Assessing Dose- and Sex-Dependent Antinociceptive Effects of Cannabidiol and Amitriptyline, Alone and in Combination, and Exploring Mechanism of Action Involving Serotonin 1A Receptors

Abstract

Inflammatory pain is caused by tissue hypersensitization and is a component of rheumatic diseases, frequently causing chronic pain. Current guidelines use a multimodal approach to pain and sociocultural changes have renewed interest in cannabinoid use, particularly cannabidiol (CBD), for pain. The tricyclic antidepressant amitriptyline (AT) is approved for use in pain-related syndromes, alone and within a multimodal approach. Therefore, we investigated sex- and dose-dependent effects of CBD and AT antinociception in the 2.5% formalin inflammatory pain model. Male and female C57BL/6J mice were pretreated with either vehicle, CBD (0.3–100 mg/kg), or AT (0.1–30 mg/kg) prior to formalin testing. In the acute phase, CBD induced antinociception after administration of 30–100 mg/kg in males and 100 mg/kg in females and in the inflammatory phase at doses of 2.5–100 mg/kg in males and 10–100 mg/kg in females. In the acute phase, AT induced antinociception at 10 mg/kg for all mice, and at 0.3 mg/kg in males and 3 mg/kg in female mice in the inflammatory phase. Combining the calculated median effective doses of CBD and AT produced additive effects for all mice in the acute phase and for males only in the inflammatory phase. Use of selective serotonin 1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635) maleate (0.1 mg/kg) before co-administration of CBD and AT reversed antinociception in the acute and partially reversed antinociception in the inflammatory phase. Administration of AT was found to enhance cannabinoid receptor type 1mRNA expression only in female mice. These results suggest a role for serotonin and sex in mediating cannabidiol and amitriptyline-induced antinociception in inflammatory pain.

SIGNIFICANCE STATEMENT

Inflammatory pain is an important component of both acute and chronic pain. We have found that cannabidiol (CBD) and amitriptyline (AT) show dose-dependent, and that AT additionally shows sex-dependent, antinociceptive effects in an inflammatory pain model. Additionally, the combination of CBD and AT was found to have enhanced antinociceptive effects that is partially reliant of serotonin 1A receptors and supports the use of CBD within a multimodal approach to pain.

Introduction

Pain consists of the sensory and emotional response to actual or potential tissue damage (Raja et al., 2020). The subtype inflammatory pain is caused by both peripheral and central tissue hypersensitization (Kidd and Urban, 2001). Inflammatory pain is associated with rheumatic diseases, impacting approximately 20% of US adults and causing chronic pain that significantly reduces quality of life (Leadley et al., 2014; Hootman et al., 2016; Von Korff et al., 2016; Barbour et al., 2017; Polston and Wallace, 2017; Dahlhamer et al., 2018). Inflammatory pain is also associated with acute pain due to infection, tissue injury, and radiation (Ji et al., 2016). Research has shown that female sex is a risk factor for chronic pain (Ruau et al., 2012; Berkley, 1997) with sex-specific mechanisms. For instance, male mice use microglia to mediate pain in the spinal cord while female mice use T-cells, with these differences attributed to differences in sex hormone functioning (Sorge and Totsch, 2017; Rosen et al., 2017). However, most pain research is exclusively male focused (Arnegard et al., 2020). Current pain management guidelines use a multimodal approach, involving a combination of nonsteroidal anti-inflammatory drugs, anticonvulsants, and antidepressants (Manworren, 2015).

Cannabis was used as an analgesic until the early 20^th century (Crocq, 2020). The non-psychoactive phytocannabinoid cannabidiol (CBD) is considered relatively safe, with its main side effects being fatigue, diarrhea, and altered weight (Romero-Sandoval et al., 2018; Iffland & Grotenhermen, 2017). However, CBD also alters liver function and has possible drug interactions (Chesney et al., 2020). Recent sociocultural changes and improved legal access to CBD increases the importance of understanding its analgesic efficacy (Sandler et al., 2019; Al-Shammari et al., 2017; Agricultural Improvement Act of 2018: https://www.congress.gov/115/plaws/publ334/PLAW-115publ334.pdf). Long-term analgesic use of cannabis is limited by tolerance to its antinociception (Häuser et al., 2018; Romero-Sandoval et al., 2018; Henderson-Redmond et al., 2021).

CBD antagonizes signaling at cannabinoid receptors CB₁ (Pertwee, 2008) and CB₂ (Thomas et al., 2007). CBD antinociception is mediated through positive allosteric modulation at serotonin 1A receptor (5-HT_1A) serotonin receptors in neuropathic pain models (Rock et al., 2012; De Gregorio et al., 2019; Jesus et al., 2019; Ward et al., 2014). CBD antinociception has also been associated with its actions at transient receptor potential cation channel subfamily V member 1 (De Petrocellis et al., 2011; Costa et al., 2007), α3 glycine receptors (Xiong et al., 2012), orphan g-protein coupled receptors 3, 6, and 12 (Laun et al., 2019), and attenuation of proinflammatory cytokines (Li et al., 2018). Prior studies examining sex differences in CBD-mediated analgesia have revealed mixed results (see Blanton et al., 2021 for a review), with results indicating either no difference (Britch et al., 2017, 2020) or greater efficacy in males (Greene et al., 2018; Linher-Melville et al., 2020). While clinical investigation into sex differences of CBD for chronic pain is lacking, one study found that men self-reported greater benefit of cannabis use in managing migraine headaches (Cuttler et al., 2016). Clinical evidence of CBD antinociception is limited by the lack of trials evaluating CBD without Δ⁹ tetrahydrocannabinol (Boyaji et al., 2020).

Amitriptyline (AT) is a tricyclic antidepressant that is approved by the Food and Drug Administration for use in multimodal analgesic frameworks (Lawson, 2017; Thour and Marwaha, 2022). AT acts at several monoaminergic and cholinergic receptors, and these actions are associated with its adverse effects of cardiotoxicity, dry mouth, dizziness, constipation, confusion, weight gain, and sedation (Lawson, 2017; McClure and Daniels, 2021; Rheker et al., 2018). AT has been shown to be efficacious in inducing antinociception in the formalin model of inflammatory pain (Sawynok et al., 2008; Liu et al., 2013). Only a single preclinical study has evaluated AT for antinociceptive sex differences, finding that male rats were more sensitive to its antinociceptive effects (O’Brien and McDougall, 2020).

To assess the utility of CBD within a multimodal analgesic framework, we assessed the effect of combining CBD and AT on antinociceptive efficacy. To understand the interaction(s) between sex, pain, CBD, and AT, we assessed the antinociceptive effects of CBD and AT in male and female mice using the formalin model of inflammatory pain. We first assessed sex differences in antinociception across a range of doses of CBD and AT to generate drug-specific dose-response curves. We then calculated the ED₅₀ for both CBD and AT based on the inflammatory phase of the formalin test. Subsequent testing using the ED₅₀ doses of CBD, AT, and their combination revealed a potential additive effect in their combination. Lastly, we evaluated the effect of selective 5-HT_1A antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY-100635) on the combination’s effect.

Materials and Methods

Animals.

These experiments were performed using 6- to 8 week-old (108 male and 108 female) wild-type C57BL6/J mice obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were group housed under the following conditions: four mice per cage, 12:12 hour light-dark cycle (light from 07:00 to 19:00), and fed standard mouse chow containing 19.8% protein ad libitum (Purina Laboratory Diet, St. Louis, MO, USA). Animal care protocols and experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center and Marshall University and in accordance with the National Institutes of Health guidelines found in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Drugs.

CBD was obtained from Sigma-Aldrich (St. Louis, MO, USA; CAT#C-045-1ML). AT was obtained from Sigma-Aldrich (St. Louis, MO, USA; CAT#A8404). WAY-100635 was obtained from Sigma-Aldrich (St. Louis, MO, USA; CAT#508146). Formalin was obtained from Thermo Fisher Scientific (Waltham, MA, USA; CAT#F79-1).

CBD was stored at -20°C and AT was stored at 4°C prior to preparation and use on the day of the experiment. CBD was diluted and administered in a solution of DMSO, Tween 80, and physiologic saline (0.9% NaCl) at a ratio of 1:1:18 (5% DMSO, 5% Tween 80, 90% physiologic saline). AT and WAY-100635 were prepared and administered in physiologic saline. Controls were administered a vehicle solution consisting of the same 1:1:18 preparation of DMSO, Tween 80, and physiologic saline except for the control for AT alone, which used physiologic saline. Vehicles and drugs were administered at a volume of 10 μl/gram of body weight, via an intraperitoneal injection, 30 minutes prior to formalin testing. The groups receiving both the CBD and AT ED₅₀ doses received separate injections with each compound, rather than a single injection containing both compounds.

Formalin Test.

The formalin test was conducted in male and female mice to assess the dose-response relationship between CBD and AT on antinociception. This test was conducted by injecting 10 µL of dilute 2.5% formalin intraplantarly into the left hind paw of the mouse. Pain behavior associated with formalin injection was scored over the following hour (Dubuisson and Dennis, 1977; Puig and Sorkin, 1996). The formalin test induces both an acute phase of pain (phase 1, 0–15 minutes) that is primarily mediated by TRPA1 peripheral nociceptors (McNamara et al., 2007) and an inflammatory phase of pain (phase 2, 15–60 minutes) that is primarily mediated by inflammation-induced peripheral and central (i.e., dorsal horn of the spinal cord) sensitization (Coderre et al., 1990; Fu et al., 2000). Pain behavior is scored according to the following system: no pain behavior (0), favoring (0; injected paw has minimal weight bearing), lifting (1; injected paw is not in contact with any surface), and licking/biting/shaking/rapid lifts (2). The aforementioned one-hour period of observation is divided into 12 5-minute intervals and the composite pain score (CPS) is computed using the composite pain score-weighted scores technique (CPS-WST 0,1,2) (Watson et al., 1997). The trapezoidal rule is used to calculate the area under the curve (AUC; CPS-WST 0,1,2 × time [min]) for both the acute (0–15 minutes) and inflammatory (15–60 minutes) phases of pain (Guindon et al., 2006). A 10 cm × 10 cm × 10 cm clear plexiglass house and glass observation table, situated over a mirror at a 45-degree angle, was used to observe behavior. Mice were euthanized with brain and spinal cord tissue being collected, flash frozen, and stored at -80°C immediately following completion of the formalin test.

Rotarod Test.

Sedation and motor impairment was assessed via the accelerating rotarod test (Model LE8205, Harvard Apparatus, Holliston, MA, USA). The rotarod was calibrated to accelerate from a rate of four rotations per minute to 40 rotations per minute over 5 minutes. Mice were placed on the rotating rod at 4 rotations per minute and the amount of time prior to falling was recorded. Mice were trained for 3 days prior to the onset of experimental testing. Three trials were conducted with ten-minute inter-trial intervals for each mouse. Values for the rotarod were measured 24 hours prior to drug administration (baseline) and at 30 and 90 minutes post injection of vehicle, saline, CBD 100 mg/kg, or AT 10 mg/kg.

Procedures.

First, the antinociceptive effects of different doses of both CBD (0.3, 1, 2.5, 10, 30, and 100 mg/kg i.p.) and AT (0.1, 0.3, 1, 3, 10, and 30 mg/kg i.p.) were assessed relative to vehicle using the formalin test (2.5% formalin solution) in different cohorts of both male and female mice. Doses of CBD were chosen based on previous research into cannabidiol-induced antinociception in the formalin test (Sepulveda et al., 2022). Additional doses of CBD were chosen to evaluate the dose-response curve for CBD. Doses of AT were chosen based on previous work using AT in the formalin test (Sawynok et al., 2008). Additional doses of amitriptyline (0.1 and 0.3 mg/kg) were used to evaluate the dose-response curve of AT. Second, the sex- and dose-dependent antinociceptive effects of both CBD and AT were used to calculate the ED₅₀ dose of both drugs in the inflammatory phase of the formalin test. Different cohorts of both male and female mice were then tested with one of the ED₅₀ dose of CBD, the ED₅₀ dose of AT, or the combination of both ED₅₀ doses. Third, the effect of 5-HT_1a antagonism on CBD and AT analgesia was evaluated following intraperitoneal injection of WAY-100635, at 0.1 mg/kg, 30 minutes prior to injection with the combination of both ED₅₀ doses. The dose of WAY-100635 was chosen based on previous work using WAY-100635 to attenuate the effects of CBD (Rock et al., 2012).

Tissue Preparation for Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis.

Male and female mice receiving vehicle or different ED₅₀ doses (CBD 17.78 mg/kg in females and 9.05 mg/kg in males, AT 3.30 mg/kg in females and 2.82 mg/kg in males, CBD 17.78 mg/kg + AT 3.30 mg/kg in females and CBD 9.05 mg/kg + AT 2.82 mg/kg in males) were euthanized following completion of the formalin test. Both brain and spinal cords from male and female mice were flash frozen using isopentane with dry ice and then stored for further processing in a -80°C freezer (Guindon et al., 2014).

Quantification of mRNA from Brain and Spinal Cord Tissue.

RT-qPCR was used to quantify mRNA levels from whole brain and spinal cord tissue (as previously described in Guindon et al., 2014). mRNA was isolated from the whole brain and spinal cords of mice treated with vehicle, individual ED₅₀ doses of CBD or AT, or combined ED₅₀ doses of CBD and AT using a RNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. The expression of cannabinoid receptors (CB₁ and CB₂) mRNAs were quantified via one-step RT-qPCR with PowerSYBR green PCR kit (Applied Biosystems, Carlsbad, CA, USA) using a Matercycler ep realplex qPCR machine (Eppendorf North America Inc., Hauppauge, NY, USA). Gyliceraldehyde-3-phosphase dehydrogenase was selected as the internal standard for mRNA level normalization using the delta-delta Ct method (Livak and Schmittgen, 2001). Primers used are as follows: mouse gyliceraldehyde-3-phosphase dehydrogenase (sense: 5′ -GGGAAGCTCACTGGCATGGC-3′, anti-sense: 5′ -GGTCCACCACCCTGTTGCT-3′); mouse CB1 (sense: 5′ -CTG ATCCTGGTGGTGTTGATCATCTG-3′, anti-sense: 5′ -CGTGTCTGTGGACA CAGACATGGT-3′); mouse CB2 (sense: 5′ -CCTGGGATAGCTCGGATGCG -3′, anti-sense: 5′ -GTGGTTTTCACATCAGCCTCTGTTTC-3′).

Data Analysis and Statistics.

All experiments were conducted in a blinded manner with animals randomly assigned to experimental conditions via random number generator. Each treatment group had pain behavior expressed as mean ± S.E.M. and data were analyzed using the ANOVA technique for repeated measures with time as a factor, two-way ANOVA, or one-way ANOVA, as appropriate. Rotarod performance was expressed as mean ± S.E.M. and data were analyzed using an independent samples T-test. Repeated factors had the Greenhouse-Geisser correction performed, though reported degrees of freedom for significant interactions are provided as uncorrected values. Significant interactions were further evaluated with one-way ANOVAs at each individual time point with Bonferroni post hoc tests. Additional evaluation was performed with a two-way ANOVA and subsequent Bonferroni post hoc tests, with the effects of both sex (male and female) and CBD (0.3, 1, 2.5, 10, 30, 100 mg/kg) on formalin pain behaviors. Levene’s test was used to assess homogeneity of variance prior to each analysis and, should this assumption be violated, Welch’s ANOVA with Games-Howell post-hoc analysis was performed. Multiple regressions analysis was used a priori to settle different components of total variation (Draper and Smith, 1998). The ED₅₀ was calculated via nonlinear regression analysis of the log dose-response curve. Analyses were performed using SPSS statistical software (version 29.0.0.0, SPSS Incorporated, Chicago, IL, USA) and using GraphPad Prism (version 9.4.1, GraphPad Software, San Diego, CA, USA). P < 0.05 was considered significant.

Results

Dose-Dependent Antinociceptive Effects of Cannabidiol (CBD) in the Formalin Test.

In female mice, significant differences between the groups (vehicle, CBD 0.3-100 mg/kg i.p.) (F_6,41 = 86.948, P < 0.00001) were found in a time- (F_11,451 = 178.695, P < 0.00001) and time-group- (F_66,451 = 7.559, P < 0.00001) dependent manner (Fig. 1A). The highest dose of CBD (100 mg/kg) suppressed the CPS relative to the vehicle in both the acute phase (at 5 minutes, P < 0.001) and in the inflammatory phase (at 20–35 minutes, P < 0.001) (Fig. 1A). Additionally, this highest dose suppressed CPS relative to all other doses (0.3–30 mg/kg) in the acute phase (at 5 minutes, P < 0.001) and relative to the lower doses (0.3–2.5 mg/kg) in the inflammatory phase (at 20 to 30 minutes, P < 0.001) (Fig. 1A). The second highest dose (30 mg/kg) also suppressed CPS relative to the vehicle in the inflammatory phase (at 25 to 35 minutes, P < 0.002) and suppressed CPS relative to the lowest doses (0.3–1 mg/kg) in the inflammatory phase (at 25 to 30 minutes, P < 0.006) (Fig. 1A). Analysis of the peak pain value, i.e., highest composite pain score, within the inflammatory phase in female mice revealed significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) (F_{6, 14.797} = 467.013, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain at 10 mg/kg (P < 0.008), 30 mg/kg (P < 0.001), and 100 mg/kg (P < 0.001). Analysis of the area under the curve (AUC) of pain behavior revealed significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) in both Phase 1 (F_6,41 = 14.329, P < 0.0001; Fig. 1C) and Phase 2 (F_6,41 = 76.403, P < 0.0001; Fig. 1D) of the formalin test. Only 100 mg/kg of CBD produced antinociception relative to the vehicle in both Phase 1 (P < 0.001; Fig. 1C) and Phase 2 (P < 0.001; Fig. 1D) of the formalin test; 10 and 30 mg/kg did, however, produce antinociception relative to the vehicle in Phase 2 (P < 0.001) of the formalin test (Fig. 1D). The 100 mg/kg dose of CBD was found to provide antinociception relative to all other groups (0.3–30 mg/kg) in both Phase 1 (Fig. 1C) and Phase 2 (Fig. 1D) of the formalin test (P < 0.001), while 10 and 30 mg/kg were additionally found to provide antinociception relative to the lower groups (0.3–2.5 mg/kg) in Phase 2 of the formalin test (P < 0.001). There was no difference between vehicle and lower CBD doses (0.3–10 mg/kg) in Phase 1 of the formalin test (P = 1.00; Fig. 1C) and no difference between vehicle and lower CBD doses (0.3–2.5 mg/kg) in Phase 2 of the formalin test (P < 0.512; Fig. 1D). Assessment of the AUC differences between the early inflammatory phase (15–25 minutes post-formalin injection) and late inflammatory phase (25–35 minutes post-formalin injection) in female mice revealed significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) (F_{6, 14.681} = 7.738, P < 0.001). Post-hoc analysis, however, revealed no groups with significant differences relative to vehicle.

Fig. 1.

Dose-dependent antinociceptive effects of CBD in the formalin test in wild-type mice. CBD suppressed formalin-induced pain behavior in a dose-dependent manner in both female (A) and male (B) wild-type mice. In female mice, CBD decreased the AUC at 100 mg/kg in Phase 1 (C) and at 10–100 mg/kg in Phase 2 (D). In male mice, CBD decreased the AUC at 30–100 mg/kg in Phase 1 (E) and at 2.5-100 mg/kg in Phase 2 (F). Data are expressed as mean ± S.E.M. (vehicle: n = 12 females and n = 12 males; CBD 0.3 mg/kg, 1 mg/kg, 2.5 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg: n = 6 females and n = 6 males). (A) Time post formalin (min) versus composite pain score (CPS) in female wild-type mice. (B) Time post formalin (min) versus CPS in male wild-type mice. (C) Compound versus AUC during Phase 1 in female wild-type mice. (D) Compound versus AUC during Phase 2 in female wild-type mice. (E) Compound versus AUC during Phase 1 in male wild-type mice. (F) Compound versus AUC during Phase 2 in male wild-type mice. Repeated measure one-way ANOVA with Bonferroni post-hoc used to calculate significance for (A) and (B). One-way ANOVA with Bonferroni post-hoc used to calculate significance for (C), (D), (E), and (F). Relative to vehicle: *P < 0.05, **P < 0.01, ***P < 0.001.

In male mice, significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) (F_6,41 = 81.797, P < 0.00001) were found in a time- (F_11,451 = 228.299, P < 0.00001) and time-group- (F_66,451 = 9.821, P < 0.00001) dependent manner (Fig. 1B). The highest doses of CBD (30–100 mg/kg) suppressed CPS relative to the vehicle in both the acute phase (at 5–10 minutes, P < 0.045; 100 mg/kg also significant at 15 minutes, P < 0.001) and the inflammatory phase (30 mg/kg at 20–35 minutes and at 45 minutes, P < 0.028; 100 mg/kg at 30–45 minutes, P < 0.044) (Fig. 1B). This highest dose (100 mg/kg) also suppressed CPS relative to more moderate groups (0.3–10 mg/kg) in both the acute phase (at 5 and 15 minutes, P < 0.019) and the inflammatory phase (at 20–25 minutes, P < 0.019) (Fig. 1B). Similarly, the second highest dose (30 mg/kg) suppressed CPS relative to the lower groups (0.3–2.5 mg/kg) in both acute (at 5 minutes, P < 0.024) and inflammatory (at 25 minutes, P < 0.047; also significant relative to 0.3 and 1 mg/kg at 20 and 30 minutes, P < 0.001) phases of the formalin test (Fig. 1B). Medium doses of CBD (2.5–10 mg/kg) suppressed CPS relative to the vehicle in only the inflammatory phase (2.5 mg/kg at 30 and 45 minutes, P < 0.041; 10 mg/kg at 25–35 minutes and 45 minutes, P < 0.041) (Fig. 1B). Analysis of the peak pain value within the inflammatory phase in male mice revealed significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) (F_{6, 14.684} = 289.156, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain at 10 mg/kg (P < 0.009), 30 mg/kg (P < 0.001), and 100 mg/kg (P < 0.001). Analysis of the AUC of pain behavior revealed significant differences between the groups (vehicle, CBD 0.3-100 mg/kg i.p.) in both Phase 1 (F_6,41 = 38.346, P < 0.00001; Fig. 1E) and Phase 2 (F_6,41 = 63.107, P < 0.00001; Fig. 1F) of the formalin test. In male mice, both 30 and 100 mg/kg produced significant antinociception relative to the vehicle in Phase 1 of the formalin test (P < 0.0001) (Fig. 1E). In Phase 2 of the formalin test, 2.5, 10, 30, and 100 mg/kg all produced significant antinociception relative to the vehicle (P < 0.0001) (Fig. 1F). The 100 mg/kg dose of CBD was found to provide antinociception relative to all other groups (0.3 to 30 mg/kg) in both Phase 1 (P < 0.001; Fig. 1E) and Phase 2 (P < 0.009; Fig. 1F) of the formalin test. The 30 mg/kg dose of CBD was similarly found to provide significant antinociception relative to low and medium groups (0.3, 1, 2.5, and 10 mg/kg) in Phase 1 (P < 0.013; Fig. 1E) and relative to lower groups (0.3, 1, and 2.5 mg/kg) in Phase 2 (P < 0.0001; Fig. 1F) of the formalin test. Additionally, no significant difference was found between the vehicle and low/medium doses of CBD (0.3, 1, 2.5, and 10 mg/kg) in Phase 1 (P = 1.00; Fig. 1E) and no significant difference was found between vehicle and the lowest doses of CBD (0.3 and 1 mg/kg) in Phase 2 (P = 1.00; Fig. 1F) of the formalin test. Assessment of the AUC differences between the early inflammatory phase (15–25 minutes post-formalin injection) and late inflammatory phase (25–35 minutes post-formalin injection) in male mice revealed significant differences between the groups (vehicle, CBD 0.3–100 mg/kg i.p.) (F_{6, 14.672} = 36.221, P < 0.001). Post-hoc analysis revealed a significant increase in this difference, representing a greater proportion of the pain occurring in the early inflammatory phase, relative to vehicle at 0.3 mg/kg (P < 0.044) and 10 mg/kg (P < 0.012).

Dose-Dependent Antinociceptive Effects of AT in the Formalin Test.

In female mice, significant differences between the groups (saline, AT 0.1–30 mg/kg i.p.) (F_6,37 = 131.779, P < 0.00001) were found in a time- (F_11,407 = 459.665, P < 0.00001) and time-group- (F_66,407 = 27.867, P < 0.00001) dependent manner (Fig. 2A). The lower doses of AT (0.1–1 mg/kg) managed to suppress the CPS relative to the vehicle in both the acute phase (at 10 minutes, P < 0.001) and the inflammatory phase (at 35–40 minutes, P < 0.001) (Fig. 2A). At 3 mg/kg of AT, CPS was suppressed relative to the vehicle in both the acute phase (at 10 minutes, P < 0.001) and the inflammatory phase (at 25–40 minutes, P < 0.001) (Fig. 2A). Additionally, the 3 mg/kg dose of AT suppressed CPS relative to the lower doses (0.1–1 mg/kg) in the inflammatory phase (at 25 minutes, P < 0.001) (Fig. 2A). The highest doses (10–30 mg/kg) of AT produced the most significant antinociception, suppressing CPS relative to the vehicle in both the acute phase (at 5–10 minutes, P < 0.001) and the inflammatory phase (at 20–40 minutes, P < 0.001) (Fig. 2A). These highest doses of AT also suppressed CPS relative to all lower doses (0.1–3 mg/kg) in both the acute phase (at 5 minutes, P < 0.001, and at 15 minutes, P < 0.050) and the inflammatory phase (at 20–25 minutes, P < 0.001, and relative to 0.1–1 mg/kg at 30 minutes, P < 0.021) (Fig. 2A). Analysis of the peak pain value within the inflammatory phase in female mice revealed significant differences between the groups (saline, AT 0.1–30 mg/kg i.p.) (F_{6, 14.432} = 656.189, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain at 10 mg/kg (P < 0.001) and 30 mg/kg (P < 0.001). Interestingly, a significant increase relative to vehicle in peak pain was observed at 0.1 mg/kg (P < 0.018), 0.3 mg/kg (P < 0.011), and 1 mg/kg (P < 0.046). Analysis of the AUC of pain behavior revealed significant differences between the groups (saline, AT 0.1–30 mg/kg, i.p.) in both Phase 1 (F_6,37 = 38.794, P < 0.0001; Fig. 2C) and Phase 2 (F_6,37 = 98.414, P < 0.0001; Fig. 2D) of the formalin test. Both 10 and 30 mg/kg of AT were found to produce antinociception relative to the vehicle in both Phase 1 (P < 0.001; Fig. 2C) and Phase 2 (P < 0.001; Fig. 2D) of the formalin test. Additionally, 3 mg/kg was found to produce antinociception relative to the vehicle in Phase 2 only (P < 0.001) of the formalin test (Fig. 2D). Both the 10 and 30 mg/kg dose were found to produce significant antinociception relative to all other groups (0.1, 0.3, 1, and 3 mg/kg) in both Phase 1 (P < 0.003; Fig. 2C) and Phase 2 (P < 0.001; Fig. 2D) of the formalin test; the 3 mg/kg dose was similarly found to produce significant antinociception relative to the lower doses (0.1, 0.3, and 1 mg/kg) in Phase 2 (P < 0.001; Fig. 2D) of the formalin test. Moreover, no difference relative to vehicle was noted within the low and medium doses (0.1, 0.3, 1, and 3 mg/kg) in Phase 1 of the formalin test (P = 1.00; Fig. 2C) and no difference relative to vehicle was noted within the lower doses (0.1, 0.3, and 1 mg/kg) in Phase 2 of the formalin test (P < 0.072; Fig. 2D). Assessment of the AUC differences between the early inflammatory phase (15-25 minutes post-formalin injection) and late inflammatory phase (25-35 minutes post-formalin injection) in female mice revealed significant differences between the groups (saline, AT 0.1-30 mg/kg i.p.) (F_{6, 14.091} = 40.131, P < 0.001). Post-hoc analysis revealed a significant increase in this difference, representing a greater proportion of the pain occurring in the early inflammatory phase, relative to vehicle at 0.1 mg/kg (P < 0.037), 0.3 mg/kg (P < 0.041), 1 mg/kg (P < 0.001), and 3 mg/kg (P < 0.001).

Fig. 2.

Dose-dependent antinociceptive effects of AT in the formalin test in wild-type mice. AT suppressed formalin-induced pain behavior in a dose-dependent manner in both female (A) and male (B) wild-type mice. In female mice, AT decreased the AUC at 10-30 mg/kg in Phase 1 (C) and at 3–30 mg/kg in Phase 2 (D). In male mice, AT decreased the AUC at 10-30 mg/kg in Phase 1 (E) and at 0.3–30 mg/kg in Phase 2 (F). Data are expressed as mean ± S.E.M. (saline: n = 8 females and n = 8 males; AT 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg: n = 6 females and n = 6 males). (A) Time post formalin (min) versus CPS in female wild-type mice. (B) Time post formalin (min) versus CPS in male wild-type mice. (C) Compound versus AUC during Phase 1 in female wild-type mice. (D) Compound versus AUC during Phase 2 in female wild-type mice. (E) Compound versus AUC during Phase 1 in male wild-type mice. (F) Compound versus AUC during Phase 2 in male wild-type mice. Repeated measure one-way ANOVA with Bonferroni post-hoc used to calculate significance for (A) and (B). One-way ANOVA with Bonferroni post-hoc used to calculate significance for (C), (D), (E), and (F). Relative to vehicle: *P < 0.05, **P < 0.01, ***P < 0.001.

In male mice, significant differences between the groups (saline, AT 0.1-30 mg/kg i.p.) (F_6,37 = 117.742, P < 0.00001) were found in a time- (F_11,407 = 300.487, P < 0.00001) and time-group- (F_66,407 = 14.818, P < 0.00001) dependent manner (Fig. 2B). The lowest dose of AT (0.1 mg/kg) managed to suppress CPS relative to the vehicle in the acute phase (at 10 minutes, P < 0.001) while the second lowest dose (0.3 mg/kg) managed to suppress CPS relative to the vehicle in both the acute phase (at 10 minutes, P < 0.001) and in the inflammatory phase (at 30 minutes, P < 0.007) (Fig. 2B). Similarly, 1 mg/kg of AT suppressed CPS relative to the vehicle in both the acute phase (at 10 minutes, P < 0.001) and in the inflammatory phase (at 30–35 minutes, P < 0.001) (Fig. 2B). The 3 mg/kg dose of AT provided even more prolonged suppression of CPS relative to the vehicle, with significance achieved in the acute phase (at 10 minutes, P < 0.001) and in the inflammatory phase (at 25–35 minutes, P < 0.001, and at 40 minutes, P < 0.010) (Fig. 2B). The highest doses of AT (10 and 30 mg/kg) produced the most significant reduction of CPS relative to the vehicle in both the acute phase (at 5–10 minutes, P < 0.001, and at 15 minutes for 30 mg/kg only, P < 0.013) and in the inflammatory phase (at 20–35 minutes, P < 0.001, and at 40 minutes, P < 0.009) (Fig. 2B). These highest doses also provided suppression of CPS relative to all other doses in both the acute phase (at 5 minutes, P < 0.001) and in the inflammatory phase (at 25 minutes, P < 0.032); additionally, these highest doses suppressed CPS relative to the lower doses (0.1 and 0.3 mg/kg) in both the acute phase (at 15 minutes, P < 0.037) and in the inflammatory phase (at 30 minutes, P < 0.002) (Fig. 2B). Analysis of the peak pain value within the inflammatory phase in male mice revealed significant differences between the groups (saline, AT 0.1–30 mg/kg i.p.) (F_{6, 14.091} = 293.577, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain at 10 mg/kg (P < 0.001) and 30 mg/kg (P < 0.001). Analysis of the AUC of pain behavior revealed significant differences between the groups (saline, AT 0.1–30 mg/kg) in both Phase 1 (F_6,37 = 51.082, P < 0.00001; Fig. 2E) and Phase 2 (F_6,37 = 102.896, P < 0.00001; Fig. 2F) of the formalin test. Both 10 and 30 mg/kg were found to produce antinociception relative to the vehicle in both Phase 1 (P < 0.001; Fig. 2E) and Phase 2 (P < 0.001; Fig. 2F) of the formalin test. Additionally, 0.3, 1, and 3 mg/kg were found to produce antinociception relative to the vehicle in Phase 2 (P < 0.001; Fig. 2F) of the formalin test. Both the 10 and 30 mg/kg dose were found to produce significant antinociception relative to all other groups in both Phase 1 (P < 0.001; Fig. 2E) and Phase 2 (P < 0.001; Fig. 2F) of the formalin test. The 3 mg/kg dose was also found to suppress pain relative to the lower doses (0.1 and 0.3 mg/kg) in Phase 2 of the formalin test (P < 0.009; Fig. 2F). No differences relative to vehicle was noted for the low and medium doses (0.1, 0.3, 1, and 3 mg/kg) in Phase 1 of the formalin test (P = 1.00; Fig. 2E) and no differences relative to the vehicle was noted for the lowest dose (0.1 mg/kg) in Phase 2 of the formalin test (P = 1.00 Fig. 2F). Assessment of the AUC differences between the early inflammatory phase (15–25 minutes post-formalin injection) and late inflammatory phase (25–35 minutes post-formalin injection) in male mice revealed significant differences between the groups (saline, AT 0.1–30 mg/kg i.p.) (F_{6, 14.035} = 24.346, P < 0.001). Post-hoc analysis revealed a significant increase in this difference, representing a greater proportion of the pain occurring in the early inflammatory phase, relative to vehicle at 1 mg/kg (P < 0.005) and 3 mg/kg (P < 0.001).

Assessment of Sex- and Dose-Dependent Differences of CBD and AT Antinociception in the Formalin Test.

Comparing the effects of CBD at different doses (0.3–100 mg/kg i.p.) on pain behaviors between males and females in Phase 1 of the formalin test, we found a significant effect of dose (F_6,82 = 41.791, P < 0.001) but a non-significant effect of sex (F_1,82 = 2.035, P = 0.157) and a non-significant sex x dose interaction (F_6,82 = 0.190, P = 0.979) (Fig. 3A). In this phase, no sex differences were observed at any of the doses (0.3–100 mg/kg, P < 0.979) (Fig. 3A). When comparing the effects of CBD at different doses (0.3–100 mg/kg i.p.) on pain behaviors between male and female mice in Phase 2 of the formalin test, we found a significant effect of dose (F_6,82 = 137.188, P < 0.001) and of sex (F_1,82 = 12.025, P < 0.001), but a non-significant effect of sex x dose interaction (F_6,82 = 1.753, P = 0.119) (Fig. 3B). Significant sex differences were observed at 2.5 mg/kg, with males showing a significant reduction in pain behaviors compared with females (P < 0.001) (Fig. 3B). No significant sex differences were noted in this phase for any of the other doses (0.3–1 mg/kg and 10–100 mg/kg, P < 0.986) (Fig. 3B).

Fig. 3.

Assessment of sex- and dose-dependent differences of CBD and AT in the formalin pain test in wild-type mice. CBD suppressed pain in a dose-dependent manner in both Phase 1 (A) and Phase 2 (B) of the formalin test. CBD suppressed pain in a sex-dependent, but not sex x dose-dependent, manner in Phase 2 (B) of the formalin test. CBD suppressed pain behavior at doses of 30 mg/kg or higher in males versus 100 mg/kg in females during Phase 1 (A) and suppressed pain behavior at doses of 2.5 mg/kg or higher in males versus 10 mg/kg or higher in females during Phase 2 (B). AT suppressed pain in a dose-dependent manner in both Phase 1 (C) and Phase 2 (D) of the formalin test. AT suppressed pain in a sex and sex x dose interaction-dependent manner in Phase 2 (D) of the formalin test. AT suppressed pain behavior at doses of 10 mg/kg or higher in males and females during Phase 1 (C) and suppressed pain behavior at doses of 0.3 mg/kg or higher in males versus 3 mg/kg or higher in females during Phase 2 (D). Data are expressed as mean ± S.E.M. (vehicle: n = 12 females and n = 12 males; saline: n = 8 females and n = 8 males; CBD 0.3 mg/kg, 1 mg/kg, 2.5 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg: n = 6 females and n = 6 males; AT 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg: n = 6 females and n = 6 males). Relative to vehicle in male versus male, female versus female: *P < 0.05, **P < 0.01, ***P < 0.001. Relative to equivalent dose in female versus male: @P < 0.05, @@P < 0.01, @@@P < 0.001. Two-way ANOVA with Bonferroni post-hoc used to calculate significance.

Comparing the effects of AT at different doses (0.1–30 mg/kg i.p.) on pain behaviors between males and females in Phase 1 of the formalin test, we found a significant effect of dose (F_6,74 = 112.290, P < 0.001) but a nonsignificant effect of sex (F_1,74 = 2.404, P = 0.125) and a non-significant sex x dose interaction (F_6,74 = 0.869, P = 0.522) (Fig. 3C). In this phase, sex differences were only noted at 0.3 mg/kg (P < 0.047) with males showing less pronounced pain behaviors compared with female mice; neither male nor female mice, however, had a statistically significant reduction in pain behavior relative to vehicle at this dose (Fig. 3C). No significant sex differences were noted in this phase for any of the other doses (0.1 and 1-30 mg/kg, P < 0.814). When comparing the effects of AT at different doses (0.1–30 mg/kg i.p.) on pain behaviors between male and female mice in Phase 2 of the formalin test, we found a significant effect of dose (F_6,74 = 195.935, P < 0.001), sex (F_1,74 = 10.499, P < 0.002), and sex x dose interaction (F_6,74 = 4.532, P < 0.006) (Fig. 3D). Significant sex differences were observed at 0.1 mg/kg (P < 0.043), 0.3 mg/kg (P < 0.001), and 1 mg/kg (P < 0.002), with males showing a significant reduction in pain behaviors compared with females at each of these doses (Fig. 3D). No significant sex differences were noted in this phase for any of the other doses (3–30 mg/kg, P < 0.966) (Fig. 3D).

Assessment of Sedative Effects of CBD and AT in the Rotarod Test.

In female mice, no significant difference was observed for CBD 100 mg/kg relative to vehicle at baseline (P < 0.125), 30 minutes post-injection (P < 0.161), or at 90 minutes post-injection (P < 0.850) (Fig. 4A). Similarly, no significant differences were observed in male mice for CBD 100 mg/kg relative to vehicle at baseline (P < 0.410), 30 minutes post-injection (P < 0.296), or at 90 minutes post-injection (P < 0.428) (Fig. 4A).

Fig. 4.

Assessment of sedative effects of CBD (A) and AT (B) in female and male wild-type mice in the rotarod test. No significant differences were observed. Data are expressed as mean ± S.E.M. (vehicle: n = 7 females and n = 8 males; CBD 100 mg/kg: n = 8 females and n = 6 males; saline: n = 8 females and n = 8 males; AT 10 mg/kg: n = 8 females and n = 7 males). Independent samples T-test used to calculate significance.

In female mice, no significant differences were observed for AT 10 mg/kg relative to saline at baseline (P < 0.107), 30 minutes post-injection (P < 0.362), or at 90 minutes post-injection (P < 0.387) (Fig. 4B). In male mice, no significant differences were observed for AT 10 mg/kg relative to saline at baselines (P < 0.079), 30 minutes post-injection (P < 0.230), or at 90 minutes post-injection (P < 0.170) (Fig. 4B).

Dose Response Curves and ED₅₀ Calculation for CBD and AT Antinociception in the Formalin Test.

The median effective dose (ED₅₀) for CBD was calculated to be 17.783 mg/kg (R²=0.911) in female mice and 9.047 mg/kg (R²=0.873) in male mice (Fig. 5A). The ED₅₀ for AT was calculated to be 3.298 mg/kg (R²=0.942) in female mice and 2.824 mg/kg (R²=0.889) in male mice (Fig. 5B).

Fig. 5.

Dose-response curves for CBD (A) and AT (B) in female and male wild-type mice in Phase 2 of the formalin test. (vehicle: n = 12 females and n = 12 males; saline: n = 8 females and n = 8 males; CBD 0.3 mg/kg, 1 mg/kg, 2.5 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg: n = 6 females and n = 6 males; AT 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg). Dotted lines represent 95% confidence interval.

Comparison of ED₅₀ Drug Antinociceptive Effects of CBD and AT, With and Without 5-HT_1A Antagonism, in the Formalin Test.

In female mice, significant difference between the groups (vehicle, CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg) (F_3,26 = 47.481, P < 0.00001) were found in a time- (F_11,286 = 92.557, P < 0.00001) and time-group- (F_33,286 = 5.525, P < 0.00001)dependent manner (Fig. 6A). The CBD ED₅₀ dose suppressed CPS relative to the vehicle in the inflammatory phase (at 25–35 minutes, P < 0.001, and at 40 minutes, P < 0.023) (Fig. 6A). The AT ED₅₀ dose also suppressed CPS relative to the vehicle in the inflammatory phase (at 25–35 minutes, P < 0.001, and at 40 minutes, P < 0.023) (Fig. 6A). The combination CBD ED₅₀ + AT ED₅₀ dose suppressed CPS relative to the vehicle in both the acute phase (at 5 minutes, P < 0.042) and in the inflammatory phase (at 25–35 minutes, P < 0.001, and at 40 minutes, P < 0.023) (Fig. 6A). Additionally, this combination dose suppressed CPS relative to the CBD ED₅₀ dose in both the acute phase (at 5 minutes, P < 0.026) and the inflammatory phase (at 20 minutes, P < 0.027) (Fig. 6A). Analysis of the AUC of pain behavior revealed significant differences between the groups (vehicle, CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg) in both Phase 1 (F_3,26 = 4.044, P = 0.017; Fig. 6C) and Phase 2 (F_3,26 = 64.301, P < 0.00001; Fig. 6D) of the formalin test. Only the combination ED₅₀ dose was found to produce significant antinociception relative to the vehicle in Phase 1 of the formalin test (P = 0.011; Fig. 6C) while all three ED₅₀ doses (CBD, AT, and combination CBD+AT) produced antinociception relative to the vehicle in Phase 2 of the formalin test (P < 0.001; Fig. 6D). In females, no significant difference was found between the combination ED₅₀ dose and either the CBD or AT ED₅₀ doses in Phase 1 (Fig. 6C) or Phase 2 (Fig. 6D) of the formalin test (P < 0.147).

Fig. 6.

Drug-dependent antinociceptive effects of CBD and AT, with and without 5-HT_1A antagonism, in the formalin test in wild-type mice. ED₅₀ doses were calculated from Phase 2 and were 17.78 mg/kg for CBD in female mice, 9.05 mg/kg for CBD in male mice, 3.30 mg/kg for AT in female mice, and 2.82 mg/kg for AT in male mice. ED₅₉ doses of CBD and AT, alone and in combination, suppressed pain behavior in both female (A) and male (B) wild-type mice. This suppression was partially reversed by 5-HT_1A antagonism by WAY-100635 in both female (A) and male (B) wild-type mice. In both female (C) and male (E) mice, the combination CBD ED₅₀ + AT ED₅₀ dose suppressed AUC of pain behavior in Phase 1 while individual CBD ED₅₀ and AT ED₅₀ doses failed to do so and this suppression was fully reversed by 5-HT_1A antagonism. In female (D) mice, the combination CBD ED₅₀ + AT ED₅₀ dose suppressed pain behavior in Phase 2 at a level not significantly different from the individual CBD ED₅₀ and AT ED₅₀ doses and this suppression was partially reversed by 5-HT_1A antagonism. In male (F) mice, the combination CBD ED₅₀ + AT ED₅₀ dose suppressed pain behavior in Phase 2 to a significantly greater degree than the antinociception produced by the individual CBD ED₅₀ and AT ED₅₀ doses and this effect was partially reversed by 5-HT_1A antagonism. Data are expressed as mean ± SEM. (vehicle: n = 12 females and n = 12 males; CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg: n = 6 females; CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg: n = 6 males). (A) Time post formalin (min) versus CPS in female wild-type mice. (B) Time post formalin (min) versus CPS in male wild-type mice. (C) Compound versus AUC during Phase 1 in female wild-type mice. (D) Compound versus AUC during Phase 2 in female wild-type mice. (E) Compound versus AUC during Phase 1 in male wild-type mice. (F) Compound versus AUC during Phase 2 in male wild-type mice. Repeated measure one-way ANOVA with Bonferroni post-hoc used to calculate significance for (A) and (B). One-way ANOVA with Bonferroni post-hoc used to calculate significance for (C), (D), (E), and (F). Relative to vehicle: *P < 0.05, **P < 0.01, ***P < 0.001. Relative to combination CBD ED₅₀ + AT ED₅₀: @P < 0.05, @@P < 0.01, @@@P < 0.001.

When assessing the effects of 5-HT_1A receptor antagonism by WAY-100635 in female mice, significant differences were found between the groups (vehicle, WAY-100635 0.1 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg) (F_3,26 = 36.123, P < 0.00001) were found in a time- (F_11,286 = 106.239, P < 0.00001) and time-group (F_33,286 = 4.843, P < 0.00001) dependent manner (Fig. 6A). WAY-100635 alone caused a temporal shift in the inflammatory phase, with CPS being increased relative to the vehicle at 20 minutes (P < 0.009) and decreased relative to the vehicle at 35 minutes (P < 0.002) (Fig. 6A). When WAY-100635 was followed by the combination CBD ED₅₀ + AT ED₅₀ dose, the antinociceptive effect produced by the combination CBD ED₅₀ + AT ED₅₀ dose was reversed in both the acute phase (at 5 minutes, P < 0.005) and the inflammatory phase (at 20 minutes, P < 0.012, and at 25 minutes, P < 0.001) (Fig. 6A). However, this reversal was not complete as WAY-100635 followed by the combination CBD ED₅₀ + AT ED₅₀ dose still reduced CPS relative to the vehicle in the inflammatory phase (at 30–35 minutes, P < 0.002, and at 40 minutes, P < 0.036) (Fig. 6A). Analysis of the AUC revealed significant differences between the groups (vehicle, WAY-100635 0.1 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg) in both Phase 1 (F_3,26 = 4.696, P = 0.009; Fig. 6C) and Phase 2 (F_3,26 = 44.272, P < 0.00001; Fig. 6D) of the formalin test. No significant difference was noted between vehicle and WAY-100635 alone in either Phase 1 (P = 1.00; Fig. 6C) or Phase 2 (P = 0.458; Fig. 6D). Meanwhile, pretreatment with WAY-100635 followed by the combination CBD ED₅₀ + AT ED₅₀ reversed antinociception mediated by the combination CBD ED₅₀ + AT ED₅₀ dose in both Phase 1 (P = 0.027; Fig. 6C) and Phase 2 (P < 0.001; Fig. 6D). Although this reversal was complete in Phase 1 (Fig. 6C), it was only partial in Phase 2 (Fig. 6D) with a statistically significant antinociceptive effect persisting relative to both vehicle (P < 0.001) and WAY-100635 alone (P = 0.045).

Analysis of the peak pain value within the inflammatory phase in female mice revealed significant differences between the groups (vehicle, CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg) (F_{5, 15.307} = 18.290, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain only at the combination of CBD and AT ED₅₀ doses (P < 0.005). Assessment of the AUC differences between the early inflammatory phase (15–25 minutes post-formalin injection) and late inflammatory phase (25–35 minutes post-formalin injection) in female mice revealed significant differences between the groups (vehicle, CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg) (F_{5, 15.307} = 18.290, P < 0.001) (F_{5, 14.021} = 14.662, P < 0.001). Post-hoc analysis revealed a significant increase in this difference, representing a greater proportion of the pain occurring in the early inflammatory phase, relative to vehicle at the CBD ED₅₀ dose (P < 0.001), AT ED₅₀ dose (P < 0.001), the combination of CBD and AT ED₅₀ doses (P < 0.015), and WAY-100635 followed by the combination of CBD and AT ED₅₀ doses (P < 0.001).

In male mice, significant difference between the groups (vehicle, CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg) (F_3,26 = 90.142, P < 0.00001) were found in a time- (F_11,286 = 111.917, P < 0.00001) and time-group- (F_33,286 = 6.761, P < 0.00001) dependent manner (Fig. 6B). The CBD ED₅₀ dose suppressed CPS relative to the vehicle in the inflammatory phase (at 25–35 minutes, P < 0.006, and at 40 minutes, P < 0.040) (Fig. 6B). The AT ED₅₀ dose also suppressed CPS relative to the vehicle in the inflammatory phase (at 25–35 minutes, P < 0.002) (Fig. 6B). The combination CBD ED₅₀ + AT ED₅₀ dose suppressed CPS relative to the vehicle in both the acute phase (at 5 minutes, P < 0.001) and in the inflammatory phase (at 20–35 minutes, P < 0.006, and at 40 minutes, P < 0.040) (Fig. 6B). This combination CBD ED₅₀ + AT ED₅₀ dose additionally suppressed CPS relative to both of individual ED₅₀ doses in both the acute phase (at 5 minutes, P < 0.001) and in the inflammatory phase (at 20 minutes, P < 0.001) (Fig. 6B). Analysis of the AUC of pain behavior revealed significant differences between the groups (vehicle, CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg) in both Phase 1 (F_3,26 = 17.425, P < 0.00001; Fig. 6E) and Phase 2 (F_3,26 = 74.530, P < 0.00001; Fig. 6F) of the formalin test. Only the combination ED₅₀ dose was found to produce significant antinociception relative to the vehicle in Phase 1 of the formalin test (P < 0.001; Fig. 6E) while all three ED₅₀ doses (CBD, AT, and combination) produced antinociception relative to the vehicle in Phase 2 of the formalin test (P < 0.001; Fig. 6F). In males, the combination ED₅₀ dose produced significant antinociception relative to both individual ED₅₀ doses (AT and CBD) in both Phase 1 (P < 0.001; Fig. 6E) and Phase 2 (P < 0.001; Fig. 6F) of the formalin test.

When assessing the effects of 5-HT_1A antagonism by WAY-100635 in male mice, significant differences were found between the groups (vehicle, WAY-100635 0.1 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg) (F_3,26 = 112.340, P < 0.00001) were found in a time- (F_11,286 = 135.297, P < 0.00001) and time-group- (F_33,286 = 8.233, P < 0.00001) dependent manner (Fig. 6B). WAY-100635 alone caused a temporal shift in the acute phase, with CPS being increased relative to the vehicle at 15 minutes (P < 0.001) (Fig. 6B). When WAY-100635 was followed by the combination CBD ED₅₀ + AT ED₅₀ dose, the antinociceptive effect produced by the combination CBD ED₅₀ + AT ED₅₀ dose was reversed in both the acute phase (at 5 minutes, P < 0.001, and at 15 minutes, P < 0.028) and the inflammatory phase (at 20 minutes, P < 0.001) (Fig. 6B). However, this reversal was not complete as WAY-100635 followed by the combination CBD ED₅₀ + AT ED₅₀ dose still reduced CPS relative to the vehicle in the inflammatory phase (at 25–35 minutes, P < 0.001) (Fig. 6B). AUC analysis revealed significant differences between the groups (vehicle, WAY-100635 0.1 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg) in both Phase 1 (F_3,26 = 27.104, P < 0.00001; Fig. 6E) and Phase 2 (F_3,26 = 82.360, P < 0.00001; Fig. 6F) of the formalin test. No significant difference was noted between vehicle and WAY-100635 alone in either Phase 1 (P = 0.585; Fig. 6E) or Phase 2 (P = 0.271; Fig. 6F). Pretreatment with WAY-100635 followed by the combination CBD ED₅₀ + AT ED₅₀ dose reversed the antinociception mediated by the combination CBD ED₅₀ + AT ED₅₀ dose in both Phase 1 and Phase 2 (P < 0.001 for both; Fig. 6, E and F). This reversal was complete in Phase 1 (Fig. 6E), but in Phase 2 (Fig. 6F) it was only partial, with a statistically significant antinociceptive effect persisting relative to both vehicle and WAY-100635 alone (P < 0.001 for both).

Analysis of the peak pain value within the inflammatory phase in male mice revealed significant differences between the groups (vehicle, CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg) (F_{5, 15.433} = 30.348, P < 0.001), with post-hoc analysis revealing a significant decrease relative to vehicle in peak inflammatory pain only at the combination of CBD and AT ED₅₀ doses (P < 0.001). Assessment of the AUC differences between the early inflammatory phase (15–25 minutes post-formalin injection) and late inflammatory phase (25–35 minutes post-formalin injection) in male mice revealed significant differences between the groups (vehicle, CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg) (F_{5, 15.247} = 14.777, P < 0.001). Post-hoc analysis revealed a significant increase in this difference, representing a greater proportion of the pain occurring in the early inflammatory phase, relative to vehicle at the CBD ED₅₀ dose (P < 0.001), AT ED₅₀ dose (P < 0.001), and WAY-100635 followed by the combination of CBD and AT ED₅₀ doses (P < 0.001).

Assessment of Sex and ED₅₀ Drug Antinociceptive Differences for CBD and AT in the Formalin Test.

When comparing the effects of the different ED₅₀ doses (vehicle, CBD 17.78 mg/kg in females and 9.015 mg/kg in males, AT 3.30 mg/kg in females and 2.82 mg/kg in males, CBD 17.78 mg/kg + AT 3.30 mg/kg in females and CBD 9.05 mg/kg + AT 2.82 mg/kg in males, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg in females and CBD 9.05 mg/kg + AT 2.82 mg/kg in males) on pain behaviors between female and male mice in Phase 1 of the formalin test, we found a significant effect of group (F_5,72 = 15.536, P < 0.00001) but a non-significant effect of sex (F_1,72 = 0.044, P = 0.835) and a non-significant sex x group interaction (F_5,72 = 0.528, P = 0.755) (Fig. 7A). No sex differences were observed between females and males at the CBD ED₅₀ dose (P = 0.790), the AT ED₅₀ dose (P = 0.584), the combination CBD + AT ED₅₀ dose (P = 0.872), and WAY-100635 followed by the combination CBD + AT ED₅₀ dose (P = 0.212) (Fig. 7A). When comparing the effects of the different ED₅₀ doses (vehicle, CBD 17.78 mg/kg in females and 9.015 mg/kg in males, AT 3.30 mg/kg in females and 2.82 mg/kg in males, CBD 17.78 mg/kg + AT 3.30 mg/kg in females and CBD 9.05 mg/kg + AT 2.82 mg/kg in males, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg in females and CBD 9.05 mg/kg + AT 2.82 mg/kg in males) on pain behaviors between female and male mice in Phase 2 of the formalin test, we found a significant effect of group (F_5,72 = 84.532, P < 0.00001) and sex (F_1,72 = 5.025, P = 0.028) but a non-significant sex x group interaction (F_5,72 = 1.527, P = 0.192) (Fig. 7B). Sex differences were observed between females and males for the combination CBD + AT ED₅₀ dose (P = 0.028) and WAY-100635 followed by the combination CBD + AT ED₅₀ dose (P = 0.014) (Fig. 7B). No sex differences were observed between females and males at the CBD ED₅₀ dose (P = 0.905) or the AT ED₅₀ dose (P = 0.701) (Fig. 7B).

Fig. 7.

Assessment of sex- and ED₅₀ dose-dependent differences of CBD and AT in the formalin test in wild-type mice. ED₅₀ doses were calculated from Phase 2 and were 17.78 mg/kg for CBD in female mice, 9.05 mg/kg for CBD in male mice, 3.30 mg/kg for AT in female mice, and 2.82 mg/kg for AT in male mice. Both the individual CBD ED₅₀ and AT ED₅₀ doses and the combination CBD ED₅₀ + AT ED₅₀ dose suppressed pain behavior in Phase 2 (B) of the formalin test while only the combination CBD ED₅₀ + AT ED₅₀ dose suppressed pain behavior in Phase 1 (A) of the formalin test. Prior administration of WAY-100635 reversed the combination CBD ED₅₀ + AT ED₅₀ mediated antinociception fully in Phase 1 (A) of the formalin test and partially in Phase 2 (B) of the formalin test, with Phase 2 reversal being greater in female than male mice. Data are expressed as mean ± S.E.M. (vehicle: n = 12 females and n = 12 males; CBD 17.78 mg/kg, AT 3.30 mg/kg, CBD 17.78 mg/kg + AT 3.30 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 17.78 mg/kg + AT 3.30 mg/kg: n = 6 females; CBD 9.05 mg/kg, AT 2.82 mg/kg, CBD 9.05 mg/kg + AT 2.82 mg/kg, WAY-100635 0.1 mg/kg, WAY-100635 0.1 mg/kg followed by CBD 9.05 mg/kg + AT 2.82 mg/kg: n = 6 males). Relative to vehicle in male versus male, female versus female: *P < 0.05, **P < 0.01, ***P < 0.001. Relative to equivalent dose in female versus male: @P < 0.05, @@P < 0.01, @@@P < 0.001. Two-way ANOVA with Bonferroni post-hoc used to calculate significance.

Assessment of mRNA Changes in Brain and Spinal Cord Cannabinoid Receptors.

In female mice, there were significant changes in mRNA levels for CB₁ (F_3,17 = 13.209, P < 0.001) with an increase in CB₁ mRNA levels for AT ED₅₀ relative to vehicle (P < 0.002) in whole brain tissue (Fig. 8A). No significant differences for CB₁ mRNA levels were noted for CBD ED₅₀ or CBD ED₅₀ + AT ED₅₀ relative to vehicle (P = 1.00). These changes were also seen in assessment of female whole spinal cord tissue mRNA levels, with significant changes in CB₁ (F_3,17 = 10.166, P < 0.001), with CB₁ mRNA levels once again being increased for AT ED₅₀ relative to vehicle (P < 0.001), while no significant differences for CB₁ mRNA levels were noted for CBD ED₅₀ or CBD ED₅₀ + AT ED₅₀ relative to vehicle (P > 0.065) (Fig. 8B).

Fig. 8.

Administration of the AT ED₅₀ dose (3.30 mg/kg in female mice, 2.82 mg/kg in male mice) increased expression of CB₁ mRNA expression levels in whole brain and whole spinal cord tissue of female [(A) brain, (B) spinal cord] but not male [(C) brain, (D) spinal cord] mice. Data are expressed as mean ± S.E.M. (female, whole brain: vehicle n = 5, CBD ED₅₀ n = 5, AT ED₅₀ n = 5, CBD ED₅₀ + AT ED₅₀ n = 6; female, whole spinal cord: vehicle n = 4, CBD ED₅₀ n = 6, AT ED₅₀ n = 5, CBD ED₅₀ + AT ED₅₀ n = 6; male, whole brain: vehicle n = 4, CBD ED₅₀ n = 6, AT ED₅₀ n = 6, CBD ED₅₀ + AT ED₅₀ n = 6; male, whole spinal cord: vehicle n = 5, CBD ED₅₀ n = 5, AT ED₅₀ n = 6, CBD ED₅₀ + AT ED₅₀ n = 5). One-way ANOVA with Bonferroni post-hoc used to calculate significance. Relative to vehicle: *P < 0.05, **P < 0.01, ***P < 0.001.

Of note, these changes were not observed in male mice as there were no significant changes in mRNA levels for CB₁ in whole brain tissue (F_3,18 = 1.987, P < 0.152) (Fig. 8C) or in whole spinal cord tissue (F_3,17 = 0.345, P < 0.793) (Fig. 8D) and no significant differences were noted between any of the groups relative to vehicle tissue (P > 0.628).

Measurement of CB₂ in the brain and spinal cord tissue of both male and female mice yielded results below the threshold for expression (data not shown). No significant differences in gyliceraldehyde-3-phosphase dehydrogenase expression were noted between any of the groups.

Discussion

Inflammatory pain is associated with chronic rheumatic disease pain and cute pain due to tissue injury, infection, and radiation (Barbour et al., 2017; Ji et al., 2016). Cannabinoid compounds represent an important target in pain management due to the endocannabinoid system’s role in pain modulation and the established preclinical utility of cannabinoids in antinociception (Guindon and Hohmann, 2009; Blanton et al., 2021; Crocq, 2020). Chronic pain prevalence is rising and is a significant detriment to the quality of life of those it impacts, meaning new analgesic therapies are needed (Dahlhamer et al., 2018; Leadley et al., 2014; Gaskin and Richard, 2012).

The phytocannabinoid CBD has been found to enhance the activity of the endocannabinoid anandamide (Mechoulam et al., 2002; Elmes et al., 2015; De Petrocellis et al., 2011). and has proven antinociceptive in several acute and chronic pain models (Philpott et al., 2017; Britch et al., 2020; Blanton et al., 2022; Linher-Melville et al., 2020). While legal restrictions have limited clinical research of CBD, there have been a number of promising results in its topical use even while systemic results have been lacking, possibly due to its pharmacokinetics (Bebee et al., 2021; Schneider et al., 2022; Dieterle et al., 2022; Eskander et al., 2020). Oral CBD has a bioavailability of 25–30%, with significant first-pass metabolism, and a half-life of between 2 to 5 days (Perucca and Bialer, 2020; Millar et al., 2018). AT is Food and Drug Administration approved for management of chronic pain and has proven effective in preclinical pain models, although few have assessed for any antinociceptive sex differences (Thour and Marwaha, 2022; Sawynok et al., 2008; Liu et al., 2013; Thompson et al., 2019).

Here, we demonstrated that CBD antinociception occurred at lower doses in male mice than in female mice in the formalin model of inflammatory pain. While male mice required lower doses, significant sex differences were only noted in the inflammatory phase at the 2.5 mg/kg dose. Previous studies investigating CBD antinociceptive sex differences found either similar results (Greene et al., 2018; Linher-Melville et al., 2020) or no significant sex differences (Britch et al., 2017, 2020). Conversely, pharmacokinetic analysis has revealed that females have higher CBD concentrations following acute or repeated dosing (Matheson et al., 2022). Lacking further pharmacokinetic and pharmacodynamic studies, it is premature to speculate as to why CBD was more effective in male mice (Lucas et al., 2018).

We found similar results when assessing the antinociceptive effect of AT alone in the formalin model of inflammatory pain. As with CBD, AT was more effective in male than in female mice. Unlike CBD, significant sex differences were observed at multiple doses with differences noted in both the acute and inflammatory phase at 0.3 mg/kg and in the inflammatory phase at 0.1 mg/kg and 1 mg/kg. Despite AT’s approval for chronic pain management, the majority of research was performed only in males as it preceded National Institutes of Health policy updates that required sex to be considered. One recent study did find that male rats achieved antinociception at lower doses of AT than females (O’Brien and McDougall, 2020). However, previous preclinical findings of AT antidepressive sex differences (Caldarone et al., 2003) were followed by clinical research that found insignificant pharmacokinetic and antidepressive sex differences (Kokras et al., 2011; Sramek et al., 2016). Our preclinical findings of AT antinociceptive sex differences thus support the need for confirmatory clinical studies.

Current pain management guidelines use a multimodal approach to limit opioid use and adverse effects from the analgesics used (Manworren, 2015). To assess CBD’s utility within this approach, we combined the ED₅₀ doses of CBD and AT. If effective, CBD could provide benefit for acute and inflammatory pain. Combining these ED₅₀ doses produced acute phase antinociception, which was not observed with either CBD or AT alone, and produced a significant additive effect in the inflammatory phase of male mice only. We hypothesized this effect was due to AT increasing synaptic serotonin to enhance the antinociceptive effects of CBD positively modulating the 5-HT_1A receptor (Rock et al., 2012; De Gregorio et al., 2019; Jesus et al., 2019; Ward et al., 2014; Lawson, 2017). 5-HT_1A is a G_i/o protein-coupled receptor that acts as a presynaptic autoreceptor, regulating negative feedback for serotonin release, and as a postsynaptic heteroreceptor, potentially mediating antinociceptive and anti-depressive effects (Albert and Vahid-Ansari, 2019; Haleem, 2019). Although CBD has been proposed to act at both locations, its actions at the postsynaptic heteroreceptor has been associated with its antidepressive and catalepsy-reversing effects (Zanelati et al., 2010; Linge et al., 2016; Sonego et al., 2016). Furthermore, targeting of CBD to postsynaptic heteroreceptors resulted in similar effects to systemic injections, further supporting the importance of these receptors in CBD’s effects (Campos and Guimarães, 2008). We identified this receptor as a potential focus for the combined effects of CBD and AT, as AT’s enhancement of synaptic serotonin would offset negative feedback by the autoreceptors while enhancing the effect of CBD at postsynaptic heteroreceptors (Fig. 9). The importance of this receptor was supported by our finding that pretreatment with 5-HT_1A antagonist WAY-100635 reversed the antinociception mediated by the combination of CBD and AT ED₅₀ doses.

Fig. 9.

Mechanism of proposed combined CBD and AT antinociception. CBD acts as a positive allosteric modulator at 5-HT1A receptors, which are G_i/o coupled receptors located both as presynaptic autoreceptors and postsynaptic heteroreceptors. These receptors are associated with enhanced potassium conductance, depressed calcium conductance, inhibition of PLCβ, inhibition of adenylyl cyclase, and indirect alteration of the extracellular signal-regulated kinase 1/2 signaling pathway. The autoreceptor attenuates release of serotonin while activation of the heteroreceptor is associated with analgesic and anti-depressive effects. AT is associated with, among other effects, inhibition of the serotonin reuptake transporter to increase synaptic serotonin. The use of AT with CBD offsets the attenuation of serotonin release by 5-HT_1A autoreceptors to allow for enhanced synaptic serotonin acting at 5-HT_1A postsynaptic heteroreceptors that have been enhanced by CBD’s actions as a positive allosteric modulator. Created with BioRender.com.

Other possible explanations for this combined effect warrant further investigation. Firstly, CBD and AT are both known to produce a net anti-inflammatory effect that could attenuate inflammatory pain (Li et al., 2018; Kubera et al., 2000). Secondly, CBD and AT both use a variety of antinociceptive mechanisms. In addition to 5-HT_1A modulation, CBD exerts antinociceptive effects through positive modulation of α3 glycine receptors, activation of the transient receptor potential V1 channel receptors, and inverse agonism of orphan g-protein coupled receptors 3, 6, and 12 (Xiong et al., 2012; De Petrocellis et al., 2011; Costa et al., 2007; Eggerickx et al., 1995; Tanaka et al., 2007; Laun et al., 2019). Meanwhile, AT antinociception occurs through its inhibition of serotonin and norepinephrine reuptake, inhibition of H₁ histamine receptors, inhibition of muscarinic acetylcholine receptors, and through enhanced norepinephrine-mediated activation of α_2A adrenoceptors (Lawson, 2017; Sawynok et al., 2001; Nekovarova et al., 2014; Ghelardini et al., 2000; Özdoğan et al., 2004). Lastly, there are possible pharmacokinetic interactions between CBD and AT as CBD inhibits the cytochrome P450 enzymes 2C19 and 2D6 that metabolize AT (Wilson-Morkeh et al., 2020). It is important to note that this interaction does not preclude usage of this combination. Recently, a clinical trial evaluated the possible benefit of CBD in the management of migraines, with one group receiving both CBD and AT (Nicolodi et al., 2017). Additionally, a preclinical study evaluated this combination in a canine model of osteoarthritis and found a significant benefit to using the drugs in combination (Brioschi et al., 2020). As this interaction could positively or negatively influence the side effects of AT, further research to confirm this interaction and its magnitude should be performed. While CBD (Iffland and Grotenhermen, 2017) and AT (McClure and Daniels, 2021) have both been associated with sedation, no significant differences in rotarod performance were observed and so reduction in pain behaviors in the formalin test can be attributed solely to antinociceptive effects.

We observed that several low doses of CBD in males and AT in both females and male caused an increased proportion of inflammatory pain in the early versus late inflammatory phase. Further, we observed that low doses of AT in females caused an increase in peak inflammatory pain relative to the vehicle, while these doses caused no change in overall inflammatory pain. Further experimentation to better understand this effects and its implications are needed.

Assessing the ED₅₀ doses (CBD ED₅₀, AT ED₅₀, and CBD ED₅₀ + AT ED₅₀) of mRNA expression changes revealed that AT ED₅₀ alone enhanced expression of CB₁ in female mice only. This is possibly explained by AT-dependent stimulation of lysophosphatidic acid receptor 1, which co-immunoprecipitates with CB₁ (Olianas et al., 2021). While CBD had no effect on CB₁ expression, despite its antagonism (Pertwee, 2008), this is consistent with previous findings (Winstone et al., 2022). With regards to the CBD ED₅₀ + AT ED₅₀ dose, it is possible that CBD blunted the effects of AT on CB₁ receptor expression. Expression of CB₂ was below the level of detection for all groups. These mRNA findings highlight the need for further research into interactions between AT and cannabinoid receptors and lend further support to cannabinoid receptor-independent actions of CBD.

In summary, this study identifies sex as an important factor in CBD- and AT-induced antinociception and supports the use of CBD within a multimodal approach due to the enhancement of its efficacy when combined with AT. Additionally, we provide evidence for the role of 5-HT_1A in combined CBD and AT antinociception through the actions of its antagonist, WAY-100635. Further research is needed into the mechanism of combined antinociception, especially regarding possible combined anti-inflammatory effects, possible pharmacokinetic interactions, and evaluation of this effect within chronic pain models. Finally, the presence of AT sex differences in this in vivo model indicates the need for clinical studies looking into possible sex differences, particularly considering its frequent use for chronic pain conditions.

Abbreviations

α3 GlyR

α3 glycine receptor

AT

amitriptyline

AUC

area under the curve

CB₁

cannabinoid receptor type 1

CB₂

cannabinoid receptor type 2

CBD

cannabidiol

CPS

composite pain score

GPR

orphan g-protein coupled receptor

MPF

minutes post formalin

5-HT_1A

serotonin 1A receptor

RT-qPCR

real-time quantitative polymerase chain reaction

WAY-100635

N-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate

Authorship Contributions

Participated in research design: Barnes, Guindon.

Conducted experiments: Barnes, Banjara, Henderson-Redmond, Castro-Piedras.

Performed data analysis: Barnes.

Wrote or contributed to the writing of the manuscript: Barnes, McHann, Almodovar, Henderson-Redmond, Morgan, Castro-Piedras, Guindon.