CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity

Abstract

While the opioid crisis has justifiably occupied news headlines, emergency rooms are seeing many thousands of visits for another cause: cannabinoid toxicity. This is partly due to the spread of cheap and extremely potent synthetic cannabinoids that can cause serious neurological and cardiovascular complications-and deaths-every year. While an opioid overdose can be reversed by naloxone, there is no analogous treatment for cannabis toxicity. Without an antidote, doctors rely on sedatives, with their own risks, or 'waiting it out' to treat these patients. We have shown that the canonical synthetic 'designer' cannabinoids are highly potent CB1 receptor agonists and, as a result, competitive antagonists may struggle to rapidly reverse an overdose due to synthetic cannabinoids. Negative allosteric modulators (NAMs) have the potential to attenuate the effects of synthetic cannabinoids without having to directly compete for binding. We tested a group of CB1 NAMs for their ability to reverse the effects of the canonical synthetic designer cannabinoid JWH018 in vitro in a neuronal model of endogenous cannabinoid signaling and also in vivo. We tested ABD1085, RTICBM189, and PSNCBAM1 in autaptic hippocampal neurons that endogenously express a retrograde CB1-dependent circuit that inhibits neurotransmission. We found that all of these compounds blocked/reversed JWH018, though some proved more potent than others. We then tested whether these compounds could block the effects of JWH018 in vivo, using a test of nociception in mice. We found that only two of these compounds-RTICBM189 and PSNCBAM1-blocked JWH018 when applied in advance. The in vitro potency of a compound did not predict its in vivo potency. PSNCBAM1 proved to be the more potent of the compounds and also reversed the effects of JWH018 when applied afterward, a condition that more closely mimics an overdose situation. Lastly, we found that PSNCBAM1 did not elicit withdrawal after chronic JWH018 treatment. In summary, CB1 NAMs can, in principle, reverse the effects of the canonical synthetic designer cannabinoid JWH018 both in vitro and in vivo, without inducing withdrawal. These findings suggest a novel pharmacological approach to at last provide a tool to counter cannabinoid toxicity.

Keywords: JWH018; antidote; cannabinoid toxicity; overdose; synthetic cannabinoid.

Figure 1

Negative allosteric modulators of CB1 receptors and their potential to reverse acute synthetic designer cannabinoid-mediated toxicity. (A) Competitive antagonists compete directly at the orthosteric site. (B) Powerful synthetic “spice-class” cannabinoids may bind tightly to the orthosteric site and strongly activate the receptor. This tight binding may prevent a competitive antagonist from reversing the effect (indicated by red cross). (C) A negative allosteric modulator targets a secondary ‘allosteric’ site, reducing the activity of the receptor (red crosses) without directly competing at the orthosteric site.

Figure 2

Structure of JWH-018 progenitor synthetic designer cannabinoid.

Figure 3

A high concentration of PSNCBAM1 reverses JWH018 inhibition of neurotransmission. (A) Sample time showing inhibitory effect of JWH018 (40 nM) and partial reversal by PSNCBAM1. (B) Summary bar graph shows a partial reversal of JWH018 (40 nM) effect on EPSCs (circles) by PSNCBAM1 at 10 μM (triangles), but not 2 μM (squares). ns, not significant; *, p < 0.05, by one-way ANOVA with Dunnett’s post hoc vs. JWH018.

Figure 4

ABD1085 inhibits DSE and reverses JWH018 effects on neurotransmission in autaptic neurons. (A) Negative allosteric modulator ABD1085 has no effect on EPSCs at 1 μM. (B) ABD1085 (squares) partly blocks DSE (circles) at 1 μM. (C) Sample DSE time courses after treatment with increasing concentrations of ABD1085. (D) Sample time course shows a full reversal of JWH018 (40 nM) effect on EPSCs by ABD1085 (1 μM). (E) Summary graph shows EPSC inhibition by JWH018 (squares) and reversal by ABD1085 (1 μM, triangles). *, p < 0.05 by paired t-test. **, p < 0.01 by paired t-test.

Figure 5

RTICBM189 inhibits DSE and reverses JWH018 effects on neurotransmission in autaptic neurons. (A) Negative allosteric modulator RTICBM189 has no effect on EPSCs at 1 μM. (B) RTICBM189 does not block DSE (circles) at 100 nM (squares), but does at 1 μM (triangles). (C) Sample DSE time courses before and after treatment with RTICBM189. (D) Sample time course shows a full reversal of JWH018 (40 nM) effect on EPSCs by RTICBM189 (1 μM). (E) Summary graph shows EPSC inhibition by JWH018 (circles) and reversal by RTICBM189 (1 μM, squares). ns, not significant; **, 0 < 0.01 one-way ANOVA with Dunnett’s post hoc test (B), by paired t-test (D).

Figure 6

CB1 NAMs vs. JWH018 effects on nociception. (A) Dose response for JWH018 in tail flick assay. (B) Time course shows that pretreatment with RTICBM189 at 10 mg/kg blocks the effect of JWH018 (0.5 mg/kg). (C) ABD1085 is ineffective at 10 mg/kg. (D) Pretreatment with PSNCBAM1 blocks effect of JWH018 at 4 and 10 mg/kg. (E) PSNCBAM1 (10 mg/kg) is still effective when co-treated with JWH018 (0.5 mg/kg). (F) PSNCBAM1 (10 mg/kg) rapidly reverses the effect of JWH018 when applied 45 min after JWH018. *, p < 0.05, **, p < 0.01, ***, p < 0.005, two-way ANOVA with Bonferroni post hoc test.

Figure 7

PSNCBAM1 does not induce withdrawal in JWH018-treated mice. Mice treated for 7 days with daily JWH018 injections (0.5 mg/kg) did not see withdrawal symptoms 45 min after treatment with PSNCBAM1 (10 mg/kg). A subset of mice subsequently treated with the competitive antagonist SR141716 showed an increase in withdrawal symptoms. ***, p < 0.005, one-way ANOVA with Dunnett’s post hoc test vs. JWH018.