CB2 receptor ( Ki = 0.6 nM )

SR144528 possesses a Ki of 0.6 nM and is a strong and specific CB2 receptor clamp antagonist. Forskolin-sensitive adenylyl cyclase activity in CHO-CB2 cells can be stimulated by SR144528 alone in a concentration-dependent manner (EC50=26±6 nM, two experiments), with the largest effect at 1 μM (4-fold stimulation). It doesn't significantly affect CHO-CB1 (15% inhibition) at this cell concentration [1]. With an IC50 value of 3.6±1.1 μM, the original 264.7 giant SR144528 supplemented with SR144528 inhibits phospholiposomal phospholipidyl-CoA:phospholipidyltransferase (ACAT) activity in a concentration-regulated manner. Approximately 68% of ACAT activity is fluoresced by SR144528 at 10 μM [2].

SR144528 After icv (10 μg/animal) or brain (10 mg/kg) administration, no binding of [³H]-CP 55,940 to particular brain sites was seen. SR144528 After powder formulation at 3 mg/kg, spleen engagement of brain cannabinoid receptors is time-dependent and significant for at least 18 hours [1]. On its own, SR144528 has no discernible impact on gastrointestinal (GI) motility. The delayed gastric emptying that is facilitated by SR144528 is not accompanied by a burst[3].

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Low, analgesic doses of WIN delayed intestinal transit, but high, psychoactive doses were required to delay gastric emptying. Acute WIN effects on GI motility were confined to the first few hours after administration. AM251 partially counteracted the effect of WIN on GI motility. Surprisingly, SR144528 (but not AM630) enhanced WIN-induced delayed gastric emptying. Conclusions & inferences: X-ray analyses confirm that cannabinoids inhibit GI motility via CB1 receptors; in addition, cannabinoids could influence motility through interaction with a SR144528-sensitive site. Further studies are needed to verify if such site of action is the CB2 receptor[3].

It measures the activity of MAP kinase. In short, 24 hours before ligands are applied, cells that have reached 80% confluence are kept in culture medium containing 0.5% foetal calf serum. After being cleaned with PBS, CHO-CB1 or -CB2 cells are cultured for 20 minutes at 37°C either without SR144528 (10^-9 to 3×10-6 M) or with it present. Following a 4°C wash, cells are lysed for 15 minutes in buffer A supplemented with 1% triton X-100, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Buffer A contains 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM ethyleneglycol-bis-(β-aminoethyl ether) N,N,N′,N-tetraacetic acid, and 1 mM Na₃PO₄. The cell extracts that have been solubilized are subsequently separated using centrifugation at 14,000 x g for 15 minutes at 4°C. Before using, remove the 15 μL aliquots and store them at -80°C. Using γ-[³³P]ATP and the p42/p44 MAP kinase enzyme system, phosphorylation assays are run for 30 minutes at 30°C (linear assay conditions). Through the use of liquid scintillation counting, the radioactivity incorporated is ascertained[1].

In CHO-CB1 or -CB2 cells, cAMP accumulations are performed. PBS is used to wash the cells, and they are then incubated for 15 minutes at 37°C in 1 mL of PBS either with or without SR144528 (3×10^-9 to 10^-5M) present. Next, cells are incubated for 20 minutes at 37°C with the addition of forskolin (3 μM final concentration). Once the assay medium has been quickly aspirated, 1.5 mL of ice-cold 50 mM Tris-HCl, pH 8, and 4 mM ethylenediaminetetraacetic acid are added to stop the reaction. The extracts are then moved to a glass tube after the dishes are kept on ice for five minutes. The extracts undergo boiling and a 10-minute, 3500 g centrifugation to remove any remaining cell debris. The concentration of cAMP is measured by radioimmunoassay using aliquots from the supernatant that have been dried and the scintillant proximity assay system. Without forskolin, the basal activity is calculated[1].

In this study, male Wistar rats weighing between 240 and 300 grams are utilized. Three distinct sets of experiments are conducted a week after the animals were brought to the lab. Rats receive an intraperitoneal injection of SR144528 (1 mg/kg) in the third series of experiments. Rats given vehicles are also used to examine the impact of SR144528. The maximum volume for SR144528 is set at 4 to 5 mL/kg[3].

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Male Wistar rats received different doses of WIN and both psychoactivity (cannabinoid tetrad) and GI motility (radiographic analysis) were tested. The duration of WIN effect on GI motility was also radiographically analyzed. Finally, the involvement of the different cannabinoid receptors on WIN-induced alterations of GI motility was analyzed by the previous administration of selective CB1 (AM251) and CB2 (SR144528 or AM630) antagonists. After administration of contrast medium, alterations in GI motility were quantitatively evaluated in serial radiographs by assigning a compounded value to each region of the GI tract. [3]

[1]. SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. J Pharmacol Exp Ther. 1998 Feb;284(2):644-50.

[2]. AM-251 and SR144528 are acyl CoA:cholesterol acyltransferase inhibitors. Biochem Biophys Res Commun. 2009 Apr 3;381(2):181-6.

[3]. The cannabinoid antagonist SR144528 enhances the acute effect of WIN 55,212-2 on gastrointestinal motility in the rat. Neurogastroenterol Motil. 2010 Jun;22(6):694-e206.

SR 144528 is a secondary carboxamide resulting from the formal condensation of the carboxy group of 5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1H-pyrazole-3-carboxylic acid with the amino group of (1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-amine. A potent and selective cannabinoid receptor type 2 (CB2 receptor) inverse agonist (Ki = 0.6 nM). It has a role as a CB2 receptor antagonist and an EC 2.3.1.26 (sterol O-acyltransferase) inhibitor. It is a member of pyrazoles, a secondary carboxamide, a member of monochlorobenzenes and a bridged compound.

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Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response. [1]

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Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2(+/+) and CB2(-/-) peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have anti-atherosclerotic activities independent of their affect on cannabinoid signaling. [2]

C29H34CLN3O

476.06

475.239

C, 73.17; H, 7.20; Cl, 7.45; N, 8.83; O, 3.36

192703-06-3

192703-06-3

3081355

White to off-white solid powder

1.2±0.1 g/cm3

627.7±55.0 °C at 760 mmHg

333.4±31.5 °C

0.0±1.8 mmHg at 25°C

1.633

7.05

1

2

5

34

750

3

O=C(C1=NN(CC2=CC=C(C)C=C2)C(C3=CC=C(Cl)C(C)=C3)=C1)N[C@H]4[C@@](C5)(C)CC[C@@]5([H])C4(C)C

SUGVYNSRNKFXQM-XRHWURSXSA-N

InChI=1S/C29H34ClN3O/c1-18-6-8-20(9-7-18)17-33-25(21-10-11-23(30)19(2)14-21)15-24(32-33)26(34)31-27-28(3,4)22-12-13-29(27,5)16-22/h6-11,14-15,22,27H,12-13,16-17H2,1-5H3,(H,31,34)/t22-,27-,29+/m1/s1

5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethyl-2-bicyclo[2.2.1]heptanyl]pyrazole-3-carboxamide

SR-144528; SR 144528; 192703-06-3; CHEMBL381791; 1H-Pyrazole-3-carboxamide, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-; SR144,528; 5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-N-((1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl)-1H-pyrazole-3-carboxamide; SR144528

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: ~50 mg/mL (~105.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.25 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 25.0 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: ≥ 2.5 mg/mL (5.25 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 25.0 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.)