AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner

Abstract

Background and purpose: Cannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies.

Experimental approach: The covalent nature of AM841 was determined in vitro using electrophysiological and receptor internalization studies on isolated cultured hippocampal neurons. Mouse models were used for behavioural analysis of analgesia, hypothermia and hypolocomotion. The motility of the small and large intestine was assessed in vivo under normal conditions and after acute stress. The brain penetration of AM841 was also determined.

Key results: AM841 behaved as an irreversible CB1 receptor agonist in vitro. AM841 potently reduced GI motility through an action on CB1 receptors in the small and large intestine under physiological conditions. AM841 was even more potent under conditions of acute stress and was shown to normalize accelerated GI motility under these conditions. This compound behaved as a peripherally restricted ligand, showing very little brain penetration and no characteristic centrally mediated CB1 receptor-mediated effects (analgesia, hypothermia or hypolocomotion).

Conclusions and implications: AM841, a novel peripherally restricted covalent CB1 receptor ligand that was shown to be remarkably potent, represents a new class of potential therapeutic agents for the treatment of functional GI disorders.

Figure 1

The structure of (-)-7′-isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol – AM841.

Figure 2

AM841 irreversibly inhibits EPSCs and internalizes CB₁ receptors. (A) AM841 (100 nM) robustly inhibited EPSCs in autaptic hippocampal neurons. Time course shows the integral of the EPSCs (charge, in pC) in response to AM841. The inhibition was not reversed by the CB₁ antagonist SR141716 (200 nM). (B) EPSCs at time points 1, 2 and 3 from (A). (C) Summary bar graph showing average EPSC inhibition for AM841 alone and with SR141716 (P > 0.05 paired t-test) as well as inhibition resulting from depolarization-induced suppression of excitation (3 s depolarization) before and after AM841 treatment. n = 5 per group. ***P < 0.001 – paired t-test. (D) SR141716 (100 nM) readily reversed inhibition by the CB₁ agonist WIN55212-2 (100 nM). (E) EPSCs at time points 1, 2 and 3 from (D). (F) Summary bar graph for data from (D) showing average EPSC inhibition for WIN55,212-2 alone and with SR141716. n = 4 per group. **P < 0.01 – paired t-test. (G) Surface CB₁ receptors were detected after incubation with the indicated concentration of AM841 for the indicated times. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline levels of CB₁ receptors before incubation with AM841 – two-way anova with Dunnett's test for multiple comparison. (H) Cell surface CB₁ receptors were measured as in (G). Thirty minutes treatment with 100 nM WIN55,212-2 (WIN-2) or 100 nM AM841 internalized ∼50% of surface CB₁ receptors. SR141716 (100 nM) caused rapid recycling of CB₁ receptors to the cell surface in WIN55,212-2-treated cells but had no effect in AM841-treated cells. n = 12 per group. ***P < 0.001 compared with WIN55,212-2 – two-way anova with Sidak's test for multiple comparison.

Figure 3

The effects of AM841 on locomotion (A), analgesia (B) and thermoregulation (C). (A) AM841 (0.1 and 1 mg·kg⁻¹) produced no significant reduction in locomotion at the doses tested. In contrast, WIN55,212-2 (1 mg·kg⁻¹) significantly reduced locomotor activity compared with the vehicle-treated mice. n = 5–7 per group. **P < 0.01 – one-way anova with Dunnett's test for multiple comparison. (B) AM841 (0.1 mg·kg⁻¹) had no effect on paw withdrawal latency compared with vehicle controls whereas WIN55,212-2 (1 mg·kg⁻¹) significantly increased the time to withdrawal, indicative of analgesia. n = 6–13 per group. *P < 0.05 – one-way anova with Dunnett's multiple comparison. (C) The thermoregulatory effects of AM841 (0.1 mg·kg⁻¹) were tested in wild-type C57Bl/6 mice and CB₁^−/− mice. There was a rapid increase in body temperature on initial injection because of the acute stress of injection; this was seen in both groups of mice. AM841 had virtually no effect on core body temperature over ∼7 h. Inset in (C) is the maximum change in body temperature from baseline (100 min before injection) over the time monitored post-injection (400 min). n = 7–8 per group, P > 0.05 t-test.

Figure 4

The effects of AM841 on small intestinal transit. (A) AM841 and WIN55,212-2 dose-dependently reduced small intestinal transit. Note that AM841 significantly slowed transit at doses as low as 0.001 mg·kg⁻¹. n = 4–21 per group. *P < 0.05,***P < 0.001 – one-way anova with Sidak's multiple comparison, compared with vehicle-treated mice. At equimolar doses (1 mg·kg⁻¹), AM841 was more potent than WIN55,212-2 in slowing small intestinal transit. ##P < 0.01 – one-way anova with Sidak's multiple comparison. (B) The effects of AM841 (1 mg·kg⁻¹) on small intestinal transit in wild-type (WT), CB₁^−/− mice and CB₂^−/− mice. Note that AM841 had no effect in CB₁^−/− mice, whereas motility was reduced in both WT and CB₂^−/− mice to the same extent. n = 6–12 per group. ***P < 0.001 – t-tests for each genotype of mice tested. (C) The effects of CB₁ (AM251) and CB₂ (AM630) receptor antagonists on the actions of AM841. Pretreatment with AM251 completely blocked the effects of 0.1 mg·kg⁻¹ AM841 at 5 mg·kg⁻¹. n = 3–8 per group. ***P < 0.001 between treatment groups indicated. AM251 5 mg·kg⁻¹, but not at a 10-fold lower dose, reversed the effect of AM841 alone. AM630 had no significant effect on the action of AM841, one-way anova with Sidak's multiple comparison.

Figure 5

The effects of AM841 on colonic bead expulsion. (A) AM841 and WIN55,212-2 dose-dependently reduced colonic transit. Note that AM841 significantly slowed transit at doses as low as 0.1 mg·kg⁻¹. n = 6–23 per group. *P < 0.05, ***P < 0.001 compared with vehicle – one-way anova with Sidak's multiple comparison. (B) The time-dependent effects of AM841 on colonic transit were compared with those of WIN55,212-2. Note that at the lower doses (0.1 mg·kg⁻¹ AM841, 0.3 mg·kg⁻¹ WIN55,212-2) WIN55,212-2 had no significant effect on colonic transit, whereas AM841 significantly reduced transit over nearly 4 h. n = 6–26 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle at the same time point – one-way anova with Dunnett's multiple comparison. (C) The effects of AM841 (1 mg·kg⁻¹) on colonic transit in wild-type (WT), CB₁^−/− mice and CB₂^−/− mice. Note that 1 mg·kg⁻¹ AM841 had no effect in CB₁^−/− mice, whereas motility was reduced in both WT and CB₂^−/− mice to a similar extent. n = 5–11 per group. *P < 0.05, ***P < 0.001 compared with vehicle – t-test for each genotype of mice tested. (D) The effects of CB₁ (AM251) and CB₂ (AM630) receptor antagonists on the actions of AM841. Pretreatment with AM251 completely blocked the effects of 0.1 mg·kg⁻¹ AM841 at 2 mg·kg⁻¹. n = 6–21 per group. ***P < 0.001 between groups indicated – one-way anova with Sidak's multiple comparison. AM630 had no significant effect on the action of AM841.

Figure 6

The effects of AM841 on small intestinal transit and colonic bead expulsion in acutely stressed mice. (A) AM841 dose-dependently reduced small intestinal transit in normal and stressed mice. There was a slight leftward shift of the dose-response curve in the stressed mice. n = 3–21 per group. (B) The effects of AM841 (0.001 mg·kg⁻¹) on small intestinal transit in non-stressed and stressed mice in the presence and absence of AM251 (5 mg·kg⁻¹) and AM630 (5 mg·kg⁻¹). Vehicle-treated stressed mice had a significantly increased small intestinal transit when compared with vehicle-treated non-stressed mice. n = 3–24 per group. *P < 0.05 – t-test. Note that at this dose, AM841 had no significant effect in non-stressed mice, but significantly reduced transit in stressed animals. **P < 0.01 compared with vehicle-treated stressed mice – two-way anova with Sidak's multiple comparison. The effects of AM841 were blocked by AM251 but unaffected by AM630. **P < 0.01 compared to vehicle-treated stressed mice – one-way anova with Sidak's multiple comparison. (C) The effects of AM841 on stress-enhanced faecal pellet output. Faecal pellet output was increased ∼3-fold by placing the animals in a novel environment. Pretreatment with AM841 (0.01 mg·kg⁻¹) normalized the stress-enhanced faecal pellet output. n = 10–11 per group. *P < 0.05 between groups as indicated – two-way anova with Sidak's multiple comparison.