Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers

Abstract

Background and purpose: Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB(2))-selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo.

Experimental approach: Competition binding assays were performed using membranes from cell lines expressing recombinant human, rat, and mouse CB(2) receptors. Inhibition of cAMP was assayed using intact CB(2)-expressing cells. A mouse model of visceral pain (para-phenylquinone, PPQ) and a rat model of acute inflammatory pain (carrageenan) were employed to characterize the compounds in vivo.

Key results: In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB(2), but an inverse agonist at rat and mouse CB(2) receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB(2) receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB(2) 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 CB(2) receptors.

Conclusions and implications: These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB(2) 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 CB(2) agonist enantiomer of AM1241, is consistent with previous observations that CB(2) agonists are effective in relief of pain.

Figure 1

Radioligand displacement curves for R,S-AM1241, R-AM1241 and S-AM1241 at the human (a), rat (b) and mouse (c) CB₂ receptor. Membranes prepared from CHO-K1 cells expressing the human, rat or mouse CB₂ receptor were incubated in the presence of drug and 1–3 nM [³H]-CP55,940 for 1 h. Membranes were filtered onto Whatman GFB paper and radioactivity determined. K_i values were calculated using the Cheng–Prusoff equation. Data are expressed as mean±s.e.m. for three independent experiments.

Figure 2

Effects of the non-selective cannabinoid agonist WIN55,212-2 (a) and the CB₂-selective antagonist/inverse agonist SR144528 (b) on cAMP levels in cells expressing the human, rat or mouse CB₂ receptor. Cells were stimulated in the presence of 1 μM forskolin for 30 min. Data are expressed as mean±s.e.m. from a representative dose–response curve.

Figure 3

Effects of R,S-AM1241 (a) and its enantiomers, R-AM1241 (b) and S-AM1241 (c) on cAMP accumulation in CHO-K1 cells expressing the human, rat or mouse CB₂ receptor. Cells were stimulated in the presence of 1 μM forskolin for 30 min. Data are expressed as mean±s.e.m. for three independent experiments. cAMP, cyclic adenosine monophosphate.

Figure 4

Effects of R,S-AM1241 and its enantiomers, R-AM1241 and S-AM1241, on visceral pain and thermal hyperalgesia associated with chemical irritants. (a) Male CD-1 mice (25–30 g; n=10 per group) were pretreated with vehicle or compound (s.c.) 30 min before PPQ administration and tested 10 min post-administration. Data (mean±s.e.m.) are expressed as percent blockade relative to vehicle-treated mice. (b) Male SD rats (220–250 g; n=8 per group) received an intraplantar injection of 50 μl of saline or 2% carrageenan into the hindpaw, followed 2.5 h later by i.p. administration of vehicle, R,S-AM1241, R-AM1241 or S-AM1241. Data (mean±s.e.m.) are expressed as percent reversal relative to vehicle-treated rats. (c) Male SD rats (220–250 g; n=10 per group) received an intraplantar injection of 50 μl of saline or 2% carrageenan into the hindpaw, followed 2.5 h later by i.p. administration of either vehicle or 10 mg kg⁻¹ S-AM1241 and either vehicle or 1 mg kg⁻¹ AM630. Data (mean±s.e.m.) are expressed as paw withdrawal latencies; pre-carrageenan baseline data not shown. I.p., intraperitoneal; SD rat, Sprague–Dawley rat.