AM630

6-Iodopravadoline (AM630) is a selective CB2 antagonist with Ki of 31.2 nM, and displays 165-fold selectivity over CB1 receptors.

Research Areas >> Neurological Disease

Signaling Pathways >> GPCR/G Protein >> Cannabinoid Receptor

Ki: 31.2 nM (CB2)

The AM251 and 6-Iodopravadoline (AM630)-evoked Ca2+ influxes into TG sensory neurons aere concentration-dependent, and fitted. The EC50 for AM251 and 6-Iodopravadoline are 7.37 μM and 15.6 μM, respectively. AM251 and 6-Iodopravadoline activate TRPA1 in TG sensory neurons. 6-Iodopravadoline is comparable in value in both TRPA1 and TRPV1/TRPA1 expressing CHO cells (2 and 4.6 μM, respectively). AM251 and 6-Iodopravadoline activation of TRPA1 is modulated by TRPV1[2]. 6-Iodopravadoline (0, 50, 100, and 200 nM) is not toxic to RAW264.7 cells. 6-Iodopravadoline (100 nM) substantially inhibits osteoclastogenesis in cultures with RANKL and Ti particles in a dose-dependent manner[3]. 6-Iodopravadoline (1 μM) blocks the CP-55,940 dose response with EC50 of 170 nM at human and EC50 of 110 nM at rat cannabinoid CB2 receptor[4].

6-Iodopravadoline (AM630) (2, 3 mg/kg, i.p.) significantly reduces the time spent in the light box compared with vehicle group. 6-Iodopravadoline (AM630) (1, 2 or 3 mg/kg, i.p., twice a day) produces a significant anxiolytic effect, increasing the time spent in the light box at all of the doses used[1].

HEK cells stabLy expressing human or rat cannabinoid CB2 receptors are grown until a confLuent monoLayer is formed. The ceLLs are harvested and homogenized in Tris-EDTA (TE) buffer (50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM EDTA) using a poLytron for 2×10 s bursts in the presence of protease inhibitors, followed by centrifugation at 45,000×g for 20 min. The final membrane pellet is re-homogenized in storage buffer (50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM EDTA and 10% sucrose) and frozen at −80°C until used. Saturation binding reactions are initiated by the addition of membrane preparation (protein concentration of 5 μg/well for the human CB2and 20 μg/well for the rat CB2) into wells of a deep-well pLate containing ([3H]CP-55,940 (120 Ci/mmol)) in assay buffer (50 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 5 mM MgCl2, and 0.5 mg/mL fatty acid-free bovine aLbumin serum). After incubation at 30°C for 90 min, binding reaction is terminated by the addition of 300 μL/well of coLd assay buffer followed by rapid vacuum filtration through a UniFilter-96 GF/C filter pLates (pre-soaked in 1 mg/mL bovine serum aLbumin for 2 h). The bound activity is counted in a TopCount using Microscint-20. In some experiments with endogenous ligands, experiments are performed in the presence of AEBSF (50 μM). Saturation experiments are conducted with 12 concentrations of [3H]CP-55,940 ranging from 0.01 to 8 nM. Competition experiments are conducted with 0.5 nM [3H]CP-55,940 and five concentrations (1 nM to 10 μM) of displacing Ligands. Nonspecific binding is defined by the addition of 10 μM unLabeLed CP-55,940 in both saturation and competition binding assays. Kd vaLues from saturation binding assays and Ki vaLues from the competition binding assays are determined with one site binding or one site competition curve fitting equations using Prism software.

The effect of AM630 and Ti particles on RAW cell viability is examined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. RAW 264.7 (1×103 cells/well) is cultured in the presence of AM630 (0, 50, 100, and 200 nM) for 24, 48, and 72 h, with or without Ti particles, and then incubated with MTT (0.5 mg/mL) at 37°C for 2 h. The culture medium is then replaced with equal volumes of DMSO to dissolve formazan crystals, followed by shaking at room temperature for 10 min. Absorbance at 490 nm is measured using a microplate reader.

[1]. Garcia-Gutierrez, Maria S., et al. Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABAA receptors. British Journal of Pharmacology (2012), 165(4), 951-964.

[2]. Patil, Mayur., et al. Cannabinoid receptor antagonists AM251 and AM630 activate TRPA1 in sensory neurons. Neuropharmacology (2011), 61(4), 778-788.

[3]. Geng, D. C., et al. Inhibition of titanium particle-induced inflammatory osteolysis through inactivation of cannabinoid receptor 2 by AM630. Journal of Biomedical Materials Research, Part A (2010), 95A(1), 321-326.

[4]. Mukherjee, Sutapa., et al. Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors. European Journal of Pharmacology (2004), 505(1-3), 1-9.

[5]. Sun L, et al. Endocannabinoid activation of CB1 receptors contributes to long-lasting reversal of neuropathic pain by repetitive spinal cord stimulation. Eur J Pain. 2017 May;21(5):804-814.