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. 2011 Nov 17:7:89.
doi: 10.1186/1744-8069-7-89.

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Haitao You  1 , Vinicius M Gadotti, Ravil R Petrov, Gerald W Zamponi, Philippe Diaz
Affiliations

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Haitao You et al. Mol Pain. .

Abstract

Background: Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity.

Results: Novel compounds were characterized in radioligand binding assays and in vitro functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compounds on transient expressed human T-type calcium channels were examined in tsA-201 cells using standard whole-cell voltage clamp techniques, and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, intraperitoneally) assessed in the formalin model. A series of compounds were synthesized and evaluated for channel and receptor activity. Compound NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compounds completely inhibited peak Cav3.2 currents with IC50 values in the low micromolar range. All compounds mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response.

Conclusions: Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects in vivo. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.

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Figures

Figure 1
Figure 1
NMP compounds selected for this study. Carbazole derivatives NMP-4, NMP-7 and NMP-139, β-carboline derivatives NMP-140 and NMP-141 and γ-carboline derivative NMP-144 were selected and used in this study.
Figure 2
Figure 2
Inhibition of T-type calcium channels by NMP compounds. . (A) The histogram summarizes the inhibitory effects of 10 μM NMP compounds on three subtypes of recombinant T-type calcium channels expressed in tsA-201 cells. The number of cells that have been tested is indicated on the top of the bars. (B) Further characterization of compounds NMP-4, NMP-7 and NMP-144 on Cav3.2 channels. The IC50 values obtained from the fit to the dose response relation were 2.47 μM, 1.84 μM 5.59 μM, respectively, for Cav3.1, Cav3.2 and Cav3.3. Data represent the mean ± SEM from 3 - 8 cells of each concentration of compounds. (C) Representative traces from a single cell showing the inhibitory effects of NMP-144 on Cav3.2 with different concentrations and recovery upon washout.
Figure 3
Figure 3
Steady-state inactivation curves obtained from Cav3.2 channels before and after application of 10 μM NMP-4 (A), NMP-7 (B) or NMP-144 (C). The half-inactivation potentials before and after the treatment with drugs were as follows: NMP-4 -52.9 ± 1.4 mV and -72.9 ± 4.7 mV (n = 4, P < 0.01, paired t test); NMP-7: -50.5 ± 0.6 mV and -63.3 ± 3.5 mV (n = 4, P < 0.05, paired t test); NMP-144: -53.7 ± 1.6 mV and -55.8 ± 2.2 mV (n = 4, P > 0.05, paired t test).
Figure 4
Figure 4
Effect of NMP-4 administered by i.t. (A and B) or i.pl. (C and D) (co-administered with formalin) routes on the first (A, C) and second (B, D) phases of formalin-induced pain. Each bar represents the mean responses from 4-7 animals and the error bars indicate the S.E.M. Control values (indicated by "C") are from animals injected with 5% of DMSO and the asterisks denote the significance relative to the control group. *P < 0.05, **P < 0.01, ***P < 0.001. (one-way ANOVA followed by Dunnett' test).
Figure 5
Figure 5
Effect of NMP-144 administered by i.t. (A and B) or i.pl. (C and D) (co-administered with formalin) routes on first (A, C) and second (B, D) phases of formalin-induced pain. Each bar represents the mean response of 4-6 animals and the error bars indicate the S.E.M. Control values (indicated by "C") are from animals injected with 5% of DMSO and the asterisks denote the significance relative to the control group. *P < 0.05, **P < 0.01. (one-way ANOVA followed by Dunnett' test).
Figure 6
Figure 6
Effect of NMP-7 administered by i.t. (A and B), or i.pl. (C and D) (co-administered with formalin) routes on first (A, C) and second (B, D) phases of formalin-induced pain. Each bar represents the mean response from 4-7 animals and the error bars indicate the S.E.M. Control values (indicated by "C") are from animals injected with 5% of DMSO and the asterisks denote the significance relative to the control group. *P < 0.05, **P < 0.01, ***P < 0.001. (one-way ANOVA followed by Dunnett' test).
Figure 7
Figure 7
Effect of i.pl. pre-treatment with NMP-4 (0.03 μg/i.pl.) on the antinociceptive effect of NMP-7 (1 μg/i.pl.) on first phase of formalin-induced pain in mice (A). Each bar represents the mean response from 5-7 animals and the error bars indicate the S.E.M. Vehicle values are from animals injected with PBS solution with 5% of DMSO. The asterisks denote the significance level: *P <0.05 (two-way ANOVA followed by the Newman-Keuls' test). B, C. Effect of systemically delivered NMP-7 (1 mg/kg, i.p.) on both phases of formalin induced pain. Each bar represents the mean response from 5-6 animals and the error bars indicate the S.E.M. Control values (indicated by "C") are from animals injected with 5% of DMSO and the asterisks denote the significance relative to the control group. *P < 0.05, **P < 0.01, ***P < 0.001. (one-way ANOVA followed by Dunnett' test).
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