test compounds (20 µl/well) were transferred into an OptiPlate. 30 µl/well of buffer containing 50 mM Tris, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA and 1 mM DTT were added. In order to determine the total binding 20 µl of buffer was used and for the nonspecific binding 20 µl of 3 GTP at 10 µM. For agonist testing 100 µl of mix M and 50 µl of [35S] GTPγS were added. The OptiPlate was then incubated for 30 min at RT under shaking at 350 rpm/min and centrifuged for 3 min at 3000 rpm. Radioactivity was counted using a TopCount Microplate Scintillation Counter. For antagonist testing 100 µl of mix M and 50 µl of [35S] GTPγS were added. The OptiPlate was then incubated for 5 min at RT under shaking at 350 rpm/min. 30 µl norepinephrine at 20 µM was added to the wells containing the compounds. The plate was then incubated for 25 min at RT under shaking at 350 rpm/min and centrifuged for 3 min at 3000 rpm. Radioactivity was counted using a TopCount Microplate Scintillation Counter. 50 µl of the 35S-GTPγS stock were counted in 5 ml of ReadySafe scintillation cocktail to determine the total counts added to the respective assays.

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Formation of Glutathione (GSH) adducts with selected compounds [3]
Microsomal suspensions were diluted in 100 mM potassium phosphate buffer, pH7.4 to make 2 mg/ml solutions. One portion of this was heat denatured at 95 °C for 10 minutes, whilst the other remained on ice. The denatured protein solutions were then cooled before use. Each incubation was made up using 220.5 µl of pretreated protein solution (see above, final concentration 1 mg/ml) and 4.5 µl substrate (final concentration 10 µM). This was warmed to 37 °C for 10 minutes before addition of 225 µl prewarmed glutathione solution in phosphate buffer (final concentration 5 mM). Incubations were stopped after 10 or 20 minutes incubation time (at 37 °C) by addition of 150 µl quench reagent (150:100:1 mix of 10% trichloroacetic acid : acetonitrile : 30% hydrogen peroxide). Samples were chilled on ice for 1 hour before centrifugation (20,000x g, 10 minutes). The supernatant was removed and 200 µl analysed by HPLC. Mass spectrometric analysis was performed on a LCQ mass spectrometer equipped with the Xcalibur 1.2 software package.

Antibodies and Western Blot Analyses [3]
The antiphospho-ERK1/2 (Thr-202/Tyr-204), anti-ERK, anti-phospho-CREB (Thr-34) and anti-CREB antibodies were used. To analyze effect of RO5263397 on TAAR1-mediated intracellular signaling events in HEK-293 cells, hTAAR1 or mTAAR1 was transiently expressed in the cells. After 24 of transfection, cells were treated with RO5263397 at concentration ranging from 0.01 to 100 nM (for concentration response experiment) or at the same concentration and then lysed at different time points (for time course experiments). Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. After 10 min of incubation on ice, lysates were centrifuged for 10 min at 13000 rpm and supernatants were collected for protein concentration assay. 25 μg of protein extract were separated on 10% SDS/PAGE and transferred on nitrocellulose membrane. All primary antibodies were incubated overnight at 4°C. Appropriate peroxidase-conjugate secondary antibodies and chemiluminescent reagents were used. For quantitative analysis, total proteins were used as loading controls for phosphoprotein signals. Results obtained with RO5263397 were normalized to respective vehicle controls.

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Biological assay section [1]
Results were obtained from at least three independent experiments. Experiments were run at least in duplicates. EC50 values are given as a mean in nM. The Emax value for the functional activity data at TAAR1 receptor describes the degree of functional activity compared to 100% for the natural ligand and full agonist phenethylamine. Compounds with Emax<85% are regarded as partial agonists. The functional selectivity ratio of hTAAR1 vs. hα2A is determined by dividing the hα2A EC50 or IC50 value by the hTAAR1 EC50 value.

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cAMP assay for determining functional activity at the human and rat TAAR1 receptor [3]
Recombinant HEK293 cells expressing human, mouse or rat TAAR1 were grown at 37 °C and 5% CO2 / 95% air in Falcon culture flasks in 30 ml culture medium. The cell culture medium contained DMEM high glucose, fetal calf serum (10%, non-dialysed, heat inactivated for 30 min at 56 °C), geneticin (375 µg/ml, Gibco), and penicillin/streptomycin (1%). Cells were harvested when 80 - 90% confluent. The culture medium was removed from the culture flasks and cells were washed once with 5 ml of PBS. After removing the PBS, 5 ml of trypsin/ EDTA solution were added for 5 min at 37 °C. Afterwards, 45 ml of culture medium was added to the 5 ml detached cell solution and the total of 50 ml was transferred into a Falcon tube. The tube was centrifuged at 900 rpm for 5 min at RT and the supernatant was removed. The cell pellet was resuspended in fresh culture medium and brought to 5 x 10 E5 cells per ml. Then the cells were plated in 96-well plates with a multipipette (100 µl/ well, 50’000 cells/well) and incubated for 20 h at 37 °C.

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Stimulation of the cells: [3]
The cell culture medium was removed, 100 µl PBS (AMIMED endotoxine free) was added and after 5 min under shaking at RT, PBS was removed and 90 µl PBS containing 1 mM IBMX was added. After shaking the cells for 2 min they were incubated for 10 min at 37 °C and 5% CO2 / 95% air. All compounds were tested at a broad range of concentrations (100 pM to 10 µM) in duplicates. Typically, 10 µl of a compound solution in PBS and 1 mM IBMX or 10 µl of a 0.3 mM β-phenylethylamine solution (as maximal response) or 10 µl of a 2% DMSO solution (as basal level) were then added and after 10 min shaking the cells 2 were incubated for 30 min at 37 °C. Afterwards, the solutions were removed and the cells were lysed with 150 µl of lysis buffer. The plates were then shaken for 30 min and stored at -20 °C.

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cAMP Assay (Millipore cAMP kit): [3]
In the 96-well rabbit anti-cAMP antibody coated plates, 50 µl of cAMP standards (8 standards from 1 pmol/µl to 0.0039 pmol/µl and one without cAMP) or 50 µl of samples from the cell plates were added. A standard curve was performed on each plate. 25 µl of diluted cAMP alkaline phosphatase conjugated tracer was added to all wells followed by 50 µl of diluted rabbit anti-cAMP antibody. After sealing, the plates were incubated for 30 min at RT under shaking followed by removal of the supernatant from each well with an automated plate washer and by washing 5 times using 1 x wash buffer. Then, 100 µl of diluted alkaline phosphatase substrate was added, the plates sealed and incubated for 30 min at RT under shaking. Finally, the plates were read for 1 s with a luminometer (1420 Multilabel counter).

Animal/Disease Models: Adult 4-5 month old male WT littermates (C57BL/6 background) [2]
Doses: 0.1, 0.3, 1 mg/kg Mode of
Route of Administration: po; almost completely suppressed for 6 hrs (hrs (hours)) [3]. Mid-light phase dosing (ZT6)
Experimental Results: increased wake time at 0.3 and 1 mg/kg. TAAR1 agonism. All mice received p.o. RO5263397 (0.1, 0.3, and 1 mg/kg), Caf (10 mg/kg), or vehicle (0.3% Tween-80) in the mid-light phase (ZT6) in balanced order with at least 3 days between treatments. Mice were acclimated to oral dosing with vehicle (Veh) for at least 3 days before data collection.[2]

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Locomotor Activity [3]
Effect of RO5263397 on spontaneous locomotor activity of DAT-KO or WT mice was tested as described previously (Revel et al., 2011). Briefly, DAT-KO or WT mice were placed in the locomotor activity chambers for 30 min and then were treated with either saline or RO5263397 at different doses and total distance traveled was measured by analyzing infrared beam interruptions for another 90 min.

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Forced Swim Test [3]
Male Spraque-Dawley rats (230–270 g) were used in the FST. The test was performed in a glass cylinder (46 cm × 20 cm), with tap water 25 cm deep and +24–25°C. Water was refreshed after each test and it was performed over two consecutive days. On the first day, the rats were forced to swim for a 15-min period and immediately after that the first dose of test compound or vehicle was administered (5 ml/kg p.o.). The second drug or vehicle administration was on the next day, 23 h after the first dosing. One hour after the second administration and 24 h after the first swimming session, the rats were subjected to a second 5-min swimming session and immobility, climbing and swimming time were registered. Rats were considered to show immobility when animals were inactive and displaying minor movements with one limb only; climbing behavior was registered when the rats were actively climbing at the walls of the cylinder; finally, time spent swimming (horizontal activity) was measured as the remaining time after immobility and climbing have been subtracted from total test time. After each FST session, the rats were dried with towel and allow rest under heating lamp. RO5263397 (0.1, 1.0, and 10 mg/kg) formulations were prepared in 5% Tween 80 in sterile H2O. Each formulation was freshly prepared each day of dosing. In experiments examining the effects of dopamine D1 (D1) receptor antagonist SCH23390 (0.1 mg/kg, s.c.), glutamate AMPA receptor antagonist NBQX (10 mg/kg, s.c.), and serotonin 5-HT1A antagonist WAY100635 (1 mg/kg, s.c.) the antagonists were administered 10 min prior to RO5263397. The dose selection for SCH23390, WAY100635, and NBQX was based on previous work with these agents (Jordan et al., 2005; Snigdha et al., 2011). Statistical analysis was done by using one-way ANOVA followed by Dunnett’s post hoc test.

Pharmacokinetic analyses in rat, mouse, and cynomolgous monkey revealed very favorable in vivo properties, which have already been reported elsewhere. [Mol. Psychiatry 2013, 18, 543–556]

[1]. Discovery and Characterization of 2-Aminooxazolines as Highly Potent, Selective, and Orally Active TAAR1 Agonists.ACS Med Chem Lett. 2015 Dec 30;7(2):192-7.

[2]. Trace Amine-Associated Receptor 1 Regulates Wakefulness and EEG Spectral Composition.Neuropsychopharmacology. 2017 May;42(6):1305-1314.

[3]. Biochemical and Functional Characterization of the Trace Amine-Associated Receptor 1 (TAAR1) Agonist RO5263397.Front Pharmacol. 2018 Jun 21;9:645.

2-Aminooxazolines were discovered as a novel structural class of TAAR1 ligands. Starting from a known adrenergic compound 1, structural modifications were made to obtain highly potent and selective TAAR1 ligands such as 12 (RO5166017), 18 (RO5256390), 36 (RO5203648), and 48 (RO5263397). These compounds exhibit drug-like physicochemical properties, have good oral bioavailability, and display in vivo activity in a variety of animal models relevant for psychiatric diseases and addiction. [1]

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Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor, which signals through elevating intracellular cAMP levels, and expressed in most vertebrates, including rodents and humans. In recent years, several lines of evidence indicated the role of TAAR1 in the regulation of dopaminergic system and its importance in physiological processes such as locomotion, control of emotional states and cognition. In our study, we used RO5263397, a selective TAAR1 agonist, as a tool and characterized its pharmacology in vitro in HEK293 cells and its effects in vivo in tests assessing potential antidepressant and antipsychotic actions. We found that RO5263397 not only increases cAMP levels at very low concentrations but also can induce the phosphorylation of ERK and CREB in a concentration- and time-dependent manner. Like other TAAR1 agonists, RO5263397 potently suppressed high dopamine-dependent hyperactivity in mice lacking the dopamine transporter. Moreover, RO5263397 produced a strong antidepressant-like effect in the forced swim test comparable to fluoxetine. Furthermore, the antidepressant-like activity was blocked by pretreatment with SCH23390 (dopamine D1 receptor antagonist) or NBQX (glutamate AMPA receptor antagonist) but only in part by WAY100635 (serotonin 5HT1A receptor antagonist). In conclusion, our study confirms some previous in vitro and in vivo findings in relation to the pharmacological effects of RO5263397 but more importantly provides new insight on intracellular signaling pathway and other neurotransmitter receptors modulated by TAAR1 receptor activation.[3]
Taken together, in this study we demonstrated that TAAR1 activation not only induce the increase of cAMP levels inside the cells, but also can induce the phosphorylation of two important signaling proteins, the MAP Kinase ERK and the transcription factor CREB. RO5263397 is highly effective in vivo in mice and rats demonstrating potential antipsychotic and antidepressant activity. Moreover, the antidepressant-like effect of the TAAR1 agonist RO5263397 relies on the engagement of other neurotransmitter systems, particularly involving the D1 receptor and the AMPA glutamate receptor.[3]

C10H11FN2O

194.205545663834

194.085

C, 44.96; H, 4.91; Cl, 26.54; F, 7.11; N, 10.49; O, 5.99

1357266-05-7

1357266-05-7;1357266-80-8

56835991

White to off-white solid powder

1.4

1

3

1

14

244

1

FC1=CC=CC(=C1C)[C@H]1COC(N)=N1

IOHOUWIYOVWGHV-SECBINFHSA-N

InChI=1S/C10H11FN2O/c1-6-7(3-2-4-8(6)11)9-5-14-10(12)13-9/h2-4,9H,5H2,1H3,(H2,12,13)/t9-/m1/s1

(4S)-4-(3-fluoro-2-methylphenyl)-4,5-dihydro-1,3-oxazol-2-amine

RO-5263397 HCl; RO 5263397 hydrochloride; RO5263397

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~260 mg/mL (~1338.76 mM)

Solubility in Formulation 1: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)