Cannabidiol Attenuates Chronic and Acute Hyperalgesia in Sickle Mice By Reducing Inflammation and Oxidative Stress

Pain is a major comorbidity of sickle cell disease (SCD) leading to hospitalization, opioid use and reduced survival. It can present as both chronic pain and/or unpredictable episodes of intense acute pain. Hydroxyurea (HU) is the mainstream FDA approved treatment for SCD, but its effect on reducing pain remains to be understood. A larger number of people with SCD use cannabis products for pain relief compared to those with other conditions. Cannabidiol (CBD) is one of the major constituents of Cannabis sativa, second to tetrahydrocannabinol, and has gained attention for its ability to treat pain without intoxicating side effects. CBD has been shown to decrease inflammation, oxidative stress, and inhibit mast cell activation, all of which occur in SCD. Thus, we hypothesize that CBD will ameliorate both chronic and acute hyperalgesia in humanized homozygous BERK sickle mice (called sickle) by inhibiting mast cell activation and decreasing inflammation and oxidative stress.

To simulate the clinical condition, we treated female sickle mice intraperitoneally with [i] vehicle (1:1:18 DMSO:Tween-20:saline), [ii] HU (50 mg/kg/d HU), [iii] CBD (20 mg/kg/d) or [iv] HU + CBD and determined their effect on chronic and acute hyperalgesia (pain). Sickle mice treated with synthetic CBD alone or cotreatment with HU+CBD daily showed significantly reduced chronic mechanical (p<0.001) and cold (p<0.001) hyperalgesia compared to baseline after 7 days of treatment, which was maintained for up to 14 days. HU decreased mechanical (p<0.01) but not cold hyperalgesia after 14 days compared to baseline. Thus, CBD independently attenuates chronic hyperalgesia, whereas HU may have a modest effect on chronic pain.

Following the initial 14 days of treatment above, we incited acute hyperalgesia by treating sickle mice with hypoxia/reoxygenation (H/R; 3 hours of hypoxia at 8% O₂ and 92% N₂, followed by 1 hour of reoxygenation at normoxia) on days 15 (H/R-1) and 18 (H/R-2). We observed that CBD, HU, and HU+CBD treatments were able to prevent an increase in H/R-incited hyperalgesia (acute response). CBD alone and HU+CBD significantly reduced mechanical (p<0.001) and cold hyperalgesia (p<0.001) compared to baseline following incitement of both H/R-1 and H/R-2, and up to 25 days, the last period of observation. HU alone did not show a significant decrease in mechanical or cold hyperalgesia compared to baseline following incitement of either H/R event, except for cold hyperalgesia 72h post-H/R-2 (p<0.05). CBD and HU+CBD treatments also reduced non-evoked cold avoidance compared to baseline in sickle mice, which spent significantly more time (p<0.01) in a cold chamber (4°C) 7d after H/R-2 (day 25), but HU alone did not. Thus, HU has a modest or no effect on different features of sickle pain, while CBD is able to ameliorate both chronic and acute pain.

We next investigated the mechanism of CBD's analgesic effect in sickle mice. Following 9 days of daily treatment, we observed that CBD decreased total and degranulating mast cells (p<0.05 and p<0.01, respectively) in the skin compared to vehicle, and a reduction in tryptase (p<0.05), a marker of mast cell activation, in the skin secretagogue. CBD treatment decreased substance P (p=0.05) and malondialdehyde, (p<0.05), markers of neuroinflammation and oxidative stress respectively, in the skin secretagogue, and serum amyloid P (p<0.05), a marker of inflammation, in plasma. CBD significantly decreased inflammatory cytokines IL-3, which promotes mast cell proliferation, TNFα, a hallmark feature of cytokine storm, and MIP-1α and RANTES, which contribute to leukocyte recruitment. Thus, CBD attenuates inflammation, neuroinflammation and oxidative stress in sickle mice and may ameliorate leukocyte activities, a hallmark feature of SCD.

We demonstrate that CBD is effective in attenuating chronic hyperalgesia and preventing acute pain by decreasing inflammation and oxidative stress. The effect of HU on reducing pain following H/R and chronic pain are modest and specific to certain characteristic features of sickle pain. Importantly, HU does not appear to interfere with the analgesic effect of CBD in sickle mice, which shows promise for CBD treatment in persons with SCD on HU. The availability of a >99% CBD-extract (Epidiolex) approved by the FDA to treat some forms of epilepsy may help facilitate clinical trials for assessing the effects of CBD on pain in persons with SCD.

Argueta:Cayenne Foundation: Honoraria; Cyclerion: Honoraria. Gupta:Novartis: Honoraria, Research Funding; CSL Behring: Honoraria; Cyclerion: Research Funding; 1910 Genetics: Research Funding; Grifols: Research Funding; Zilker LLC: Research Funding; UCI Foundation: Research Funding; SCIRE Foundation: Research Funding; Tautona Group: Honoraria.