Select Minor Cannabinoids from Cannabis sativa Are Cannabimimetic and Antinociceptive in a Mouse Model of Chronic Neuropathic Pain
Department of Pharmacology, College of Medicine (A.M.S., D.K., J.A.M., V.M.-R., C.S., T.B., A.W. J.C., J.M.S.) and Comprehensive Center for Pain and Addiction (J.M.S.), University of Arizona, Tucson, Arizona
Abstract
Chronic pain conditions affect nearly 20% of the population in the United States. Current medical interventions, such as opioid drugs, are effective at relieving pain but are accompanied by many undesirable side effects. This is one reason increased numbers of chronic pain patients have been turning to Cannabis for pain management. Cannabis contains many bioactive chemical compounds; however, current research looking into lesser-studied minor cannabinoids in Cannabis lacks uniformity between experimental groups and/or excludes female mice from investigation. This makes it challenging to draw conclusions between experiments done with different minor cannabinoid compounds between laboratories or parse out potential sex differences that could be present. We chose five minor cannabinoids found in lower quantities within Cannabis: cannabinol (CBN), cannabidivarin (CBDV), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabivarin (THCV). These compounds were then tested for their cannabimimetic and pain-relieving behaviors in a cannabinoid tetrad assay and a chemotherapy-induced peripheral neuropathy (CIPN) pain model in male and female CD-1 mice. We found that the minor cannabinoids we tested differed in the cannabimimetic behaviors evoked, as well as the extent. We found that CBN, CBG, and high-dose Δ8-THC evoked some tetrad behaviors in both sexes, while THCV and low-dose Δ8-THC exhibited cannabimimetic tetrad behaviors only in females. Only CBN efficaciously relieved CIPN pain, which contrasts with reports from other researchers. Together these findings provide further clarity to the pharmacology of minor cannabinoids and suggest further investigation into their mechanism and therapeutic potential.
SIGNIFICANCE STATEMENT
Minor cannabinoids are poorly studied ligands present in lower levels in Cannabis than cannabinoids like THC. In this study, we evaluated five minor cannabinoids (CBN, CBDV, CBG, THCV, and Δ8-THC) for their cannabimimetic and analgesic effects in mice. We found that four of the five minor cannabinoids showed cannabimimetic activity, while one was efficacious in relieving chronic neuropathic pain. This work is important in further evaluating the activity of these drugs, which are seeing wider public use with marijuana legalization.
Article notes
Untitled section
Received 2024 Mar 3; Accepted 2024 May 30; Issue date 2024 Nov.
Introduction
Despite the increased use and accessibility of Cannabis, most research investigating the potential therapeutic benefits and drawbacks of this plant has focused on the most abundant cannabinoids: cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). THC is the primary psychotropic compound in Cannabis and has therapeutic effects such as antinausea, appetite stimulation, and pain relief. The psychotropic effects of THC can also lead to increased anxiety, memory impairment, and fatigue (Pagano et al., 2022; Stella, 2023).
However, there are far more chemical constituents in Cannabis than THC and CBD. There are over 550 unique chemical compounds in Cannabis, many of which are bioactive and contribute to its overall effect (Russo and Marcu, 2017; Mudge et al., 2019; Rock and Parker, 2021). Different chemovars of Cannabis elicit varying intensities of behavioral outcomes (Devsi et al., 2020), underlining the need to understand the pharmaceutical effects of the different constituents of the whole plant. These constituents include cannabinoid-like ligands related to THC but found in generally lower abundance, termed minor cannabinoids.
In comparison with THC and CBD, less is known about minor cannabinoids; this includes the five investigated in the present study, cannabinol (CBN), cannabidivarin (CBDV), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabivarin (THCV). Previous studies have conflicting results regarding the cannabimimetic effects of CBN. This is likely due to differences in the route of administration; injection of CBN-induced catalepsy, analgesia, and hypolocomotion, key cannabimimetic behaviors, while oral administration did not (Takahashi and Karniol, 1975; Formukong et al., 1988; El-Alfy et al., 2010; Kulpa et al., 2023). We do not yet know how CBN affects chronic pain behaviors.
CBDV has some activity at cannabinoid receptor type 1 (CB1) but is a more potent CB2 agonist and at lower doses does not elicit cannabimimetic behaviors (Navarro et al., 2020; Zagzoog et al., 2020). A small clinical study found that CBDV was safe but did not help to relieve HIV-induced neuropathy in humans (Eibach et al., 2021). However, other work suggests CBDV is effective for reducing neuroinflammation and may thus improve analgesia when combined with morphine (Wang et al., 2022).
More research has been done on CBG, a weak partial agonist for CB1 and CB2 and found in higher abundance (0.1–0.9 ng/mL) than CBN in the Cannabis plant (Happyana et al., 2013; Zagzoog et al., 2020). CBG has been shown to relieve neuropathy in mice via systemic injection, but, similar to CBN, the effects of CBG are dependent on the route of administration (Formukong et al., 1988; Sepulveda et al., 2022; Nachnani et al., 2023). Injections (10 mg/kg, i.p.) of CBG have previously been shown to increase tail-flick latency but not other cannabimimetic behaviors (Zagzoog et al., 2020).
THCV relieves neuropathic pain on its own and had an additive effect on pain relief when combined with CBD during CIPN (Kumar Kalvala et al., 2022). When injected, THCV has been shown to elicit cannabimimetic behaviors in male mice, though to a lower degree than THC (Zagzoog et al., 2020). As previously mentioned with the other minor cannabinoids, the route of administration is essential as cannabimimetic behaviors were not observed when THCV was given orally (Kulpa et al., 2023).
Δ8-THC is an isomer of Δ9-THC. Δ8-THC is found in meager amounts in Cannabis, and commercially available Δ8-THC is often derived from CBD (Food and Drug Administration, 2022). Both Δ8-THC and THC bind to the orthosteric site of CB1 and CB2. However, Δ8-THC has less affinity to CB1 than THC, while both compounds have a similar affinity for CB2 (Tagen and Klumpers, 2022). Δ8-THC has also been shown to induce the cannabinoid tetrad in rodents when injected systemically but not when given orally; these studies are limited since the effects of Δ8-THC on female rodents were not assessed (Dewey et al., 1970; El-Alfy et al., 2010; Tagen and Klumpers, 2022; Kulpa et al., 2023). Despite the breadth of research on Δ8-THC, there has yet to be an investigation into its effects on chronic pain and cannabimimetic behaviors in female mice.
While the previous studies noted here have tested these cannabinoids, there are limitations to these studies, such as the use of only male test subjects in some studies, varying doses and routes of administration, and a lack of testing in chronic pain models for some minor cannabinoids. We thus sought in this study to comprehensively test these five minor cannabinoids using a uniform protocol of cannabinoid tetrad and chemotherapy-induced peripheral neuropathy testing using male and female mice.
Methods
Drugs.
Cannabinol isolate (KND Laboratories, Lot #CBN-3014), cannabidivarin isolate (KND Laboratories, Lot #CBDV22-001), crystalline cannabigerol isolate (KND Laboratories, Lot #CBG-014), Δ9-tetrahydrocannabivarin (KND Laboratories, Lot #D9THCV22-009), and Δ8-tetrahydrocannabinol (Terpene Belt Farms) were diluted into a 10% DMSO, 10% Tween80, and 80% USP saline vehicle for mouse injections. All experimental drugs were stored at 4°C, except Δ9-THCV, which was held at –20°C. Stocks of 6 mg/mL paclitaxel were dissolved in 1:1 cremophor:ethanol and stored at 4°C. These stocks were diluted to a final concentration of 2 mg/mL paclitaxel in 16.6% cremophor, 16.6% ethanol, and 66.6% USP saline for mouse injections, as described in previous work from our laboratory (Stine et al., 2020).
Animals.
Both male and female CD-1 (a.k.a. ICR) mice 5 to 8 weeks old from Charles Rivers Laboratory were used for all experiments. Unless otherwise specified, equal numbers of male and female mice were used in every experiment. After shipment, the mice were given at least 5 days to acclimate to the temperature- and humidity-controlled vivarium at the University of Arizona. Mice were on a 12-hour conventional light (7 am–7 pm) and dark cycle and given ad libitum access to standard chow and water. The mice were given 30 minutes to acclimate to the experiment room before any experimental manipulation. Naïve mice were then randomly assigned to treatment groups to which the experimenters were blinded via coded drug vials. Unblinding only occurred after all data for an experiment had been collected. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Arizona and carried out according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the International Association for Pain Guidelines for the Use of Animals in Research.
Cannabinoid Behavior Tetrad.
The cannabinoid tetrad of behaviors is used to evaluate typical behaviors associated with Cannabis consumption as described in our laboratory’s previous work (LaVigne et al., 2021). The mice were given time to acclimate to the experiment room and then given drug treatment (minor cannabinoid; intraperitoneal) or vehicle (10% DMSO, 10% Tween80, 80% saline; intraperitoneal). After injection, the mice had either their locomotion tested via open field test or cataleptic behavior tested via ring test. The open field box had opaque, white walls with a black floor and dimensions of 30 cm × 28 cm. Mouse movement was recorded by a video camera 1.5 m above the open field box. For the ring test, the mice were placed on a ring stand suspended ∼30 cm above the tabletop, with their total time spent cataleptic (no movement) recorded using a stopwatch. The behavior for both tests was observed for 5 min, and mice were then switched to have the other behavior (locomotion or catalepsy) measured at 15 min post-injection. Twenty minutes post-injection, nociception behavior was assessed via tail flick assay by submerging the mouse’s tail into a 52°C water bath and recording the latency to withdraw using a stopwatch (10-s cutoff time). Finally, the core body temperature of the mice was measured 30 min post-injection by inserting a lubricated thermometer ∼1 cm into the mouse’s rectum.
Anxiety (Open Field Test).
The open field test was used to assess anxiety-like behaviors during the locomotor test described previously. The field was divided into eight 5 × 10 cm square sections in the periphery and a ninth center square (10 × 10 cm) by black marks on the chamber floor. The mouse’s time spent in the center square was assessed using AnyMaze software during analysis of the locomotor test.
Chemotherapy-Induced Peripheral Neuropathy.
This neuropathic pain model was induced as reported in our previous work (Stine et al., 2020). The baseline mechanical sensitivity of the mice was recorded using the up-down method using von Frey filaments, outlined in (Chaplan et al., 1994) and as we reported in our previous work (Stine et al., 2020). The mice were then given 2 mg/kg of paclitaxel via intraperitoneal injection on days 1, 3, 5, and 7 of the experiment. On day 8, the mice had their post-chemotherapy-induced peripheral neuropathy (CIPN) baselines measured to establish that they had developed neuropathy. Animals that did not exhibit sufficient pain behaviors (< 0.5 g mechanical threshold) were excluded from the study. The mice were then given the drug under investigation via intraperitoneal injection, and mechanical sensitivity was recorded over a 3-h time course.
Data Analysis.
All data are reported as the mean ± S.E.M. and raw, without normalization. Post hoc video analysis was used to determine the distance traveled, time in the chamber’s center, and immobile time for the locomotor/open field test, which was analyzed via AnyMaze software. The center of the animal’s body was used to track its movement. The animals were not habituated to the locomotor boxes before baseline measurements, as we have found that such habituation suppresses all locomotor activity (LaVigne et al., 2021). For cannabinoid tetrad experiments with two groups, a Student’s unpaired two-tailed t-test was used to determine significant differences. Tetrad data with three or more groups was analyzed via one-way ANOVA with Tukey’s post hoc test. For CIPN experiments, data were analyzed via two-way ANOVA followed by Sidak’s post hoc test when two treatment groups were compared or Tukey’s test for three or more groups. For all statistical analyses, P ≤ 0.05 was considered a significant difference between groups. All graphing and statistical analysis was completed using GraphPad Prism 10.1.2. Male and female mice were included in every experiment; if no sex difference was found, then both sexes were combined for analysis. When such differences were found, males and females were reported separately.
Results
As noted, we sought to test five minor cannabinoids in a consistent paradigm of cannabinoid tetrad and CIPN assays. All drugs were delivered by the intraperitoneal route in male and female mice.
Cannabinol.
Previous research showed that injections of CBN elicit the cannabimimetic tetrad of behaviors: catalepsy, analgesia, hypothermia, and hypolocomotion (Takahashi and Karniol, 1975). This study used a dose of 10 mg/kg, which we started with for our work. This dose failed to evoke cannabimimetic behaviors or antinociception in CIPN (Supplemental Fig. 1). Increasing the dose by a half-log step to 32 mg/kg produced a small but significant increase in catalepsy but no other tetrad behaviors and no antinociception in CIPN (Supplemental Fig. 2). We thus further increased our dose of CBN (100 mg/kg, i.p.), which elicited some cannabimimetic behaviors (hypothermia and hypolocomotion) with the notable exception of catalepsy. Furthermore, analgesia, measured by tail-flick assay, was only observed in male mice (Fig. 1A). The amount of time the mice spent in the center section during the open field test, a mark of anxiolytic behavior, remained unchanged between CBN-treated and control mice (Fig. 1A) (La-Vu et al., 2020).
CBN had not been previously tested in a chronic pain model. We found that both male and female mice displayed significant antinociception in the CIPN model at 100 mg/kg (Fig. 1B). Interestingly, we detected a mild sex difference in the CIPN response, with female mice showing an earlier onset of antinociception at 20 min with the male mice not showing a response until 40 min (Fig. 1B). These observations suggest that CBN may produce analgesia in chronic pain. The observations overall suggest that CBN is cannabimimetic and antinociceptive, although at a higher dose than previously reported for the tetrad assay.
Cannabidivarin.
A previous report (Zagzoog et al., 2020) showed no tetrad activity in male mice at a 10 mg/kg dose. We used this same dose in males and females, and in agreement with this earlier literature, we found no tetrad activity in either sex (Supplemental Fig. 3). We thus increased the dose to 100 mg/kg, the same used for CBN, and again found no tetrad activity in either sex (Fig. 2A). These observations agree with the earlier literature and suggest no cannabimimetic behavior for this minor cannabinoid.
To our knowledge, CBDV has not been previously tested in a chronic pain model. We thus tested the same dose of CBDV (100 mg/kg, i.p.) in our CIPN model. We found that female mice, but not male mice, showed a small but significant antinociceptive effect (Fig. 2B). The effect does not suggest that this ligand would have strong therapeutic efficacy, but it does show that the drug has some biologic effect in females.
Cannabigerol.
Previous reports showed that CBG evoked antinociception in neuropathic pain in male and female mice (Sepulveda et al., 2022; Nachnani et al., 2023) and tail-flick antinociception but not other tetrad behaviors in male mice (Zagzoog et al., 2020), all at a 10 mg/kg dose. We thus started with this 10 mg/kg dose in our study and did not observe any changes in tetrad behavior (Supplemental Fig. 4A) or CIPN antinociception (Supplemental Fig. 4B) in either sex. We also did not observe any changes in CIPN antinociception at 20 mg/kg (Supplemental Fig. 4C).
We thus increased our dose of CBG (100 mg/kg, IP). This increased dose evoked two of the cannabimimetic tetrad in both sexes: hypothermia and tail-flick antinociception (Fig. 3A). Since CBG relieved acute thermal pain, we anticipated it would also induce CIPN pain relief; however, injections of CBG (100mg/kg, i.p.) did not affect CIPN antinociception in either sex (Fig. 3B). To confirm that our observations were not due to altered potency of CBG, we increased the dose again to 320 mg/kg. At this high dose, we found that the hypothermia was increased over the 100 mg/kg dose, and we also detected an increase in catalepsy and immobile time (Supplemental Fig. 5A). In contrast, we could not detect tail-flick antinociception at this high dose (Supplemental Fig. 5A). However, like the 100 mg/kg dose, we detected absolutely no increase in CIPN antinociception (Supplemental Fig. 5B). We thus observed some tetrad effects as in a previous report (Zagzoog et al., 2020) but were unable to replicate the neuropathic pain antinociception observed in other studies even at a very high dose (Sepulveda et al., 2022; Nachnani et al., 2023).
Δ9-Tetrahydrocannabivarin.
Previous testing showed tetrad effects with 10 mg/kg THCV in male mice alone (Zagzoog et al., 2020). We thus started with this dose and only observed an increase in catalepsy but not other tetrad behaviors (Supplemental Fig. 6). Since this dose was sufficient to induce one tetrad behavior, we only increased the dose by a half-log step to 32 mg/kg i.p. At this increased dose, we were able to observe three of the four tetrad behaviors but only in female mice (Fig. 4A). This finding was in contrast to the earlier report (Zagzoog et al., 2020), as we did not observe tetrad effects in male mice, nor could we detect differences in locomotor activity or anxiolytic behavior.
This same dose of 32 mg/kg was unable to evoke any CIPN antinociception in either sex, despite the tetrad effects shown previously (Fig. 4B). A previous report did show that 15 mg/kg THCV was able to evoke antinociception in female mice alone (Kumar Kalvala et al., 2022). Notably, the THCV in this study was injected twice a week for 6 weeks prior to measuring antinociception, instead of once acutely as performed here. The action of THCV thus may represent a chronic mechanism such as the modulation of inflammation and not acute antinociception.
Δ8-Tetrahydrocannabinol.
Previous studies in male mice given injections of Δ8-THC (10 and 20 mg/kg, i.p.) exhibited the four primary cannabimimetic tetrad behaviors (Dewey et al., 1970; El-Alfy et al., 2010; Tagen and Klumpers, 2022). We thus started with the same dose of 10 mg/kg, i.p. in males and females. In contrast to this previous work, we observed injections of Δ8-THC affected only female mice, increasing cataleptic behavior and lowering rectal temperature (Supplemental Fig. 7A). At this dose, we observed that neither male nor female mice treated with Δ8-THC had CIPN antinociception significantly different from vehicle-treated animals (Supplemental Fig. 7B). To our knowledge, this is the first test of Δ8-THC in a chronic pain model.
Given the modest effects of Δ8-THC at this dose, we increased the dose by a half-log step to 32 mg/kg. At this higher dose, we were able to detect increases in tail-flick antinociception, hypothermia, and catalepsy, which is more in line with the literature reports cited earlier (Fig. 5A). In contrast to our lower dose studies, this higher dose caused tetrad behaviors in both male and female mice, suggesting a potential dose dependence on the sex difference observed at 10 mg/kg. However, despite this increased tetrad effect at 32 mg/kg, we also did not observe any detectable CIPN antinociception (Fig. 5B). Our studies thus suggest that Δ8-THC has cannabinoid-like effects in the tetrad but does not relieve chronic neuropathic pain.
Discussion
The increased availability, interest, and use of Cannabis extracts and isolated minor cannabinoids by consumers makes it essential to assess the side effects and therapeutic benefits of these compounds in isolation. We tested five minor cannabinoids (CBN, CBDV, CBG, Δ8-THC, and THCV) to determine if they elicit similar adverse side effects that are associated with cannabis use: catalepsy, hypothermia, hypolocomotion, and anxiety. Additionally, we tested their analgesic effects in acute and chronic pain models to determine if these compounds had pain-relieving therapeutic potential. We found that CBN, CBG, and high-dose Δ8-THC evoked some tetrad behaviors in both sexes, while THCV and low-dose Δ8-THC evoked some tetrad behaviors only in females. Of the minor cannabinoids tested, only CBN efficaciously relieved CIPN pain.
In terms of tetrad testing, our observations differed from past reports in key respects. CBN was shown to induce tetrad behaviors at 10 to 40 mg/kg, albeit not when administered by the oral route (Takahashi and Karniol, 1975; Formukong et al., 1988; El-Alfy et al., 2010; Kulpa et al., 2023). In contrast, we found some tetrad behaviors induced at 100 and 32 mg/kg but not 10 mg/kg, with catalepsy unaffected by CBN treatment except for a small but significant effect at 32 mg/kg. For CBG, a previous study found increased tail-flick antinociception but not other behaviors at 10 mg/kg (Zagzoog et al., 2020); we found both tail-flick antinociception and hypothermia at 100 mg/kg and hypothermia, catalepsy, and immobility at 320 mg/kg. Similarly, THCV was found to induce tetrad behaviors at 10 mg/kg in male mice (Zagzoog et al., 2020), while we were only able to detect tetrad effects in females, not males, and only at 32 mg/kg. For Δ8-THC, previous studies found tetrad effects in males at 10 to 20 mg/kg (Dewey et al., 1970; El-Alfy et al., 2010; Tagen and Klumpers, 2022); while we did detect tetrad effects at 10 mg/kg, we could only do so in females. This sex selectivity was not observed at 32 mg/kg, which induced tetrad effects in both sexes, suggesting some of these sex effects could be dose dependent. Only for CBDV did we find complete agreement with the literature, with no detectable tetrad behaviors at 10 to 100 mg/kg (Navarro et al., 2020; Zagzoog et al., 2020; Eibach et al., 2021). Overall, we found that tetrad behaviors required significantly higher doses and for two minor cannabinoids could not be replicated in male mice in at least some doses.
There are key differences between our study and past literature that could explain these discordant results. For one, the past studies used different mouse strains, mostly C57Bl6 and in one case Swiss Webster mice. On the other hand, we used CD-1 mice. Pain and analgesic responses can differ strongly by mouse strain or even vendor for the same mouse strain (Leo et al., 2008; Smith, 2019). Notably, a previous study found that CD-1 mice expressed a novel, non-CB1 cannabinoid receptor, while C57 mice did not (Hoffman et al., 2005). Strain differences could thus explain the relative insensitivity of our mice to the minor cannabinoids when compared with other reports. Other experimental differences include composition of the drug vehicle (e.g., 1:1:18 ethanol:emulphor:saline in Zagzoog et al. [2020] versus 1:1:8 DMSO:Tween80:saline here), small differences in the timing of the tetrad tests, animal husbandry differences, and other variables that could have impacted our results. Another potential factor could be a potency shift of these minor cannabinoids in our hands. While we tested several doses of each cannabinoid, this does not rule out a shift in potency rather than efficacy/activity. Extended dose-response analysis could help test this possibility. Further testing will help iron out these differences and help to establish the true cannabimimetic activity of these minor cannabinoids in the tetrad assay.
Our experiments were also informative in terms of the minor cannabinoid activity in the CIPN chronic pain assay. CBN, CBDV, and Δ8-THC have not been previously tested in any chronic pain model to our knowledge. Our finding that CBN produced robust antinociception in CIPN thus represents a possible new therapeutic approach to manage chronic pain, especially since the tetrad effects at that dose were modest (no catalepsy), suggesting the drug might be better tolerated than full cannabinoid ligands like THC. One note of caution: CBN also produced motor effects in the tetrad assay, which could be confounding in the von Frey paw withdrawal assay in CIPN. Further nonmotor pain testing can rule out this possibility, although we note that CBN had no impact on tail-flick latency in females, which is also a reflexive motor-based pain assay. The other minor cannabinoids produced very little (CBDV) to no (Δ8-THC) antinociception in this model, which is also informative and suggests these ligands might not be effective therapies for chronic pain. This is especially interesting given that Δ8-THC is widely commercially available to the public, who often take cannabinoids to manage their pain (Kosiba et al., 2019; Mahabir et al., 2020). Further investigation into the activity of Δ8-THC could thus be impactful to public health.
For CBG and THCV, previous studies have shown that these drugs produce pain relief in chronic and neuropathic pain models, while we observed no such activity in our study (Kumar Kalvala et al., 2022; Sepulveda et al., 2022; Nachnani et al., 2023). Again, there are differences between our studies that could explain these different results. For the CBG studies, the authors also used C57Bl6 mice, which, as explained earlier, could be more sensitive than our CD-1 mice (Hoffman et al., 2005; Leo et al., 2008; Smith, 2019). Both of these studies also used chronic cisplatin to induce their neuropathic pain state, while we used shorter-duration treatment with paclitaxel. One study found that paclitaxel was effective in suppressing mechanoreceptors in a sustained manner that was not observed for cisplatin, which could explain why CBG was ineffective in reducing mechanical hypersensitivity in our study (Zhang and Tuckett, 2008). Our high-dose studies with 320 mg/kg CBG produced strong tetrad effects but still no CIPN antinociception, suggesting our results are not just due to a potency shift for CBG. For the THCV study, the drug was delivered chronically over a 6-week treatment period, as opposed to the acute delivery we used here (Kumar Kalvala et al., 2022). The authors of this study also used C57Bl6 mice and further used a much higher dose of 8 mg/kg paclitaxel, in contrast to the 2 mg/kg we used in our study. These differences could similarly impact the lack of effect we observed for THCV. As with the tetrad assay, further study will be needed to determine the impact of minor cannabinoids in chronic pain. Few studies overall have investigated this topic, highlighting the need for further testing.
Lastly, we observed interesting sex differences in our study, namely that THCV and low-dose Δ8-THC induced tetrad behaviors only in females. Previous work suggested these two ligands induced tetrad behaviors in males (Dewey et al., 1970; El-Alfy et al., 2010; Zagzoog et al., 2020; Tagen and Klumpers, 2022). Our lack of replication in males as explored earlier could be due to mouse strain or other differences. Selective activity in females on the other hand is new. A recent review of sex differences in cannabinoid antinociception in a variety of pain models analyzed 27 total studies; of these, 14 showed enhanced cannabinoid antinociception in females while only 4 showed enhanced activity in males (Blanton et al., 2021). This analysis as well as other work on sex differences in the endocannabinoid system and metabolism of cannabinoids suggests females may be more sensitive to cannabinoid neuropharmacology. This hypothesis is further supported by our Δ8-THC observations, which showed female-only activity at 10 mg/kg, but both sexes were impacted at 32 mg/kg. This could explain why we were able to detect female-only tetrad behaviors with THCV and low-dose Δ8-THC. Future work will need to examine sex differences in minor cannabinoids in more detail, especially since many of the studies we cited here used only male rodents.
In conclusion, the minor cannabinoids tested in the present study have varying activity in the cannabimimetic tetrad assay and CIPN pain assay. Our observations differ from the literature in key respects, which may help to define the activity of these compounds. We also identified potential sex differences, as well as identified new activities for these minor cannabinoids in chronic neuropathic pain. Future work will need to investigate these compounds further and determine the therapeutic potential of these drugs in light of the limited literature available on these ligands, as well as determine their molecular mechanisms of action.
Acknowledgments
The authors acknowledge the University of Arizona Comprehensive Center for Pain and Addiction for support of this work. They also acknowledge Terpene Belt Farms for their generous provision of the experimental ligands. J.M.S. is an equity holder in Teleport Pharmaceuticals, LLC and Botanical Results, LLC, a local cannabidiol company. Neither company was involved in this study in any way, nor do they develop minor cannabinoids as products.
Data Availability
Any data needed to interpret the results are presented in this manuscript or the Supplemental Material. However, the raw data is available upon request to the corresponding author.
Abbreviations
- Δ8-THC
- Δ8-tetrahydrocannabinol
- CB1/2
- cannabinoid receptor type 1/2
- CBD
- cannabidiol
- CBDV
- cannabidivarin
- CBG
- cannabigerol
- CBN
- cannabinol
- CIPN
- chemotherapy-induced peripheral neuropathy
- THC
- Δ9-tetrahydrocannabinol
- THCV
- Δ9-tetrahydrocannabivarin
Footnotes
Footnote Group
References
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