Cannabinoid-2 Agonism with AM2301 Mitigates Morphine-Induced Respiratory Depression

Abstract

Introduction: An escalating number of fatalities resulting from accidental opioid overdoses typically attributed to respiratory depression continue to define the opioid epidemic. Opioid respiratory depression results from a decrease in reflexive inspiration within the preBötzinger complex in the brainstem. Objective: Cannabinoid receptor agonism is reported to enhance opioid analgesia, yet whether cannabinoids enhance or inhibit opioid-induced respiratory depression is unknown. Methods: Studies herein sought to define the roles of cannabinoid-1 receptor (CB1R) and cannabinoid-2 receptor (CB2R) on respiratory depression using selective agonists alone and in combination with morphine in male mice. Results: Using whole body plethysmography, the nonselective CB1R and CB2R agonist (Δ⁹-tetrahydrocannabinol) and the CB1R synthetic cannabinoid, AM356, induced respiratory depression, whereas the well-published selective CB2 agonist, JWH 133, and the novel CB2 agonist (AM2301) did not. Moreover, a selective CB2R agonist (AM2301) significantly attenuated morphine sulfate-induced respiratory depression. Conclusion: Notably, findings suggest that attenuation of opioid-induced respiratory depression relies on CB2R activation, supporting selective CB2R agonism as an opioid adjunct therapy.

Keywords: cannabinoid receptor 1; cannabinoid receptor 2; mu opioid receptor; opioid-induced respiratory depression; preBötzinger complex.

FIG. 1.

Morphine depresses respiration frequency. (A) Morphine dose-dependent suppression of respiration. Data represent minute bins of respiration frequency, or breaths per minute. (B) Morphine AUC suppression of respiration. Data represent additional view of same data from (A) in means of MS. AUC, area under the curve; BL, baseline; BPM, breaths per minute; CO₂, carbon dioxide/oxygen; MS, morphine sulfate; SEM, standard error of the mean. Color images are available online.

FIG. 2.

CB1R/CB2R differentially contribute to respiratory control. (A) THC, JWH 133, and AM2301 do not induce respiratory depression under the room air condition, whereas AM365 does decrease respirations. (B) JWH 133 and AM2301 do not induce respiratory depression, whereas THC and AM365 do decrease respirations in response to the CO₂ challenge condition. CB1R, cannabinoid-1 receptor; CB2R, cannabinoid-2 receptor; THC, Δ-tetrahydrocannabinol. Color images are available online.

FIG. 3.

AM2301 influence on respirations with MS. (A) Attenuation of morphine-induced respiratory depression through CB2 activation. The combination of AM2301 (10 mg/kg) and MS (10 mg/kg) significantly prevented respiratory depression compared with MS, whereas AM2301 (10 mg/kg) failed to decrease 30 or 100 mg/kg MS-induced respiratory depression in either CO₂ condition. Increasing the AM2301 dose (30 mg/kg) failed to decrease 30 mg/kg MS-induced respiratory depression and an increase to 100 mg/kg failed to overcome 100 mg/kg MS-induced respiratory depression in either CO₂ condition as well. (B) Room air respiratory dose–response curves. Dose–response curves for AM2301, MS, and AM2301 10 mg/kg plus escalating doses of MS were developed using this nonlinear regression equation Y=100/(1+10^[{LogEC50-X}×HillSlope]). (C) 5% CO₂ respiratory dose–response curves. Dose–response curves for AM2301, MS, and AM2301 10 mg/kg plus escalating doses of MS were developed using this nonlinear regression equation Y=100/(1+10^[{LogEC50-X}×HillSlope]). Color images are available online.

FIG. 4.

Effects of CB inverse agonists on respirations and reversal of morphine-induced respiratory depression mitigation. (A) Inverse agonist timeline. After completion of the initial timeline, an injection of the inverse agonist is administered and followed by a repeat of the CO₂ conditions to evaluate reversal of the previously administered compound. (B) CB2 inverse agonism induces respiratory depression. SR-144528 10 mg/kg, a CB2 inverse agonist, induced respiratory depression on its own. AM630 10 mg/kg, a CB2 inverse agonist, induced respiratory depression under the 5% CO₂ condition, but not under room air. Administration of SR-144528 10 mg/kg after JWH 133 10 mg/kg was able to induce respiratory depression in both air conditions. (C) MS respiratory depression not impacted by administration of CB2 inverse agonism. Administration of the CB2 inverse agonist, SR-144528 10 mg/kg or AM630 10 mg/kg, after administration of MS (from Fig. 1B) had no impact on the MS-induced respiratory depression. (D) AM356-induced respiratory depression reversed by administration of CB1 inverse agonism. Administration of the CB1 inverse agonist, SR-141718A 10 mg/kg, after administration of AM356 10 mg/kg significantly reversed the respiratory depression induced by AM356 in both CO₂ conditions. Administration of the CB2 inverse agonist, SR-144528 10 mg/kg, after administration of AM356 increased the mean BPM in both CO₂ conditions, but was only significantly higher than AM356 alone in room air. (E) Reversal of AM2301 mitigation of opioid-induced respiratory depression with CB2 inverse agonists. AM630 (10 mg/kg; N=7/group) and SR-144528 (10 mg/kg; N=8/group) significantly reversed the AM2301 mitigation of MS-induced respiratory depression. Color images are available online.

FIG. 5.

Total expression of Mu, CB1R, and CB2R in the preBötzinger. (A) Using qPCR to measure mRNA levels, CNR2 and CNR1 were present in the pBc, as well as OPRM (B) Relative mRNA expression showed the presence of CNR1, CNR2, and OPRM in the PAG. (C) mRNA levels within the spleen were assessed using qPCR, and CNR1, CNR2, and OPRM were all found. (D) Membrane images of the Western blots are analyzed in the next panel. (E) Western blots were used to measure protein levels of MOR, CB1, and CB2 in pBc samples (n=3). Protein expression of MOR and CB1 was compared with PAG-positive control tissue, whereas CB2 was compared with spleen. Protein levels showed a spleen-equivalent concentration of CB2R expression, as well as MOR and CB1R expression. MOR, mu opioid receptor; mRNA, messenger RNA; PAG, periaqueductal gray; pBc, preBötzinger complex; qPCR, quantitative PCR. Color images are available online.

FIG. 6.

AM2301, a brain penetrant CB2R agonist retains antinociceptive and antiallodynic properties in combination with morphine. (A) Tail-flick antinociception. AM2301+MS showed equiefficacious antinociception versus MS alone. All groups returned to baseline tail-flick latencies 120 min postdrug administration with peak drug effect between 45 and 60 min. (B) Tail flick area under the curve. AM2301 in combination with MS showed equal increases in statistical significance in the AUC as MS compared with vehicle or AM2301 alone. (C) Postoperative pain evaluation of AM2301 dose–response after paw incision through von Frey assay. After postinjury baseline, AM2301 (N=6–7/group) demonstrated a significant increase in thresholds. (D) Postoperative pain evaluation of AM2301 area under the curve. Compared with vehicle, AM2301 at all doses showed statistically significant increases in AUC. (E) Postoperative pain evaluation of AM2301+morphine and morphine alone after paw incision through von Frey assay. MS administration (N=6) significantly reversed postoperative mechanical hypersensitivity. A combination of MS (10 mg/kg) and AM2301 (10 mg/kg; N=6) retained antihypersensitive effects for the testing duration. (F) Postoperative pain evaluation of AM2301+morphine and morphine alone area under the curve. Compared with vehicle from (D), the combination AM2301 with MS and MS alone showed statistically significant increases in AUC. (G) Reversal of AM2301 antinociception with a CB2 inverse agonist. Pretreatment with a CB2R inverse agonist, SR-144528, (10 mg/kg), 10 min before AM2301 alone or in combination with MS, (N=10/group) significantly mitigated the antihyperalgesia of AM2301, yet, the AM2301+MS antihyperalgesia was not significantly prevented. (H) CB2 inverse agonist reversal of AM2301 antinociception area under the curve. Administration of SR-144528 10 mg/kg before drug administration showed a prevention for AM2301-induced antinociception back to postincision baseline while having no impact on the antinociception induced by the combination of AM2301 and MS. Color images are available online.