Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35 S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35 S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.
Cannabinoid receptor agonists produce antinociception in a variety of animal models, and the majority of these effects appear to be mediated by CB1 receptors. Interactions between cannabinoid and opioid receptor systems remain an area of intense research, especially in light of the mounting importance of identifying safer and more effective pain therapies that may be able to reduce opioid use and associated harms. Acutely, the non-selective CB agonists Δ9-tetrahydrocannabinol (THC) and CP-55,940 have been shown to increase morphine antinociceptive effects (Smith et al., 1998; Manzanares et al., 1999; Finn et al., 2004; Tham et al., 2005; Vigano et al., 2005; Maguire and France 2018). We and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance (Cichewicz et al., 2001; Cichewicz and Welch 2003; Fischer et al., 2010). The CB1 receptor is abundantly expressed throughout the central nervous system and identified as the cannabinoid receptor responsible for the “psychoactive” effects of non-selective cannabinoid agonists such as THC; therefore, it is presumed that these CB agonist effects on morphine tolerance are associated with their actions on the CB1 receptor. However, this remains to be demonstrated empirically.
Relative to CB1 receptors, detection of CB2 receptors in the CNS of naïve animals remains relatively low to absent, and by and large CB2 receptor activation does not lead to the range of CNS effects associated with CB1 receptor activation, such as euphoria, changes in mood, and alterations in cognition. However, CB2 receptor expression is upregulated within the CNS in animal models of chronic inflammatory or neuropathic pain (Zhang et al., 2003; Wotherspoon et al., 2005; Beltramo et al., 2006), and activation of CB2 receptors reverses allodynia and hyperalgesia in these models (Guindon and Hohmann 2008; Rahn et al., 2011). In addition, co-administration of morphine with CB2 receptor selective agonists synergistically inhibits inflammatory, post-operative and neuropathic pain in rodent models (Grenald et al., 2017; Yuill et al., 2017; Iyer et al., 2020) and reduces morphine-induced thermal hyperalgesia in rats (Tumati et al., 2012). While a preponderance of studies has demonstrated that tolerance is associated with a significant reduction in functional surface _µ_ opioid receptors (Williams et al., 2013). Other studies have suggested that morphine tolerance is due at least in part to direct microglial activation and the release of proinflammatory cytokines (Hutchinson et al., 2007, see Hutchinson et al., 2011 for review). Our laboratory has extensively characterized the protective and anti-inflammatory effects of the CB2 receptor agonist O-1966 in several rodent models of CNS injury (Zhang et al., 2007; Adhikary et al., 2011; Elliott et al., 2011; Amenta et al., 2012; Ramirez et al., 2012; Ronca et al., 2015). As CB2 receptor activation has been shown to significantly modulate inflammatory responses, including inhibition of microglial activation, we hypothesized that CB2 receptor activation may lead to attenuation of morphine antinociceptive tolerance.
In the present set of experiments, we tested both the acute and chronic interactions between morphine and O-1966 treatments alone and in combination on antinociception and antinociceptive tolerance and hyperalgesia in C57Bl6 mice using a standard hot plate assay. Based on previous research, we hypothesized that O-1966 would be devoid of acute antinociceptive effects but would attenuate morphine antinociceptive tolerance. Because our first results from our acute hotplate experiments revealed an unpredicted attenuating effect of O-1966 on acute morphine antinociception, we proceeded in these acute studies as well as the tolerance studies to test administration of morphine and O-1966 under three dosing regimens: concurrent administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. Based on the results of these experiments revealing that the order of drug administration had dramatic effects on how these two drugs affected morphine analgesia and analgesic tolerance, we further tested the hypothesis that select interactive effects between O-1966 and morphine were a result of direct effects of this CB2 receptor agonist on the µ opioid receptor. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35 S]GTPγS binding assays. Lastly, as our behavioral data revealed that O-1966 could attenuate morphine antinociception but also facilitate morphine tolerance, we tested the hypothesis that O-1966 was interfering with mu-opioid receptor homodimerizationvia FRET analysis linked to flow cytometry.
For _in vivo_ experiments, O-1966 (Organix Laboratories, Massachusetts, USA) and SR144528 (RTI) were prepared in ethanol:Cremophor:Saline (1:1:18). Morphine was dissolved in 0.9% saline. All injections were given i. p. in a volume of 10 ml/kg. For _in vitro_ experiments, O-1966 and SR144528 were dissolved in DMSO (final concentration 2% in assays) and morphine was dissolved in Milli-Q water. The affinity of O-1966 for CB1 and CB2 cannabinoid receptors was reported previously to be 5055 ± 984 and 23 ± 2.1 nmol/L, respectively (Wiley et al., 2002).
All experiments were conducted in 7 to 8-week-old male C57BL/6 mice weighing 18–23 g (Taconic Laboratories, New York, USA). Studies were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at Temple University. Animals were housed under a 12 h light/dark cycle with lights on at 07:00 h and maintained on a regular chow diet and had access to food and water _ad libitum_ throughout the study. All experimental groups were n = 8/treatment condition.
Nociception was analyzed by means of a hot plate analgesia meter (Columbus Instruments, Columbus, OH). Mice were placed on a hot plate maintained at 54.0 ± 0.5°C. The latency to hind paw lick, hind paw lift, hind paw flutter, mouse shuffle, or mouse jump was measured to the nearest 0.1 s as described in Fischer et al., 2010. A maximal cutoff of 30 s was utilized to prevent injury to the paw tissue. Immediately after the end of the trial, mice were returned to their home cage. The latency to respond at 54°C was measured twice at 2 and 1.5 h prior to the beginning of drug administration, and these data were averaged to yield one baseline value. Following baseline latency measurements, multiple 30 min cycles were run and drugs and drug mixtures were administered cumulatively. During this procedure, cumulative doses of morphine, O-1966, or their combination were administered during the first min of each cycle (i.e., 30-min inter-injection interval), increasing in one-half log unit increments, and antinociceptive measurements were determined during the last minute of each cycle. Latencies obtained following drug administration were reported as Percent Maximal Possible Effect (%MPE). The following formula was utilized to calculate such:
%M P E=(E x p e r i m e n t a l L a t e n c y−A v e r a g e B a s e l i n e L a t e n c y)(M a x i m a l C u t O f f T i m e−A v e r a g e B a s l i n e L a t e n c y)×100
The antinociceptive effects of 1) morphine alone, 2) O-1966 alone, 3) their simultaneous administration, and 4) their simultaneous administration following CB2 antagonist treatment, were assessed in the same group of mice, with a 1 week washout period separating each drug or drug combination testing. In a separate group of mice, the antinociceptive effects of 1) morphine alone, 2) O-1966 administration followed 15 min later by morphine administration, and 3) morphine administration followed 15 min later by O-1966 administration were assessed with a 1-week washout period separating each drug or drug combination testing.
One day following assessment of hot plate withdrawal latencies and the generation of baseline morphine dose-response curves, separate groups of mice were treated twice daily separated by 10 h for 5 days, as described in Fischer et al., 2010, with two vehicle regimens (saline, cremophor vehicle), two morphine alone dosing regimens (32 mg/kg, 100 mg/kg), and three morphine + O-1966 dosing regimens (simultaneous, O-1966 followed 15 min by morphine, morphine followed 15 min by O-1966). Reassessment of hot plate withdrawal latencies and morphine dose response curves began 14 h after the last tolerance regimen injections.
[Tyrosyl-3, 5-3 H(N)]-DAMGO (56 Ci/mmol) and [35 S]GTPγS (1,250 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA); sucrose, bovine serum albumin (BSA), phenylmethylsulfonyl fluoride, GDP and GTPγS were purchased from Sigma-Aldrich (St. Louis, MO). DMEM/F12, trypsin and penicillin/streptomycin were purchased from Gibco Life Technologies (Grand Island, NY). The following reagents were purchased from the indicated companies: geneticin (G418), Cellgro Mediatech, Inc. (Herndon, VA); EcoScint scintillation fluid, National Diagnostics (Atlanta, GA); fetal bovine serum (FBS), Atlanta Biologicals (Atlanta, GA). Naloxone and morphine were generously provided by the National Institute on Drug Abuse (Bethesda, MD).
The following is a modified procedure from Wang et al. (Wang et al., 2005).
