NMP-7 inhibits chronic inflammatory and neuropathic pain via block of Cav3.2 T-type calcium channels and activation of CB2 receptors

Abstract

Background: T-type calcium channels and cannabinoid receptors are known to play important roles in chronic pain, making them attractive therapeutic targets. We recently reported on the design, synthesis and analgesic properties of a novel T-type channel inhibitor (NMP-7), which also shows mixed agonist activity on CB1 and CB2 receptors in vitro. Here, we analyzed the analgesic effect of systemically delivered NMP-7 (intraperitoneal (i.p.) or intragstric (i.g.) routes) on mechanical hypersensitivity in inflammatory pain induced by Complete Freund's Adjuvant (CFA) and neuropathic pain induced by sciatic nerve injury.

Results: NMP-7 delivered by either i.p. or i.g. routes produced dose-dependent inhibition of mechanical hyperalgesia in mouse models of inflammatory and neuropathic pain, without altering spontaneous locomotor activity in the open-field test at the highest active dose. Neither i.p. nor i.g. treatment reduced peripheral inflammation per se, as evaluated by examining paw edema and myeloperoxidase activity. The antinociception produced by NMP-7 in the CFA test was completely abolished in CaV3.2-null mice, confirming CaV3.2 as a key target. The analgesic action of intraperitoneally delivered NMP-7 was not affected by pretreatment of mice with the CB1 antagonist AM281, but was significantly attenuated by pretreatment with the CB2 antagonist AM630, suggesting that CB2 receptors, but not CB1 receptors are involved in the action of NMP-7 in vivo.

Conclusions: Overall, our work shows that NMP-7 mediates a significant analgesic effect in a model of persistent inflammatory and chronic neuropathic pain by way of T-type channel modulation and CB2 receptor activation. Thus, this study provides a novel therapeutic avenue for managing chronic pain conditions via mixed CB ligands/T-type channel blockers.

Figure 1

Chemical structure of NMP-7.

Figure 2

Effect of intraperitoneal (A, C) or intragastric (B) treatment with NMP-7 on inflammatory pain and neuropathic pain. Each point represents the mean ± SEM responses of 6–8 mice in Panels A, B and 6–12 mice in Panel C. Asterisks indicate significance relative to vehicle-treated (PBS +5% DMSO) control group. *p < 0.05, **p < 0.01 and ***p < 0.001 when compared to vehicle-treated controls, ^### p < 0.001 when comparing vehicle-treated control to sham group (Two-way ANOVA followed by a Tukey’s test).

Figure 3

Effect of intraperitoneal (A) and intragastric (B) NMP-7 treatment in the open field test. Bars represent means ± SEM of total number of crossings of 10–14 animals. Control values (black bars) represent vehicle-treated group (PBS +5% DMSO) (Student’s t-test, ns = non-significant).

Figure 4

Effect of intraperitoneal (A, C) or intragastric (B, D) treatment with NMP-7 on tissue myeloperoxidase (MPO) activity (Panel A and B) and paw volume (Panel C and D). Bars represent mean ± SEM MPO units per mg of tissue (Panel A, B) and mean ± SEM of paw volumes (Panel C, D) of 7–10 mice when NMP-7 was delivered i.p. and 4–5 mice when delivered i.g. ^### p < 0.001 when compared to the non-inflamed group (20 μl of PBS injected intraplantarily), ns = non significant relative to the inflamed vehicle-treated controls (PBS +5% DMSO).

Figure 5

Time-dependent effect (A) and bar representation (B) of NMP-7 delivered intraperitoneally to Ca _V 3.2-null mice on inflammatory pain. Each point (Panel A) represents the time dependent mean ± SEM of mechanical withdrawal thresholds of 7–14 wild type or Ca_V3.2 null mice. Bars (Panel B) represent mean ± SEM responses of mechanical withdrawal threshold measurements taken 30 minutes after i.p. treatment of wild type and Ca_V3.2 null mice with vehicle (PBS +5% DMSO, black bars) or NMP-7 (white bars) (n = 7-14). Asterisks indicate significance relative to vehicle control group, ***p < 0.001, ns = non significant (Three-way ANOVA for panel A and Two-way ANOVA for panel B, followed by a Tukey’s test).

Figure 6

Effect of pre-treatment of mice with selective CB ₂ (A) and CB ₁ (B) antagonists on the analgesic action of NMP-7. Each bar represents mean ± SEM responses of 5–9 mice. Asterisks denote significance relative to vehicle-treated controls (PBS +5% DMSO), *p < 0.05, **p < 0.01 , ***p < 0.001. ^# p < 0.05, ^### p < 0.001 and ns = non significant when compared to antagonist-treated groups (Two-way ANOVA, followed by a Tukey’s test).