Brain Penetrant, but not Peripherally Restricted, Synthetic Cannabinoid 1 Receptor Agonists Promote Morphine-Mediated Respiratory Depression

Abstract

Introduction:

Cannabis acceptance and use continues to rise despite the gaps in knowledge regarding the mechanisms of cannabinoids and the endocannabinoid system in many physiological functions, including respiratory influence.

Methods:

With recent evidence of cannabinoid receptor 1 (CB1R) presence in the collection of respiratory neurons in the brainstem, as well as in the peripheral lung tissue, it is vital that the mechanisms involved in central and peripheral CB1R modulation of respiratory function be delineated. In this study we sought to define the roles of central versus peripheral CB1R activation on respiratory depression alone and in combination with morphine using whole body plethysmography.

Results:

We show that the peripherally restricted CB1 agonist (4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine [PrNMI] 0.3, 0.6, and 1 mg/kg) does not induce respiratory depression, while our previous studies showed that a central penetrating synthetic cannabinoid does induce respiratory depression. Significantly, the combination of morphine with the peripheral CB1 agonist, PrNMI, attenuated morphine-induced respiratory depression.

Conclusions:

These studies support that a peripherally restricted CB1R agonist may be a unique strategy to attenuate the respiratory depression associated with opioid therapy.

Introduction

Cannabis use continues to increase with more legalization across the United States. Yet, legal barriers and restrictions on cannabis research have kept scientists from being able to fully understand the mechanisms influenced by cannabinoids,¹ including the influence on respiratory function. Recent evidence has shown that cannabis and cannabinoids may substitute as viable analgesics, on their own² or in combination with opioids.^3–5 Pre-clinical studies have indicated that cannabinoid receptor 1 (CB1R) activation induces pain relief,^2,6 and clinical studies have shown that CB1R activation is accompanied by an increase in quality of life for the patient.⁷ Furthermore, the impact of cannabinoid and opioid combinations on respiratory function remains understudied.⁸ While fatalities from cannabis overdoses have not been reported,¹ complete understanding of the mechanisms involved in resulting adverse effects, including changes in respiration, from synthetic cannabinoid use is not well understood.

Synthetic cannabinoids, such as the selective agonist WIN 55,212-2 and other synthetic cannabis-like products such as K2 or spice, have reportedly produced respiratory depression in both animals and humans.^9–12 These commonly used brain penetrant synthetic cannabinoids have been shown to influence respiratory function by increasing airway resistance,⁹ decreasing blood pressure, and circulating noradrenaline, resulting in sympathetic inhibition and increasing vagal tone.^11,13 Inhibition of the excitatory effects of noradrenaline in the airways using synthetic cannabinoids can suppress cough and bronchospasms, which may provide an explanation for respiratory depression through vagal transmission.¹⁴ Yet, no studies have looked at receptor specific mechanism(s) (i.e., type, as well as location) for synthetic cannabinoid influence on respiration.

In addition, synthetic cannabinoids have been associated with a variety of other adverse effects such as tachycardia, paranoia, acute kidney injury, seizures, nausea, and vomiting¹⁵ that have resulted in over 2600 calls to the poison control hotline during 2016 and more than 28,000 emergency room visits during 2011,¹⁶ as well as death.^15,17–19 Often these synthetic cannabinoids are potent agonists at the CB1R,²⁰ but since many phytocannabinoids, as well as mixed cannabinoid agonists, do not induce respiratory depression,⁸ understanding how CB1R activation drives respiratory depression is vital to ensuring safe consumption of these opioid adjuncts.

This investigation explored the role of selective CB1R activation peripherally versus centrally on respiratory activity in vivo. Studies utilized both peripherally restricted and brain penetrant selective CB1R agonists alone and in coadministration with morphine using an acute dosing paradigm in mice. Data suggest that central nervous system activation of CB1Rs induces respiratory suppression, whereas peripheral activation of CB1Rs does not. Most excitingly, peripheral activation of CB1Rs mitigated morphine-induced respiratory depression and may provide a therapeutic strategy for widening the safety profile of opioid therapies.

Materials and Methods

Animals

Male CD1 mice weighing 25–35 g were obtained from Charles River Laboratories (Wilmington, MA). All procedures conform to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the University of Arizona Animal Care and Use Committee (Approval 06-110). Animals were provided with food and water ad libitum and maintained in a climate-controlled room on a 12-h light/12-h dark cycle. A total of 96 animals were used to complete these experiments.

Drugs

Morphine sulfate, a mu opioid receptor (MOR) agonist (K_i=1.1 nM²¹), was obtained from the National Institute of Drug Abuse drug supply program (Rockville, MD). AM356 (CB1R K_i=37 nM, CB2 K_i > 1000 nM)^22,23 was gifted by Dr. Alexandros Makryiannis (Boston, MA). 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine (PrNMI), a peripherally restricted CB1 agonist (CB1R K_i=1.18 nM, CB2R K_i=1 nM),²⁴ was provided by Dr. Igor Spigelman (UCLA, Los Angeles, CA). SR141716A, a CB1 inverse agonist (CB2 K_i=>1000 nM, CB1 K_i=5.6 nM),²⁵ was obtained from Cayman Chemical (Ann Arbor, MI). All drugs were dissolved in a vehicle solution of 10% dimethyl sulfoxide, 10% Tween-80, and 80% saline (Sigma, St Louis, MO) and injected intraperitoneally (i.p.) at a volume of 10 mL/kg.

Respiratory depression

Respiratory depression was measured in freely moving conscious mice using whole body plethysmography chambers (Data Sciences International, St Paul, MN). Chambers were maintained at room temperature, and flow and composition of the gas were set by mass flow controllers. Vehicle (10% dimethyl sulfoxide [DMSO], 10% Tween-80, 80% Saline), morphine (10 mg/kg), PrNMI (0.3 mg/kg), PrNMI (0.6 mg/kg), PrNMI (1 mg/kg), PrNMI (0.3 mg/kg)+morphine (10 mg/kg), PrNMI (0.6 mg/kg)+morphine (10 mg/kg), PrNMI (1 mg/kg)+morphine (10 mg/kg), AM356 (1 mg/kg)+morphine (10 mg/kg), or AM356 (10 mg/kg)+morphine (10 mg/kg) was administered i.p. at time 0 min following a 30 min baseline.

Mice remained in the chambers following injection for a 7-min 0% carbon dioxide (CO₂) room air reading, followed by a 7-min 5% concentration of CO₂/oxygen mixture challenge to uncover possible respiratory compensation mechanisms that are associated with hypercapnia.^8,26 Animals administered the CB1R inverse agonist, SR141716A (10 mg/kg), followed the completion of the initial paradigm with additional 0% CO₂ and 5% CO₂ readings repeated. Minute ventilation, tidal volume, and respiratory rate were recorded for each condition; baseline, room air, and 5% CO₂ concentration challenge.

Statistical analysis

GPower 3.1 was used to determine the numbers needed for statistical power (80%) for all experiments. GraphPad Prism 8.0 software was used to analyze for statistical significance. All data were expressed as mean±standard error of mean. A multiple comparisons two-way analysis of variance (ANOVA) was conducted to analyze differences between groups to assess for respiratory depression, with a Bonferroni test applied post hoc.

Results

Peripheral CB1R influence on respiratory function

Supporting our previous studies⁸ that CB1R activation mediated the observed respiratory adverse events, we determined if respiratory depression was driven by activation of the peripheral and/or central CB1 receptors through administration of a peripherally restricted CB1 agonist, PrNMI (0.3, 0.6, 1 mg/kg, i.p., n=7–8/group) (Fig. 1).

FIG. 1.

Peripherally restricted CB1 agonism does not induce respiratory depression. PrNMI 0.3, 0.6, and 1 mg/kg did not significantly decrease frequency of respirations under either condition. CB1, cannabinoid 1; PrNMI, 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine. Color images are available online.

A multiple comparisons two-way ANOVA was conducted to compare the effect of PrNMI to vehicle on respiratory depression in room air and a CO₂ challenge condition. PrNMI 0.3 mg/kg, p=0.1375, 0.6 mg/kg, p=0.1199, and 1 mg/kg, p=0.9996, did not significantly decrease frequency of respirations, or breaths per minute, under the room air condition or 5% CO₂ challenge: PrNMI 0.3 mg/kg, p=0.9289, 0.6 mg/kg, p=0.8771, and 1 mg/kg, p=0.9994, [F(12, 264)=16.60, p<0.0001]. These doses were chosen to represent the therapeutic window of peripheral restriction with 0.3 and 0.6 mg/kg and the beginning brain penetrance at 1 mg/kg.^2,24,27 These data suggest that central, rather than peripheral, CB1R activation underscores the observed respiratory depression seen with selective synthetic agonists as observed previously.⁸

Actions of CB1R inverse agonists on peripheral CB1R influence on respiratory depression

To further evaluate the role of CB1R activation on respiratory function, a CB1R inverse agonist was administered following completion of the initial timeline for PrNMI 0.6 mg/kg administration (Fig. 2). A multiple comparisons two-way ANOVA was conducted to compare the effect of SR-141716A following PrNMI 0.6 mg/kg administration to vehicle on respiratory function. PrNMI 0.6 mg/kg followed by SR-141716A 10 mg/kg did not significantly decrease frequency of respirations under room air conditions, p=0.0690, or under a 5% CO₂ challenge, p>0.9999, [F(3, 66)=19.16, p<0.0001], suggesting that tonic activation of the peripheral CB1R is not influencing basal respiratory rates.

FIG. 2.

Respiration influence by a CB1 inverse agonist. Administration of the selective CB1R inverse agonist SR141718A (10 mg/kg) after treatment with PrNM1 (0.6 mg/kg) did not induce respiratory depression or impact respiratory function. CB1R, cannabinoid receptor 1. Color images are available online.

Central CB1R agonism and morphine-induced respiratory depression

As cannabinoids and opioids are commonly used together, the brain penetrant CB1 agonist, AM356, at doses of 1 and 10 mg/kg were evaluated in combination with morphine to determine if morphine-induced respiratory depression was altered following coadministration (Fig. 3). A multiple comparisons two-way ANOVA was conducted to compare the effect of the central CB1 agonist, AM356, 1 and 10 mg/kg, combination with morphine (10 mg/kg, i.p.) to morphine on respiratory depression in room air and CO₂ challenge conditions. AM356 1 mg/kg in combination with morphine 10 mg/kg did significantly decrease frequency of respirations, or breaths per minute, under the room air condition, p<0.0001, compared to morphine, but not under the 5% CO₂ challenge, p=0.4282 compared to morphine. AM356 10 mg/kg in combination with morphine 10 mg/kg significantly decreased breaths per minute under room air conditions, p<0.000, compared to morphine, but not in the 5% CO₂ condition, p=0.7312 compared to morphine. These data support that the brain penetrant CB1R agonist, AM356, does induce respiratory depression, as well as enhances respiratory depression in combination with morphine under room air conditions.

FIG. 3.

Brain penetrant CB1 receptors enhance opioid induced respiratory depression. AM356 (1 mg/kg) and morphine (10 mg/kg) significantly decreased breaths per minute under both conditions compared to morphine alone. AM356 10 mg/kg in combination with morphine 10 mg/kg also significantly decreased breaths per minute under both air conditions. Color images are available online.

Peripheral CB1R agonism and morphine-induced respiratory depression

To determine if the results of CB1R agonism were consistent regardless of receptor location, PrNMI, 0.3, 0.6, and 1 mg/kg were administered in combination with morphine (10 mg/kg, i.p., n=7–12/group; Fig. 4). A multiple comparisons two-way ANOVA was conducted to compare the effect of PrNMI 0.3, 0.6, and 1 mg/kg combinations with morphine (10 mg/kg, i.p.) to morphine alone on respiratory depression in both conditions. PrNM1 0.3 and 0.6 mg/kg in combination with morphine 10 mg/kg attenuated morphine induced respiratory suppression under room air conditions, 0.3 mg/kg p<0.0001, 0.6 mg/kg p=0.0200, 1 mg/kg p=0.7462, and CO₂ challenge conditions, 0.3 mg/kg p<0.0001, 0.6 mg/kg p=0.025, 1 mg/kg p=0.4429, compared to morphine, [F(5, 88)=3.246, p=0.0098]. Together, these data suggest that peripheral activation of CB1Rs prevents morphine-induced respiratory depression, whereas central CB1R agonism exacerbates opioid induced respiratory depression.

FIG. 4.

Peripheral CB1 receptors mitigate opioid induced respiratory depression. PrNMI (0.3 and 0.6 mg/kg) in combination with morphine (10 mg/kg) attenuated morphine-induced respiratory suppression, while PrNMI (1 mg/kg) coadministered with morphine (10 mg/kg) did not. Color images are available online.

Discussion

There are no reports of fatal cannabis overdose compared to hundreds of thousands who have succumbed to a lethal accidental opioid overdose. Fatal opioid overdoses are typically attributed to respiratory depression. Cannabis or cannabinoid compounds have been proposed as a potential solution to reduce opioid related harms, for example, through reduced opioid consumption, for review see Okusanya et al.,²⁸ Wiese and Wilson-Poe,²⁹ but the endocannabinoid mechanisms, specifically selective CB1R activation, on respiratory function have not been clearly delineated. Given the previously published data from our laboratory demonstrating the effect of tetrahydrocannabinol (THC) 10 mg/kg decreasing respirations during the CO₂ challenge condition only,⁸ other phytocannabinoids, such as cannabidiol (CBD) 10 mg/kg, did not reduce respirations under room air, 86.86% of baseline, p=0.0924, or the CO₂ condition, 87% of baseline, p>0.9999 (data not shown), suggests that not all cannabinoids will act the same on respiratory function. Moreover, previous studies suggest that the CB1R must play some role in cannabinoid-induced respiratory suppression since the highly selective, brain penetrable CB1R agonist, AM356, significantly decreased the frequency of respirations under both tested conditions and was reversed by systemic administration of the CB1 inverse agonist, SR-141716A, but not by the CB2 inverse agonist, SR-144528.⁸

To better understand the individual roles of central versus peripheral CB1R activation on respiration and morphine-induced respiratory depression, in the present studies the CB1R was pharmacologically manipulated using selective CB1R agonists, alone and in combination with morphine with in vivo effects monitored using whole body plethysmography. Application of the peripherally restricted CB1R agonist, PrNMI, did not induce respiratory depression and was not impacted by the administration of a CB1 inverse agonist, which is contrary to our previous findings, where the administration of the CB2 inverse agonist, SR-144528, alone induced respiratory depression under both conditions.⁸ Most excitingly, data here indicated that PrNMI prevents morphine-induced respiratory depression when coadministered at doses of 0.3 and 0.6 mg/kg.²

Interactions between the endocannabinoid and opioidergic systems are widespread from reward enhancement, mitigation of opioid withdrawal symptoms,^30–36 analgesic synergy, and their colocalization in many regions of the brain.²⁹ This interconnected relationship and vast distribution of CB1Rs and MORs^30,37–43 provide clear evidence to suggest interfaces between the opioid and cannabinoid systems as a strategy for potential therapeutics. With the similarities in action of CB1Rs and MORs to reduce the neuron ability to depolarize,^44,45 it is understandable that selective central CB1R activation with AM356 induces reductions in respiratory rates,^9,11,46 but with the location of CB1Rs to only be found presynaptically, compared to MORs that are found on both pre- and postsynaptic terminals,^30,40,47 explaining why fatal respiratory depression does not result from central CB1R activation compared to MOR activation in this region.

Furthermore, the peripherally restricted CB1 agonist, PrNMI, has been shown to induce the cannabinoid tetrad² at the 1 mg/kg dose, suggesting that at this dose it crosses the blood–brain barrier, explaining the observations that this increased dose did not prevent morphine-induced respiratory depression. Additional research has suggested PrNMI to have a possible affinity for the CB2R as well²⁴ suggesting possible promiscuity of the compound that could be influencing our results, but given that the administration of the CB1 agonist is peripherally restricted based on studies performed by Zhang et al., we do not believe this compound to be activating central CB2Rs. CB1Rs have been found in the periphery on bronchiolar smooth muscle cells, nerve fibers that innervate bronchioles,¹⁴ termini of lung tissue,^14,48 and vagal afferents¹³ that all feed forward messages to the central nervous system to influence respirations using the vagal and glossopharyngeal nerves,^{9,11,13,14,48} suggesting that peripheral CB1Rs may play a homeostatic role in respiratory modulation and bronchial contractility.¹⁴ This explanation of peripheral CB1Rs ability to promote homeostatic respiratory activity may account for the observations that PrNMI prevented opioid-induced respiratory depression at the 0.3 and 0.6 mg/kg dose, while the 1 mg/kg dose did not.

This study does have limitations. The respiratory chambers are not the animals' home cage which could create heightened alertness or stress to a new environment. To control for this potential stressor, an extended baseline period was used to allow for time to habituate adequately to the new chamber, and vehicle treated animals to determine controls were performed in all studies. Furthermore, this study was performed under acute administration conditions. Therefore, it is critical to assess the impact of chronic cannabinoid and opioid administration on these interactions. In addition, these studies were just the beginning of our investigation and were conducted in male mice. These findings will need to be investigated in female mice to look for possible sex differences that could exist. Finally, it is unknown if this administration of cannabinoids alongside opioids could also aid in the opioid sparing effect that is seen across other cannabinoid studies citing cannabinoids as a potential exit strategy to opioid use.^4,49,50

Clinical significance

With the increase in individuals utilizing cannabis, and all the evidence supporting cannabis and cannabinoids, as adjunct to current opioid therapies (for review see Wiese and Wilson-Poe²⁹), it is important that we delineate the influence of cannabinoids and the endocannabinoid system on respiratory function when administered alone and/or in combination with opioids. Studies conducted here showed that central mediated CB1R activation is necessary to induce respiratory depression and can enhance morphine-induced respiratory depression when coadministered. Furthermore, peripherally restricted CB1R activation does not induce respiratory depression and when coadministered with morphine was able to mitigate morphine-induced respiratory depression.

Conclusions

As cannabis legalization persists, research into cannabinoids and the endogenous cannabinoid system has illuminated gaps in our understanding of how the endocannabinoid system influences physiological mechanisms, especially respiratory function. These studies provide additional understanding for how selective synthetic cannabinoids, alone and in combination with morphine, influence respirations, respectively. The brain penetrant synthetic cannabinoid, AM356, further suppressed morphine-induced respiratory depression supporting that central CB1R activation can suppress respiratory function.

More importantly, studies here identified that a peripherally restricted CB1R agonist, PrNMI, previously shown to inhibit pain² did not induce respiratory depression and significantly attenuated morphine-induced respiration at therapeutic doses in the CD1 male mouse model. These data provide evidence that distinct targeting of peripheral CB1Rs is a potential method of reducing opioid related harms while maintaining or increasing analgesic efficacy. In conclusion, central and peripheral CB1R activation play opposing roles in regulating respirations, which should be considered in both mechanistic evaluations and therapeutic discoveries moving forward.

Abbreviations Used

ANOVA

analysis of variance

CB1

cannabinoid 1

CB1R

cannabinoid receptor 1

CBD

cannabidiol

CO₂

carbon dioxide

CPAC

Comprehensive Pain and Addiction Center

DMSO

dimethyl sulfoxide

i.p.

intraperitoneally

MOR

mu opioid receptor

PrNMI

4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3yl]ethyl}morpholine

THC

tetrahydrocannabinol

Authors' Contributions

Project was conceptualized by T.W.V., T.M.L.-M., and B.M.W. Experiments were performed by B.M.W. and S.A.C.; compounds were developed and supplied by S.P.N., L.J., Y.L., A.M., and I.S. Writing was performed by B.M.W. with revisions offered by S.P.N., E.L.-B., I.S., T.W.V., and T.M.L.-M.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Supported by grant funding from the National Institutes of Health/National Institute of Drug Abuse to T.W.V. (1P01DA041307-01), National Cancer Institute (R01CA142115-02), departmental funding from the University of Arizona Medical Pharmacology, and with support of the Comprehensive Pain and Addiction Center (CPAC) at the University of Arizona.

Cite this article as: Wiese BM, Liktor-Busa E, Couture SA, Nikas SP, Ji L, Liu Y, Makriyannis A, Spigelman I, Vanderah TW, Largent-Milnes TM (2022) Brain penetrant, but not peripherally restricted, synthetic cannabinoid 1 receptor agonists promote morphine-mediated respiratory depression, Cannabis and Cannabinoid Research 7:5, 621–627, DOI: 10.1089/can.2021.0090.