5-HT1B Receptor

Cho1p is not inhibited by SB-224289 hydrochloride, however it does have a particular toxin swelling ability. The hydrochloride of SB-224289 (100 μM–25 μM) consistently showed effectiveness in giving resistance against Pap-A. Additionally, SB-224289 hydrochloride inhibited the activity of babuamide in this in vitro experiment, but not that of other film-disrupting compounds. While SB-224289 hydrochloride can offer protection against TPap, it is unable to preserve wild-type cell scaffold KF connections. Compared to the nearly comparable 5-HT1B receptor, the human cloned 5-HT1B receptor shows a higher pKi of 8 for SB-224289 hydrochloride. -Over 80x axis is displayed by the HT1D receiver and a number of other receivers. Hydrochloride SB-224289 is a strong antagonist with a pEC50 of 7.9±0.1. The hydrochloride of SB-224289 shifts the 5-HT concentration curve parallel to the right, with a pA2 of 8.4±0.2. In guinea pig cerebral cortex slices, SB-224289 hydrochloride (100 nM and 1 μM) also markedly enhanced [3H]-5HT release [3].

SB-224289 hydrochloride (SB 224289) may either when used alone or in conjunction with cocaine, exacerbate anxiety-like behaviors. When cocaine is used, SB 224289 dramatically lowers locomotor activity. SB 224289 Compared to animals treated with a vehicle, animals treated in purple stayed considerably longer. [2]. SB 224289 is a strong antagonist that causes guinea pigs to become hypothermic when exposed to SK&F-99101; its bone ED50 is 3.6 mg/kg. Additionally, SB 224289 (4 mg/kg, bone) reversed the suppression of 5-HT release mediated by sumatriptan, suggesting that it is a strong in vivo interstitial 5-HT autoreceptor antagonist. Among guinea pigs, SB 224289 (2-16 mg/kg, bay) does not raise 5-HT in prefrontal potentials. Nonetheless, guinea pigs' dentate gyrus significantly increases 5-HT levels when exposed to SB 224289 (4 mg/kg, wall). [3].

Phosphatidylserine synthase assay[1]
This procedure was done as described in with minor alterations. Cultures were grown overnight and then diluted to approximately 0.1 OD600/ml in 1 L YPD, and were shaken at 30°C for 6 to 10 hours. Cells were harvested by centrifugation at 6,000 x g for 20 minutes. Pellets were then transferred to 50 ml conical tubes and washed with water and re-pelleted. Supernatant was removed and the wet weight of the samples was taken. Cell pellets were stored overnight at -80°C. The following day, a cold mixture of 0.1 M Tris-Cl pH 7.5, 5 mM β-mercaptoethanol (BME), 10% glycerol, and protease inhibitors 1.7 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin was added to the frozen pellets (1 ml/g [wet weight]) and allowed to thaw on ice. Cells were lysed using a French press (three passes at approximately 13,000 lb/in2). The homogenate was centrifuged at 4°C for 5 minutes at 3,000 rpm to clear unbroken cells and heavy material. Supernatant was then spun again at 27,000 x g for 10 minutes at 4°C. For some experiments, the resulting supernatant was then spun at 100,000 x g to collect the lower density membranes. Pellets were resuspended in 500 μl to 1 ml of 0.1 M Tris-Cl pH 7.5, 5 mM BME, 10% glycerol, and protease inhibitors. This mixture was aliquoted into microcentrifuge tubes and homogenized to break apart clumps, keeping on ice as much as possible. Total crude protein concentration was determined using a Bradford Assay. The optimal assay mixture contained 50 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 0.5 mM MnCl2, 0.1 mM CDP-DAG added as a suspension in 1% - 20% Triton X-100, and 0.4–0.5 mg protein in a total volume of 0.1 ml. SB-224289 and MG-624 were added to the reaction mixture at varying concentrations to monitor their ability to inhibit [3H]-PS production. The PS synthase assay was performed by monitoring the incorporation of 0.5 mM l-serine spiked with 5% [3H]-l-serine (or 0.02 μM) into the chloroform-soluble product at 37°C for a predetermined amount of time. The reaction was terminated by the addition of 1 ml chloroform: methanol (2:1). Following a low-speed spin, 800–1000 μl of the supernatant was removed to a fresh tube and washed with 200 μl 0.9% NaCl. Following a second low-speed spin, 400–500 μl of the organic phase was removed to a new tube and washed with 500 μl of chloroform: methanol: 0.9% NaCl (3:48:47). Following a third low-speed spin, 200–300 μl was transferred into scintillation vials. Tubes were dried under the chemical hood and 2.5 ml Cytoscint-ES liquid scintillation cocktail.was added to each tube and counted in a Packard TriCarb 2900TR Liquid Scintillation Analyzer.

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SB-224289 has a pKi of 8.1 at human cloned 5-HT1B receptors and displays ≥80 fold selectivity over the closely related 5-HT1D receptor and a range of other receptors. In [35S]GTPγS radioligand binding studies in CHO cells expressing human cloned 5-HT1B receptors, SB-224289 behaves as a potent inverse agonist with pEC50 = 7.9 ± 0.1 (5-HT, pEC50 = 7.8 ± 0.1, -GR127935, pEC50 = 8.1 ± 0.1). In antagonist studies in this system SB-224289 evoked a parallel rightward shift in the 5-HT concentration response curve with pA2 of 8.4 ± 0.2. At concentrations of 100 nM and 1 mM, SB-224289 significantly increased [3H]5-HT release in electrically stimulated guinea-pig brain cortex slices demonstrating that it is a terminal 5-HT autoreceptor antagonist in vitro[3].

Papuamide A resistance assay[1]
Strains were grown overnight in liquid YPD shaking at 30°C to saturation, and cultures were diluted to 2 x 104 cells/ml in YPD. Compounds of interest were diluted to twice the working concentration by serial dilution in a 96 well plate or by preparing separately and adding to the wells directly, in a volume of 37.5 μl of YPD. Then 37.5 μl of cells at 2 x 104 cells/ml in YPD were added. Plates were incubated at 37°C for 6 hours or 3 hours depending on the experiment, and then 75 μl of YPD containing depsipeptide (Pap-A at 8 μg/ml, VA at 6 μg/ml, KF at 30 μg/ml, or TPap-A at 12 μg/ml) were added to each well, diluting those concentrations by half. This addition was followed by a 37°C overnight incubation.
Cell survival was measured the next day by fluorescence intensity or optical density. For fluorescence intensity, Alamar Blue was added to the wells at a 1:10 dilution. Plates were allowed to incubate again at 37°C for 30 minutes to 2 hours until color change was apparent. Fluorescence was then read at excitation 550 nm and emission 590 nm. For optical density, plates were removed from overnight incubation and absorbance was read in a plate reader at a wavelength of 600 nm. All measurements were performed on a Cytation3 BioTek plate reader using Gen 5 software.

In an in vivo model of 5-HT1B receptor function, SK&F-99101-induced hypothermia in the guinea-pig, SB-224289 was a potent antagonist with an ED50 of 3.6 mg/kg p.o. In microdialysis studies in the frontal cortex of the freely moving guinea-pig, SB-224289 (4 mg/kg p.o.) reversed sumatriptan-induced inhibition of 5-HT release demonstrating that it is also a potent terminal 5-HT autoreceptor antagonist in vivo. In the guinea-pig frontal cortex SB-224289 alone (2–16 mg/ kg p.o.) did not increase 5-HT levels. In contrast, in the guinea-pig dentate gyrus, SB-224289 evoked a significant increase in levels of 5-HT at 4 mg/kg p.o. These results may reflect regional differences in terminal 5-HT autoreceptor tone[3].

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SB-224289 and cocaine experiment[2]
Day 1: Each animal was injected with sterile saline (1 ml/kg ip) immediately before it was placed in the start box.[2]

Day 2: Ninety minutes before each animal was tested, it received an ip injection of either 5 mg/kg SB 224289 in a vehicle of 10% Trappsol in sterile water or vehicle alone (total volume 1 ml/kg). This dosage of this drug has been shown to be effective as a pharmacological agent in previous studies. The rat was put back in its home cage until just before it was to be tested. Just prior to testing, each rat was injected with cocaine (15 mg/kg ip) dissolved in sterile saline (15 mg/ml) or with saline alone as a control.

Validation experiment
To validate the usefulness of this procedure as a measure of anxiety, a validation test was performed using diazepam. Seven animals were used in each experimental group. The animals were unique to this study.

Day 1: Each animal was injected with sterile saline (1 ml/kg ip) immediately before it was placed in the start box.

Day 2: Fifteen minutes before each animal was tested, it received an ip injection of either 0.5 mg/kg diazepam or vehicle alone (total volume 1 ml/kg). This dosage has been shown to have anxiolytic effects without seriously impairing motor function. The animal was put back in its home cage until just before it was to be tested.

Analysis: The validation experiments were analyzed using a two-tailed t-test. All the SB 224289/cocaine data were analyzed using a 2×2 ANOVA (SB 224289×cocaine). Scheffe's post hoc test was used where appropriate. All analysis was performed with the Statistica™ software package. Analysis of latency to exit was performed using Fischerˈs exact test. Analysis excluded any effects of time or order of testing on any results on either day.

[1]. Cassilly CD, et al. SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A. PLoS One. 2016 May 16;11(5):e0154932.
[2]. Hoplight BJ, et al. The effects of SB 224289 on anxiety and cocaine-related behaviors in a novel object task. Physiol Behav. 2005 Apr 13;84(5):707-14.
[3]. Gaster LM, et al. The selective 5-HT1B receptor inverse agonist SB-224289, potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo. Ann N Y Acad Sci. 1998 Dec 15;861:270-1.

SB 224289 is a member of the class of benzamides obtained by formal condensation of the carboxy group of 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxylic acid with the secondary amino group of 1'-methyl-6,7-dihydro-5H-spiro[furo[2,3-f]indole-3,4'-piperidine]. Selective 5-HT1B receptor antagonist (pKi = 8.2). Displays >60-fold selectivity over 5-HT1D, 5-HT1A, 5-HT1E, 5-HT1F, 5-HT2A and 5-HT2C receptors in radioligand binding and functional assays. Centrally active following oral administration in vivo. It has a role as a serotonergic antagonist. It is an organic heterotetracyclic compound, an azaspiro compound, a 1,2,4-oxadiazole and a member of benzamides. It is a conjugate base of a SB 224289(1+).
SB-224289 is a small molecule with a high affinity for human 5-HT1B receptors.

C32H32N4O3

520.62148

556.25

C, 73.82; H, 6.20; N, 10.76; O, 9.22

180083-23-2

SB-224289 hydrochloride;180084-26-8

3378093

Typically exists as solid at room temperature

1.34g/ml

280℃

6.384

0

6

3

39

885

0

CC1=CC(=CC=C1C2=CC=C(C=C2)C(=O)N3CCC4=C3C=C5C(=C4)OCC65CCN(C)CC6)C7=NOC(=N7)C

ATQMRMGXINTJHV-UHFFFAOYSA-N

InChI=1S/C32H32N4O3/c1-20-16-25(30-33-21(2)39-34-30)8-9-26(20)22-4-6-23(7-5-22)31(37)36-13-10-24-17-29-27(18-28(24)36)32(19-38-29)11-14-35(3)15-12-32/h4-9,16-18H,10-15,19H2,1-3H3

(1'-Methyl-6,7-dihydro-5H-spiro(furo(2,3-f)indole-3,4'-piperidin)-5-yl)(2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl)methanone

SB 224289; SB-224289; (2'-Methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-yl)(1'-methyl-6,7-dihydrospiro[furo[2,3-f]indole-3,4'-piperidin]-5(2H)-yl)methanone; SB224289; J854.001B; CHEMBL281350; (1'-Methyl-6,7-dihydro-5H-spiro(furo(2,3-f)indole-3,4'-piperidin)-5-yl)(2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl)methanone;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)