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Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response [nih.gov]:

RESULTS

To test the effect of CBD on SARS-CoV-2 replication, we pretreated A549 human lung carcinoma cells expressing exogenous human ACE-2 receptor (A549-ACE2) for 2 hours with 0–10 μM CBD prior to infection with SARS-CoV-2. After 48 hours, we monitored cells for expression of the viral spike protein (S). For comparison, we also treated cells over a similar dose range with an MLK inhibitor (URMC-099) previously implicated as an antiviral for HIV () and KPT-9274, a PAK4/NAMPT inhibitor () that our analysis suggested might reverse many changes in gene expression caused by SARS-CoV-2. All three inhibitors potently inhibited viral replication under non-toxic conditions with EC50s ranging from 0.2–2.1 μM (Fig. 1A [nih.gov]). CBD inhibited SARS-CoV-2 replication in Vero E6 monkey kidney epithelial cells as well (fig. S1A). No toxicity was observed at the effective doses (fig. S1B). We also determined that CBD suppressed replication of a related beta-coronavirus, mouse hepatitis virus (MHV), under non-toxic conditions with an EC50 of ~5 μM using A549 cells that express the MHV receptor (A549-MHVR), indicating the potential for more broader viral efficacy (fig. S1C,D).

When isolated from its source plant, natural non-synthetic CBD is typically extracted along with other cannabinoids, representing the unavoidable residual complexity of natural products. To verify that CBD is indeed responsible for the viral inhibition, we analyzed a CBD reference standard as well as CBD from three different sources for purity using 100% quantitative NMR (qNMR). These sources included two chemical vendors (Suppliers A and B) and one commercial vendor that used natural materials (Supplier C). The striking congruence between the experimental 1H NMR and the recently established quantum-mechanical HiFSA (1H Iterative Full Spin Analysis) profiles observed for all materials confirmed that 1) the compounds used were indeed CBD with purities of at least 97% (Fig. 1B [nih.gov]) and 2) congeneric cannabinoids were not present at levels above 1.0% (). Analysis of these different CBD preparations in the viral A549-ACE2 infection assay showed similar EC50s with a range from 0.6–1.8 μM likely reflecting the intrinsic biological variability of the assay (Fig. 1C [nih.gov]). No toxicity was observed for any of the CBD preparations at the doses used to inhibit viral infection (fig. S1 E–G).

CBD is often consumed as part of a C. sativa extract, particularly in combination with psychoactive THC enriched in marijuana plants. We therefore determined whether congeneric cannabinoids, especially analogues with closely related structures and polarities produced by the hemp plant, are also capable of inhibiting SARS-CoV-2 infection. Remarkably, only CBD was a potent agent, while limited or no antiviral activity was exhibited by the structurally closely related congeners that share biosynthesis pathways and form the biogenetically determined residual complexity of CBD purified from C. sativa: THC, cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabichromene (CBC), or cannabigerol (CBG) (Fig. 2 A [nih.gov]–B [nih.gov]; see Methods). None of these compounds were toxic to the A549-ACE2 cells in the dose range of interest (fig. S2). Notably, combining CBD with THC (1:1) significantly suppressed CBD efficacy consistent with competitive inhibition by THC.

CBD is rapidly metabolized in the liver and gut into two main metabolites, 7-carboxy-cannabidiol (7-COOH-CBD) and 7-hydroxy-cannabidiol (7-OH-CBD). Although the levels of 7-COOH-CBD are 40-fold higher than 7-OH-CBD in human plasma, 7-OHCBD is the active ingredient for the treatment of epilepsy (). Like CBD but unlike the other cannabinoids, 7-OH-CBD effectively inhibited SARS-CoV-2 replication in A549-ACE2 cells (EC50 3.6 μM; Fig. 2C [nih.gov]) and was non-toxic to cells (fig. S2H). Analysis of blood plasma levels in healthy patients taking FDA-approved CBD (Epidiolex®) shows a maximal concentration (Cmax) for CBD in the nM range whereas 7-OH-CBD had a Cmax in the μM range, similar to that observed in cultured cells (15). These results suggest that CBD itself is not present at the levels needed to effectively inhibit SARS-CoV-2 in people. By contrast, the plasma concentrations of its metabolite 7-OH-CBD, whose Cmax can be increased several-fold by co-administration of CBD with a high-fat meal, are sufficient to potentially inhibit SARS-CoV-2 infection in humans (15).

CBD could be acting to block viral entry to host cells or at later steps following infection. As CBD was shown to decrease ACE2 expression in some epithelial cells including A549 (), we first determined whether CBD suppressed the SARS-CoV-2 receptor in our A549-ACE2 overexpressing cells. No decrease in ACE2 expression was observed (Fig. 3A [nih.gov]). Furthermore, analysis of lentiviruses pseudotyped with either the SARS-CoV-2 spike protein or the VSV glycoprotein () showed that antibody to the spike protein effectively blocked viral infection of the SARS-CoV-2, but not VSV-G expressing viruses. However, 10 μM CBD only partially inhibited cell entry by spike-expressing virus, suggesting that other mechanisms were largely responsible for its antiviral effects (Fig. 3B [nih.gov], and figs. S3 A and B). By contrast, antibodies to the spike protein effectively blocked viral infection of the SARS-CoV-2 but not VSV-G expressing viruses. Consistent with this, CBD was also effective at inhibiting SARS-CoV-2 spike protein expression in host cells even 2 hours after infection in the presence of antibodies to the spike protein to prevent reinfection during this time period (Fig. 3C [nih.gov],​,D).D [nih.gov]). To assess whether CBD might be preventing viral protein processing by the viral proteases Mpro or PLpro, we assayed their activity in vitro (fig. S4). CBD did not affect the activity of either protease, raising the possibility that CBD targets host cell processes.

Consistent with this interpretation, RNA-seq analysis of infected A549-ACE2 cells treated with CBD for 24 hours shows a striking suppression of SARS-CoV-2-induced changes in gene expression. CBD effectively eradicated viral RNA expression in the host cells, including RNA coding for spike, membrane, envelope and nucleocapsid proteins (Figs. 4 A [nih.gov] and ​andB).B [nih.gov]). Both SARS-CoV-2 and CBD each induced significant changes in cellular gene expression, including a number of transcription factors (figs. S5 and S6). Principal component analysis of host cell RNA shows almost complete reversal of viral changes but, rather than returning to a normal cell state, the CBD+virus infected cells resemble those treated with CBD alone (Fig. 4C [nih.gov]). Clustering analysis using Metascape reveals some interesting patterns and associated themes (Fig. 4D [nih.gov], figs. S7, and S8). For example, viral induction of genes associated with chromatin modification and transcription (Cluster 1) is reversed by CBD although CBD alone has no effect. Similarly, viral inhibition of genes associated with ribosomes and neutrophils (Cluster 3) is largely reversed by CBD, but the drug alone has no effect. This contrasts with Clusters 5 and 6 where CBD alone induces strong activation of genes associated with the host stress response. Together these results suggest that CBD acts to prevent viral protein translation and associated cellular changes.

One potential mechanism by which CBD could suppress viral infection and promote degradation of viral RNA is through induction of the interferon signaling pathway. Interferons are among the earliest innate immune host responses to pathogen exposure (). SARS-CoV-2 infection suppresses the interferon signaling pathway () (Fig. 5A [nih.gov], and fig. S9). Some genes that are induced by CBD in both the absence and presence of the virus include receptors for interferons beta and gamma as well as mediators of the signaling pathway such as STATs 1 and 2 (Fig. 5A [nih.gov] and fig. S10). Other genes in the pathway like OAS1, an interferon-induced gene that leads to activation of RNase L and RNA degradation (), are not significantly induced by CBD unless in the presence of the virus (Fig. 5A [nih.gov] and fig. S11). These latter results are consistent with the possibility that CBD lowers the effective viral titer sufficiently to enable normal host activation of the interferon pathway. At the same time, CBD effectively reverses viral induction of cytokines that can lead to the deadly cytokine storm at later stages of infection (Fig. 5B [nih.gov]). Collectively, these results suggest that CBD inhibits SARS-CoV-2 infection in part by activating the interferon pathway leading to degradation of viral RNA and subsequent viral-induced changes in host gene expression, including cytokines.

Given that CBD preparations containing substantial amounts of CBD are taken by a large number of individuals, we examined whether CBD exposure might correlate to a decreased risk of SARS-CoV-2 infection. Analysis of over 93,000 patients tested for SARS-CoV-2 at the University of Chicago Medical Center showed that 10.0% tested positive overall, but only 5.7% of the ~400 who had any cannabinoid in their medical record tested positive (Fig. 6 [nih.gov]). Patients taking CBD versus other cannabinoids had an even lower rate of testing positive (1.2% in 85 CBD patients versus 7.1% in 113 patients taking other cannabinoids, p=0.08). This finding that patients taking other cannabinoids had less protection against viral infection is consistent with our cell culture studies. Since multiple potential confounding factors could explain these findings, including age, race, clinical morbidities, and sex, we matched 82 patients who were prescribed oral, FDA-approved CBD (Epidiolex®) before COVID-19 testing to patients who had no indication of taking any cannabinoids but had comparable other characteristics including similar demographic characteristics, clinical comorbidities, and records of other medications in the two years before COVID-19 testing (table S1). Of the patients prescribed oral CBD before their COVID-19 test, the most common morbidity categories were hypertension and conditions with immunosuppression. Strikingly, only 1.2% of the patients prescribed CBD contracted SARS-CoV-2 whereas 12.2% of the matched, non-cannabinoid patients tested positive (p=0.009), suggesting a potential reduction in SARS-CoV-2 infection risk of approximately an order of magnitude.

from the everybody-must-get-stoned dept.

Journal Reference:
Error: DOI Prefix [dx.doi.org] Not Found, (DOI: //dx.doi.org/10.1101%2F2021.03.10.432967 [doi.org])
Error: DOI Prefix [dx.doi.org] Not Found, (DOI: //dx.doi.org/10.1101%2F2021.03.10.432967 [doi.org])

Original Submission