Preprint Article Version 1 This version is not peer-reviewed

In Search of Preventative Strategies: Novel Anti-Inflammatory High-CBD Cannabis Sativa Extracts Modulate ACE2 Expression in COVID-19 Gateway Tissues

Version 1 : Received: 17 April 2020 / Approved: 19 April 2020 / Online: 19 April 2020 (02:45:50 CEST)

How to cite: Wang, B.; Kovalchuk, A.; Li, D.; Ilnytskyy, Y.; Kovalchuk, I.; Kovalchuk, O. In Search of Preventative Strategies: Novel Anti-Inflammatory High-CBD Cannabis Sativa Extracts Modulate ACE2 Expression in COVID-19 Gateway Tissues. Preprints 2020, 2020040315 (doi: 10.20944/preprints202004.0315.v1). Wang, B.; Kovalchuk, A.; Li, D.; Ilnytskyy, Y.; Kovalchuk, I.; Kovalchuk, O. In Search of Preventative Strategies: Novel Anti-Inflammatory High-CBD Cannabis Sativa Extracts Modulate ACE2 Expression in COVID-19 Gateway Tissues. Preprints 2020, 2020040315 (doi: 10.20944/preprints202004.0315.v1).

Abstract

With the rapidly growing pandemic of COVID-19 caused by the new and challenging to treat zoonotic SARS-CoV2 coronavirus, there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. Inhibition of viral entry and thereby spread constitute plausible therapeutic avenues. Similar to other respiratory pathogens, SARS-CoV2 is transmitted through respiratory droplets, with potential for aerosol and contact spread. It uses receptor-mediated entry into the human host via angiotensin-converting enzyme II (ACE2) that is expressed in lung tissue, as well as oral and nasal mucosa, kidney, testes, and the gastrointestinal tract. Modulation of ACE2 levels in these gateway tissues may prove a plausible strategy for decreasing disease susceptibility. Cannabis sativa, especially one high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been proposed to modulate gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. Working under the Health Canada research license, we have developed over 800 new Cannabis sativa lines and extracts and hypothesized that high-CBD C. sativa extracts may be used to modulate ACE2 expression in COVID-19 target tissues. Screening C. sativa extracts using artificial human 3D models of oral, airway, and intestinal tissues, we identified 13 high CBD C. sativa extracts that modulate ACE2 gene expression and ACE2 protein levels. Our initial data suggest that some C. sativa extract down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV2 entry into host cells. While our most effective extracts require further large-scale validation, our study is crucial for the future analysis of the effects of medical cannabis on COVID-19. The extracts of our most successful and novel high CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the treatment of COVID-19 as an adjunct therapy. They can be used to develop easy-to-use preventative treatments in the form of mouthwash and throat gargle products for both clinical and at-home use. Such products ought to be tested for their potential to decrease viral entry via the oral mucosa. Given the current dire and rapidly evolving epidemiological situation, every possible therapeutic opportunity and avenue must be considered.

Subject Areas

COVID-19; SARS-CoV2; ACE2 receptor; medical cannabis; CBD

Comments (28)

Comment 1
Received: 15 May 2020
Commenter: Mix Rix
The commenter has declared there is no conflict of interests.
Comment: Nice.
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Response 1 to Comment 1
Received: 23 May 2020
Commenter: Jane carville
The commenter has declared there is no conflict of interests.
Comment: Fantastic research thats derserves recognition now !!!!!!!
Comment 2
Received: 15 May 2020
Commenter: Pablo Bermudez
The commenter has declared there is no conflict of interests.
Comment: Cannabis is not confirmed to help fight the coronavirus only that it has relaxing effects for people in the final phase of AIDS or cancer.
Foros Peru
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Response 1 to Comment 2
Received: 19 May 2020
Commenter: Debi daniels
The commenter has declared there is no conflict of interests.
Comment: Did you even read the abstract? It specifically mentions Ace 2 receptors being modulated. It has zilch to do with relaxation.
Response 2 to Comment 2
Received: 20 May 2020
The commenter has declared there is no conflict of interests.
Comment: I'm skeptical because CBD is just a chemical. One strain might produce more CBD and less THC than another but it makes no sense that two of them would stand out from the others. If you were able to relate CBD level or concentration to the ACE2 level then this work might have some value. But you don't even say what differentiates these two strains from the others. So now people worried about covid need to contact you to find out which weed variety will protect them? This is why the FDA was created in the first place.
Response 3 to Comment 2
Received: 22 May 2020
The commenter has declared there is no conflict of interests.
Comment: Concentration of CBD is not the question. Rather, it blocks the ACE2 receptor site on tissues targeted by COVID-19. This is a MULTI-ORGAN receptor! In viral cancers like prostate, CBD also directly contacts the cancer cells & turns off their Biological Clock, so they die young before replication.

Levels of CBD & COVID-19 aren't the same.
As to the species, treatment has to be monitored. How would the patient know what to test or measure as proof of effectiveness?
Response 4 to Comment 2
Received: 22 May 2020
Commenter: Ken g
The commenter has declared there is no conflict of interests.
Comment: Since medical tinctures and smokables are available in many places, it would be very helpful to know the dosages of cbd / thc that showed encouraging results. I'm sure many people already taking these would be willing to augment for potential anti-covid benefits.
Comment 3
Received: 19 May 2020
Commenter: Garth Brookes
The commenter has declared there is no conflict of interests.
Comment: Just a couple puffs. Downregulate it and then another couple. Get those receptors down. Seems like a good idea to me. Take a ‘nother couple puffs, and see what it do to your Aces. Read ‘em and weep.
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Comment 4
Received: 21 May 2020
Commenter: B. Ricemahn
The commenter has declared there is no conflict of interests.
Comment: I think that is extremely important that all possible studies and theories for discovering safe treatments to treat Covid-19 should be explored. We cannot leave this issue to trillion dollar pharmaceutical companies with special interests. The human body produces a natural form of endogenous cannabinoids similar to CBD. CBD does not make you high. If there is any chance we can help the body fight off the virus or alleviate some of the most severe symptoms, we must be supportive of the effort. CBD derivatives have been used in balms as an anti-inflammatory for over a century, and many medical specialists in prestigious institutes, including in the US, Europe and Canada are studying their medicinal benefits. It was not that long ago that the world at large thought medicine discovered through observing growing mold was bizarre. That resulted in the development of penicillin, a remedy that changed the world of medicine and cured the “ incurable”. Prior to that for centuries, various forms of plant mold was used by many all over the world as folk medicine to heal wounds, not yet the refined form of penicillin later developed in 1928. We must have an open mind and be grateful that so many people are willing to spend all their time and efforts to explore various known and unknown possible ways to alleviate suffering.
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Comment 5
Received: 21 May 2020
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Comment: Good work guys, looking forward to seeing more work in the area of CBD. If there is anything Cannubu can do to help out please let us know.
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Comment 6
Received: 21 May 2020
Commenter: Anon Ymouse
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Comment: I admittedly have only scanned the paper so far, but I'm not able to see precisely which strains were found to be effective. Is this intentional, or would you be willing to share that information?
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Response 1 to Comment 6
Received: 22 May 2020
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Comment: I am sure the strains are proprietary. The authors are all employed by Pathway Rx, a cannabis therapy research company, and Swysh Inc, a cannabinoid-based research company. I’d bet these companies are trying to prove clinical utility of their proprietary strains to then make money off of.
Response 2 to Comment 6
Received: 22 May 2020
Commenter: Igor Kovalchuk
The commenter has declared there is no conflict of interests.
Comment: Thank you for your questions Anon. These are unique varieties. There is no intention to hide them, but they are not available commercially yet because there should be a clinical trial to validate our finding. Hope this helps. Igor
Response 3 to Comment 6
Received: 22 May 2020
Commenter: Anon Lee
The commenter has declared there is no conflict of interests.
Comment: Why not study strains already available to the public? False hope is not helpful.
Comment 7
Received: 22 May 2020
Commenter: Paulo Jorge Morgado Crespo
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Comment: Read about this yesterday, a lot of studies still to be done but looks promising.
Mate you are a saint to be answer to all this ridiculous questions and comments.

Thank you for sharing
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Comment 8
Received: 22 May 2020
The commenter has declared there is no conflict of interests.
Comment: Thanks for your research

Which Sativa varieties were successful? Can you name them?
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Response 1 to Comment 8
Received: 25 May 2020
Commenter: Igor Kovalchuk
The commenter has declared there is no conflict of interests.
Comment: All varieties were hybrids produced in our lab; they don't have common names and are not commercially available yet.
Comment 9
Received: 22 May 2020
Commenter: Amanda Mealer-McArtor
The commenter has declared there is no conflict of interests.
Comment: Looking at the results, it is incorrect to state that the effects seen on protein expression were due to THC or CBD, as opposed to other components in the Sativa extract. The 3 lines that demonstrated significantly lower ACE2 protein levels in both oral and intestinal models (#1, 7, 169) were at different ends of the spectrum for CBD and THC percentages and ratios. All 3 had about the same percentage of THC (which appeared about mid-range of all samples), but #1 & 7 had 6.8% & 7.2% CBD respectively, while #169 had 1.9%. As far as THC:CBD ratios, #1 & 7 were 1:27 & 1:34 respectively, while #169 was 1:9.

Given that these lines, with much different CBD compositions, are the only ones w/significantly less ACE2 protein in oral & intestinal models, it makes a strong argument that it is something other than the CBD or THC responsible for the observed effects. So it is misleading to imply CBD caused the results, rather than the full extract itself.

Additionally, as pointed out by the authors, certain lines caused INCREASED expression of ACE2. In theory, this would imply that these other lines would be HARMFUL if taken for COVID. This point should have been more prominent in the paper, bc I fear at face-value, people will take this study to mean that any sativa plant has potential to help, w/o being aware some may actually do harm.

Another finding not discussed by the authors was that totally different lines caused decreased levels of ACE2 protein in respiratory cells (#5, 10, 31) versus intestinal/oral cells (#1, 7, 169). What is concerning about the data presented is that oral/intestinal models’ results of lines 5, 10, and 31 were not shown. And respiratory model data for lines 1 and 7 was not shown. It would be important to know whether they have increased amounts of ACE2 protein in the models not shown.

Seeing that the hypothesis was based on CBD being responsible for decreasing ACE2 protein, and the results indicated it was not likely due to CBD alone, future research should be directed at comparing different strains of Cannabis.
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Response 1 to Comment 9
Received: 25 May 2020
Commenter: Igor Kovalchuk
The commenter has declared there is no conflict of interests.
Comment: Thank you for your comments Amanda, I appreciate you took time to read the paper. Few things to get straight. We have never stated that CBD is being responsible for decreasing ACE2 protein, as we simply don't know it. What you are right about is that the effect is likely not due to CBD alone and yes, future research will include more lines, with broader spectrum of cannabinoids.
On your comment why not all extracts were used on all tissues. We wrote in the Discussion section: "Our original experiments were designed for screening of the biological activities of novel cannabis extracts in human 3D tissue models." We meant to say that tissue treatment and gene expression analysis were done prior to COVID for the purpose to understand broad changes in biological activity in response to cannabis extract. When COVID started, we were not able to do more experiments, and just analyzed the available data and done some Western blotting under the lock-down situation. We will definitely do cross-comparisons of all active extracts on all tissues.
Thanks and regards, Igor
Comment 10
Received: 22 May 2020
Commenter: Matthew Boyle
The commenter has declared there is no conflict of interests.
Comment: Is there a commonality of terpine profiles in the effective strains ?
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Comment 11
Received: 22 May 2020
The commenter has declared there is no conflict of interests.
Comment: It is not obvious why an ACE2 inhibitor would be helpful to someone with COVID-19. According to other articles, COVID attacks ACE2, leading to hypertension and lung failure. Other proposed therapies increase, not decrease, the levels of ACE2 in the lungs.

Also, what is a “strain” of CBD? Isn’t one CBD molecule the same as the next???
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Response 1 to Comment 11
Received: 25 May 2020
Commenter: Igor Kovalchuk
The commenter has declared there is no conflict of interests.
Comment: Thanks for your comment. We never stated it is ACE2 inhibitor. Our data show that ACE2 expression (number of receptors per cell) decrease - it does not mean that ACE2 is inhibited - there are just fewer number of molecules per cell after the treatment with extracts, And it is not a "strain" of CBD, but extracts high in CBD, meaning that CBD is a dominant molecule in the extract. Hope this helps. Igor
Comment 12
Received: 23 May 2020
The commenter has declared there is no conflict of interests.
Comment: It is very interesting that this organization points out this was a high CBD extract from a chemovar of cannabis SATIVA. They say they screened 1000s of plants of which only a a few had that characteristic. My guess they found some phenotype higher in "minor" cannabinoids and don't want to say, look at the analytics they show THC/CBD only. Aside from this being a blatant advertising plug, it also doesn't factor in terpene content or other cannabinoids which are known to have synergistic effects together with THC and CBD.
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Comment 13
Received: 24 May 2020
Commenter: CeeJay
The commenter has declared there is no conflict of interests.
Comment: I love it. I had the idea that cannabis could help in this fight because I recalled how cannabis can signal to cancer cells to stop growing or to die. I'm happy to hear that this is happening and look forward to hearing more about this in the immediate future. Good work fellas.
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Comment 14
Received: 24 May 2020
Commenter: Dennis Jarvis
The commenter has declared there is no conflict of interests.
Comment: THC is immunsuppressive so why not test CBD isolate directly? Thanks for the research!
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Response 1 to Comment 14
Received: 25 May 2020
Commenter: Igor Kovalchuk
The commenter has declared there is no conflict of interests.
Comment: Yes, we are working on single cannabinoids now.
Comment 15
Received: 26 May 2020
Commenter: Alexandre Benzi
The commenter has declared there is no conflict of interests.
Comment: Chemotypes is the key. They interacts with human behaviors, and those behaviors aren’t same in all humans. Depending the emotions that chemotype is working on a specific human will differ from each to other strain. Immune system is depending of human emotions adequacy and behaviors related.
Terpenes and related emotions are the key on cannabis, much more that those questions of CBD/THC ratio.
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Comment 16
Received: 27 May 2020
Commenter: Jordi Hans
The commenter has declared there is no conflict of interests.
Comment: Greatt research and excellent comments.
This is going to get very interesting to see and understand the upcoming new or final results.
Fantastic the idea to show the world with this new phenomena, that such a world known famous natural plant the cannabis sativa will be key factor for healing and preventing against this new pathogen.
Many alreadh conducted cientifically proven studies are coming to the conclusion or have come to conclusion that cannabis sativa essencial oils can treat so many and different pathologies attacking the human body as epilepsy, anorexia , most type of cancers, dementia, parkinson, high blood pressure, anxiety, insomnia, as many others.

Thank you so much for sharing and best of luck with your research project.

Jordi Hans
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