A CB2 receptor inverse agonist
Ki= 0.6 nM
SR 144528 is a CB2 receptor inverse agonist.
The CB2 receptor is principally expressed in immune tissue where it is involved in cannabinoid-mediated immune responses, but CB2 mRNA has been detected in mouse cerebellar cells and cerebellum and in rat microglial cells.
In vitro: SR 144528 displayed subnanomolar affinity for both the rat spleen and cloned human CB2 receptors and had a 700-fold lower affinity for both the rat brain and cloned human CB1 receptors. Moreover, SR 144528 showed no affinity for more than 70 receptors, ion channels or enzymes tested. SR 144528 could antagonize the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines expressing the human CB2 receptor but not in cells expressing the human CB1. In addition, SR 144528 could selectively block the mitogen-activated protein kinase activity that was induced by CP 55,940 in cell lines expressing human CB2 while an IC50 value of more than 1 μM was observed in cells expressing human CB1 [1].
In vivo: Animal study showed that oral administration of SR 144528 could totally displace the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes with a long action duration. Whereas, after the oral route SR 144528 did not interact with the cannabinoid receptor expressed in the mouse brain CB1 [1].
Clinical trial: Up to now, SR 144528 is still in the preclinical development stage.
Reference:
[1] Rinaldi-Carmona M et al.? SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. J Pharmacol Exp Ther. 1998 Feb;284(2):644-50.
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Kinase experiment: |
MAP kinase activity is measured. Briefly, cells grown to 80% confluence are maintained in culture medium containing 0.5% foetal calf serum for 24 hour prior to the application of ligands. CHO-CB1 or -CB2 cells previously washed with PBS are incubated at 37°C in the absence (basal activity) or in the presence of SR144528 (10-9 to 3×10-6 M) for 20 min. Cells are then washed at 4°C with 0.5 mL of buffer A [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM ethyleneglycol-bis-(β-aminoethyl ether) N,N,N′,N-tetraacetic acid, 1 mM Na3PO4] and lysed for 15 min 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. The solubilized cell extracts are then clarified by centrifugation at 14,000× g for 15 min at 4°C. Aliquots (15 μL) are removed and stored at -80°C until use. Phosphorylation assays are carried out at 30°C for 30 min (linear assay conditions) with γ-[33P]ATP by using the p42/p44 MAP kinase enzyme system. The radioactivity incorporated is determined by liquid scintillation counting[1]. |
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Cell experiment: |
cAMP accumulations are carried out in CHO-CB1 or -CB2 cells. Cells are washed with phosphate-buffered saline (PBS) and incubated for 15 min at 37°C in 1 mL of PBS in the absence or in the presence of SR144528 (3×10-9 to 10-5M). Forskolin (3 μM final concentration) is added and cells are incubated for another 20 min at 37°C. The reaction is terminated by rapid aspiration of the assay medium and addition of 1.5 mL of ice-cold 50 mM Tris-HCl, pH 8, 4 mM ethylenediaminetetraacetic acid. Dishes are placed on ice for 5 min and then the extracts are transferred to a glass tube. Extracts are boiled and centrifuged for 10 min at 3500 g to eliminate cell debris. Aliquots from supernatant are dried and the cAMP concentration is determined by radioimmunoassay by using the scintillant proximity assay system. The basal activity is determined in the absence of forskolin[1]. |
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Animal experiment: |
Male Wistar rats (240 to 300 g) are used in this study. One week after the animals arrived at the laboratory, three different sets of experiments are carried out. In the third set of experiments, SR144528 (1 mg/kg i.p.) is administered in rats. The effect of SR144528 is also analyzed in vehicle-treated rats. SR144528 volume is adjusted to a maximum of 4 to 5 mL/kg[3]. |
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参考文献: [1]. Rinaldi-Carmona M, et al. SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. J Pharmacol Exp Ther. 1998 Feb;284(2):644-50. |
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