β adrenergic receptor

SR59230A (100 nM-50 μM; 24 hours) decrease cell viability in a dose-dependent manner in Neuro-2A, BE(2)C and SK-N-BE(2) NB cell lines[3].

Hyperthermia caused by MDMA (20 mg/kg) develops gradually and reaches a maximum of 1.8°C 130 minutes after injection. SR59230A (0.5 mg/kg) causes the gradually escalating hyperthermia in response to MDMA to be slightly but significantly attenuated. SR59230A (5 mg/kg) indicates that MDMA causes a notable and early hypothermic response[4].

The SS-enantiomer 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahy dronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A) is proposed to be the first beta 3-adrenergic receptor antagonist. The present work shows that SR 59230A, unlike its inactive RR-enantiomer (SR 59483), antagonized a typical beta 3-adrenergic response in vitro, i.e., SR 58611A, the ethyl-[(7s)-7-[[(2R)-2-(3- chlorophenyl)-2-hydroxethyl]amino]-5,6,7,8-tetrahydronaphth- 2- yl]oxyacetate hydrochloride- or (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177)-stimulated synthesis of cAMP in rat brown adipose tissue membranes, with pKB values of 8.87 +/- 0.12 and 8.20 +/- 0.15 [1].

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Competitive radioligand binding [4]
Competition binding assays were carried out in duplicate in 5 mL polypropylene test tubes. Membrane aliquots of 100 µL were incubated with 100 µL of [3H]-prazosin (2 nmol·L−1; Specific activity: 85 Cinmol−1), and 100 µL of unlabelled test ligand (concentrations from 1 nmol·L−1 to 0.1 mmol·L−1), incubation buffer (vehicle) or phentolamine (10 µmol·L−1). Assays were performed at 25oC for 30 min. Following the 30 min incubation period, bound and free radioligand were separated by vacuum filtration. The assays were terminated by the addition of 5 mL ice-cold wash buffer (Tris-HCl 50 mmol·L−1, EDTA 5 mmol·L−1: pH 7.4 at 4oC) to all tubes. This was followed by rapid filtration through Whatman GF/C glass fibre filters using a Brandell Call Harvester. Filters and tubes were then washed four times with 5 mL of ice-cold wash buffer. Each filter was placed in a standard polypropylene scintillation vial and 5 mL of organic liquid scintillation medium was added to each vial. The vials were left overnight before being counted on a LKB 1214 Rack Beta counter.

Cell Line: Three different neuroblastoma (NB) cell lines, one murine (Neuro-2A) and two human (SK-N-BE(2), BE(2)C)
Concentration: 100 nM, 1 μM, 5 μM, 10 μM, and 50 μM
Incubation Time: 24 hours
Result: Reduced cell viability in a dose-dependent manner, with significant effect at a concentration limit over 1 µM for Neuro-2A cells and 5 µM for SK-N-BE(2) and BE(2)C).

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MTT assay [3]
Viability of tumor cells was evaluated using an MTT assay. NB cells were treated for 24 h with different concentration of SR59230A and then maintained in MTT for 1 h at 37 °C before lysis with an equal volume of DMSO. The absorbance of the solubilized dye was evaluated at 570 nm using a spectrophotometer.

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Neurosphere assay[3]
For neurosphere formation assay, 24-well plates were coated with 1,2% of Poly(2-hydroxyethyl methacrylate) diluted in 95% ethanol. Then, cells were plated (5.000/well) in Neurosphere basic medium composed of DMEM:F12 supplemented with 2% B27, 1% N2, 20 ng/ml FGF and 20 ng/ml EGF. After 24 h, cells were treated with 1 μM SR59230A and 1 μM BRL37344 alone, or in combination with 1 μM ABC294640 and 10 μM CYM5520. Once formed, spheres were disrupted and cells re-plated for a second passage (P2). After 7 days, neurosphere were counted and the diameter size measured using the ImageJ software (National Institutes of Health, U.S.). Neurosphere were then disrupted and stained for a flow cytometry analysis.

Male C-57BL6J wild-type mice (22-35 g)
0.5 or 5 mg/kg
Injected s.c.; administered 30 min prior to the injection s.c. of MDMA (20 mg/kg).

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Tumor syngeneic model [3]
Female NCI A/JCr mice 4-weeks-old were used. Neuro-2A cells were subcutaneously implanted in A/J recipient mice by injecting 1 × 106 cells in 100 µl of PBS in the right flank. When Neuro-2A cells formed a palpable tumor (about 6 days), treatments started. The treatments were administrated twice a day for SR59230A and Vehicle, and once a day for ABC294640 and CYM5520. SR59230A was delivered at 10 mg/kg of physiological solution via intraperitoneal (i.p.); ABC294640 was delivered at 30 mg/kg in 0,375% of Polysorbate 80 in PBS via per os (p.o); CYM5520 was delivered at 5 mg/kg in 3.6% DMSO in PBS via i.p. Tumor growth rate was evaluated by measuring tumor mass with a caliber, and tumor mass volume calculated as Volume = [(length × width)2/2]. Mice were sacrificed after 8 days of treatment.

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Animals were injected s.c. with the β3-adrenoceptor antagonist SR59230A (0.5 or 5 mg·kg−1) or SR59230A (5 mg·kg−1) plus prazosin (0.1 mg·kg−1). Antagonists or vehicle were administered 30 min prior to the injection s.c. of vehicle (1 mL·kg−1) or MDMA (20 mg·kg−1).[4]

[1]. Functional studies of the first selective beta 3-adrenergic receptor antagonist SR 59230A in rat brown adipocytes.Mol Pharmacol. 1996 Jan;49(1):7-14.

[2]. Involvement of β3-adrenergic receptors in the control of food intake in rats.Braz J Med Biol Res. 2011 Nov;44(11):1141-7.

[3]. β3-adrenoreceptor blockade reduces tumor growth and increases neuronal differentiation in neuroblastoma via SK2/S1P2 modulation.Oncogene. 2020 Jan;39(2):368-384.

[4]. Role of alpha 1- and beta 3-adrenoceptors in the modulation by SR59230A of the effects of MDMA on body temperature in the mouse. Br J Pharmacol. 2009 Sep;158(1):259-66.

SS-enantiomer 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphthyl-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) is considered the first β3-adrenergic receptor antagonist. This study demonstrates that SR 59230A, unlike the inactive RR enantiomer (SR 59483), antagonizes in vitro the typical β-adrenergic response, namely, the synthesis of cAMP in rat brown adipose tissue membrane stimulated by SR 58611A ethyl-[(7S)-7-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-5,6,7,8-tetrahydronaphthyl-2-yl]oxyacetate hydrochloride-or (-)-4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one (CGP 12177), with pKB values of 8.87 +/- 0.12 and 8.20 +/- 0.15, respectively. Furthermore, SR 59230A did not antagonize the accumulation of cAMP in rat interscapular brown adipose tissue induced by fosclerin. Unlike the selective β1 and β2 adrenergic receptor antagonists (+/-)[2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol and erythro-(+/-)-1-(7-methylindan-4-oxy)-3-isopropylaminobutyric acid-2-ol hydrochloride, SR 59230A does not antagonize cAMP production induced by (-)-isoproterenol or norepinephrine (NE) in rat brain regions rich in β1 or β2 adrenergic receptors, such as the frontal cortex and cerebellum. Furthermore, in proliferating brown adipocytes, the β1-adrenergic receptor is the only β-adrenergic subtype coupled with cAMP production; SR 59230A does not affect NE-induced cAMP production, while CGP 12177 does. In fused brown adipocytes, the β3-adrenergic receptor is a functional β-adrenergic subtype coupled with adenylate cyclase; SR 59230A antagonizes NE-induced cAMP accumulation and glycerol release but does not affect baseline levels; while CGP 12177 itself stimulates cAMP accumulation and glycerol release but does not alter the NE-induced increase in these two parameters. Finally, SR 59230A antagonizes, in a concentration-dependent manner, the synthesis of norepinephrine (NE)-stimulated uncoupling protein genes in fused brown adipocytes, which is primarily attributed to selective stimulation of the β3-adrenergic receptor. These results indicate that this novel selective β3-adrenergic receptor antagonist can greatly enhance the functional characterization of β3-adrenergic receptors. [1]

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This study examined changes in food intake in rats (adult male Wistar rats, 200–350 g, 6 rats per group) after intraventricular (icv) injection of a selective β3-adrenergic receptor agonist (BRL37344, 2 and 20 nmol) or antagonist (SR59230A, 10 and 50 nmol) after fasting for 24 hours. To determine the selectivity of BRL37344 in terms of food intake and the selectivity of SR59230A in terms of risk assessment (RA) behavior, animals were pretreated with intraventricular injection of saline (SAL) or SR59230A (50 nmol) followed by BRL37344 (20 nmol) or SAL. The highest dose of BRL37344 (N = 7) reduced food intake 1 hour after administration (6.4 ± 0.5 g in the SAL treatment group and 4.2 ± 0.8 g in the drug treatment group). Although neither dose of SR59230A affected food intake (5.1 ± 1.1 g in the 10 nmol group and 6.0 ± 1.8 g in the 50 nmol group), this treatment reduced the frequency of recurrent respiratory arrest (RA) (number of times within 30 minutes) (4 ± 2 in the saline group, 1 ± 1 in the 10 nmol SR59230A group, and 0.5 ± 1 in the 50 nmol group), RA frequency being a behavioral indicator associated with anxiety. SR59230A pretreatment (7.0 ± 0.5 g) eliminated the food intake reduction induced by BRL37344 (3.6 ± 0.9 g), while BRL37344 inhibited the RA frequency reduction induced by SR59230A. These results suggest that the reduced food intake caused by BRL37344 is selectively mediated by β3-adrenergic receptors in the central nervous system. Furthermore, they propose that these receptors are involved in the regulation of anxiety. [2] Neuroblastoma (NB) is the most common type of extracranial solid tumor in children. It exhibits strong clinical heterogeneity, particularly in presentation at diagnosis and treatment response, which often depends on the differentiation/steminess of cancer cells. Elevated levels of catecholamines in the blood and urine are common in NB patients, suggesting that understanding the adrenergic system is crucial for a better understanding of this cancer. β3-adrenergic receptors (β3-AR) are the most recently discovered members of the adrenergic receptor family and are associated with a variety of oncological diseases, such as melanoma. Multiple studies have shown that dysregulation of the metabolism and signaling of the bioactive lipid sphingosine-1-phosphate (S1P) is associated with a variety of pathological diseases, including cancer. However, whether S1P is crucial for the progression and aggressiveness of NB remains under investigation. The experimental evidence presented in this article demonstrates that β3-adrenergic receptors (β3-AR) are expressed in both human specimens and cell lines of neuroblastoma (NB) and play a crucial role in NB cell proliferation activation and stemness/differentiation regulation through functional crosstalk with the sphingosine kinase 2 (SK2)/sphingosine-1P receptor 2 (S1P2) axis. The specific antagonistic effect of SR59230A on β3-AR, by specifically blocking the SK2/S1P2 signaling pathway, inhibits NB cell growth and tumor progression both in vivo and in vitro, thereby promoting the transition of cells from a stem state to a differentiated state. [3]

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Background and Objective: We investigated the effects of the β(3)-adrenergic receptor antagonist 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthyl]amino]-(2S)-2-propanol hydrochloride (SR59230A) on MDMA-induced hyperthermia in conscious mice, and whether α(1)-adrenergic receptor antagonism was involved. Methods: Under anesthesia, temperature probes were implanted in mice and allowed to recover for 2 weeks. MDMA (20 mg/kg) was subcutaneously injected 30 minutes after the injection of the excipient or test antagonist, and its effect on body temperature was monitored by telemetry. Main Results: Following the injection of the excipient, MDMA induced a slowly developing hyperthermia, reaching a maximum increase of 1.8°C at 130 minutes post-injection. Low concentrations of SR59230A (0.5 mg/kg) slightly but significantly attenuated the slow-developing hyperthermia induced by MDMA. High concentrations of SR59230A (5 mg/kg) caused a significant and pronounced early hypothermia response to MDMA, an effect similar to that of the α1-adrenergic receptor antagonist prazosin. Functional and ligand binding studies revealed the effect of SR59230A on α(1)-adrenergic receptors. Conclusions and significance: High concentrations of 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthyl]amino]-(2S)-2-propanol hydrochloride regulate the hyperthermic effect of MDMA in mice in two ways: first, by blocking the early α(1)-adrenergic receptor-mediated component, resulting in hypothermia; second, by slightly weakening the late hyperthermic component, which may be mediated by β(3)-adrenergic receptors (this phenomenon can be observed even at low concentrations of SR59230A). Therefore, the main mechanism by which SR59230A regulates the thermogenic effect of MDMA is by antagonizing α(1)-adrenergic receptors. [4]

C23H29NO6

415.4795

415.199

C, 66.49; H, 7.04; N, 3.37; O, 23.10

1932675-95-0

SR59230A;174689-39-5; (2R)-SR59230A; 1932675-95-0; SR59230A hydrochloride; 1135278-41-9; 174689-38-4

White to off-white solid powder

CCC1=CC=CC=C1OC[C@@H](CN[C@H]2CCCC3=CC=CC=C23)O.C(=O)(C(=O)O)O

XTBQNQMNFXNGLR-VDWUQFQWSA-N

InChI=1S/C21H27NO2.C2H2O4/c1-2-16-8-4-6-13-21(16)24-15-18(23)14-22-20-12-7-10-17-9-3-5-11-19(17)20;3-1(4)2(5)6/h3-6,8-9,11,13,18,20,22-23H,2,7,10,12,14-15H2,1H3;(H,3,4)(H,5,6)/t18-,20+;/m1./s1

(2R)-1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydronaphthalen-1-yl]amino]propan-2-ol;oxalic acid

(2R)-SR59230A; (2R)-SR-59230A; 1932675-95-0;

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: 31.25 mg/mL (75.21 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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