Evidence For Cannabidiol Modulation of Serotonergic Transmission in a Model of Osteoarthritis via in vivo PET Imaging and Behavioral Assessment

Abstract

Background: Preclinical studies indicate that cannabidiol (CBD), the primary nonaddictive component of cannabis, has a wide range of reported pharmacological effects such as analgesic and anxiolytic actions; however, the exact mechanisms of action for these effects have not been examined in chronic osteoarthritis (OA). Similar to other chronic pain syndromes, OA pain can have a significant affective component characterized by mood changes. Serotonin (5-HT) is a neurotransmitter implicated in pain, depression, and anxiety. Pain is often in comorbidity with mood and anxiety disorders in patients with OA. Since primary actions of CBD are analgesic and anxiolytic, in this first in vivo positron emission tomography (PET) imaging study, we investigate the interaction of CBD with serotonin 5-HT_1A receptor via a combination of in vivo neuroimaging and behavioral studies in a well-validated OA animal model.

Methods: The first aim of this study was to evaluate the target involvement, including the evaluation of modulation by acute administration of CBD, or a specific target antagonist/agonist intervention, in control animals. The brain 5-HT_1A activity/availability was assessed via in vivo dynamic PET imaging (up to 60 min) using a selective 5-HT_1A radioligand ([¹⁸F]MeFWAY). Tracer bindings of 17 ROIs were evaluated based on averaged SUVR values over the last 10 min using CB as the reference region. We subsequently examined the neurochemical and behavioral alterations in OA animals (induction with monosodium iodoacetate (MIA) injection), as compared to control animals, via neuroimaging and behavioral assessment. Further, we examined the effects of repeated low-dose CBD treatment on mechanical allodynia (von Frey tests) and anxiety-like (light/dark box tests, L/D), depressive-like (forced swim tests, FST) behaviors in OA animals, as compared to after vehicle treatment.

Results: The tracer binding was significantly reduced in control animals after an acute dose of CBD administered intravenously (1.0 mg/kg, i.v.), as compared to that for baseline. This binding specificity to 5-HT_1A was further confirmed by a similar reduction of tracer binding when a specific 5-HT_1A antagonist WAY1006235 was used (0.3 mg/kg, i.v.). Mice subjected to the MIA-induced OA for 13-20 days showed a decreased 5-HT_1A tracer binding (25% to 41%), consistent with the notion that 5-HT_1A plays a role in the modulation of pain in OA. Repeated treatment with CBD administered subcutaneously (5 mg/kg/day, s.c., for 16 days after OA induction) increased 5-HT_1A tracer binding, while no significant improvement was observed after vehicle. A trend of increased anxiety or depressive-like behavior in the light/dark box or forced swim tests after OA induction, and a decrease in those behaviors after repeated low-dose CBD treatment, are consistent with the anxiolytic action of CBD through 5HT_1A receptor activation. There appeared to be a sex difference: females seem to be less responsive at the baseline towards pain stimuli, while being more sensitive to CBD treatment.

Conclusion: This first in vivo PET imaging study in an OA animal model has provided evidence for the interaction of CBD with the serotonin 5-HT_1A receptor. Behavioral studies with more pharmacological interventions to support the target involvement are needed to further confirm these critical findings.

Keywords: Anxiety; Cannabidiol; Osteoarthritis; PET imaging; Pain; Serotonin receptor.

Figure 1:

Synthesis Scheme of [¹⁸F]MeFWAY: Synthesis scheme of [¹⁸F]MeFWAY: (i) methyl trans-4-(chlorocarbonyl)cyclohexane-1-carboxylate, CH₂Cl₂, rt, 69%; (ii) NaBH₄, EtOH, rt, 2 days, 40–60%; (iii) TsOTs, Et₃N, CH₂Cl₂, rt, 50–55%; (iv) DAST, CH₂Cl₂, rt, 20–40%; (V) [¹⁸F]fluoride, Kryptofix222, K₂CO₃

Figure 2:

Brain segmentation to determine regional 5-HT_1A tracer binding

Figure 3:

Representative images of [¹⁸F]MeFWAY studies in mice (coronal and sagittal views; top level displayed CT and PET co-registered images and bottom level displayed PET images alone): (A) baseline (injection with tracer only); (B) pretreatment with acute CBD (1 mg/kg, iv.); (C) pretreatment with acute WAY100635 (0.3 mg/kg, i.v.). The brain area, as indicated with arrows in both coronal and sagittal views in panel C, is the area delineated as the “black” area due to the significant reduction of tracer binding after pretreatment with WAY100635. As seen, there was a significant reduction of tracer binding in the brain area in panel B and C, as indicated by the black area with lower brain uptake of the radiotracer when compared with the corresponding brain area in panel A of the baseline study. For comparison purposes, comparative PET images were displayed as SUV images (SUV_25–30, kBq/cc) for individual animals [derived from the normalized SUV_max values based on the SUV(CB) intensity values of individual animals and scaled to the same SUVR scale range (0–6.5) for all studies].

Figure 4:

Bar graphs of averaged regional brain 5-HT_1A availability (SUVR values for the last 10 min calculated using cerebellum as the reference region) at baseline (blue bar), and after pretreatment with either CBD (orange) or WAY100635 (grey). (n = 3–4 each group)

Figure 5:

Representative images (coronal and sagittal views) to indicate changes of 5-HT_1A tracer binding at d13 (B) and d20 (C) after the OA induction, as compared to baseline (A). A larger reduction in tracer binding was seen in day 13 animals. For comparison purposes, comparative PET images were displayed as SUV images (SUV_25–30min, kBq/cc) for individual animals [derived from the normalized SUV_max values based on the SUV(CB) intensity values of individual animals and scaled to the same SUVR scale range (0–4) for all studies].

Figure 6:

Representative images (coronal and sagittal views) to indicate changes of 5-HT_1A tracer binding at d13 after the OA induction (A) vs. an increased tracer binding after repeated low-dose of CBD treatment (5 mg/kg, s.c.) for 16 days (B). For comparison purposes, comparative PET images were displayed as SUV images (SUV_25–30min, kBq/cc) for individual animals [derived from the normalized SUV_max values based on the SUV(CB) intensity values of individual animals and scaled to the same SUVR scale range (0–4) for all studies].

Figure 7:

Bar graphs to indicate averaged regional changes in 5-HT_1A tracer binding (SUVR values for the last 10 min calculated using cerebellum as the reference region) of 17 ROIs for male mice across various time points; Baseline, OA_d13, OA_d20, OA_S and OA_C [OA_d13 and OA_d20 are groups of mice post-injection of MIA at day 13 and day 20, respectively. OA_S and OA_C are groups of mice treated for 16 days with CBD (5 mg/kg, s.c.) and saline, respectively.] (n = 3–4 for each group)

Figure 8:

Bar graphs to indicate averaged regional changes in 5-HT_1A tracer binding (SUVR values for the last 20 min calculated using cerebellum as the reference region) of 17 ROIs for female mice across various time points; Baseline, OA_d20, OA_S and OA_C [OA_d20 are groups of mice post-injection of MIA at day 20. OA_S and OA_C are groups of mice treated for 16 days with CBD (5 mg/kg, s.c.) and saline, respectively.] (n = 3–4 for each group)

Figure 9:

Von Frey tests: A. Comparison of R-paw vs. L-paw during the entire study: values for electronic Von Frey (Auto VF) withdraw threshold (g) for R-paw (MIA injected) vs. L-paw (vehicle injected) of male and female mice were compared. A progressive increase in mechanical sensitivity (decrease pain threshold) of the right hind paw following OA surgery, with no significant changes in sensitivity in the left hind paw, was observed. Figures 9B–9D are presented by using averaged paw (L and R) and pooling all data from baseline and OA (under the same paradigm) and separate them based on the different drug treatment. Plots (avg paw 50% withdrawal) are presented when both sex are included (B) and separately (C), to clearly indicate the sex/gender difference. The averaged paw 50% threshold measurement appeared to be decoupled in males as compared to females (CBD vs. vehicle), but not with the averaged paw filament measurement (Figure D). (data were derived from 5 M and 5 F).

Figure 10:

Light/Dark box tests: A (%light preference) & B (total distance traveled) when both males and females were included (top panel); C (%light preference) & D (total distance traveled) when males and females are separated in data analysis (data were derived from 5 M and 5 F). Less preference to light (A & C; did not reach significance) or increased distance traveled (B & D; *p < 0.05) were observed for both male and female mice post-OA surgery, as compared to baseline, suggesting a potential increase in anxiety after OA induction. The extent of decreased distance traveled (reduction of anxiety) was higher after CBD treatment than that after vehicle treatment. Females appeared to exhibit less anxiety-like behaviors (less reduction of %light preference) after OA induction, and were more sensitive to the CBD treatment, as compared to males (C & D).

Figure 11:

Forced Swim tests: A (immobility) & B (total distance traveled) when both males and females were included (top panel); C (immobility) & D (total distance traveled) when males and females are separated in data analysis (data were derived from 5 M and 5 F; *denoted for p < 0.05, and ** denoted for p < 0.005). When both M & F were included, immobility in FST showed significantly different (**p < 0.005) between baseline vs. OA, and baseline vs. post-treatment, while distance traveled showed significantly different (**p < 0.005) between baseline vs. post-treatment, and OA vs. post-treatment. In M vs. F separate analysis, male mice showed significant difference in immobility between baseline and OA; while female mice showed significant immobility differences among baseline vs. OA, OA vs. Veh, and CBD vs. Veh.