Based on molecular docking studies of the binding of 32 cannabinoids to the SARS-CoV-2 protease 3CLpro followed by antiviral assays using Vero cells, Raj et al. [21] identified CBD as the most potent anti-SARS-CoV-2 among these compounds and that it inhibited virus replication with an IC₅₀ value of 7.91 μM (Table 1). Inhibition of SARS-CoV-2 3CLpro was determined to be a mechanism of action of CBD by Pitakbut et al. [22] who reported that CBD inhibited 3CLpro with an IC₅₀ value of 1.86 ± 0.04 μM (Table 1). In addition to inhibition of 3CLpro, Pitakbut et al. [22] reported that CBD can inhibit human ACE2 with an IC₅₀ value of 14.65 ± 0.47 μM. CBD and hemp extracts rich in CBD can also reduce the expression of ACE2 [25] and TMPRSS2 [26] (both of which are human cell surface receptors that facilitate infection by SAR-CoV-2), and block induction of inflammatory mediators cyclooxygenase-2, IL-6, and IL-8 via the protein kinase B (AKT) pathway [25,26] (Figure 2).

In 2022, Nguyen et al. [27] reported that CBD can inhibit SARS-CoV-2 replication in A549 human lung carcinoma cells expressing the human ACE2 receptor. The effective concentration (EC₅₀) of CBD in this assay was 1.24 µM, which was non-toxic to the cells (Table 1). An abundant Phase 1 metabolite of CBD in humans, 7-hydroxy-CBD (Figure 1), was also found to inhibit viral replication in cell culture with an EC₅₀ value of 3.60 µM. In a mouse model expressing human ACE2 (K18-hACE2), CBD treatment inhibited SARS-CoV-2 replication in a dose-dependent manner. Based on changes in mRNA levels caused by CBD in cell culture, Nguyen et al. [27] concluded that anti-SARS-CoV-2 mechanisms of action might include inducing stress in the endoplasmic reticulum of the host cell as well as stimulating innate immune responses (Table 1 and Figure 2). Unlike Pitakbut et al. [22], they did not identify inhibition of SARS-CoV-2 3CLpro as a target for CBD.

In a retrospective analysis of hospital records, Nguyen et al. [27] found a negative association between patients taking CBD for control of epileptic seizures and positive diagnosis of SARS-CoV-2 infection (Table 1). They also found that patients reporting frequent use of medicinal or recreational marijuana had a lower risk of COVID-19. Note that consumption of 1500 mg daily of the CBD drug epidiolex has been reported to produce CBD concentrations as high as 1.7 µM [28], which is equivalent to the IC₅₀ value of CBD for the inhibition of viral 3CLpro [22].

In another retrospective clinical study, Shover et al. [29] analyzed clinical outcomes of 1831 patients who had been hospitalized in Southern California for COVID-19. Active cannabis users (n=69) had significantly better outcomes than non-users including a lower NIH COVID-19 Severity Score (5.1 vs 6.0, P<0.001), reduced need for supplemental oxygen, reduced intensive care unit admission (12% vs 31%, P<0.001), less need for mechanical ventilation (6% vs 17%, P=0.027), and shorter length of hospitalization (4 days vs 6 days, P<0.001) (Table 1). Cannabis users hospitalized for COVID-19 also had lower levels of inflammatory markers, although their overall survival rate was not statistically different from non-users. The authors acknowledged that conclusions from retrospective studies need to be interpreted with caution and that prospective studies on the anti-COVID-19 benefits of cannabis are needed.

In the first prospective clinical trial of CBD as a treatment for COVID-19, Crippa et al. [30] evaluated the safety and efficacy of CBD (300 mg/day for 14 days) for treating patients with mild-to-moderate COVID-19. Using a randomized, double-blind design, participants diagnosed with mild-to-moderate COVID-19 receiving the standard of care were randomized to either CBD (n=49) or placebo (n=42). With respect to safety, CBD was well tolerated, no adverse events were reported, and the side effects of CBD and placebo were indistinguishable. Based on the primary outcomes of recovery time and prevention of worsening clinical status, there were no significant differences between the CBD and placebo treatment groups (Table 1). The authors concluded that further trials are needed to evaluate the efficacy of higher doses of CBD and that efficacy in patients with severe COVID-19 should be investigated. The design of this clinical trial was criticized by Vitella et al. [31] who suggested that because plasma levels only ranged from 10.2 to 17.1 nM, the dosages were too low to be effective. They also suggested that sublingual administration might have produced higher plasma levels than per oral dosing, and that the high percentage of participants with comorbidities (35%), co-administered pharmaceuticals (18%), and alcohol abuse (24%) might have adversely affected intestinal absorption.

In a 28-day randomized, single-blind clinical trial of the safety and efficacy of cannabinoids on post COVID conditions (also known as long-COVID), Young et al. [32] tested a hemp inflorescence extract supplemented with terpenes. Instead of a placebo, the hemp extract was compared inadvertently with a cannabinoid-free mixture of terpenes. The initial dosage of both products was 0.25 ml/day but was titrated up thereafter to optimize effect. The hemp extract contained 40.7 mg/ml CBD, 2.91 mg/ml Δ⁹-THC, 2.28 mg/ml cannabichromene, 3.63 mg/ml CBDA, and 0.94 mg/ml cannabigerol (Figure 1). Out of 23 participants completing the study, 11 participants received the cannabinoid-rich extract while 12 participants received a terpene product. Post COVID conditions improved equally in both treatment groups (Table 1). After 28 days, the study was extended to 70 days in an open label format in which all participants received the study extract containing cannabinoids. No adverse events were reported. The authors acknowledged the omission of a placebo and the small study size as limitations.

In 2020, Khodadadi et al. [33] demonstrated anti-inflammatory effects of CBD in mice with acute respiratory distress syndrome resembling COVID-19 (Table 1). Acute respiratory distress syndrome was induced by intranasal administration of polyinosinic-polycytidylic acid, which stimulates the Toll-like receptor 3. Treatment with CBD (5 mg/kg on days 1, 3, and 5) reduced the levels of the proinflammatory cytokines IL-6 and TNF-α as well as lowered lung infiltration by macrophages and neutrophils.

In a similar study using mice treated with lipopolysaccharide (LPS) intranasally to induce lung inflammation resembling COVID-19 or intraperitoneally to cause systemic inflammation, Aswad et al. [34] found that a high-CBD hemp extract (35% CBD, 0.3% Δ⁹-THC, and 0.3% cannabigerol) reduced inflammation (Table 1). In the lung inflammation model, intravenously injected high-CBD hemp extract (100-150 mg/kg) reduced neutrophil migration to the lungs and decreased IL-1β, IL-6, TNF-α, and monocyte chemoattractant protein-1. In another experiment using mice with systemic inflammation, the high-CBD extract injected intravenously reduced the proinflammatory cytokines IL-6 and TNF-α while increasing the anti-inflammatory cytokine IL-10. Note that Aswad et al. [34] selected this high-CBD extract for evaluation after screening several high-CBD and high-Δ⁹-THC extracts for suppression of IL-6 secretion in LPS-stimulated RAW 264.7 mouse macrophages.

Using human THP-1 macrophages and primary human bronchial epithelial cells, Suryavanshi et al. [35] showed that CBD as well as Δ⁹-THC could inhibit LPS-induced inflammatory responses (Table 1 and Figure 2). Specifically, treatment of cells with CBD or Δ⁹-THC reduced levels of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α. The mechanism of action was found to be inhibition of activation of the nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 inflammasome, and down-regulation of phosphorylation of STAT3. In addition, CBD attenuated phosphorylation of nuclear factor-κB, and both CBD and Δ⁹-THC prevented LPS-induced oxidative stress. The anti-inflammatory activities of CBD and Δ-⁹THC were discussed as of potential benefit to COVID-19 patients.

Santos et al. [36] treated several cell lines infected with SARS-CoV-2 with CBD and proprietary mixtures of terpenes derived in part from C. sativa. They found that CBD as well as the terpene mixtures inhibited viral replication and that the combination of both had an additive effect (Table 1). Although it was suggested that CBD prevented the release of proinflammatory cytokines by functioning as a partial agonist of the cannabinoid receptor-2, no cytokine measurements were reported.

By measuring mRNA changes in lung alveolar A549 cells that had been stably overexpressed with human ACE2 and infected by SARS-CoV-2 with or without CBD treatment (2.5 µM), Nguyen et al. [27] concluded that CBD can inhibit the stimulation of cytokines associated with COVID-19 and that a mechanism of action might include activation of serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α. In a review of anti-inflammatory effects of cannabinoids, Janecki et al. [37] observed that activation of the cannabinoid receptor-1 and especially cannabinoid receptor-2 by cannabinoids could inhibit the release of proinflammatory cytokines as well as stimulate production of anti-inflammatory cytokines (Figure 2). Note that Δ⁹-THC has high affinity for both cannabinoid receptor-1 and cannabinoid receptor-2 [38].

Using human monocytic THP-1 cells and primary human macrophages infected with HIV, Tomer et al. [39] measured mRNA changes induced by CBD treatment (10 µg/ml). CBD was found to decrease interferon-regulated gene expression while increasing autophagy (Table 1 and Figure 2). Interferon-regulated gene expression decreased due to attenuated cGAS-STING-mediated activation and was probably responsible for an increase in HIV RNA expression 24 h post-infection. However, up-regulation of the autophagy receptor p62/SQSTM1 resulted in reduced HIV viral spread during 8-day incubations.