Cannabidiol: Effect on ECS and Inflammation

Unlike the tricyclic chemical structure of THC, which is stable in a planar conformation, the bicyclic structure of CBD prefers the lowest energy conformation whereby the two ring structures are anti-periplanar, or perpendicular to each-other, ascribing important steric properties that differentiate the pharmacological profile of CBD from other phytocannabinoids (Burstein 2015). Where THC demonstrates partial agonism to CB1 and CB2, CBD has consistently demonstrated weak antagonism for both receptors and recent research further suggests CBD is a negative allosteric modulator (NAM) at CB1 while it retains partial agonist effects on CB2 (Morales et al. 2017, Tham et al. 2019). Nevertheless, CB1 has been indicated as central to the psychoactive properties of cannabinoids and the inherent non-psychoactive properties of CBD has supported interest in its therapeutic potential, especially as a modulator of pain and inflammation. In vitro, CBD has shown indirect effects on the endocannabinoid system (ECS) suggesting the prolongation of AEA via inhibition of the degradative enzyme fatty acid-amide hydrolase (FAAH) and antagonism at the putative membrane transporter responsible for AEA reuptake, thus enhancing ECS inflammatory and analgesic properties (DePetrocellis et al. 2011; see Figure 5E)[TG1] . Interestingly, FAAH has been indicated in the biosynthesis of lipoamines, such as N-arachidonoyl glycine (NAGly), with similar activity as eCBs and recent work has shown a divergent lipidomic phenotype produced by FAAH inhibitor URB597 and CBD in BV2 microglial cell lines such that URB597 upregulated NAEs and downregulated lipoamines to a much greater extent than CBD (Leishman et al. 2018). Further, in mice lacking N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), one of the primary biosynthetic enzymes for NAEs, treatment with CBD had no effect on NAE expression supporting the hypothesis that CBD potentiates NAPE-PLD to increase eCB production rather than inhibiting FAAH (Leishman et al. 2018).

Cytokines, such as interleukins (IL1), interferons (INFγ), and tumor necrosis factors (TNFα), are pro-inflammatory signals secreted for the recruitment and differentiation of immune cells in response to pathogenic assault or injury and cells of the mononuclear phagocytic system, such as macrophages and microglia, share a critical role in cytokine processing (Bhat et al. 2018, Parameswaran et al. 2010, Akids et al. 2011). As biomarkers, cytokines directly relate induced inflammation via in vivo assaying of lipopolysaccharide (LPS), complete Freund’s adjuvant (CFA) injection, or other pharmacological or invasive procedure as well as stimulating measurable downstream events such as activation of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is induced by TNFα and has been shown to be enhanced by INFγ and sustained by INFβ (Parameswaran et al. 2010). The anti-inflammatory effects of CBD are acknowledged as practical enlistment of its therapeutic potential. Equilibrative nucleoside transporter 1 (ENT), which reuptakes adenosine, was found to be inhibited by CBD and follow-up studies showed reduction of TNFα due to prolonged A_2A activity under LPS treatment in mice (Carrier et al. 2006). In a recent study focusing on gender-associated response to CFA induced inflammation on Sprague-Dawley rats, Britch et al. found CBD to reduce hind-paw edema as well as decrease IL-10 and IL-1β in healthy subjects, TNFα in CFA-treated males, and INFγ in CFA-treated males and females, while THC showed ineffective in reducing edema and did not alter levels of TNFα or INFγ significantly (Britch et al. 2020).

Related to inflammation, oxidative stress is produced by reactive oxygen species (ROS) that may generate unsaturated aldehydes 4-hydroxynenenal (4-HNE), malonodialdehyde (MDA), and acrolein as byproducts of lipid peroxidation, which may form adducts with important cellular constituents, such as nucleic acids and proteins, to elicit cell dysfunction and eventual apoptosis (Atalay et al. 2019). CBD has demonstrated several antioxidative qualities stemming from a potential cation free-radical stabilization by the inductive effect from the alkyl moiety and resonance effects from the phenol moiety such that it has shown to reduce MDA, glutathione (GSH) peroxidase and reductase levels as well as reduce nitric oxide, an immunological free radical, despite an inability to alter its biosynthetic enzyme inducible nitric oxide synthase (iNOS) in epithelial tissue of CFA assay (Costa et al. 2007, Kathia et al. 2013). In cardiomyopathy of induced type I diabetic mice, CBD showed to attenuate oxidative stress response by restoring MDA, 4-HNE, GSH/GSSG, and super oxide dismutase (SOD) levels to baseline, while also reducing NF-κB activity, TNFα release, and iNOS expression in cardiomyocytes (Rajesh et al. 2010). Additionally, CBD demonstrated a similar reversal of doxorubicin induced oxidative stress and inflammatory response in myocardial cells (Fouad et al. 2013). Interestingly, the metabolic derivative cannabidiol hydroxyquinone possesses cytotoxic activity via adduct formation to intracellular thiols, aiding in the depletion of antioxidant GSH (Wu et al. 2010). While contrary to the neuroprotective and anti-inflammatory qualities of CBD, the toxicity has shown potential as an anti-cancer therapeutic by potentiating ROS formation in tumorigenic cells leading to cell death, which may be directed by a caspase-dependent mechanism (Massi et al. 2006).

Cannabidiol: Neuropathic Pain[TG2]

While some in vivo assays, such as LPS or CFA, may be indicative of inflammatory-induced pain, several researchers have utilized the spared neural injury (SNI) assay to simulate chronic pain via ligation of one sciatic nerve of an animal subject to assess nociception in terms of mechanical allodynia (i.e. Von-Frey) and thermal hyperalgesia (i.e. Hargreaves, hot plate test; Cichon et al. 2018).

In a pioneering work, Comelli et al. found that cannabis extract high in CBD (enriched CBD) was able to restore withdrawal latency and mechanical thresholds of SNI rats, while pure CBD showed greater efficacy than THC; TRPV1 antagonist, capsazepine (CPZ), was able to reverse its effect (Comelli et al. 2008). Further studies indicate CBD and CBDV possess anticonvulsant properties via the rapid activation and desensitization of rTRPV1, rTRPV2 and rTRPA1 channels inferred by an observed dose-dependent bi-directional current evocation in patch-clamp assays of HEK293 cells, as well as CBDV showing CPZ-type dephosphorylation of TRPV1 in rat hippocampal slices in epileptic-life (Mg²⁺-free) conditions (Ianotti et al. 2014). The interactions of CBD and receptors related to pain have been further defined measuring repression of dorsal raphe nucleus (DRN) activity that is reversed by CPZ and 5HT_1A antagonists (De Gregorio et al. 2019). De Gregorio et al. subjected SNI rats to a panel of behavioral tests including the open field test (OFT), forced swim test (FST), elevated plus maze test (EPMT), and novelty-suppressed feeding test (NSFT), in addition to Hargreaves and Von Frey assays, to determine accompanying anxiolytic effects of CBD, which indicated low-doses of CBD (5mg/kg) may alleviate pain-induced anxiety through a 5HT_1A mechanism, not TRPV1, as only the 5HT_1A antagonist (WAY100635) was able to reverse the increase in distance traveled in OFT, time in the open arm of EPMT, and decreased latency to feed to in the NSFT by CBD (De Gregorio et al. 2019). In a novel self-reinforcement assay, Abraham et al. allowed SNI mice ad libitum access to THC, CBD, and morphine laced gelatin and found that during a three-week Von Frey and hot plate assessment morphine had returned to control (non-treated) levels after completely relieving allodynia and hyperalgesia six days after surgery, while THC and CBD persistently decreased pain response; results inferred cannabinoids may be optimal in treating pain over opioids due to the relevant tolerance observed and that CBD may be more applicable over THC as CBD did not induce the symptomatic ‘cannabinoid-tetrad’ (hypothermia, hypo-locomotion, catalepsy, analgesia) of THC in pain-naïve mice, leading the investigators to hypothesize the anti-inflammatory and analgesic capacity of CBD resides in its ability to modulate pro-inflammatory responses to pain (Abraham et al. 2019)[TG3] .

Modulation of membrane potential is central to mitigating the transmission of pain stimulus in neurons and CBD has been found to interact with both cell surface receptors as well as intracellular receptors to potentiate and/or attenuate electrochemical gradients. In recent work by A. Bouron, cannabinoids AEA, NAGly, and CBD were found to inhibit store operated calcium channels (SOCC), present on the plasma membrane, from compensatory store operated Ca²⁺ entry (SOCE) after pharmacological depletion of endoplasmic reticulum (ER) Ca²⁺ stores in embryonic neural tissue (Bouron 2018). It was also reported that CBD depleted ER Ca²⁺ stores by either the similar sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibition as thaspsigargin or other potentiation, however activation of G_i/o induced calcium release had been ruled out as pertussis toxin (G_i/o inhibitor), AM251 (CB1 antagonist), and AM630 (CB2 antagonist) did not produce similar effects (Bouron 2018). The synthetic isomer abnormal CBD (abn-CBD) and NAGly have been shown to activate Ca²⁺ -activated potassium channels of large conductance (BK_Ca) to hyperpolarize vascular endothelial tissue as a mechanism of vasodilation (Bondarenko et al. 2018). Glycine receptors (GlyR) are hetero-pentameric chloride (Cl^-) channels consisting of three or four α_1-4-subunits and one or two β-subunit(s) primarily activated by the amino acid glycine for influx Cl^- ions to hyperpolarize resting membrane potential. Proinflammatory lipid mediator prostaglandin E2 (PGE2) is suggested to inhibit α₃-subunit containing GlyRs and, in HEK293 cell co-expressing PGE2 receptors and α₃-GlyR knock-out mice, Xiong et al. confirmed the interaction and further showed CBD and dehydroxyl-CBD (DH-CBD) agonism at α₃-GlyR to have analgesic effect in CFA model pain (Xiong et al. 2012). NMR analysis indicated the interaction of residue S296 on α₃-GlyR to be responsible for cannabinoid agonism and Lu et al. developed knock-in α₁-GlyR^S296Amutant mice to support the claim as well as demonstrating α₁-GlyRs are a target for DH-CBD analgesic effect (Xiong et al. 2012, Lu et al. 2018). Thus, the indications for CBD therapy will ultimately depend on specific pathologies whereby neural transmission may be modulated by several discreet mechanisms or in combination with specific pharmaceuticals.