Binding assays

Membrane samples were prepared from HEK cells stably expressing the human CB2 receptors previously generated, or the CHO cell line that stably expresses the human CB1 receptor. Briefly, the cells were harvested and homogenized using a Polytron for 2 × 10s bursts in a buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM EDTA in the presence of protease inhibitors followed by centrifugation at 45000g for 20min. The membrane pellets were washed and frozen at -80℃ in aliquots until use. Saturation binding reactions were performed at 30℃ for 90min using [3H]CP 55,940 (0.01–8nm) in an assay buffer containing 50mm Tris-HCl, pH 7.4, 2.5mM EDTA, 5mM MgCl2, and 0.05% fatty acid free bovine serum albumin (BSA) and the reactions were terminated by rapid vacuum filtration through UniFilter-96 GF/C filter plates and four washes with cold assay buffer. Competition experiments were conducted using 0.5nM [3H]CP 55,940 in the presence of test compounds (0.1nM–10μM).

Cell lines

Human embryonic kidney (HEK) cells stably expressing the human CB2 receptor, Chinese hamster ovary (CHO) cell line stably expressing the human CB1 receptor

Preparation method

The solubility of this compound in DMSO is >25.2 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

Ki:～7 nM (human CB2 receptor)

Applications

In HEK cells stably expressing the human CB2 receptor, AM1241 exhibited antagonist activity, blocking the agonist CP 55,940-evoked Ca2+ response in a concentration dependent manner with a Kb value of 63nM. In [3H]CP 55,940 competition binding assays, AM-1241 displayed high affinity at the human CB2 receptor with a Ki value of ～7 nM, whereas its affinity at the human CB1 receptor was more than 80-fold weaker, using membrane preparations from stable HEK and CHO cell lines expressing the recombinant human CB2 and CB1 receptors, respectively.

Animal models

Adult male Sprague–Dawley rats

Dosage form

Intraperitoneal injection, 100, 330 μg/kg

Application

AM1241 (100, 330 μg/kg i.p.) suppressed the development of carrageenan-evoked thermal and mechanical hyperalgesia and allodynia. Intraplantar (ipl) administration of AM1241 (33 μg/kg ipl) suppressed hyperalgesia and allodynia following administration to the carrageenan-injected paw but was inactive following administration in the contralateral (noninflamed) paw.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

IC50: AM-1241 is a selective cannabinoid CB2 receptor agonist with Ki of 3.4 nM, exhibits 82-fold selectivity over CB1 receptor. [1].
The cannabinoid receptor type 2, abbreviated as CB2, is a G protein-coupled receptor from the cannabinoid receptor family that in humans is encoded by the CNR2 gene. CB2 receptors may have possible therapeutic roles in the treatment of neurodegenerative disorders such as Alzheimer's disease. AM-1241, a chemical from the aminoalkylindole family, acts as a potent and selective agonist for the cannabinoid receptor CB2, has analgesic effects in animal studies, particularly against "atypical" pain such as hyperalgesia and allodynia.
In vitro: The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo. In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB2, but an inverse agonist at rat and mouse CB2 receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB2 receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB2 receptors. In pain models, S-AM1241 was more efficacious than either R-AM1241 or the racemate. Antagonist blockade demonstrated that the in vivo effects of S-AM1241 were mediated by CB2 receptors. These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB2 receptors and the first characterization of the AM1241 enantiomers in recombinant cell systems and in vivo. The greater antinociceptive efficacy of S-AM1241, the functional CB2 agonist enantiomer of AM1241, is consistent with previous observations that CB2 agonists are effective in relief of pain [2].
In vivo:
Effective treatment or amyotrophic lateral sclerosis (ALS) remains elusive. Motor neuron degeneration is the primary pathology in ALS; however non-neuronal cells contribute to the disease process. In particular, inflammatory processes have been shown to play an important role. AM1241 is a cannabinoid CB2 receptor selective agonist that has been shown to be effective in models of inflammation and hyperalgesia. Here we report that treatment with AM1241 was effective at slowing signs of disease progression when administered after onset of signs in an ALS mouse model (hSOD1G93A transgenic mice). Administration at the onset of tremors delayed motor impairment in treated mice when compared to vehicle controls. Three conditions of ALS, the loss of motor function, paralysis scoring and weight loss, were analyzed using a mathematical model. Loss of motor function (as assessed by performance on a rotarod) was delayed by 12.5 days in male mice by AM1241. In female mice, AM1241 extended rotarod performance by 3 days, although this was not statistically significant. In male mice, AM1241 also extended by 5 days the time to reach the 50%point on a visually-assessed performance scale.AM1241 did not affectweight loss or survival (129.8±1.7 days, vehicle; 129.1±7.0 days, AM1241, n=16). As AM1241 was well tolerated by the animals, cannabinoid CB2 receptor-selective compounds may be the basis for developing new drugs for the treatment of ALS and other chronic neurodegenerative diseases [3].
Clinical trial: AM1241 is still in preclinical development phase and no clinical study has been found.
Reference:
[1] Ibrahim MM, Deng H, Zvonok A, Cockayne DA, Kwan J, Mata HP, Vanderah TW, Lai J, Porreca F, Makriyannis A, Malan TP Jr. Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS. Proc Natl Acad Sci U S A. 2003;100(18):10529-33.
[2] B Bingham, PG Jones, AJ Uveges, S Kotnis, P Lu, VA Smith, S-C Sun, L Resnick, M Chlenov, Y He. Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers. British Journal of Pharmacology (2007) 151, 1061–1070
[3] Kathline Kim, Dan H. Moore, Alexandros Makriyannis, Mary E. Abood. AM1241, a cannabinoid CB2 receptor selective compound, delays disease progression in a mouse model of amyotrophic lateral sclerosis. European Journal of Pharmacology 542 (2006) 100-105