website stat Compound Find in Hemp Block Corona Virus Via Spike Proteins
Compound Find in Hemp Block Corona Virus Via Spike Proteins
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Originally Published at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987002/

Cannabidiol inhibits replication of SARS-CoV-2 and promotes the host`s innate immune response

Long Chi Nguyen,*,1 Dongbo Yang,*,1 Vlad Nicolaescu,*,2,9 Thomas J. Best,3 Takashi Ohtsuki,4,^ Shao-Nong Chen,4 J. Brent Friesen,4 Nir Drayman,6 Adil Mohamed,6 Christopher Dann,1 Diane Silva,5 Haley Gula,2,9 Krysten A. Jones,7 J. Michael Millis8 Bryan C. Dickinson7 Savas Tay6 Scott A. Oakes5 Guido F. Pauli4,4 David O. Meltzer3, Glenn Randall#,2,9 and Marsha rich Rosner#.1

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Associated Data

Abstract

COVID-19 has spread rapidly, highlighting the need to find new treatments. We report here that CBD, a compound found in cannabis plants, inhibits SARS/CoV-2 infection. CBD and its metabolite 7-OH-CBD but not congeneric cannabis cannabinoids potently inhibit SARS-CoV-2 replication within lung epithelial cell lines. CBD works after cellular infection, blocking viral gene expression and reverseing many of the effects of SARS-2 on host gene transcript. CBD increases interferon production and regulates its antiviral signaling pathway. The SARSCoV-2 incidence in a cohort of patients who had previously taken CBD was significantly lower than that of matched pairs or the general public. This study highlights CBD and its active metabolite 7-OH-CBD as possible therapeutic treatments and preventative agents for SARS-CoV-2 in the early stages.

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Summation:

The potential of cannabidiol, a compound found in cannabis plants, to stop and inhibit SARS-CoV-2 is well known.

The severe acute respiratory syndrome coronavirus 2 coronavirus 2 is responsible for the coronavirus epidemic 2019 (COVID-19), which has ravaged the globe in the past year. SARS-CoV-2 is a coronavirus that causes severe acute respiratory syndrome-related coronavirus. (SARS–CoV) is the seventh known species of coronavirus to infect humans. These coronaviruses include SARS-CoV (CoV), 229E, NL63 and OC43. They can cause a variety of symptoms, from the common cold to more serious pathologies ( ). SARS-CoV-2 continues to spread rapidly despite recent vaccine availability ( 2). This highlights the need for other treatments, especially for those with limited access. Few therapies have been found to block SARS-CoV-2 viral replication.

CBD With CBDA & CBGA

SARS-CoV-2, a single-stranded positive-sense RNA (+ssRNA), enveloped virus, is composed of a bilayer of lipids and four structural proteins that drives viral particle formation. The virus membrane`s integral proteins, the spike (S), membrane(M), and the envelope (E), drive virion budding and recruit the nucleocapsid protein (N) and the viral genome RNA to nascent viruses. SARS-CoV-2, like SARS, enters human cells through the binding of the virus S protein to the angiotensin converting protein 2 (ACE2) receptor (3-5). After that, the S protein is proteolyzed by transmembrane protease 2 (TMPRSS2), or other proteases, into two non-covalently bound peptides S1, S2, which facilitate entry into host cells. The Nterminal binds to the ACE2 receptor. The C_terminal SM2 mediates viral cell membrane fusion after proteolytic cleavage with TMRSS2 and other proteases. Depending on the cell type, viral entry may also occur following ACE2 binding. After cell entry, the SARS/CoV-2 genome is translated to two large polypeptides, which are cleaved using two viral proteases MPro and PLPro (9, 10, 15). Subgenomic RNAs, which encode 10 additional proteins, as well as the four structural proteins, then produce 15 proteins. These proteins allow viral assembly, replication, and budding. We tested the antiviral properties of several small molecules that target stress-related pathways in order to reduce infection by the SARS-2 beta-coronavirus and other emerging pathogenic viruses.

Cannabidiol, a member the cannabinoid cannabinoid group of natural products, is a potential regulator of host stress and antiviral inflammation responses ( ). Cannabis Sativa (Cannabaceae) produces CBD. Hemp is cannabis plants and materials derived from them that contain 0.3% to less psychotropic tetrahydrocannabinol, (THC). These cannabis plants have a high CBD content. Cannabis, on the other hand, refers to C. Materials containing more than 0.3% THC per dry weight. THC is a cannabinoid receptor that acts by binding to CBD. CBD potentiates this interaction ( 12). Although there are many studies and unsubstantiated claims about CBD-containing products ( 11,), the biology of CBD is not clear. An oral CBD solution is FDA-approved. It is used primarily to treat epilepsy ( 13). CBD is a drug and can be used as a therapeutic. It cannot, however, be sold as a dietary supplement in the United States. Some studies have shown that CBD has antiviral properties against HCV and other viruses ( ).

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RESULTS

We pretreated A549 cells expressing the exogenous human ACE-2 receptor (A549ACE2) for 2 hours with 0-10 mM CBD before infecting them with SARS. We monitored cells for the expression of viral spike protein (S) after 48 hours. We also treated cells with an MLK inhibitor (URMC099), previously identified as an antiviral for HIV ( 12), and KPT-9274 (13), which our analysis indicated might reverse many of the gene expression changes caused by SARS/CoV-2. The three inhibitors were effective in inhibiting viral replication under non-toxic conditions, with EC50s ranging between 0.2 and 2.1 mM (Fig. 1A). CBD also inhibited SARS-2 replication in Vero E6 monkey kidney epithelial cell (fig. S1A). Effective doses were safe (fig. S1B). S1B). We also found that CBD inhibited the replication of mouse hepatitisvirus (MHV) under non-toxic conditions. This was done using A549 cells that express MHV receptor (A549 -MHVR). This indicates the potential for greater viral efficacy (fig. S1C,D).


Fig. 1.

Cannabidiol, or CBD (CBD), is an effective inhibitor of SARS-CoV-2 in vitro .

(A) A549 cells overexpressing ACE2 (A549 ACE2) were treated with the indicated doses KPT-9274 or URMC099, followed by 48-hour infection with SARS/CoV-2 at a multiplicity (MOI) 0.5. The cells were stained with spike protein, and the percentage of spike protein-expressing cells in each condition was plotted. The EC50 values are indicated. (B) The 1H QNMR spectra for CBD from a reference material. Also, CBD samples from A, B, & C. (C). A549-ACE2 cells were infected with SARS-CoV-2 at a MOI of 0.5 for 48-hours. The cells were stained with spike protein, and the percentage of spike protein-expressing cells in each condition was plotted. The EC50 values are indicated.

Natural non-synthetic CBD can be extracted from the source plant. This is to preserve the inherent complexity of natural products. We used 100% quantitative NMR to verify that CBD was indeed responsible for viral inhibition. Two chemical vendors (Suppliers B and C) were involved, as well as one commercial vendor who used natural materials (Supplier A). The remarkable congruence of the 1H NMR profile and the newly established quantum-mechanical HiFSA (1H iterative full spin analysis) profiles for all materials showed that 1) the compounds used were CBD with at least 97% purity (Fig. 1B and 2 congeneric cannabisoids did not exceed 1.0% (11). Analyses of these CBD preparations in the viral A549 ACE2 infection assay revealed similar EC50s, with a range of 0.6 to 1.8 mM. This is likely due to the inherent biological variability of this assay (Fig. 1C). None of the CBD preparations were found to cause toxicity at the doses that are used to infuse viral infection (fig. S1 E-G).

CBD can be consumed in combination with THC. CBD is often consumed as part of a C. Congeneric cannabinoids (especially analogues that are produced in the hemp plant) were also tested for their ability to inhibit SARS-CoV-2. Surprisingly, CBD was the only active agent. The structurally related congeners, which share biosynthesis pathways, and make the biogenetically determined residual complex of CBD extracted from C., had limited or no antiviral activity. sativa: THC, cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabichromene (CBC), or cannabigerol (CBG) (Fig. 2 A-B; refer to Methods. These compounds did not cause any harm to A549-ACE2 cells at the doses of interest (fig. S2). S2.


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Fig. 2.

Cannabinoids other that CBD have a limited or non-existent effect on SARS-CoV-2 infections.

(A) A549 ACE2 cells were infected with SARS-CoV-2 at a MOI of 0.5 for 48 hrs with the indicated cannabinoids. The cells were stained with spike protein, and the percentage of spike protein-expressing cells in each condition was plotted. Methods describes how all cannabinoids were extracted from hemp extract. (B) Chemical structure of cannabinoids. 7-OH CBD. (C) A549 ACE2 cells were infected with SARS-CoV-2 at a MOI of 0.0.5 and given the indicated 7-OH CBD doses. The cells were stained with spike protein, and the percentage of spike protein-expressing cells in each condition was plotted. For comparison, the following data are taken from Figure1C (Supplier B). The EC50 values are indicated.

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). The levels of CBD in the blood are 40 times higher than that of 7-OH-CBD, but 7-OHCBD is still the active ingredient for treating epilepsy ( 14). 7-OHCBD, unlike CBD and other cannabinoids in the same way, inhibited SARS-2 replication in A549 ACE2 cells (EC50 3.6 mM; Figure. 2C and was not toxic to cells (fig. S2H). Analyses of blood plasma levels from healthy patients who have taken FDA-approved CBD (Epidiolex(r),) show that CBD has a maximum concentration (Cmax), while 7-OH-CBD has a Cmax in a mM range. This is similar to the results in cultured cells (15). These results indicate that CBD is not sufficient to inhibit SARS-CoV-2 infection in humans. However, the plasma levels of 7-OH-CBD (whose C max is several-fold higher when combined with high-fat meals) are sufficient to possibly inhibit SARS-CoV-2 in humans ( 15 ).

CBD may be acting to prevent viral entry into host cells, or in later stages after infection. We first tested whether CBD inhibited the SARS-2 receptor in A549-ACE2-overexpressing cells. No decrease in ACE2 expression (Fig. 3A). 3A). However, 10mM CBD did not completely block spike-expressing virus cell entry, suggesting other mechanisms may be responsible (Fig. 3B, and figs. S3 A, and B. However, antibodies to spike protein prevented viral infection of the SARS/CoV-2 virus but not VSVG-expressing viruses. In accordance with this, CBD also inhibited the expression of the SARS-CoV-2 spike proteins in host cells up to 2 hours after infection. This was in spite of the presence antibodies to the spike protein. 3C,,D).D). We tested their activity in vitro to determine if CBD could be preventing viral protein processing with the viral proteases Mpro and PLpro (fig. S4). S4).


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Fig. 3.

After SARS-CoV-2 enters the host cell, CBD inhibits viral replication.

(A) Immunoblots showing ACE2 protein expression in A549-ACE2 cell lysates. Cell lysates were either untreated (n=3) or treated with CBD or vehicle at the indicated doses. The blots were probed using antibodies against ACE2 or tubulin. The tubulin signal in each sample was normalized to the ACE2 protein levels. The ACE2 levels were plotted against untreated samples. One-way ANOVA was used to compare expression levels with vehicle. (B) 293T ACE2 cells were infected with spike or VSV–G pseudovirus for 72 hours. The indicated CBD doses were administered to infected cells and the percentage of infected cells was plotted. (C) A549 ACE2 cells were pre-treated with 10 mg CBD or not for 2 hours. After that, they were infected by SARS-CoV-2 at a MOI of 0.25 for 2 hours. To prevent reinfection, cells were treated with 10 mM CBD and DMSO for 16 hrs. The virus-infected sample was then normalized for spike positive cells. (D) Validation and validation of the neutralizing antibody`s efficacy. 400 pfu SARS-CoV-2 viruses were incubated for one hour with or without 100mM neutralizing antibody. The mixture was incubated for 16 hours in A549-ACE2 cell lines. Spike positive cells were then quantified.

This interpretation is supported by RNA-seq analysis on infected A549 ACE2 cells that were treated with CBD for 24 hrs. It shows a remarkable suppression of SARS/CoV-2-induced gene expression changes. CBD effectively eliminated viral RNA expression from the host cells. This included RNA coding to spike, membrane and nuclecapsid proteins (Figs. 4 A and andB. SARS-CoV-2, CBD and CBD both induced significant changes to cellular gene expression. S5 and S6. Principal component analysis of host cells RNA shows nearly complete reverse of viral changes, but the CBD+virus infected cells do not return to normal. Instead, they resemble those that were treated with CBD (Fig. 4C). Metascape cluster analysis reveals interesting patterns and related themes (Fig. 4D, figs. S7 and S8. CBD, however, has no effect on viral induction of genes involved with chromatin modification or transcription (Cluster 1) but it can reverse this. CBD is also able to reverse viral inhibition of genes related with ribosomes, neutrophils (Cluster 3), but it does not work as a standalone drug. Contrary to Clusters 5, and 6, where CBD alone causes strong activation genes related to the host stress response. These results indicate that CBD can prevent viral protein translation as well as cellular changes.


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Fig. 4.

After CBD treatment or SARS-CoV-2 infection, there are changes in the transcription of host cells and viral genes.

A549-ACE2 cells infected by SARS-CoV-2 at MOI 3 with or without CBD treatment at ten mM for 24 hour. Methods describes how RNA-seq was done. (A) Heatmap showing relative levels of SARS-2 genes based on RNA-seq data. (B) Levels of expression for SARS-CoV-2 nucleocapsid and spike genes. Each gene is indicated with the percentage of changes in gene expression between cells infected and cells CBD-treated. (C) Principal component analysis of RNA-seq data. It shows control (veh_mock), SARS CoV-2 infected cells (veh_infect), CBD treatment (CBD_mock) and SARS CoV-2 infected with CBD (CBD_infect). These plots show the first and second principal components (PC1 & PC2) for each sample. (D) Heatmap showing the normalized expression levels for 5,000 genes from all RNA-seq sample. It is clustered into six groups based upon differential expression between treatment conditions.

Induction of interferon signaling pathways is one potential way CBD could suppress viral infection. Interferons are part of the earliest immune host responses to pathogen exposure ( ). SARS-CoV-2 infections suppress the interferon signaling pathway (19). 5A and fig. S9). CBD can induce certain genes in the presence or absence of the virus, such as receptors for interferons beta (Fig. 5A and fig. S10). OAS1, an interferon induced gene that activates RNase L, and RNA degradation (20), is not significantly induced in this pathway by CBD unless the virus is present (Fig. 5A and fig. S11). These results support the possibility that CBD reduces the viral titer enough to allow normal host activation for the interferon pathway. CBD reverses the viral induction of cytokines, which can lead to the deadly Cytokine Storm at later stages (Fig. 5B). These results indicate that CBD may inhibit SARS-CoV-2 infections in part because it activates the interferon pathway, which leads to viral RNA degradation and subsequent changes in host gene expression including cytokines.


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Fig. 5.

CBD stimulates interferon response in host cells and inhibits viral cytokine production.

(A) Heatmap showing the normalized expression levels for genes from the Interferon Response Canonical Pathway. This includes all RNA-seq samples, including control (veh_mock), SARS-2 infected(veh_infect), CBD treatment (CBD_mock), SARS-2 infected + CBD treated (CBD_infect). The genes were clustered using hierarchical clustering. (B) Heatmap showing the normalized expression levels for GO Cytokine activity genes. These genes were up-regulated in response to viral infection, but were down-regulated after CBD treatment. All RNA-seq samples are as described in (A).

We examined the possibility that CBD-rich CBD products might be associated with a lower risk of SARS-CoV-2 infections, given the fact that CBD preparations containing large amounts of CBD are commonly taken by many people. An analysis of more than 93,000 patients who were tested positive for SARS-CoV-2 infection at the University of Chicago Medical Center revealed that only 5.7% of those with any cannabinoid in the medical records tested positive (Fig. 6). CBD was more common than other cannabinoids in patients who tested positive (1.2 percent in 85 CBD patients, 7.1% in 113 CBD patients, p=0.08). Our cell culture studies confirmed that patients who took other cannabinoids were less protected against infection. These findings could be explained by multiple factors, such as age, race, and sexual orientation. We matched 82 patients who received oral CBD (Epidiolex ) prior to COVID-19 screening to patients who had no indication of using cannabinoids, but had similar characteristics. This included comparable demographic characteristics, clinical comorbidities and records of any other medications taken in the two years preceding COVID-19 ( table). The most common conditions for which patients were prescribed CBD were hypertension and immunosuppression. Surprisingly, CBD was prescribed to only 1.2% of patients who contracted SARS-CoV-2, while 12.2% of non-cannabinoid patients tested positive. (p=0.009) This suggests that CBD may have a significant effect on the SARS-CoV-2 infection rate.


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Fig. 6.

Patients who use high doses of CBD are significantly more likely to be positive for COVID-19.

There was a correlation between COVID-19 test results and reported cannabinoid medication usage among adults at the University of Chicago Medicine (total number n=93565). P*: The p-values for percent positivity in the specific patient population relative to percent positivity in all patients (93,565 patients were positive for COVID-19). Middle right: 85 patients used CBD before their COVID date. Upper right: 82 patients received FDA-approved CBD (Epidiolex (r)). They were then matched with 82 patients (Matched controls) using a nearest neighbor propensity scoring model. This model scores patients based on their demographics, their records and any medications they took in the two years prior to their COVID-19 test. The Fisher`s exact two-sided test was used to calculate P-values.

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DISCUSSION

Our findings suggest that CBD may block SARS CoV-2 infection at the early stages. CBD administration is associated to a lower risk of SARS CoV-2 infection in humans. The active compound found in patients is 7-OH-CBD, which is the same metabolite involved in CBD treatment for epilepsy. Patients who received FDA-approved CBD showed a significant decrease in SARS-CoV-2 infections risk by approximately one-half of an order of magnitude. This highlights the potential effectiveness of CBD in fighting SARS-CoV-2. The ability of CBD to prevent replication of MHV suggests that CBD could be effective against future pathogenic viruses.

Interferon pathways are triggered by activation of the host immune system to the virus pathogen. This is one mechanism that CBD has shown to have antiviral properties. Interferons have been clinically tested as possible treatments for COVID-19 ( 21). CBD is also known to suppress cytokine activation during viral infection. This reduces the possibility of immune cell recruitment, and subsequent cytokine storms in the lungs and other tissues affected by the virus. These findings are consistent with previous research that CBD inhibits the production of cytokines in recruited immune cells like macrophages ( 22). CBD can be used to not only protect against infection at the early stages but also as an immune suppressor at later stages.

CBD is a potent preventative agent against SARS. CBD can be found in large quantities without restriction if its THC content is below 0.3%. There are many ways to inhale CBD, including nasal delivery and inhalation. CBD inhibits viral replication once it has entered cells. It is therefore likely to be effective against mutant spike proteins and other viral variants. CBD administration is not required for injections in hospitals, unlike antiviral drugs like remdesivir and antiviral antibodies. CBD has very few side effects ( ).

Before CBD can be considered as a treatment for COVID-19 ( 11), there are many issues that need to be examined. There are many CBD products on the market. However, there are vast differences in the quality and the amount of CBD they contain. These are also different pharmacokinetic properties. The hydrophobic nature of CBD means that it forms large micellar structures in the liver. This traps and breaks down the drug, which limits the availability to other tissues. Clinically available concentrations are affected by inactive carriers. We tested flavored commercial hemp oils for CBD and found that only 0.30% of them contained CBD (fig. S12). S12). This effectively eliminates the possibility of marijuana being used as an effective source for antiviral CBD. The potential for lung damage from other methods of CBD administration, such as smoking and vaping, is also a concern.

Clinical trials and future studies will be required to confirm the potential of CBD as a treatment to stop SARS-CoV-2 infections. We recommend moving to clinical trials over preclinical studies in animals ( ). To understand the role of CBD in treating and preventing early SARS-CoV-2 infections, we recommend carefully planned placebo-controlled clinical trials using known concentrations and well-characterized formulations. It remains to be determined the human in-vivo required concentration as well as the optimal route and formulation. We caution you against taking CBD in any form currently available as a treatment or preventative therapy, especially without having completed a rigorous randomized clinical trial.

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