LY2828360 displays a delayed signaling profile at mouse CB2 receptors. (A) In CHO cells stably expressing mCB2 receptors, CP55940 recruited arrestin in a concentration-dependent manner, whereas LY2828360 failed to do so after 90-minute drug incubation. (B) In HEK cells stably transfected with mCB2, CP55940 concentration dependently internalized the mCB2; LY2828360 was less potent and efficacious. (C) In a forskolin-stimulated cAMP time course assay, CP55940 (1 µM) was efficacious and rapid in inhibiting forskolin-stimulated cAMP accumulation at 5 minutes, whereas LY2828360 (1 µM) was efficacious only after 30 minutes. (D) After PTX treatment, CP55940 (1 µM) modestly increased cAMP accumulation at 5 minutes, whereas LY2828360 (1 µM) failed to affect cyclase levels at all time points examined/tested. (E) CP55940 was potent and efficacious in inhibiting forskolin-stimulated cAMP accumulation at 5 minutes, whereas LY2828360 failed to affect cAMP levels at this time point. (F) After 30-minute incubation, however, LY2828360 concentration dependently inhibited forskolin-stimulated cAMP accumulation, and this inhibition was completely blocked by 1 µM SR144528(SR2). Forskolin-stimulated cAMP assays were performed in duplicate. All other assays were performed in triplicate. All data were plotted and analyzed using GraphPad Prism 4.

LY282360 displays a delayed CB₂ receptor– and G protein–dependent signaling profile in activating pERK1/2. (A) In HEK cells stably expressing mouse CB2 receptors, CP55940 (1 µM) increased phosphorylated ERK1/2 at 5-, 10-, 30-, and 40-minute time points, whereas LY2828360 (1 µM) had no effect at 5- and 10-minute time points but increased ERK1/2 phosphorylation at 20, 30, and 40 minutes. (B) PTX treatment abolished the 20-minute phosphorylation of ERK1/2 by LY2828360 (1 µM) and abolished the CP55940-mediated phosphorylation of ERK1/2 at the 5-minute time point, but it was retained at the 40-minute time point after PTX treatment. (C) CP55940 concentration dependently increased ERK1/2 phosphorylation at 5 minutes, whereas LY2828360 failed to affect pERK1/2 levels at this time point. (D) Conversely, after 20 minutes of treatment, CP55940 decreased ERK1/2 phosphorylation, whereas LY2828360 increased ERK1/2 phosphorylation, in a concentration- dependent manner. Both effects were blocked by the CB2 receptor antagonist SR144528 (1 µM) (SR2). All pERK1/2 assays were performed in triplicate. All the experimental data were plotted and analyzed using GraphPad Prism 4.

Paclitaxel produced hypersensitivities to mechanical (A) and cold (B) stimulation. Nonchemotherapy control mice received cremophor-based vehicle in lieu of paclitaxel. Dose response of LY2828360, administered systemically (i.p.), on the maintenance of (C) mechanical and (D) cold allodynia in paclitaxel-treated WT (C57BL/6J) mice. The time course of LY2828360, administered systemically (3 mg/kg i.p.), on the maintenance of (E) mechanical and (F) cold allodynia in paclitaxel-treated WT mice. Data are expressed as mean ± S.E.M. (n = 6/group). *P < 0.05 vs. control, one-way analysis of variance at each time point, followed by Bonferroni’s post hoc test. #P < 0.05 vs. baseline before paclitaxel, repeated measures analysis of variance. ^&P < 0.05 vs. baseline after paclitaxel, repeated measures analysis of variance. BL, pre-paclitaxel baseline; Pac, baseline after paclitaxel.

History of chronic LY2828360 treatment blocked the development of morphine tolerance in WT but not in CB2KO mice. (A) The testing scheme used to evaluate the two phases of treatment during the maintenance of neuropathic pain. History of chronic LY2828360 (3 mg/kg per day i.p. × 12 days in phase 1) treatment suppressed paclitaxel-induced (B) mechanical (C) cold allodynia in WT mice. History of chronic LY2828360 (3 mg/kg per day i.p. × 12 days in phase 1) blocked the development of tolerance to the antiallodynic effects of morphine (10 mg/kg per day × 12 days in phase 2) in WT but not in CB₂KO mice for both mechanical (D) and cold (E) modalities. Data are expressed as mean ± S.E.M. (n = 6/group). *P < 0.05 versus Veh (1)-Veh (2), one-way analysis of variance at each time point, followed by Bonferroni’s post hoc test. ^#P < 0.05 vs. baseline before paclitaxel, repeated measures analysis of variance.

Chronic LY2828360 treatment showed sustained antiallodynic efficacy in morphine-tolerant WT mice but not in CB2KO mice. (A) Testing scheme used to evaluate the two phases of treatment during the maintenance of neuropathic pain. Chronic LY2828360 (3 mg/kg per day i.p. × 12 days in phase 2) treatment suppressed paclitaxel-induced mechanical (B and D) and cold (C and E) allodynia in WT mice but not in CB₂KO mice previously rendered tolerant to morphine (10 mg/kg per day i.p. × 12 days in phase 1). Data are expressed as mean ± S.E.M. (n = 6/group). Veh (1)-Veh (2) group is replotted from Fig. 5. *P < 0.05 vs. Veh (1)-Veh (2), one-way analysis of variance at each time point, followed by Bonferroni’s post hoc test. ^#P < 0.05 vs. baseline before paclitaxel, repeated measures analysis of variance.

Chronic coadministration of low-dose LY2828360 (0.1 mg/kg per day i.p. × 12 days) with morphine (10 mg/kg per day i.p. × 12 days) blocked development of morphine tolerance in WT but not in CB₂KO mice tested for both (A) mechanical and (B) cold allodynia. Data are expressed as mean ± S.E.M. (n = 6/group). *P < 0.05 vs. WT-Veh, one-way analysis of variance at each time point, followed by Bonferroni’s post hoc test. ^#P < 0.05 vs. baseline before paclitaxel, repeated measures analysis of variance.