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. 2006 Oct;149(4):431-40.
doi: 10.1038/sj.bjp.0706888. Epub 2006 Sep 4.

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands

M G Cascio  1 , T Bisogno, E Palazzo, A Thomas, M van der Stelt, A Brizzi, V de Novellis, I Marabese, R Ross, T van de Doelen, V Brizzi, R Pertwee, S Maione, V Di Marzo
Affiliations

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands

M G Cascio et al. Br J Pharmacol. 2006 Oct.

Abstract

Background and purpose: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity.

Experimental approach: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined.

Key results: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception.

Conclusions and implications: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

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Figures

Figure 1
Figure 1
Chemical structures of CB-25 and CB-52 (Brizzi et al., 2005).
Figure 2
Figure 2
The effect of CP55940 (n=7–9), CB-25 (n=5–7) and CB-52 (n=8) on the level of [35S]GTP-γ-S binding to mouse whole brain membranes. Each symbol represents the mean percentage increase in [35S]GTP-γ-S binding ±s.e.
Figure 3
Figure 3
The effect of CP55940, CB-25 and CB-52 on forskolin-stimulated cAMP levels in CHO cells overexpressing the human recombinant CB1 (a) or CB2 (b) receptors. Data are expressed as percent inhibition and are means±s.e. of four experiments.
Figure 4
Figure 4
(a) The effect of CB-25 (n=6) or CB-52 (n=6) on the level of [35S]GTP-γ-S binding to CB2-transfected CHO cell membranes and the effect of (b) 100 nM CB-25 (n=3–4) or (c) 1 μM CB-52 (n=3–4) on the mean log concentration–response curve of CP55940 for stimulation of [35S]GTP-γ-S binding to CB2-transfected CHO cell membranes. Each symbol represents the mean percentage increase in [35S]GTP-γ-S binding ±s.e.
Figure 5
Figure 5
Nociception, expressed as percentage of the maximum possible effect (%MPE) after systemic administration of (a) CB-25 (1 and 2.5 mg kg−1, i.p.) and (b) CB-52 (1 and 5 mg kg−1, i.p.) either alone or in combination (at 1 mg kg−1, i.p.) with WIN55,212-2 (2.5 mg kg−1, i.p.) in the rat. Each data point represents the mean±s.e.m. of 10 observations. Significant differences between groups are shown as filled symbols (P<0.05; ANOVA; Student–Newman–Keuls test). For treatments with a single compound, means of the treated groups were compared to those from the relevant vehicle. For combination treatments, means were compared to those from treatment with the corresponding single compounds.
Figure 6
Figure 6
Spontaneous activities of RVM ‘ON' (a, c) and ‘OFF' (b, d) cells before and after microinjections into the ventrolateral PAG of CB-25 (4 nmol per rat), CB-52 (4 nmol per rat) or WIN55,212-2 (4 nmol per rat) alone, or of CB-25 or CB-52 (2 nmol per rat) in combination with WIN55,212-2 (4 nmol per rat). CB-25 and CB-52 at the lowest dose (2 nmol per rat) did not change pro-nociceptive ‘ON' and anti-nociceptive ‘OFF' cell firing (for clarity, these curves are not shown). CB-25 and CB-52 at the highest dose increased pro-nociceptive ON cell firing (a, c) and decreased anti-nociceptive OFF cell firing (b, d). WIN55,212-2 (4 nmol per rat) caused effects that were opposite to those induced by CB-25 and CB-52 and were blocked by co-injection with the low, inactive, doses of CB-25 and CB-52 (2 nmol per rat). The black arrow denotes the time of injections of drugs. Each point represents the mean±s.e.m. of 10 recorded neurons. Significant differences between groups are shown as filled symbols (P<0.05; Wilcoxon signed rank test). For treatments with a single compound, means of the treated groups were compared to those from the relevant vehicle. For combination treatments, means were compared to those from treatment with the corresponding single compounds.
Figure 7
Figure 7
Effect of subcutaneous formalin (1.2%, 50 μl) on the time course of the nociceptive behaviour. (a, b) Formalin was injected into the hind paw of mice 15 min after the systemic administration of vehicle (10% DMSO in 0.9% NaCl, i.p.), CB-25 (2.5 and 5 mg kg−1, i.p.) or CB-52 (1.0 and 5 mg kg−1, i.p.). CB-25 and CB-52, at highest dose (5 mg kg−1, i.p.), were also administered in combination with SR141716A (0.5 mg kg−1, i.p.), or AM630 (2 mg kg−1, i.p.), which per se did not change the formalin-induced nocifensive behaviour (not shown). (c) The effect of WIN55,212-2 (WIN, 2.5 mg kg−1, i.p.) either alone or in combination with the low dose of either CB-25 (2.5mg kg−1, i.p.) or CB-52 (1 mg kg−1, i.p.). The data represent the total time of the nociceptive responses (mean±s.e.m.)of 10 mice per group, measured every 5 min. Significant differences between groups are shown as filled symbols (P<0.05; ANOVA; Student–Newman–Keuls test). For treatments with a single compound, means of the treated groups were compared to those from the relevant vehicle. For combination treatments, means were compared to those from treatment with the corresponding single compounds.
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