WO2021191888A1 - Cannabis sativa (hemp and cannabis) products for viral disease prevention and management - Google Patents

Cannabis sativa (hemp and cannabis) products for viral disease prevention and management Download PDF

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Publication number
WO2021191888A1
WO2021191888A1 PCT/IL2021/050265 IL2021050265W WO2021191888A1 WO 2021191888 A1 WO2021191888 A1 WO 2021191888A1 IL 2021050265 W IL2021050265 W IL 2021050265W WO 2021191888 A1 WO2021191888 A1 WO 2021191888A1
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Prior art keywords
extracts
cbd
cells
extract
ace2
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PCT/IL2021/050265
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French (fr)
Inventor
Anna KOVALCHUK
Dongping LI
Bo Wang
Olga Kovalchuk
Igor Kovalchuk
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I.P. Israel Patents Ltd.
Pathway RX Inc.
Swysh Inc.
Humanithé Inc.
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Application filed by I.P. Israel Patents Ltd., Pathway RX Inc., Swysh Inc., Humanithé Inc. filed Critical I.P. Israel Patents Ltd.
Publication of WO2021191888A1 publication Critical patent/WO2021191888A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)

Definitions

  • the present invention relates generally to cannabis and hemp plant products and methods of their production, as well as in production of medicaments for preventing and treating viral diseases, such as COVID-19.
  • the basic replication number, Ro, of SARS-CoV-2 is thought to be around 2-6 [Zhao, S., et al, 2020], meaning that each infected individual, some of whom are asymptomatic infect 2-6 other people, if no interventions are taken.
  • SARS-CoV-2 shares 82% sequence identity with severe acute respiratory syndrome -related coronavirus (SARS-CoV). SARS-CoV relies on its spike protein to bind a host cell surface receptor for entry. For the SARS-CoV-2, research has shown that this receptor is the angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • Both SARS-CoV-2 and SARS-CoV encode a large spike protein (2019-nCoV: 1253 aa; SARS-CoV: 1273 aa).
  • the sequence identity of this protein between the two viruses is 76%.
  • the spike protein has three regions, SI, S2, and S3.
  • Receptor binding domain (RBD) of spike protein in SARS-CoV is majorly in SI and S2.
  • SI region in the RBD from the two viral origins shows 73.5% sequence identity but S2 RBD is almost complete different in SARS-CoV-2 and SARS-CoV. This might affect binding of SARS-CoV-2 to ACE2 as compared to SARS-CoV.
  • ACE2 is expressed in upper respiratory tract, lung, liver, intestine and kidney tissues in humans. Once SARS-CoV-2 enters the host via suggested major routes above, it further increases ACE2 expression.
  • ACE2 ACE2-induced inflammatory response
  • IL1B, IL1RA, IL7, IL8, IL9, IL10 levels were very high as well.
  • Wuhan pointed that the levels of IL-1B, IL-10 and IL-8 were significantly increased in critically ill patients with new coronavims SARS-CoV-2 infection, indicating that there were cytokine-mediated inflammatory responses in patients with new coronavims infection.
  • cytokine storm' and acute respiratory distress syndrome ARDS
  • High concentrations of cytokines were recorded in plasma of critically ill patients infected with SARS-CoV-2 [Huang, et ah, 2019, Guo et al, 2019].
  • ICU patients In the severe patients, levels of IL-6, IL-10, and TNF-a further increased[ Guo, et al., 2019]. ICU patients also had high plasma levels of IL-2, IL-7, granulocyte colony- stimulating factor (GCSF). Furthermore, patients requiring ICU admission had higher concentrations of GCSF, IP10, MCP1, MIP1A, IL-6, IL-10, TNFa and other cytokines than did those not requiring ICU admission, suggesting that the cytokine storm was associated with disease severity [Huang, et al., 2019, Guo et al, 2019].
  • GCSF granulocyte colony- stimulating factor
  • TNFa is one of the key cytokines in the cytokine storm and is likely to responsible for the escalation in severity [Liu et al., 2016].
  • IL-6 [Tanaka et al., 2016]
  • IL-1 and others are key mediators of the cytokine storm [Tisoncik et al., 2012].
  • a cytokine storm manifests before the ARDS in COVID-19 patients and the cytokine storm constitutes one of the ARDS underlying mechanisms. There thus remains an unmet need to provide a product or pharmaceutical composition effective in treating viral diseases in mammals, including humans and non-human mammals, which spread the disease.
  • anti-inflammatory therapies either prophylactically or to prevent a worsening of the underlying disease progression.
  • anti-IL-6 receptor antibody tocilizumab
  • tocilizumab anti-IL-6 receptor antibody
  • compositions for improving wellness in a human or non-human mammalian organism It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or non-human mammalian organism.
  • the present invention provides products and compositions used in reducing levels of an ACE2 receptor in oral, lung and intestinal epithelial tissues in COVID-19 patients to prevent entry thereto of the SARS-CoV-2 RNA virus and related viruses.
  • Products of the present invention are used further to treat a cytokine storm that precedes and underlies acute respiratory distress syndrome (ARDS), seen in the COVID-19 patients.
  • ARDS acute respiratory distress syndrome
  • the method includes generation of unique lines, whole plant extract preparation, treating normal EpiOral, Epilntestinal and EpiAirway human 3D tissues and human cell lines with tumor necrosis factor alpha (TNF) to induce inflammation, and then with extracts in an amount sufficient to profoundly down-regulate ACE2 expression, inflammation and molecular pathways involved in ACE2 regulation, vims entry and inflammation in the oral, intestinal and airway tissues.
  • TNF tumor necrosis factor alpha
  • Extracts of novel cannabis and hemp lines can be combined with anti-viral agents and anti inflammatory extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics to increase their efficacy.
  • the present invention provides methods of production of pharmaceutical compositions, suitable for treating viral diseases in mammalian subjects.
  • the present invention provides pharmaceutical compositions, suitable for treating viral diseases in mammalian subjects.
  • the present invention provides methods for preventing, mitigating and treating cytokine storms in in COVID-19 patients by administration of C. sativa extracts, which exhibit profound cytokine inhibition patterns.
  • the present invention further provides methods and products for ACE2 inhibition in target tissues - lung, oral and intestinal epithelia, thereby decreasing or inhibiting a SARS-CoV-2 infection in a mammalian patient.
  • the present invention further provides combination therapies including a combination of anti- viral (anti-SARS-CoV-2) and anti-cytokine agents will be an effective regimen targeting both etiology and pathogenesis of COVID-19.
  • combination therapies including a combination of anti- viral (anti-SARS-CoV-2) and anti-cytokine agents will be an effective regimen targeting both etiology and pathogenesis of COVID-19.
  • the present invention provides new Cannabis sativa lines and extracts as well as combination of single cannabinoids and terpenes and method of using them as a means to modulate ACE2 expression in oral, lung or intestinal tissues.
  • the present invention also provides methods of modulating ACE2 expression through the application cannabis extracts to the oral, lung and intestinal tissues to prevent or treat COVID-19 and other SARS-CoV virus-caused diseases.
  • the present invention provides a method for modulating ACE2 expression on mRNA and protein levels (e.g., in oral, lung and intestinal tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue, lung cell or lung epithelial tissue, intestinal cells of intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of ACE2 expression results in a prevention of disease via decreased opportunity of virus entry into the cells.
  • mRNA and protein levels e.g., in oral, lung and intestinal tissues and cells
  • the present invention also provides new Cannabis sativa lines and extracts and as well as combinations of single cannabinoids and terpenes, and methods of using them as a means to modulate cytokine levels and cytokine storm in lung tissues.
  • Extracts of novel cannabis and hemp lines or single cannabinoids and terpenes are combined with anti-viral agents and anti-inflammatory, such as, but not limited to, extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics, to increase their efficacy.
  • anti-viral agents and anti-inflammatory such as, but not limited to, extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics, to increase their efficacy.
  • the disclosure provides a means for modulating gene expression and protein levels (e.g., in oral, lung and intestinal tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue, lung cell or lung epithelial tissue, intestinal cells of intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a prevention of disease or reduction of a disease state in the aforementioned tissues.
  • a source of new Cannabis sativa hemp and cannabis
  • the present invention provides extracts of twenty nine new C. sativa lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317).
  • the present invention further combines use of extracts of these lines, as well as combination of single cannabinoids and terpenes.
  • the aforementioned extracts, single cannabinoids and terpenes have been tested for anti-inflammatory properties and/or ACE2 inhibition properties using human cells lines and 3D EpiOral, Epilntestinal and EpiAirway tissue models. This is performed by detecting the expression of genes that are associated with molecular etiology and pathogenesis of COVID-19 and ARDS and cytokine storms.
  • the present invention provides compositions having potent therapeutic ACE2 inhibitory effects in oral, intestinal and airway tissues and thus for treatment and prevention of COVID-19 and ARDS and cytokine storm.
  • the present invention provides new Cannabis sativa lines and extracts as well as combination of single cannabinoids and terpenes and method of using them as a means to modulate ACE2 gene expression in oral, lung and intestinal tissues.
  • the present invention also provides methods of modulating gene expression through the application of cannabis extracts to the mouth, airways and intestine affected by SARS-CoV-2 and other diseases and viruses.
  • the present invention further provides extracts for treating and preventing viral diseases, including SARS-CoV-2 virus and resultant COVID-19, as well as other viral diseases and ARDS.
  • the Cannabis sativa extract efficacy can further be increased by adding thereto additional Cannabidiol (CBD), Cannabigerol (CBG), Cannabinol (CBN), terpenes or combinations thereof.
  • the extract efficacy can be further potentiated and increased by adding anti inflammatory herbs such as but not limited to chamomile, sage, turmeric, thyme, ginger, rosehip, as well as probiotics and their components, or combinations thereof.
  • anti inflammatory herbs such as but not limited to chamomile, sage, turmeric, thyme, ginger, rosehip, as well as probiotics and their components, or combinations thereof.
  • the present invention provided the methods of preparation of Cannabis extracts, cannabinoids, such as but not limited to, Cannabidiol (CBD), Cannabigerol (CBG), Cannabinol (CBN), D 9 - 1 c t a h y d o c a n n a b i n o 1 (THC), and terpenes.
  • CBD Cannabidiol
  • CBG Cannabigerol
  • CBN Cannabinol
  • THC terpenes
  • the present invention provides new unique cannabis lines, extracts and methods for oral, lung and intestinal improvement and healing and reduction in virus entry into the cells and inflammation.
  • the method includes generation of unique lines, whole plant extract preparation, exposing human oral, lung and intestinal tissues to the extracts in amount sufficient to modulate gene expression in them. The modulation of gene expression then results in a reduction of the disease state- associated changes or aspects thereof in the cannabis -treated tissues.
  • improved methods and products are provided for oral application and treatment to prevent viral infection.
  • improved methods and products are provided for oral treatment.
  • improved methods and products are provided for airway application and treatment to prevent viral infection, COVID-19 and acute respiratory distress syndrome.
  • improved methods and products are provided for treatment of viral infection and/or associated diseases.
  • improved methods and products are provided for intestinal application and treatment to prevent SARS-CoV- 2 viral infection and/or COVID-19 disease.
  • new Cannabis sativa lines are provided.
  • new extracts from new Cannabis sativa lines are provided.
  • the present invention provides a method for modulating gene expression on mRNA and protein levels (e.g., in oral cells, in oral tissue, in lung cells, in lung tissue, in intestinal cells, in intestinal tissue) by providing a source of new extracts, exposing cells or tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a prevention and reduction of a disease state in the in oral, lung and intestinal cells and tissue.
  • mRNA and protein levels e.g., in oral cells, in oral tissue, in lung cells, in lung tissue, in intestinal cells, in intestinal tissue
  • the present invention provides new unique cannabis lines, extracts, dried powders from the extracts, compositions comprising the powders or parts thereof, compounds derived therefrom, pharmaceutical compositions comprising the compound(s), and methods for the treatment of COVID-19, viral diseases, ARDS, cytokine storms, others and combinations thereof.
  • the method includes generation of unique lines, whole plant extract preparation, treating human 3D oral, airway and intestinal tissues with extracts in amount sufficient to modulate gene expression in the said tissues.
  • the modulation of Angiotensin-converting enzyme 2 gene (ACE2) expression and inflammatory gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the treated tissues.
  • ACE2 Angiotensin-converting enzyme 2 gene
  • compositions and dosage forms of the present invention are useful in promoting health and preventing or treating a large number of disorders in human patients and other mammalian subjects.
  • compositions and methods are provided for treating and/or preventing viral disorders.
  • the invention provides methods and compositions for preventing ARDS and other diseases that harbor an inflammatory component.
  • the invention provides methods and compositions for preventing systemic diseases and complications related to virus-caused pathologies.
  • compositions of the present invention may be used for improving wellness of a human or mammalian subject. Additionally, the compositions of the present invention may be used to treat any disorder or ailment in a human patient or mammalian subject. Furthermore, the compositions of the present invention may be conveniently used in conjunction with a drug to treat any disorder or ailment in a human patient or mammalian subject.
  • Some embodiments of the present invention provide compounds, compositions and formulations from at least one of hemp and cannabis.
  • compositions and methods are provided for treating and/or preventing viral, inflammatory and fibrosis-related disorders.
  • compositions and methods are provided for treating and/or preventing pneumonia and ARDS, and other disorders.
  • compositions and methods are provided for inhibiting viral life cycle processes thereby treating and/or preventing viral disorders.
  • Some embodiments of the present invention provide compounds, compositions and formulations from at least one of hemp and cannabis.
  • Some further embodiments of the present invention provide methods for downregulating expression of ACE2 gene.
  • Some further embodiments of the present invention provide methods for downregulating expression of at least one inflammatory pathway gene.
  • Some further embodiments of the present invention provide methods for downregulating expression of at least TNF gene.
  • Some further embodiments of the present invention provide methods for downregulating expression of at least IL gene.
  • Some further embodiments of the present invention provide methods for downregulating expression of at least PARP-1 gene.
  • Some further embodiments of the present invention provide methods for downregulating expression of at least iNOS gene.
  • Some further embodiments of the present invention provide methods for downregulating at least one inflammatory pathway gene product.
  • compositions, compounds, formulations and extracts for preventing viral diseases in a human patient there are described methods for preparing compositions, compounds, formulations and extracts for preventing viral diseases in a human patient.
  • compositions, compounds, formulations and extracts for treating viral diseases in a human patient there are described methods for preparing compositions, compounds, formulations and extracts for treating viral diseases in a human patient.
  • a use of a solvent extract from at least one of hemp and cannabis, according to some embodiments of the present invention, is for the manufacture of a pharmaceutical composition for the prevention and treatment of ARDS.
  • Some embodiments of the present invention are directed to a method for preventing and treating a viral disease or disorder in a human patient comprising administering to said patient a pharmaceutically effective amount of the cannabis extract composition as described herein.
  • some further embodiments of the present invention are directed to a method for preventing and treating a viral disorder or disease or complication of those in a human patient comprising administering to said patient the oral, inhalation or topical dosage form as described herein.
  • liquid cannabis extracts of the present invention are suitable for oral administration, and appear to be well absorbed through the intestine by the blood and thus exhibit the potential to heal a wide range of viral and inflammatory conditions, such as, but not limited to those mentioned by Chattopadhyay et al. Current Science 87(1) July 2004, 44-53.
  • the composition or formulation further comprises at least one solvent or hydrant.
  • the hydrant is water, such as double-distilled water. In some cases, it may be at least one organic solvent, such as alcohol.
  • the at least one solvent or hydrant is present in the composition or formulation in a concentration of 10-90%, 15-80%, 20-70%, 25-50%, 30-40%, or 10-18% by weight percent.
  • the solvent or hydrant may further comprise a pH regulator, such as an acid or base.
  • a pH regulator such as an acid or base.
  • the base comprises sodium hydroxide.
  • Suitable products or compositions of the present invention may be in the form of ointments or salves, creams, emulsions, gels, foams, sprays or medicated dressings or bandages, which must be directly applied on the affected zone and must be kept in contact with the oral.
  • the compositions further comprise up to 10% of water.
  • the composition is substantially non-aqueous and/or substantially alcohol-free.
  • the present invention provides a method for inhibiting a disease in a subject comprising administering a subject a composition of the invention.
  • the present invention provides a method for inhibiting a viral disease in a subject comprising administering a subject a composition of the present invention.
  • the present invention provides a method for inhibiting a disease in a subject comprising orally administering a product of the present invention to the subject.
  • the present invention provides a method for inhibiting a disease in a subject comprising inhalation-based administering a product of the present invention to the subject.
  • the composition of the present invention is in a chewable oral dosage form.
  • the chewable oral dosage form is a chewable tablet.
  • the chewable tablet of the invention is taken slowly by chewing or sucking in the mouth.
  • the chewable tablet of the invention enables the dried cannabis extracts contained therein to be orally administered without drinking.
  • the composition further comprises a therapeutically effective concentration of one or more active agents.
  • composition of the present invention further contains a surface-active agent.
  • Surface- active agents include any agent linking oil and water in the composition, in the form of emulsion.
  • a composition of the present invention includes one or more additional components.
  • additional components include but are not limited to anti-static agents, buffering agents, bulking agents, chelating agents, cleansers, colorants, conditioners, diluents, dyes, emollients, fragrances, humectants, permeation enhancers, pH-adjusting agents, preservatives, protectants, oral penetration enhancers, softeners, solubilizers, sunscreens, sun blocking agents, sunless tanning agents, viscosity modifiers and vitamins.
  • a specific additional component may have more than one activity, function or effect.
  • a pharmaceutical composition for treating a viral disease or disorder in a mammalian subject including at least one of: a) an extract as described herein; b) at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD); and c) at least one terpene or terpene alcohol; wherein the pharmaceutical composition is suitable for treating the viral disease or the viral disorder in the mammalian subject.
  • the at least one terpene is selected from the group consisting of myrcene, humulene, pinene carophyllene and valencene.
  • the terpene alcohol includes bisabolol.
  • the at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD) includes only Cannabidiol (CBD).
  • the viral disease or disorder is caused by a corona virus, a mutant or variant thereof.
  • the viral disease is COVID 19.
  • the pharmaceutical composition is effective in treating a cytokine storm associated with the COVID 19.
  • the pharmaceutical composition is suitable for reducing a level of an ACE2 protein in the mammalian subject.
  • a method for treating a viral disease or disorder in a mammalian subject including administering a pharmaceutical composition including at least one of; an extract as described herein; at least one of Cannabigerol (CBG), Cannabinol (CBN) and
  • Cannabidiol CBD
  • terpene or terpene alcohol at least one terpene or terpene alcohol
  • the method is effective in treating a cytokine storm associated with the COVID 19.
  • the method is effective in reducing a level of an ACE2 protein in the mammalian subject.
  • a method for preventing and treating corona virus-caused disease or disorder comprising: a) combining at least one marijuana strain and at least one hemp strain to form at least one Cannabis line; b) extracting at least one compound from said at least one Cannabis line to form an extract or combining an isolated cannabidiol and adding set of terpenes; and c) treating said disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said disease or disorder.
  • said at least one Cannabis line is selected from the group consisting of a marijuana/marijuana hybrid line, hemp/hemp hybrid line and hemp/marijuana hybrid line.
  • a method according to embodiment 5, wherein said at least one line is selected from the group consisting of designated lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317) and combinations thereof.
  • the disease state is selected from the group consisting of a virus-caused disease, virus-caused disease complications, environmental factor-induced inflammation and combinations thereof.
  • An organic extract of at least one plant line said at least one plant line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain, wherein said organic extract comprises at least one compound suitable for treating a mammalian oral disease or disorder.
  • An organic extract according to embodiment 19, wherein said mammalian oral disease or disorder is selected from the group consisting of a viral disease, an oral disease, a lung disease, an intestinal disease and combinations thereof.
  • a combination therapy isolated from an organic extract of at least one hybrid line, said at least one hybrid line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain; and wherein said organic extract comprises a plurality of compounds suitable for treating a mammalian viral disease and disorder.
  • a Cannabis extract for treating and preventing a viral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
  • a Cannabis extract for treating and preventing viral disease or disorder wherein said extract efficacy can be further increased by adding extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
  • a line of Cannabis sativa formed by combining at least one marijuana strain and at least one hemp strain said line will be deposited at public culture collection, currently under designation numbers (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317, #CD10.
  • a method for treating a disease state in oral epithelial cells or oral tissue comprising the steps of: a) providing a source of unique extract from at least one line of Cannabis sativa; and b) preventing and treating the COVID-19 and virus-caused disorders with aforementioned extract(s) in an effective amount to induce modulation of ACE 2 and inflammatory gene expression in the oral, airway and intestinal tissue; wherein the modulation of gene expression results in a reduction of the disease state in the aforementioned tissues.
  • Fig. 1A is a simplified image of an EpiOral tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention
  • Fig. IB is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.lA, in accordance with an embodiment of the present invention
  • Fig. 2A is a graph showing the effects of cannabis extract 81 on the levels of ACE2 gene expression and expression of inflammation-related genes in EpiOral tissues, in accordance with an embodiment of the present invention
  • Fig. 2B is a graph showing the effects of cannabis extract 130 on the levels of ACE2 gene expression and expression of inflammation-related genes in EpiOral tissues, in accordance with an embodiment of the present invention
  • Fig. 2C is a graph showing the effects of several pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts vs DMSO) for several different extracts, in accordance with an embodiment of the present invention
  • Fig. 2D is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels in EpiOral tissues, in accordance with an embodiment of the present invention
  • Fig. 2E is a graph showing the ratio of ACE2 protein to GAPDH protein after treatment with TNF/IFN and application of different cannabis extracts to EpiOral cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment;
  • Fig. 3A is a simplified image of Epilntestinal tissues experimental set-up for testing candidate treatments for anti inflammatory properties, in accordance with an embodiment of the present invention;
  • Fig. 3B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-induccd inflammation using the set-up in Fig.3A, in accordance with an embodiment of the present invention
  • Fig. 4 A is a graph showing the effects of different cannabis extracts on the levels of ACE2 gene expression in Epilntestinal tissues, in accordance with an embodiment of the present invention
  • Fig. 4B is a graph showing the effects of extract of Cannabis Line 45 effectively decreasing levels of ACE2 gene expression in human 3D Epilntestinal tissues, in accordance with an embodiment of the present invention
  • Fig. 5A is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels in Epilntestinal tissues, in accordance with an embodiment of the present invention
  • Fig. 5B is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment;
  • Fig. 5C is a graph showing the effects of different cannabis extracts on down- regulation of inflammation-related PARP-1 and iNOS proteins in Epilntestinal cells, in accordance with an embodiment of the present invention
  • Fig. 5D is a graph showing the ratio of PARP-1 to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
  • Fig. 5E is a graph showing the ratio of iNOS to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
  • Fig. 6A is a simplified image of an EpiAirway tissue model tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention
  • Fig. 6B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.6A, in accordance with an embodiment of the present invention.
  • Fig. 7 A is a graph showing the effects of cannabis extracts of novel cannabis lines in reducing levels of the ACE2 protein in human 3D EpiAirway tissue, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment;
  • Fig. 7B is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue, in accordance with an embodiment of the present invention
  • Fig. 7C is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment;
  • Fig. 7D is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and Actin protein in human 3D EpiAirwayFT tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts, in accordance with an embodiment of the present invention
  • Fig. 7E is a graph showing the ratio of ACE2 protein to Actin protein after induction of inflammation with TNF/IFN and after application of different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment;
  • Fig. 7F is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts, in accordance with an embodiment of the present invention
  • Fig. 7G is a graph showing the ratio of ACE2 protein to GAPDH protein after induction of inflammation with TNF/IFN and after application of different cannabis extracts were applied to human 3D EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
  • Figs. 8A-8F are a set of graphs showing that cannabinoids affect processes involved in a viral life cycle -
  • Figs. 8A-8C show that different cannabinoids applied at different concentrations to an in vitro viral experiment inhibit reverse transcription- namely Fig. 8A, CBD, Fig. 8B, THC, Fig. 8B THC and Fig. 8C, CB and Figs. 8D-8F show that different cannabinoids applied at different concentrations to an in vitro viral experiment inhibit DNA replication- namely Fig. 8D, CBD, Fig. 8E, THC and Fig. 8F THC, in accordance with some embodiments of the present invention;
  • Fig. 9 is a simplified pictorial illustration showing a schematic summary of the main effects of cannabis extracts on SARS-COV-2, in accordance with an embodiment of the present invention.
  • Fig. 10A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in HSIEC cells; asterisks indicated significant (p ⁇ 0.01) difference;
  • Fig. 10B shows the dose-dependent increase in relative expression of COX2 protein as measured by western blot 24h after treatment with TNFa/IFNy in HSIEC cells;
  • Fig. IOC shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in HSIEC.
  • Fig. 10D is a graph showing ImageJ measured (from Fig. IOC) time-dependent increase in relative expression of COX2 in response to 10 ng/ml of TNFa/IFNy in HSIEC.
  • Fig. 11A shows the dose-dependent increase in relative expression of COX2 (as measured by qRT-PCR) in response to TNFa/IFNy in WI-38 human lung fibroblast cells;
  • Fig. 11B shows the dose-dependent increase in relative expression of COX2 (as measured by western blot) in response to TNFa/IFNy in WI-38 cells;
  • Fig. llC is a graph showing ImageJ measured dose-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells;
  • Fig. 11D shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to TNFa/IFNy in WI-38 cells;
  • Fig. 11E is a graph showing ImageJ measured time-dependent increase in relative expression of COX2 (done by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
  • Fig. 12A is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
  • Fig. 12B is a graph showing ImageJ measured expression of COX2 relative to GAPDH in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
  • Fig. 12C is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
  • Fig. 12D is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in WI-38 cells;
  • Fig. 12E is a graph showing ImageJ measured expression of COX2 relative to GAPDH in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells;
  • Fig. 12F is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells;
  • Fig. 13A is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
  • Fig. 13B is a graph showing ImageJ measured expression of IL8 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
  • Fig. 13C is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
  • Fig. 13D is a graph showing ImageJ measured expression of IL8 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
  • Fig. 13E is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
  • Fig. 13F is a graph showing ImageJ measured expression of IL-6 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
  • Fig. 13G is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IRNg alone, or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
  • Fig. 13H is a graph showing ImageJ measured expression of IL-6 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IRNg alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
  • Fig. 14A is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169;
  • Fig. 14B is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169;
  • Fig. 14C is a graph showing qRT-PCR analysis of COX-2 expression in WI- 38 in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169;
  • Fig. 14D is a graph showing qRT-PCR analysis of IL-6 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169;
  • Fig. 14E is a graph showing qRT-PCR analysis of IL-8 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IRNg alone, or followed by treatment with extracts #1, #5, #7 and #169;
  • Fig. 14F is a graph showing qRT-PCR analysis of COX-2 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IRNg alone or followed by treatment with extracts #1, #5, #7 and #169.
  • Fig. 15A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 10 mM CBD or 0.015 pg of various extracts;
  • Fig. 15B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 10 pM CBD or 0.015 pg of various extracts;
  • Fig. 16A is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #1;
  • Fig. 16B is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #1;
  • Fig. 16C is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #5;
  • Fig. 16D is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #5.
  • Fig. 17A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of extracts #1 and #5;
  • Fig. 17B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to different concentrations of extracts #1 and #5;
  • Fig. 18A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
  • Fig. 18B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
  • Fig. 18C is a western blot image showing ACE2 and GAPDH protein levels in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
  • Fig. 18D is a graph showing the ratio of ACE2 protein to GAPDH protein in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
  • Fig. 19A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
  • Fig. 19B is a graph showing the ratio of ACE2 protein to GAPDH protein in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
  • Fig. 19C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
  • Fig. 20A shows graphs of the fold change in the expression of selected pro- inflammatory genes in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
  • Fig. 20B shows a graph of the fold change in the expression of PTGS2 (COX2) gene in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
  • Fig. 20C is a western blot image showing IL-6 and COX-2 protein levels in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
  • Fig. 21 A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 2 IB is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 21C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 22A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 22B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 22C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
  • Fig. 23A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 23B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 23C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 24A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 24B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 24C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
  • Fig. 25A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 25B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 25C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 26A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 26B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 26C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
  • Fig. 27A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 27B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 27C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 28A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 28B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 28C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
  • Fig. 29A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 29B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 29C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 30A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 30B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 30C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
  • Fig. 31A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
  • Fig. 3 IB is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
  • Fig. 31C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
  • Fig. 32A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
  • Fig. 32B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
  • Fig. 32C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene.
  • Fig. 33A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b-caryophyllene, a-bisabolol, a- humulene, a combination of all these components or to the extract #1 itself.
  • Fig. 33B shows the same for COX-2, IL-6, IL-8 and GAPDH.
  • Fig. 33C and D show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 33A.
  • Fig. 34A shows the western blot in HSIEC cells exposed for 24h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #1 or its individual components.
  • Fig. 34B, C and D show ImageJ quantification of COX2, IL6 or IL8, respectively.
  • Fig. 35A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
  • Fig. 35B and C show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35A.
  • Fig. 35D shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in Bj-5ta cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
  • Fig. 35E and F show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35D.
  • Fig. 36A shows the western blot image of IL6, COX2 and GAPDH proteins in WI-38 cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
  • Fig. 36B and C show ImageJ quantification of IL6 and COX2 from Fig. 36A.
  • Fig. 36D shows the western blot image of IL8 and GAPDH proteins in HSIEC cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
  • Fig. 36E shows ImageJ quantification of IL8 from Fig. 36D.
  • Fig. 1A is a simplified image of an EpiOral tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention.
  • EpiOral tissues 110 consist of normal, human- derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes.
  • Fig. IB is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNY-induced inflammation using the set-up in Fig.lA, in accordance with an embodiment of the present invention
  • 3D EpiOral tissues 110 were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
  • an extract treatment step 130 all crude extracts were diluted from a 60 mg/mL stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uL in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uL of 0.01 ug/uL extract solution or control (DMSO alone) were applied to the tissues.
  • an analysis step 140 the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
  • Fig. 2 A and 2B represent the graphs showing the relative expression of ACE2 gene in EpiOral 3D tissues in response in response to treatment with cannabis extract #81 (2A) and #130 (2B), in accordance with an embodiment of the present invention; Extracts #81 and #130 down-regulate the levels of ACE2 in EpiOral tissues.
  • Fig. 2C is a graph showing the cumulative effect of several (as per Embodiment 11) pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts vs DMSO) in EpiOral tissues treated with TNF/IFN followed by treatment with several different extracts (as per Fig. 1), in accordance with an embodiment of the present invention.
  • Fig. 2D is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels using the same EpiOral tissues as per Fig. 1, in accordance with an embodiment of the present invention.
  • Fig. 2E is a graph showing the ImageJ-based measurement of the band intensity from Fig.2D, representing the ratio of ACE2 protein to GAPDH protein after treatment with TNF/IFN and application of different cannabis extracts to EpiOral cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
  • Fig. 3A is a simplified image of Epilntestinal tissues 310 experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention.
  • Epilntestinal tissues are 3D highly differentiated tissue models produced from normal, human cell-derived small intestine epithelial and endothelial cells and fibroblasts.
  • Fig. 3B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-induccd inflammation using the set-up in Fig.3A, in accordance with an embodiment of the present invention.
  • Fig. 3B is one simplified illustration of a scheme of a TNFa/IFNy-induccd inflammation experiment. 3D Epilntestinal tissues of oral epithelial cells 310 from Fig. 3A were used.
  • 3D tissues 310 were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
  • an extract treatment step 330 all crude extracts were diluted from a 60 mg/mF stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uF in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uF of 0.01 ug/uF extract solution or control (DMSO alone) were applied to the tissues.
  • an analysis step 340 the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
  • Fig. 4 A is a graph showing the relative expression of ACE2 gene in Epilntestinal tissues in response to treatment with different cannabis extracts, in accordance with an embodiment of the present invention.
  • Fig. 4B is a graph showing the effects of extract of Cannabis Line 45 effectively decreasing levels of ACE2 gene expression in human 3D Epilntestinal tissues, in accordance with an embodiment of the present invention.
  • Fig. 5A is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein in Epilntestinal tissues treated with TNF/IFN (as per Fig. 3), in accordance with an embodiment of the present invention.
  • Fig. 5B is a graph showing the ImageJ-based measurement of the band intensity from Fig. 5A showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
  • Fig. 5C is a graph showing the effects of different cannabis extracts on down- regulation of inflammation-related PARP-1 and iNOS proteins in Epilntestinal tissues treated with TNF/IFN, in accordance with an embodiment of the present invention.
  • Fig. 5D is a graph showing the ratio of PARP-1 to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
  • Fig. 5E is a graph showing the ratio of iNOS to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
  • Fig. 6A is a simplified image of an EpiAirway tissue model tissue 610 experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention.
  • Fig. 6B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.6A, in accordance with an embodiment of the present invention.
  • 3D EpiAirway tissues of oral epithelial cells 610 were obtained (per Fig. 6A).
  • 3D tissues were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
  • extract treatment step 630 all crude extracts were diluted from a 60 mg/mF stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uF in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uF of 0.01 ug/uF extract solution or control (DMSO alone) were applied to the tissues.
  • an analysis step 640 the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
  • Fig. 7 A is a graph showing the effects of cannabis extracts of novel cannabis lines in reducing levels of the ACE2 protein in human 3D EpiAirway tissue (as described in Fig. 6), in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
  • Fig. 7B is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue (measured from Fig. 7A), in accordance with an embodiment of the present invention.
  • Fig. 7C is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
  • Fig. 7D is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and Actin protein in human 3D EpiAirwayFT tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts (as described in Fig. 6), in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
  • Fig. 7E is a graph showing the ratio of ACE2 protein to Actin protein measured from the image in Fig. 7D, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
  • Fig. 7F is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts (as described in Fig. 6), in accordance with an embodiment of the present invention.
  • Fig. 7G is a graph showing the ratio of ACE2 protein to GAPDH protein measured from the image in Fig. 7F, in accordance with an embodiment of the present invention. asterisks show significant difference from TNF/IFN treatment.
  • Figs. 8A-8F are a set of graphs showing that cannabinoids affect processes involved in a viral life cycle -
  • Figs. 8A-8C show that different cannabinoids applied at different concentrations inhibit reverse transcription in vitro - namely Fig. 8A, CBD, Fig. 8B, THC, and Fig. 8C, CBN and Figs. 8D-8F show that different cannabinoids applied at different concentrations inhibit DNA replication- namely Fig. 8D, CBD, Fig. 8E, THC and Fig. 8F CBN, in accordance with some embodiments of the present invention.
  • Fig. 9 is a simplified pictorial illustration showing a schematic summary 900 of the main effects of cannabis extracts on SARS-COV-2, in accordance with an embodiment of the present invention.
  • Step 910 illustrates mechanism of entry of SARS-COV-2 virus through the recognition of ACE2 receptor.
  • Step 920 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to block the viral entry by downregulating the expression of ACE2 receptor.
  • Step 930 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to block the virus life cycle, including but not limited to blocking reverse transcription and replication.
  • Step 940 illustrates mechanism of cytokine storm development in response to SARS-COV-2 vims.
  • Step 950 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to decrease the expression of pro- inflammatory molecules and prevent the development of cytokine storm.
  • Fig. 10A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in HSIEC cells; asterisks indicated significant (p ⁇ 0.01) difference.
  • Fig. 10B shows the dose-dependent increase in relative expression of COX2 protein as measured by western blot 24h after treatment with TNFa/IFNy in HSIEC cells.
  • Fig. IOC shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in HSIEC.
  • Fig. 10D is a graph showing ImageJ measured (from Fig. IOC) time-dependent increase in relative expression of COX2 in response to 10 ng/ml of TNFa/IFNy in HSIEC.
  • Fig. 11A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in WI-38 human lung fibroblast cells; asterisks indicated significant (p ⁇ 0.01) difference.
  • Fig. 1 IB shows the dose-dependent increase in relative expression of COX2 as measured by western blot 24h after treatment with TNFa/IFNy in WI-38 cells.
  • Fig. llC is a graph showing ImageJ-measured dose-dependent increase in relative expression of COX2 (from Fig. 11B) in response to TNFa/IFNy in WI-38 cells.
  • Fig. 11D shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
  • Fig. 11E is a graph showing ImageJ measured time-dependent increase in relative expression of COX2 (from Fig. 11D) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
  • Fig. 12A is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 5mM CBD or 15 uF of 0.01 ug/uF of various cannabis extracts in HSIEC cells.
  • Fig. 12B is a graph showing ImageJ measured expression of COX2 relative to GAPDH (from Fig. 12A) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in HSIEC cells; asterisks show significant difference.
  • Fig. 12C is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH (from Fig. 12 A) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in HSIEC cells; asterisks show significant difference.
  • Fig. 12D is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 5mM CBD or 15 uL of 0.01 ug/uL of various cannabis extracts in WI-38 cells.
  • Fig. 12E is a graph showing ImageJ measured expression of COX2 relative to GAPDH (from Fig.l2D) in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in WI-38 cells; asterisks show significant difference.
  • Fig. 12F is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH (from Fig.l2D) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells; asterisks show significant difference.
  • Fig. 13A is a western blot image showing the expression of IL8 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells.
  • Fig. 13B is a graph showing ImageJ measured expression of IL8 relative to GAPDH (from Fig. 13A) in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells; asterisks show significant difference.
  • Fig. 13C is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells.
  • Fig. 13D is a graph showing ImageJ measured expression of IL8 relative to GAPDH (from Fig. 13C) in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells; asterisks show significant difference.
  • Fig. 13E is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells.
  • Fig. 13F is a graph showing ImageJ measured expression of IL-6 relative to GAPDH (from Fig. 13E) in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells; asterisks show significant difference.
  • Fig. 13G is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells.
  • Fig. 13H is a graph showing ImageJ measured expression of IL-6 relative to GAPDH (from Fig. 13G) in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells; asterisks show significant difference.
  • Fig. 14 shows qRT-PCR of IL-6, IL-8 and COX-2 genes in WI-38 and HSIEC cells in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169; asterisks show significant difference.
  • FIG. 14A shows IL-6 expression in WI-38
  • Fig. 14B - IL-8 in WI-38
  • Fig. 14C - COX-2 in WI-38
  • Fig. 14D - IL-6 in HSIEC
  • Fig.l4E - IL-8 in HSIEC
  • Fig. 14F COX-2 in HSIEC.
  • Fig. 15 A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 10 mM CBD or 0.015 pg of various extracts.
  • Fig. 15B is a graph showing the ratio of ACE2 protein to GAPDH protein measured from Fig. 15A using ImageJ; asterisks show significant difference.
  • Fig. 16 shows qRT-PCR of IL-6 and IL-8 genes in WI-38 cells treated for 24- 96h with 10 ng/ml TNFa/IFNy alone or in combination with 0.015 pg of extract #1 or #5; asterisks show significant difference.
  • Fig. 16A shows IL-6 expression in response to extract #1
  • Fig. 16B - IL-8 in response to extract #1
  • Fig.l6C - IL-6 in response to ext #5
  • Fig. 16D - IL-8 in response to ext #5.
  • Fig. 17 A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of extracts #1 and #5.
  • Fig. 17B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells (calculated from Fig. 17A) in response to different concentrations of extracts #1 and #5; asterisks show significant difference.
  • Fig. 18A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
  • Fig. 18B is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig. 18A) in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
  • Fig. 18C is a western blot image showing ACE2 and GAPDH protein levels in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
  • Fig. 18D is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig. 18A) in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
  • Fig. 19A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in BJ-5ta cells in response to 24h treatment with 0.015 pg/pl concentration of CBD or various extracts.
  • Fig. 19B is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig.l9A) in BJ-5ta cells in response to 24h treatment with 0.015 pg/pl concentration of CBD or various extracts.
  • Fig. 19C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein (calculated from Fig.l9A) in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC.
  • Fig. 20A shows graphs of the fold change in the expression of selected pro- inflammatory genes in EpiDermFT human 3D tissues in response to UVC followed by 24h treatment with 0.015 pg/pl concentration of extracts #4, #6, #8, #12, #13, #14 and #15.
  • Fig. 20B shows a graph of the log2 fold change in the expression of PTGS2 (COX2) gene in EpiDermFT human 3D tissues in response to UVC f treatment with extracts #4, #6, #8, #12, #13, #14 and #15.
  • COX2 PTGS2
  • Fig. 20C is a western blot image showing IL-6 and COX-2 protein levels in EpiDermFT human 3D tissues in response to UVC or UVC followed by 24h treatment with 0.015 pg/pl concentration of extracts #4, #6, #8, #12, #13, #14 and #15; PBS and DMSO are controls; UV-PBS and UV-DMSO are samples treated with UV with PBS or DMSO added to them.
  • Fig. 21 -Fig.32 show the set of experiments to compare the effect of CBD and terpenes on the expression of ACE2 and TMPRSS2 proteins in WI-38 and HSIEC cells.
  • Cells were treated for 24h with 20 uM CBD alone, with 10-100 pg/ml of an isolated terpene or with combination of CBD and terpenes.
  • Asterisks show significant difference, either between control and any individual treatment, or between CBD and CBD + terpene, or terpene and CBD + terpene.
  • Fig. 21 A shows the western blot image of ACE2, TMPRSS2 and GAPDH protein levels
  • Fig. 2 IB and C show the ImageJ calculated expression of ACE2 and TMPRSS2 relative to GAPDH in response to CBD and myrcene in WI- 38 cells
  • Fig. 22 shows the same in HSIEC cells.
  • Fig. 23 and 24 show the same for caryophyllene in WI-38 and HSIEC cells.
  • Fig. 25 and 26 show the same for a-pinene in WI-38 and HSIEC cells.
  • Fig. 27 and 28 show the same for bisabolol in WI-38 and HSIEC cells.
  • Fig. 29 and 30 show the same for humulene in WI-38 and HSIEC cells.
  • Fig. 31 and 32 show the same for valencene in WI-38 and HSIEC cells.
  • CBD may be less efficient than whole extracts or isolated terpenes in downregulation of ACE2/TMPRSS2 and pro- inflammatory proteins COX, IL-6 and IL-8, it was attempted to check individual components of the extracts.
  • Fig. 33-36 show the results of exposure of three different cell types, WI-38, HSIEC and Bj-5ta for 24h or 48h to 15 pg/ml of two best working extracts, #1 and #7.
  • cells were exposed to the same concentrations found in the extract of three main cannabinoids, CBD, THC and CBN or 4-6 major terpenes or to a combination of all cannabinoids and major terpenes, attempting to reconstitute the effect of a whole extract.
  • Asterisks show significant difference from DMSO control.
  • Fig. 33A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-bisabolol, a-humulene, a combination of all these components or to the extract #1 itself.
  • Fig. 33B shows the same for COX-2, IL-6, IL-8 and GAPDH.
  • Fig. 33C and D show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 33A.
  • Fig. 34A shows the western blot in HSIEC cells exposed for 24h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #1 or its individual components.
  • Fig. 34B, C and D show ImageJ quantification of COX2, IL6 or IL8, respectively.
  • Fig. 35A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
  • Fig. 35B and C show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35A.
  • Fig. 35D shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in Bj-5ta cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
  • Fig. 35E and F show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35D.
  • Fig. 36A shows the western blot image of IL6, COX2 and GAPDH proteins in WI-38 cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
  • Fig. 36B and C show ImageJ quantification of IL6 and COX2 from Fig. 36A.
  • Fig. 36D shows the western blot image of IL8 and GAPDH proteins in HSIEC cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
  • Fig. 36E shows ImageJ quantification of IL8 from Fig. 36D.
  • Described herein is a method for decreasing the expression of ACE2 and decreasing the levels of cytokine storm and inflammation, but is not limited to the steps of: 1) preparation of new cannabis extracts, 2) exposing normal and disease oral, lung and intestinal models to novel extracts and 3) modulating the gene expression and protein levels to cause a reduction of a disease state, or prevent an increase in the disease state in the body tissues, such as oral cavity tissues, lung tissues and intestinal tissues.
  • the present invention provides extracts of twenty nine new C. sativa lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317) ( see Table 1).
  • the present invention further combines use of extracts of these lines, as well as combination of single cannabinoids and terpenes.
  • ACE2- inhibitory potential of these lines in oral tissues may be used to develop modalities to prevent SARS-CoV2 entry via oral and nasopharyngeal epithelial cells.
  • ACE2-inhibitory potential of these lines in lung epithelial tissues may be used to develop modalities to prevent SARS-CoV2 entry via airway cells as well as to treat COVID-19.
  • ACE2 protein in the Epilntestinal tissues (Fig. 5A), and affected the levels of PARP-1 (Fig. 5C), iNOS, (Fig. 5C), as well as inhibited pro -inflammatory gene induction by TNFa/IFNy (Fig. 5B).
  • ACE2-inhibitory and anti-inflammatory potential of these lines in intestinal epithelial tissues may be used to develop modalities to prevent SARS-CoV2 entry via intestinal cells as well as to treat COVID-19.
  • a method for COVID-19 prevention and treatment via affecting the levels of SARS-CoV2 receptor ACE2 in the oral and nasopharyngeal, lung and intestinal epithelial tissues that includes the steps of exposing a patient to novel cannabis extracts, resulting in a reduction in the disease incidence and manifestation in the patient.
  • extracts of new cannabis lines are applied to a human patient. In other embodiments, they may be applied to human oral tissue, artificial human oral tissue, or even animal oral tissue to modulate gene expression leading to a reduction, or at least prevent an increase in a disease state.
  • ACE2 pro-inflammatory genes
  • pro-inflammatory genes ABR, ACP5, ACVR1, ADA, ADORA1, ADORA2A, ADORA2B, AGER, AGTR2, AHCY, AKT1, ALOX5, APOA2, ASH1L, ASS1, ATM, AXL, B4GALT1, BACE2, BAP1, BCR, BDKRB1, BMP6, BMPR1B, BTK, C1QTNF12, C3, C5orf30, C6, CALCA, CALCRL, CCL11, CCL24, CCR4, CD28, CD40, CD96, CDK19, CELA1, CLOCK, CNR1, CNR2, CXCR2, CYP19A1, CYP26B1, CYSLTR1, DROSHA, DUOXA1, DUOXA2, DUSP10, ECM1, EDNRB, EGFR, EIF2AK1, ELANE, EPHA2, EPO, ESR1, ETS1, FABP4, F
  • sequence identity or “identity” in the context of two nucleic acid or protein sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1° C to about 20° C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)).
  • nucleic acid molecules and peptides that are substantially identical to the nucleic acid molecules and peptides presented herein.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • new extracts may modulate genes and proteins sharing a sequence identity or substantial sequence identity to those genes and proteins listed herein.
  • cannabinoids and new extracts may affect key processes of the viral life cycle - reverse transcription, replication, and other ones (Figs. 8A-8F).
  • cannabis extracts including but not limited to #1, #5, #7, #10, #169 and #317, are more potent than CBD alone in downregulation of COX2 and upregulation of SOCS3 in two different models, intestinal epithelial cells (HSIEC) and lung fibroblast cells (WI-38) (Fig. 12).
  • HSIEC intestinal epithelial cells
  • WI-38 lung fibroblast cells
  • Top performing extracts such as #1, #5, #7 and #169 were further found to substantially downregulate the expression of IL-6, IL-8 and COX-2 on both mRNA and protein levels in both models, HSIEC and WI-38 (Fig. 13-14).
  • Extracts #317, #129 and #98 also more substantially inhibited ACE2 expression as compared to CBD alone (Fig. 15).
  • Extracts #1 and #5 demonstrated time-dependent decrease in TNF/IFN- induced expression of IL-6 and IL-8 (Fig. 16). Dose-dependent decrease in ACE2 expression in response to extracts #1 and #5 was also observed (Fig. 17).
  • TMPRSS2 is a serine protease essential for activation of recognition of SARS-COV-2, namely by priming the viral spike protein to allow viral entry into the target cell.
  • Top extracts (#1, #5 and #7) were common in the composition of cannabinoids (CBD-dominant) and terpenes and were enriched in caryophyllene, myrcene, pinene, bisabolol, humulene and valencene. Therefore, the contribution of each terpene on ACE2 and TMPRSS2 expression was tested (Fig. 23A-C). Caryophyllene demonstrated comparable to CBD effect on downregulation of ACE2 and TMPRSS2, with higher concentrations being more efficient. It was further found that combination of CBD with caryophyllene results in potentiation of the effect of CBD on ACE2 and TMPRSS2 in WI-38 cells (Fig.
  • Fig. 24A-C Effect of a-pinene was less obvious, although synergistic effect of CBD and 10 pg/rnl pinene on ACE2 and TMPRSS2 expression was evident in WI-38 cells (Fig. 25A-C) and on TMPRSS2 in HSIEC cells (Fig. 26A-C). Bisabolol also downregulated ACE2 and TMPRSS2 expression in WI-38 cells, more prominent at lower concentration; no synergistic effect was observed (Fig. 27A-C). Bisbolol alone did not decrease the expression of ACE2 and TMPRSS2 expression in HSIEC cells, but synergistic effect on ACE2 expression was apparent (Fig.
  • Humulene decreased the expression of ACE2 in WI-38 cells, but without synergistic effect; no effect on TMPRSS2 was observed (Fig. 29A-C). Humulene alone did not have any effect on ACE2 expression in HSIEC cells, but showed synergistic effect with CBD (Fig. 30A-C). Valencene decreased the expression of ACE2 in WI-38 cells, but without synergistic effect; no effect of valencene alone on TMPRSS2 was observed, but synergistic effect at low concentration was evident (Fig. 31A-C). In HSIEC cells, valencene decreased expression of ACE2 with synergistic effect with CBD (Fig. 32A-C).
  • Extract Preparation 3 g of the powdered plant tissue were weighed using an analytical balance Plant material was placed inside a 250 mL Erlenmeyer flask (clean). 100 mL of Ethyl Acetate was poured into the flask containing the plant material. The flasks were then wrapped with tin foil and shaken continuously (120 rpm) in an incubator @ 21°C overnight and in the dark.
  • the stocks were prepared weighing a 3-6 mg of crude extract into a micro centrifuge tube.
  • the crude extract was dissolved in DMSO (Dimethyl sulfoxide anhydrous from Life technologies cat # D 12345) to reach 60 mg/mL final concentration and stored at -20°C.
  • DMSO Dimethyl sulfoxide anhydrous from Life technologies cat # D 12345
  • Appropriate cell culture media in our experiments RPMI 1640 medium supplemented with 10% FBS or EMEM medium supplemented with 10% FBS) were used to dilute the 60mg/mL stock. The stocks are allowed to thaw then added to the cell culture media, mixed thoroughly to ensure they are in solution and filtered through a 0.22 pm syringe filter. These filtrates were ready to be applied to cells and tested.
  • Terpenes were purchased from True Terpenes (www.trueterpenes.com) and were diluted to the assay concentrations using DMSO or media.
  • CBD, THC and CBN were purchased from MilliporeSigma (Sigma- Aldrich) and were diluted to the assay concentrations using DMSO or media.
  • a 2 pL injection volume was used for all calibration standards (THC, CBD, THC-A, CBD-A, CBG, CBG-A, all Sigma- Aldrich) and sample analysis. The compound peaks were detected for 230 nm and 280 nm.
  • Mobile phases consisted of 50 mM ammonium formate (pH 5.19) (Sigma - Aldrich) in HPLC grade water (Lisher Chemical) on the A side and 100% methanol (Lisher Chemical) on the B side, with a flow rate 0.3 ml/min. Two samples per cultivar were analyzed, with two technical repeats per each sample.
  • EpiAirway (AIR-100) tissues Mattek’s Epi Airway tissue model is a human 3D mucociliary tissue model that consists of normal, human-derived tracheal/bronchial epithelial cells, is cultured at the air-liquid interface and fully recapitulates the barrier, mucociliary responses, infection, toxicity responses of human airway tissues in vivo.
  • EpiAirwayFT (AFT-100) tissues EpiAirway FT is a ready-to-use, 3D mucociliary tissue model consisting of normal, human-derived tracheal/bronchial epithelial cells and normal human stromal fibroblasts (Mattek Life Sciences, MA). H&E-stained section of EpiAirwayFT tissue (courtesy of Mattek) exhibits a pseudostratified epithelium with ciliated cells and an extracellular matrix containing fibroblasts on a microporous membrane.
  • EpiOral (ORL-20) tissues MatTek’s EpiOral tissues consist of normal, human-derived oral epithelial cells. The cells have been cultured to form multilayered, highly differentiated models of the human buccal (EpiOral) phenotypes. The tissues are cultured on specially prepared cell culture inserts using serum free medium and attain levels of differentiation on the cutting edge of in vitro cell culture technology. The EpiOral tissue models exhibit in vivo-like morphological and growth characteristics which are uniform and highly reproducible.
  • Epilntestinal (SMI-100) tissues are 3D highly differentiated tissue models produced from normal, human cell-derived small intestine epithelial and endothelial cells and fibroblasts. Grown at the air-liquid interface, Epilntestinal tissue models are similar to in vivo human epithelial tissues and exhibit columnar shaped basal cells and Kerckring folds, as well as brush borders, functional tight junctions and mucous secreting granules.
  • Human normal foreskin fibroblasts (BJ-5ta), purchased from American Type Culture Collection (ATCC, Manassas, USA), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).
  • DMEM Modified Eagle’s Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • Human primary small intestinal epithelial cells (HSIEC), purchased from Cell Biologies, were cultured in Epithelial Cell Medium /w Kit supplemented with 1% P/S.
  • Human lung fibroblasts (WI-38), purchased from ATCC, were cultured in EMEM supplemented with 10% FBS and 1% P/S. All cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2.
  • tissues or cells were equilibrated for 24 h, after equilibration tissues were treated with 10 ng/mL TNFa /IFNy alone or in combination with 5-30 mM CBD, 5-30 mM CBN, 5-30 mM THC, or different concentrations of extracts for various time.
  • cells were treated either with CBD alone, terpenes alone or in combination of CBD with terpenes.
  • HSIEC and WI-38 cells grown to 70% confluency were exposed to either 10 ng/ml TNFa/IFNy or in combination with extracts #1 or #5 or #7 or #169 for 48 h, 1% DMSO served as a control; HSIEC and WI-38 cells grown to 80% confluency were treated with either 10 ng/ml TNFa/IFNy or in combination with extracts #1 or #5 for an indicated time-course, 1% DMSO served as a control; HSIEC cells grown to 70% confluency were exposed to either 10 ng/ml TNFa/IFNy or in combination with a range of doses of extracts #1 or #5 for 48 h, 1% DMSO served as a control; at the indicated time-point after treatment, total RNA was isolated using TRIzol RNA isolation reagent (ThermoFisher Scientific) and subjected to qRT-PCR analysis with iScript Select cDNA Synthesis kit (Bio-Rad) and SsoF
  • Tissues were harvested and solubilized in laboratory-prepared 1% sodium dodecyl sulfate (SDS) lysis buffer (BioUltraPure, Bioshop) by sonication using a Braunsonic model 1510 sonicator (B. Braun Germany) operating at 80% sonication intensity. Lysates were centrifuged at 15,000 rpm for 10 min and the supernatant was decanted for use. Protein concentrations were determined using the Bradford protein assay with bovine serum albumin as the standard protein using the Nanodrop 2000c spectrophotometer (v. 1.5). Total proteins were separated by electrophoresis on 10- 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).
  • SDS sodium dodecyl sulfate
  • PVDF polyvinyl difluoride
  • the membranes were incubated for one hour in a blocking solution (5% dry skimmed milk in PBS, 0.5% Tween 20) at room temperature, and incubated with specific primary antibodies at 4°C overnight with 1:200 to 1:1,000 dilution of polyclonal/monoclonal antibodies against cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2) (all from Abeam, Cambridge, UK) or GPR18 (from LSBio, Seattle, USA) or CDK2, cyclin El, E2F1, ERK1/2, pERKl/2, PFKFB3 (all from Cell Signaling, Danvers, USA) or AKT1, p53, pAKTl/2/3 (all from Santa Cruz Biotechnology, Dallas, USA), IL-6, IL-8, COX-2, ACE2, TMPRSS2 (Ab
  • compositions of the present invention may be provided in any suitable dosage form.
  • the dosage form is an oral dosage form.
  • Oral dosage forms comprise liquids (solutions, suspensions, and emulsions), semi-solids (pastes), and solids (tablets, capsules, powders, granules, premixes, and medicated blocks).
  • oral dosage forms in the art include, W090/04391, which discloses an oral dosage form of omega-3 polyunsaturated acids to overcome the problems of diseases. It is known to supply said acids in soft gelatine capsule shells.
  • EP 2 240 581 B1 discloses a gelatine capsule for pharmaceutical use with a controlled release of active ingredients and a process for the preparation of said gelatine capsules. During said process xylose is added to the liquid gelatine from which afterwards gelatine capsules are formed. Gelatine capsules manufactured according to the process provide retarded release of active ingredients.
  • US Patent No. 7,264,824 discloses and oral dosage form for food and food supplements, as well as dietetics comprising polyunsaturated acids in a xylose- hardened gelatine capsule with a retarded release time.
  • compositions described herein may be in a suspension or emulsion.
  • a suspension is a coarse dispersion of insoluble drug particles, generally with a diameter exceeding 1 pm, in a liquid (usually aqueous) medium.
  • Suspensions are useful for administering insoluble or poorly soluble drugs/components or in situations when the presence of a finely divided form of the material in the GI tract is required.
  • the taste of most drugs is less noticeable in suspension than in solution, due to the drug being less soluble in suspension.
  • Particle size is an important determinant of the dissolution rate and bioavailability of drugs in suspension.
  • suspensions include surfactants and thickening agents. Surfactants wet the solid particles, thereby ensuring the particles disperse readily throughout the liquid. Thickening agents reduce the rate at which particles settle to the bottom of the container. Some settling is acceptable, provided the sediment can be readily dispersed when the container is shaken. Because hard masses of sediment do not satisfy this criterion, caking of suspensions is not acceptable.
  • An emulsion is a system consisting of 2 immiscible liquid phases, one of which is dispersed throughout the other in the form of fine droplets; droplet diameter generally ranges from 0.1-100 pm.
  • the 2 phases of an emulsion are known as the dispersed phase and the continuous phase.
  • Emulsions are inherently unstable and are stabilized through the use of an emulsifying agent, which prevents coalescence of the dispersed droplets. Creaming, as occurs with milk, also occurs with pharmaceutical emulsions. However, it is not a serious problem because a uniform dispersion returns upon shaking. Creaming is, nonetheless, undesirable because it is associated with an increased likelihood of the droplets coalescing and the emulsion breaking.
  • Other additives include buffers, antioxidants, and preservatives.
  • Emulsions for oral administration are usually oil (the active ingredient) in water, and facilitate the administration of oily substances such as castor oil or liquid paraffin in a more palatable form.
  • a paste is a 2-component semi-solid in which drug is dispersed as a powder in an aqueous or fatty base.
  • the particle size of the active ingredient in pastes can be as large as 100 pm.
  • the vehicle containing the drug may be water; a polyhydroxy liquid such as glycerin, propylene glycol, or polyethylene glycol; a vegetable oil; or a mineral oil.
  • Other formulation excipients include thickening agents, co solvents, adsorbents, humectants, and preservatives.
  • the thickening agent may be a naturally occurring material such as acacia or tragacanth, or a synthetic or chemically modified derivative such as xanthum gum or hydroxypropylmethyl cellulose.
  • the degree of cohesiveness, plasticity, and syringeability of pastes is attributed to the thickening agent. It may be necessary to include a cosolvent to increase the solubility of the drug. Syneresis of pastes is a form of instability in which the solid and liquid components of the formulation separate over time; it is prevented by including an adsorbent such as microcrystalline cellulose. A humectant (eg, glycerin or propylene glycol) is used to prevent the paste that collects at the nozzle of the dispenser from forming a hard crust. Microbial growth in the formulation is inhibited using a preservative. It is critical that pastes have a pleasant taste or are tasteless.
  • a tablet consists of one or more active ingredients and numerous excipients and may be a conventional tablet that is swallowed whole, a chewable tablet, or a modified-release tablet (more commonly referred to as a modified-release bolus due to its large unit size).
  • Conventional and chewable tablets are used to administer drugs to dogs and cats, whereas modified-release boluses are administered to cattle, sheep, and goats.
  • the physical and chemical stability of tablets is generally better than that of liquid dosage forms.
  • the main disadvantages of tablets are the bioavailability of poorly water-soluble drugs or poorly absorbed drugs, and the local irritation of the GI mucosa that some drugs may cause.
  • a capsule is an oral dosage form usually made from gelatin and filled with an active ingredient and excipients.
  • Two common capsule types are available: hard gelatin capsules for solid-fill formulations, and soft gelatin capsules for liquid-fill or semi-solid-fill formulations.
  • Soft gelatin capsules are suitable for formulating poorly water-soluble drugs because they afford good drug release and absorption by the GI tract.
  • Gelatin capsules are frequently more expensive than tablets but have some advantages. For example, particle size is rarely altered during capsule manufacture, and capsules mask the taste and odor of the active ingredient and protect photolabile ingredients.
  • a powder is a formulation in which a drug powder is mixed with other powdered excipients to produce a final product for oral administration.
  • Powders have better chemical stability than liquids and dissolve faster than tablets or capsules because disintegration is not an issue. This translates into faster absorption for those drugs characterized by dissolution rate-limited absorption. Unpleasant tastes can be more pronounced with powders than with other dosage forms and can be a particular concern with in-feed powders, in which it contributes to variable ingestion of the dose. Moreover, sick animals often eat less and are therefore not amenable to treatment with in-feed powder formulations.
  • Drug powders are principally used prophylactically in feed, or formulated as a soluble powder for addition to drinking water or milk replacer. Powders have also been formulated with emulsifying agents to facilitate their administration as liquid drenches.
  • a granule is a dosage form consisting of powder particles that have been aggregated to form a larger mass, usually 2-4 mm in diameter. Granulation overcomes segregation of the different particle sizes during storage and/or dose administration, the latter being a potential source of inaccurate dosing. Granules and powders generally behave similarly; however, granules must deaggregate prior to dissolution and absorption.
  • a premix is a solid dosage form in which an active ingredient, such as a coccidiostat, production enhancer, or nutritional supplement, is formulated with excipients.
  • Premix products are mixed homogeneously with feed at rates (when expressed on an active ingredient basis) that range from a few milligrams to -200 g/ton of food/beverage
  • the density, particle size, and geometry of the premix particles should match as closely as possible those of the feed in which the premix will be incorporated to facilitate uniform mixing. Issues such as instability, electrostatic charge, and hygroscopicity must also be addressed.
  • the excipients present in premix formulations include carriers, liquid binders, diluents, anti-caking agents, and anti dust agents.
  • Carriers such as wheat middlings, soybean mill run, and rice hulls, bind active ingredients to their surfaces and are important in attaining uniform mixing of the active ingredient.
  • a liquid binding agent such as a vegetable oil, should be included in the formulation whenever a carrier is used.
  • Diluents increase the bulk of premix formulations, but unlike carriers, do not bind the active ingredients. Examples of diluents include ground limestone, dicalcium phosphate, dextrose, and kaolin.
  • Caking in a premix formulation may be caused by hygroscopic ingredients and is addressed by adding small amounts of anti-caking agents such as calcium silicate, silicon dioxide, and hydrophobic starch.
  • the dust associated with powdered premix formulations can have serious implications for both operator safety and economic losses, and is reduced by including a vegetable oil or light mineral oil in the formulation. An alternate approach to overcoming dust is to granulate the premix formulation.
  • a medicated block is a compressed feed material that contains an active ingredient, such as a drug, anthelmintic, surfactant (for bloat prevention), or a nutritional supplement, and is commonly packaged in a cardboard box. Ruminants typically have free access to the medicated block over several days, and variable consumption may be problematic. This concern is addressed by ensuring the active ingredient is nontoxic, stable, palatable, and preferably of low solubility.
  • excipients in the formulation modulate consumption by altering the palatability and/or the hardness of the medicated block. For example, molasses increases palatability and sodium chloride decreases it.
  • a binder such as lignin sulfonate
  • the hygroscopic nature of molasses in a formulation may also impact the hardness of medicated blocks and is addressed by using appropriate packaging.
  • the composition of the present invention is in a chewable oral dosage form.
  • the chewable oral dosage form is a chewable tablet.
  • the chewable tablet of the invention is taken slowly by chewing or sucking in the mouth.
  • the chewable tablet of the invention enables the dried cannabis extracts contained therein to be orally administered without drinking.
  • the composition may comprise any suitable flavor or combination of flavors.
  • composition may further comprise other additives, coloring, emulsifiers.
  • flavors and additives may be of a natural, semi-synthetic, synthetic source or combinations thereof.
  • the composition further comprises fructose, sorbitol, microcrystalline cellulose, magnesium stearate, or any combination thereof.
  • the composition further comprises chamomile.
  • the composition further comprises ginger.
  • the composition further comprises peppermint.
  • the composition further comprises anise.
  • the composition further comprises fennel.
  • the composition further comprises thyme.
  • the composition further comprises Arsenicum album.
  • the composition further comprises Carbo vegetabilis.
  • the composition further comprises Ignatia, homeopathic ipecac.
  • the composition further comprises Nux vomica.
  • the composition further comprises Zingiber officinale.
  • composition of the present invention is in the form of a chewing gum product.
  • chewing gum compositions contemplated by the present invention comprise all types of sugar and sugarless chewing gums and chewing gum formulations known to those skilled in the art, including regular and bubble gum types.
  • chewing gum compositions of the invention comprise a chewing gum base, a modifier, a bulking agent or sweetener, and one or more other additives such as, flavoring agents, colorants and antioxidants.
  • the modifying agents are used to soften, plasticize and/or compatibilize one or more of the components of the gum base and/or of the formulation as a whole.
  • the present invention provides a soft, chewable dosage form which is pliable and chewy, yet dissolves quickly in the mouth, has a long shelf life, contains little moisture which improves stability and decreases the tendency for the dosage form to dry out, does not require cooking or heating as part of the manufacturing process.
  • the dosage form is used as a matrix for dried cannabis extracts.
  • the chewable tablet of the invention comprises a metal salt such as calcium, magnesium, aluminum salt, or any mixture thereof.
  • the chewable tablet of the invention comprises hydroxyalkyl cellulose.
  • the chewable tablet of the invention comprises low viscosity hydroxyalkyl cellulose.
  • the chewable tablet of the invention comprises high viscosity hydroxyalkyl cellulose.
  • the chewable tablet of the invention comprises various additives. In another embodiment, the chewable tablet of the invention comprises sweeteners. In another embodiment, the chewable tablet of the invention comprises acidic ingredients. In another embodiment, the chewable tablet of the invention comprises taste correctives. In another embodiment, the chewable tablet of the invention comprises polymeric compounds. In another embodiment, the chewable tablet of the invention comprises essential oils.
  • the chewable tablet of the invention is a soft tablet. In another embodiment, the chewable tablet of the invention is made in a state of soft candy. In another embodiment, the chewable tablet of the invention is made in a state of jelly.
  • the chewable tablet of the invention comprises a core comprising the vitamins of the invention.
  • the chewable tablet of the invention comprises an outer layer wrapping the core which is made up of chewable base such as a gum, a soft candy or a caramel.
  • compositions of the present invention may be provided in any suitable food of a solid, semi- solid or liquid form.
  • compositions that contain a dried cannabis extract for example by mixing, granulating, or tablet-forming processes, is well understood in the art.
  • the dried cannabis extracts are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • the active ingredients of compositions of the present invention are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.
  • additional methods of administering the dried cannabis extracts, or compound(s) isolated therefrom, of the invention comprise injectable dosage forms.
  • the injectable is administered intraperitoneally.
  • the injectable is administered intramuscularly.
  • the injectable is administered intra-dermally.
  • the injectable is administered intravenously.
  • the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation.
  • suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra arterial administration.
  • the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra muscular administration.
  • additional methods of administering the dried cannabis extracts of the invention comprise dispersions, suspensions or emulsions.
  • the dispersion, suspension or emulsion is administered orally.
  • the solution is administered by infusion.
  • the solution is a solution for inhalation.
  • the pharmaceutical composition is administered as a suppository, for example a rectal suppository or a urethral suppository.
  • the pharmaceutical composition is administered by subcutaneous implantation of a pellet.
  • the pellet provides for controlled release of active compound agent over a period of time.
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs. Each possibility represents a separate embodiment of the present invention.
  • parenteral vehicles for subcutaneous, intravenous, intra-arterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the active compounds are released over a period of time after administration.
  • Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils).
  • the composition is an immediate -release composition, i.e. a composition in which all the active compound is released immediately after administration.
  • each possibility represents a separate embodiment of the present invention.
  • the pharmaceutical composition is delivered in a controlled release system.
  • the agents are administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).
  • polymeric materials are used; e.g. in microspheres in or an implant.
  • a controlled release system is placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990).
  • a controlled release system is placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990).
  • compositions also include, in another embodiment, incorporation of the active materials into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.)
  • polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • particulate compositions coated with polymers e.g. poloxamers or poloxamines
  • polymers e.g. poloxamers or poloxamines
  • the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors are also included in the present invention.
  • polymers e.g. poloxamers or poloxamines
  • Also comprehended by the invention are compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline.
  • the modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et ah, 1981; Newmark et ah, 1982; and Katre et ah, 1987).
  • Such modifications also increase, in another embodiment, the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound.
  • the desired in vivo biological activity is achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
  • compositions of the present invention may comprise one or more additional components may further include an additional component selected from the group consisting of an anti-static agent, a buffering agent, a bulking agent, a chelating agent, a colorant, a diluent, a dye, an emollient, a fragrance, an occlusive agent, a pH- adjusting agent, a preservative, and a vitamin.
  • an additional component selected from the group consisting of an anti-static agent, a buffering agent, a bulking agent, a chelating agent, a colorant, a diluent, a dye, an emollient, a fragrance, an occlusive agent, a pH- adjusting agent, a preservative, and a vitamin.
  • compositions of the present invention may comprise one or more additional active agents, selected from the group consisting of active herbal extracts, analgesics, anti-allergic agents, anti-aging agents, anti-bacterials, antibiotic agents, anticancer agents, antidandruff agents, antidepressants, anti-dermatitis agents, anti- edemics, antihistamines, anti-helminths, anti-hyperkeratolyte agents, anti inflammatory agents, anti-irritants, anti-microbials, anti-mycotic s, anti-proliferative agents, antioxidants, anti-wrinkle agents, anti-pruritic s, antiseptic agents, antiviral agents, anti-yeast agents, astringents, topical cardiovascular agents, chemotherapeutic agents, corticosteroids, dicarboxylic acids, disinfectants, fungicides, hair growth regulators, hormones, hydroxy acids, immunosuppressants, immunoregulating agents, keratolytic agents, lactams, metals, metal oxide
  • the composition may comprise one or more anti-oxidants/radical scavengers.
  • the anti-oxidant/radical scavenger may be selected from butylated hydroxy benzoic acids and their salts, coenzyme Q10, coenzyme A, gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N,N- diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid and its salts, lycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts, grape skin/seed extracts, melanin
  • the term “treating” refers to curing a disease. In another embodiment, “treating” refers to preventing a disease. In another embodiment, “treating” refers to reducing the incidence of a disease. In another embodiment,
  • treating refers to ameliorating symptoms of a disease.
  • treating refers to ameliorating symptoms of a disease.
  • treating refers to inducing remission. In another embodiment, “treating” refers to slowing the progression of a disease.

Abstract

The present invention provides Cannabis Sativa (hemp and cannabis) lines, extracts and methods of their use to reduce the levels of ACE2 receptors in oral, lung and intestinal epithelial tissues thereby preventing entry of SARS-CoV-2 and related viruses to mammalian subjects and further treating or preventing cytokine storms, which precede and underlie acute respiratory distress syndrome (ARDS) in COVID- 19 and other diseases and affect viral life cycle processes.

Description

CANNABIS SATIVA (HEMP AND CANNABIS) PRODUCTS FOR VIRAL DISEASE PREVENTION AND MANAGEMENT
FIELD OF THE INVENTION
The present invention relates generally to cannabis and hemp plant products and methods of their production, as well as in production of medicaments for preventing and treating viral diseases, such as COVID-19.
BACKGROUND OF THE INVENTION
There are many viral diseases for which there is no known or approved medicament. There is currently a Corona vims (SARS-CoV-2) pandemic causing the COVID-19 disease. Moreover, as the pandemic spreads around the world, new mutants are discovered. The limited published data on this virus suggests that it spreads easily and has human-human transmission, with a 2-5% death rate. Up to 20% of the COVID-19 disease cases are serious, requiring hospitalization and supplementary oxygen. Around 5% of COVID-19 patients require intensive care. The basic replication number, Ro, of SARS-CoV-2 is thought to be around 2-6 [Zhao, S., et al, 2020], meaning that each infected individual, some of whom are asymptomatic infect 2-6 other people, if no interventions are taken.
SARS-CoV-2 shares 82% sequence identity with severe acute respiratory syndrome -related coronavirus (SARS-CoV). SARS-CoV relies on its spike protein to bind a host cell surface receptor for entry. For the SARS-CoV-2, research has shown that this receptor is the angiotensin-converting enzyme 2 (ACE2).
Both SARS-CoV-2 and SARS-CoV encode a large spike protein (2019-nCoV: 1253 aa; SARS-CoV: 1273 aa). The sequence identity of this protein between the two viruses is 76%. The spike protein has three regions, SI, S2, and S3. Receptor binding domain (RBD) of spike protein in SARS-CoV is majorly in SI and S2. SI region in the RBD from the two viral origins shows 73.5% sequence identity but S2 RBD is almost complete different in SARS-CoV-2 and SARS-CoV. This might affect binding of SARS-CoV-2 to ACE2 as compared to SARS-CoV. Recent studies relating to SARS-CoV-2 RBD-ACE2 crystal structure concluded that changes in RBD of SARS- CoV-2 suggest evolution from SARS-CoV. Now it is well established that ACE2 binding of SARS-CoV-2 is significantly higher than SARS-CoV and it is the main route for receptor-mediated entry of the vims into the human host.
ACE2 is expressed in upper respiratory tract, lung, liver, intestine and kidney tissues in humans. Once SARS-CoV-2 enters the host via suggested major routes above, it further increases ACE2 expression.
Upon SARS-CoV-2 infection, lungs and upper respiratory tract showed very high ACE2 expression. High expression of ACE2 was related to innate and acquired immune response, cytokine secretion, and enhanced the inflammatory response. Neutrophils were significantly higher in infected patients as compared to healthy and IL1B, IL1RA, IL7, IL8, IL9, IL10 levels were very high as well. A clinical study in Wuhan pointed that the levels of IL-1B, IL-10 and IL-8 were significantly increased in critically ill patients with new coronavims SARS-CoV-2 infection, indicating that there were cytokine-mediated inflammatory responses in patients with new coronavims infection.
Higher ACE2 expression after SARS-CoV-2 infection is correlated with increased cytokine levels in the patients. Key aspects of the COVID-19 disease pathogenesis include hyperinduction of proinflammatory cytokine production, which is also known as ‘cytokine storm' and acute respiratory distress syndrome (ARDS) [Li, 2020, Channappanavar et ah, 2020]. High concentrations of cytokines were recorded in plasma of critically ill patients infected with SARS-CoV-2 [Huang, et ah, 2019, Guo et al, 2019]. It was noted that patients infected with SARS-CoV-2 had high amounts of interleukin (IL) IL-1, IL-2, IL- 4, IL-7, IL-10, IL-12, IL-13, IL-17, GCSF, macrophage colony-stimulating factor (MCSF), IP- 10, MCP-1, MIP-la, hepatocyte growth factor (HGF), IFN-g and TNF-a, probably leading to activated T- helper-1 (Thl) cell responses [Guo et al., 2019].
In the severe patients, levels of IL-6, IL-10, and TNF-a further increased[ Guo, et al., 2019]. ICU patients also had high plasma levels of IL-2, IL-7, granulocyte colony- stimulating factor (GCSF). Furthermore, patients requiring ICU admission had higher concentrations of GCSF, IP10, MCP1, MIP1A, IL-6, IL-10, TNFa and other cytokines than did those not requiring ICU admission, suggesting that the cytokine storm was associated with disease severity [Huang, et al., 2019, Guo et al, 2019].
Moreover, TNFa is one of the key cytokines in the cytokine storm and is likely to responsible for the escalation in severity [Liu et al., 2016]. Along with TNFa, IL-6 [Tanaka et al., 2016], IL-1 and others are key mediators of the cytokine storm [Tisoncik et al., 2012]. As appearing chronologically, a cytokine storm manifests before the ARDS in COVID-19 patients and the cytokine storm constitutes one of the ARDS underlying mechanisms. There thus remains an unmet need to provide a product or pharmaceutical composition effective in treating viral diseases in mammals, including humans and non-human mammals, which spread the disease. Moreover, there is an urgent unmet need to provide a product or pharmaceutical composition effective in treating corona viral diseases. Additionally, there is an urgent unmet need to provide a product or pharmaceutical composition effective in treating SARS-CoV-2 (COVID-19) viral disease and mutants thereof in human subjects.
Additionally, it would be highly advantageous to provide anti-inflammatory therapies either prophylactically or to prevent a worsening of the underlying disease progression. Currently, anti-IL-6 receptor antibody, tocilizumab, is being evaluated as a potential cytokine storm therapy [Tanaka et al., 2016]. However, potentially effective, biologies have significant side effects and are extremely expensive. Thus, there is an urgent need for new, effective, minimally invasive, easy to use, and have no systemic side effects that can inhibit cytokine storm.
SUMMARY OF THE INVENTION
It is an object of some aspects of the present invention, to provide improved methods and products for preventing, treating, improving and curing viral diseases, including SARS-CoV2 as well as other corona virus-caused diseases in mammalian subjects.
It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or non-human mammalian organism.
It is another object of some aspects of the present invention to provide compositions for preventing or treating diseases or disorders in a human or non human mammalian organism.
It is another object of some aspects of the present invention to provide compositions for preventing, mitigating or treating viral diseases or disorders in a human or non-human mammalian organism.
It is another object of some aspects of the present invention to provide compositions for preventing, mitigating or treating pathogenic coronavims diseases or disorders in a human or non-human mammalian organism.
The present invention provides products and compositions used in reducing levels of an ACE2 receptor in oral, lung and intestinal epithelial tissues in COVID-19 patients to prevent entry thereto of the SARS-CoV-2 RNA virus and related viruses. Products of the present invention are used further to treat a cytokine storm that precedes and underlies acute respiratory distress syndrome (ARDS), seen in the COVID-19 patients.
The method includes generation of unique lines, whole plant extract preparation, treating normal EpiOral, Epilntestinal and EpiAirway human 3D tissues and human cell lines with tumor necrosis factor alpha (TNF) to induce inflammation, and then with extracts in an amount sufficient to profoundly down-regulate ACE2 expression, inflammation and molecular pathways involved in ACE2 regulation, vims entry and inflammation in the oral, intestinal and airway tissues. The modulation of these pathways is a key to prevention and treatment success in COVID-19. Extracts of novel cannabis and hemp lines can be combined with anti-viral agents and anti inflammatory extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics to increase their efficacy. The present invention provides methods of production of pharmaceutical compositions, suitable for treating viral diseases in mammalian subjects.
The present invention provides pharmaceutical compositions, suitable for treating viral diseases in mammalian subjects.
The present invention provides methods for preventing, mitigating and treating cytokine storms in in COVID-19 patients by administration of C. sativa extracts, which exhibit profound cytokine inhibition patterns.
The present invention further provides methods and products for ACE2 inhibition in target tissues - lung, oral and intestinal epithelia, thereby decreasing or inhibiting a SARS-CoV-2 infection in a mammalian patient.
The present invention further provides combination therapies including a combination of anti- viral (anti-SARS-CoV-2) and anti-cytokine agents will be an effective regimen targeting both etiology and pathogenesis of COVID-19.
The present invention provides new Cannabis sativa lines and extracts as well as combination of single cannabinoids and terpenes and method of using them as a means to modulate ACE2 expression in oral, lung or intestinal tissues. The present invention also provides methods of modulating ACE2 expression through the application cannabis extracts to the oral, lung and intestinal tissues to prevent or treat COVID-19 and other SARS-CoV virus-caused diseases.
Accordingly, the present invention provides a method for modulating ACE2 expression on mRNA and protein levels (e.g., in oral, lung and intestinal tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue, lung cell or lung epithelial tissue, intestinal cells of intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of ACE2 expression results in a prevention of disease via decreased opportunity of virus entry into the cells.
The present invention also provides new Cannabis sativa lines and extracts and as well as combinations of single cannabinoids and terpenes, and methods of using them as a means to modulate cytokine levels and cytokine storm in lung tissues.
Extracts of novel cannabis and hemp lines or single cannabinoids and terpenes are combined with anti-viral agents and anti-inflammatory, such as, but not limited to, extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics, to increase their efficacy. Accordingly, the disclosure provides a means for modulating gene expression and protein levels (e.g., in oral, lung and intestinal tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue, lung cell or lung epithelial tissue, intestinal cells of intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a prevention of disease or reduction of a disease state in the aforementioned tissues.
The present invention provides extracts of twenty nine new C. sativa lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317). The present invention further combines use of extracts of these lines, as well as combination of single cannabinoids and terpenes. The aforementioned extracts, single cannabinoids and terpenes have been tested for anti-inflammatory properties and/or ACE2 inhibition properties using human cells lines and 3D EpiOral, Epilntestinal and EpiAirway tissue models. This is performed by detecting the expression of genes that are associated with molecular etiology and pathogenesis of COVID-19 and ARDS and cytokine storms.
The present invention provides compositions having potent therapeutic ACE2 inhibitory effects in oral, intestinal and airway tissues and thus for treatment and prevention of COVID-19 and ARDS and cytokine storm.
The present invention provides new Cannabis sativa lines and extracts as well as combination of single cannabinoids and terpenes and method of using them as a means to modulate ACE2 gene expression in oral, lung and intestinal tissues. The present invention also provides methods of modulating gene expression through the application of cannabis extracts to the mouth, airways and intestine affected by SARS-CoV-2 and other diseases and viruses.
The present invention further provides extracts for treating and preventing viral diseases, including SARS-CoV-2 virus and resultant COVID-19, as well as other viral diseases and ARDS. The Cannabis sativa extract efficacy can further be increased by adding thereto additional Cannabidiol (CBD), Cannabigerol (CBG), Cannabinol (CBN), terpenes or combinations thereof.
The extract efficacy can be further potentiated and increased by adding anti inflammatory herbs such as but not limited to chamomile, sage, turmeric, thyme, ginger, rosehip, as well as probiotics and their components, or combinations thereof.
The present invention provided the methods of preparation of Cannabis extracts, cannabinoids, such as but not limited to, Cannabidiol (CBD), Cannabigerol (CBG), Cannabinol (CBN), D 9 - 1 c t a h y d o c a n n a b i n o 1 (THC), and terpenes. These extracts and products are adapted to affect key molecular processes required for a virus life cycle, such as reverse transcription, transcription, viral replication, and by doing so, these exhibit anti-viral activity.
The present invention provides new unique cannabis lines, extracts and methods for oral, lung and intestinal improvement and healing and reduction in virus entry into the cells and inflammation. The method includes generation of unique lines, whole plant extract preparation, exposing human oral, lung and intestinal tissues to the extracts in amount sufficient to modulate gene expression in them. The modulation of gene expression then results in a reduction of the disease state- associated changes or aspects thereof in the cannabis -treated tissues.
In some embodiments of the present invention, improved methods and products are provided for oral application and treatment to prevent viral infection.
In some embodiments of the present invention, improved methods and products are provided for oral treatment.
In other embodiments of the present invention, a method and product is described for improving oral health.
In some embodiments of the present invention, improved methods and products are provided for airway application and treatment to prevent viral infection, COVID-19 and acute respiratory distress syndrome.
In other embodiments of the present invention, a method and product is described for improving lung health.
In some embodiments of the present invention, improved methods and products are provided for treatment of viral infection and/or associated diseases.
In some embodiments of the present invention, improved methods and products are provided for intestinal application and treatment to prevent SARS-CoV- 2 viral infection and/or COVID-19 disease.
In other embodiments of the present invention, a method and product is described for improving intestinal health.
In additional embodiments for the present invention, new Cannabis sativa lines are provided.
In additional embodiments for the present invention, new extracts from new Cannabis sativa lines are provided.
Accordingly, the present invention provides a method for modulating gene expression on mRNA and protein levels (e.g., in oral cells, in oral tissue, in lung cells, in lung tissue, in intestinal cells, in intestinal tissue) by providing a source of new extracts, exposing cells or tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a prevention and reduction of a disease state in the in oral, lung and intestinal cells and tissue.
The present invention provides new unique cannabis lines, extracts, dried powders from the extracts, compositions comprising the powders or parts thereof, compounds derived therefrom, pharmaceutical compositions comprising the compound(s), and methods for the treatment of COVID-19, viral diseases, ARDS, cytokine storms, others and combinations thereof.
The method includes generation of unique lines, whole plant extract preparation, treating human 3D oral, airway and intestinal tissues with extracts in amount sufficient to modulate gene expression in the said tissues. The modulation of Angiotensin-converting enzyme 2 gene (ACE2) expression and inflammatory gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the treated tissues.
The compositions and dosage forms of the present invention are useful in promoting health and preventing or treating a large number of disorders in human patients and other mammalian subjects.
In additional embodiments of the present invention, compositions and methods are provided for treating and/or preventing viral disorders.
Further, the invention provides methods and compositions for preventing ARDS and other diseases that harbor an inflammatory component.
Further, the invention provides methods and compositions for preventing systemic diseases and complications related to virus-caused pathologies.
The compositions of the present invention may be used for improving wellness of a human or mammalian subject. Additionally, the compositions of the present invention may be used to treat any disorder or ailment in a human patient or mammalian subject. Furthermore, the compositions of the present invention may be conveniently used in conjunction with a drug to treat any disorder or ailment in a human patient or mammalian subject.
Some embodiments of the present invention provide compounds, compositions and formulations from at least one of hemp and cannabis.
In additional embodiments of the present invention, compositions and methods are provided for treating and/or preventing viral, inflammatory and fibrosis-related disorders.
In additional embodiments of the present invention, compositions and methods are provided for treating and/or preventing pneumonia and ARDS, and other disorders.
In additional embodiments of the present invention, compositions and methods are provided for inhibiting viral life cycle processes thereby treating and/or preventing viral disorders.
Some embodiments of the present invention provide compounds, compositions and formulations from at least one of hemp and cannabis.
Some further embodiments of the present invention provide methods for downregulating expression of ACE2 gene.
Some further embodiments of the present invention provide methods for downregulating expression of at least one inflammatory pathway gene.
Some further embodiments of the present invention provide methods for downregulating expression of at least TNF gene.
Some further embodiments of the present invention provide methods for downregulating expression of at least IL gene.
Some further embodiments of the present invention provide methods for downregulating expression of at least PARP-1 gene.
Some further embodiments of the present invention provide methods for downregulating expression of at least iNOS gene.
Some further embodiments of the present invention provide methods for downregulating at least one inflammatory pathway gene product.
In particular, there are described methods for preparing compositions, compounds, formulations and extracts for preventing viral diseases in a human patient.
In particular, there are described methods for preparing compositions, compounds, formulations and extracts for treating viral diseases in a human patient.
Further embodiments of the present invention provide use of a solvent extract from at least one of hemp and cannabis, for the manufacture of a pharmaceutical composition for the treatment of a viral disease or disorder.
A use of a solvent extract from at least one of hemp and cannabis, according to some embodiments of the present invention, is for the manufacture of a pharmaceutical composition for the prevention and treatment of ARDS.
Some embodiments of the present invention are directed to a method for preventing and treating a viral disease or disorder in a human patient comprising administering to said patient a pharmaceutically effective amount of the cannabis extract composition as described herein.
Additionally, some further embodiments of the present invention are directed to a method for preventing and treating a viral disorder or disease or complication of those in a human patient comprising administering to said patient the oral, inhalation or topical dosage form as described herein.
The liquid cannabis extracts of the present invention, and/or dry powders therefrom, are suitable for oral administration, and appear to be well absorbed through the intestine by the blood and thus exhibit the potential to heal a wide range of viral and inflammatory conditions, such as, but not limited to those mentioned by Chattopadhyay et al. Current Science 87(1) July 2004, 44-53.
According to some embodiments of the present invention, the composition or formulation further comprises at least one solvent or hydrant. In some cases, the hydrant is water, such as double-distilled water. In some cases, it may be at least one organic solvent, such as alcohol.
According to some embodiments of the present invention, the at least one solvent or hydrant is present in the composition or formulation in a concentration of 10-90%, 15-80%, 20-70%, 25-50%, 30-40%, or 10-18% by weight percent.
The solvent or hydrant may further comprise a pH regulator, such as an acid or base. In some embodiments, the base comprises sodium hydroxide.
Suitable products or compositions of the present invention may be in the form of ointments or salves, creams, emulsions, gels, foams, sprays or medicated dressings or bandages, which must be directly applied on the affected zone and must be kept in contact with the oral. In one or more embodiments, the compositions further comprise up to 10% of water.
In one or more embodiments, the composition is substantially non-aqueous and/or substantially alcohol-free.
In another embodiment, the present invention provides a method for inhibiting a disease in a subject comprising administering a subject a composition of the invention.
In another embodiment, the present invention provides a method for inhibiting a viral disease in a subject comprising administering a subject a composition of the present invention.
In another embodiment, the present invention provides a method for inhibiting a disease in a subject comprising orally administering a product of the present invention to the subject.
In another embodiment, the present invention provides a method for inhibiting a disease in a subject comprising inhalation-based administering a product of the present invention to the subject.
In another embodiment, the composition of the present invention is in a chewable oral dosage form. In another embodiment, the chewable oral dosage form is a chewable tablet. In another embodiment, the chewable tablet of the invention is taken slowly by chewing or sucking in the mouth. In another embodiment, the chewable tablet of the invention enables the dried cannabis extracts contained therein to be orally administered without drinking.
In one or more embodiments, the composition further comprises a therapeutically effective concentration of one or more active agents.
The composition of the present invention further contains a surface-active agent. Surface- active agents (also termed "surfactants") include any agent linking oil and water in the composition, in the form of emulsion.
In an embodiment of the present invention, a composition of the present invention includes one or more additional components. Such additional components include but are not limited to anti-static agents, buffering agents, bulking agents, chelating agents, cleansers, colorants, conditioners, diluents, dyes, emollients, fragrances, humectants, permeation enhancers, pH-adjusting agents, preservatives, protectants, oral penetration enhancers, softeners, solubilizers, sunscreens, sun blocking agents, sunless tanning agents, viscosity modifiers and vitamins. As is known to one skilled in the art, in some instances a specific additional component may have more than one activity, function or effect.
There is thus provided according to an embodiment of the present invention, a pharmaceutical composition for treating a viral disease or disorder in a mammalian subject, the pharmaceutical composition including at least one of: a) an extract as described herein; b) at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD); and c) at least one terpene or terpene alcohol; wherein the pharmaceutical composition is suitable for treating the viral disease or the viral disorder in the mammalian subject.
Additionally, according to an embodiment of the present invention, the at least one terpene is selected from the group consisting of myrcene, humulene, pinene carophyllene and valencene.
Further, according to an embodiment of the present invention, the terpene alcohol includes bisabolol.
Moreover, according to an embodiment of the present invention, the at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD) includes only Cannabidiol (CBD).
Importantly, according to an embodiment of the present invention, the viral disease or disorder is caused by a corona virus, a mutant or variant thereof.
Additionally, according to an embodiment of the present invention, the viral disease is COVID 19.
Furthermore, according to an embodiment of the present invention, the pharmaceutical composition is effective in treating a cytokine storm associated with the COVID 19.
Further, according to an embodiment of the present invention, the pharmaceutical composition is suitable for reducing a level of an ACE2 protein in the mammalian subject.
There is thus provided according to an embodiment of the present invention, a method for treating a viral disease or disorder in a mammalian subject, the method including administering a pharmaceutical composition including at least one of; an extract as described herein; at least one of Cannabigerol (CBG), Cannabinol (CBN) and
Cannabidiol (CBD); and at least one terpene or terpene alcohol; to the mammalian subject thereby treating the viral disease or the viral disorder in the mammalian subject.
Additionally, according to an embodiment of the present invention, the method is effective in treating a cytokine storm associated with the COVID 19. Importantly, according to an embodiment of the present invention, the method is effective in reducing a level of an ACE2 protein in the mammalian subject.
EMBODIMENTS
1. A method for preventing and treating corona virus-caused disease or disorder, the method comprising: a) combining at least one marijuana strain and at least one hemp strain to form at least one Cannabis line; b) extracting at least one compound from said at least one Cannabis line to form an extract or combining an isolated cannabidiol and adding set of terpenes; and c) treating said disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said disease or disorder.
2. A method according to embodiment 1, wherein said preventing and treating step induces modulation of gene expression in at least one of oral cells and oral tissue; and wherein said modulation of ACE2 gene said expression results in a reduction of vims entry and virus disease, an inflammatory state, a disorder state and combinations thereof.
3. A method according to embodiment 1, wherein said preventing and treating step induces modulation of gene expression in at least one of airway cells and tissue; and wherein said modulation of ACE2 gene expression results in a reduction of virus entry and virus disease, an inflammatory state, a disorder state and combinations thereof.
4. A method according to embodiment 1, wherein said preventing and treating step induces modulation of gene expression in at least one of intestinal cells and tissue; and wherein said modulation of ACE2 gene expression results in a reduction of virus entry and virus disease, an inflammatory state, a disorder state and combinations thereof.
5. A method according to embodiment 1, wherein said at least one Cannabis line is selected from the group consisting of a marijuana/marijuana hybrid line, hemp/hemp hybrid line and hemp/marijuana hybrid line.
6. A method according to embodiment 5, wherein said at least one line is selected from the group consisting of designated lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317) and combinations thereof.
7. A method according to embodiment 1, wherein said extracting step comprises extracting flowers of said at least one Cannabis line.
8. A method according to embodiment 5, wherein said extracting step comprises extracting said at least one compound in at least one organic solvent.
9. A method according to embodiment 5, wherein said extracting step is performed at a temperature in the range of 15- to 60°C and at a pressure in a range of -0.5 to 1.5 bar and wherein said at least one organic solvent comprises ethyl acetate.
10. A method according to embodiment 2, wherein said modulation of ACE2 gene expression results in a reduction of a 0.1-3 log2 fold reduction.
11. A method according to embodiment 10, wherein said modulation of gene expression results in a reduction of a 0.1-3 log2 fold reduction in at least one of a gene selected from the group consisting of: ABR, ACP5, ACVR1, ADA, ADORA1, ADORA2A, ADORA2B, AGER, AGTR2, AHCY, AKT1, ALOX5, APOA2, ASH1L, ASS1, ATM, AXL, B4GALT1, BACE2, BAP1, BCR, BDKRB1, BMP6, BMPR1B, BTK, C1QTNF12, C3, C5orf30, C6, CALCA, CALCRL, CCL11, CCL24, CCR4, CD28, CD40, CD96, CDK19, CELA1, CLOCK, CNR1, CNR2, CXCR2, CYP19A1, CYP26B1, CYSLTR1, DROSHA, DUOXA1, DUOXA2, DUSP10, ECM1, EDNRB, EGFR, EIF2AK1, ELANE, EPHA2, EPO, ESR1, ETS1, FABP4, FANCA, FANCD2, FCER1G, FOXF1, FOXP3, GAL, GAT A3, GPR17, GPX1, GPX4, HAMP, HFE, HGF, HIST1H2BA, HSPD1, ICAM1, IDOl, IDOl, IGFBP4, IL10, IL12B, IL13, IL15, IL17B, IL17F, IL17RA, IL17RB, IL17RC, ILIA, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2, IL20RB, IL22RA2, IL25, IL2RA, IL31RA, IL33, IL5, IL5RA, iNOS, ITGA2, ITGB2, ITGB6, JAK2, JAM3, JUN, KDM6B, KRT1, LBP, LDLR, LIPA, LRRK2, LTA, LYN, MAP2K3, MAS1, MCPH1, NAMPT, NFE2L2, NFKB1, NFKBIZ, NLRP6, NLRX1, NOTCH1, NPFF, NPPA, NPY5R, NT5E, NUPR1, OGGI, OPRM1, PARP-1, P2RX1, P2RX7, PBK, PGLYRP1, PGLYRP2, PIK3CG, PLAA, PLP1, POLB, PPARG, PRCP, PSMB4, PTAFR, PTGES, PTGS2, PYCARD, RASGRP1, RBPJ, RHBDD3, RICTOR, S100A8, S1PR3, SCN9A, SDC1, SEH1L, SELP, SERPINC1, SERPINF1, SGMS1, SLC7A2, SMAD1, SMAD3, SMO, SOCS3, SOCS5, SPHK1, SPP1, STAT3, STAT5B, STK39, SUCNR1, TAC1, TBC1D23, TFR2, TGM2, TIMP1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TNF, TNFAIP3, TNFRSF1B, TRPV1,TRPV4, TSPAN2, TUSC2, UCN, UGT1A1, UNC13D, VCAM1, WFDC1 and ZYX and combinations thereof.
12. A method according to embodiment 1, wherein said at least one compound is provided in a concentration in a range of 0.0001-0.05 pg/pl, 0.001-0.05 pg/pl, 0.001-0.005 pg/pl, 0.003-0.03 pg/pl or 0.007-0.015 pg/pl.
13. A method according to embodiment 1, wherein said at least one compound is provided in a solvent extract and said solvent extract exhibits viral disease or disorder healing properties.
14. A method according to embodiment 1, wherein said at least one compound is provided in a solvent extract and said solvent extract exhibits ARDS and other inflammatory conditions healing properties.
15. A method according to embodiment 10, wherein said solvent extract is at least 2-20, 3-15, 4-12, 5-10 or 6-9 times as effective as at least one of THC and CBD, administered at the same concentration in treating said disease or said disorder.
16. The method of embodiment 1, wherein the disease state is a viral disease, corona virus-cause disease and complications, inflammatory disease or combinations thereof.
17. The method of embodiment 16, wherein the disease state is selected from the group consisting of a virus-caused disease, virus-caused disease complications, environmental factor-induced inflammation and combinations thereof.
18. A method according to embodiment 1, wherein said Cannabis line is a Cannabis sativa line.
19. An organic extract of at least one plant line, said at least one plant line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain, wherein said organic extract comprises at least one compound suitable for treating a mammalian oral disease or disorder.
20. An organic extract according to embodiment 19, wherein said at least one plant line comprises a Cannabis sativa line.
21. An organic extract according to embodiment 19, wherein said mammalian oral disease or disorder is selected from the group consisting of a viral disease, an oral disease, a lung disease, an intestinal disease and combinations thereof.
22. An organic extract according to embodiment 19, wherein said extract is effective against corona virus disease.
23. An organic extract according to embodiment 19, wherein said extract is effective against other virus diseases.
24. An organic extract according to embodiment 19, wherein said extract is effective against ARDS and other cytokine-induced diseases and disorders.
25. An organic extract according to embodiment 19, wherein said organic extract is at least 2-20, 3-15, 4-12, 5-10 or 6-9 times as effective as at least one of THC and CBD, administered at the same concentration in treating said disease.
26. A combination therapy, isolated from an organic extract of at least one hybrid line, said at least one hybrid line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain; and wherein said organic extract comprises a plurality of compounds suitable for treating a mammalian viral disease and disorder.
27. A Cannabis extract for treating and preventing a viral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
28. A Cannabis extract for treating and preventing viral disease or disorder, wherein said extract efficacy can be further increased by adding extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
29. A line of Cannabis sativa formed by combining at least one marijuana strain and at least one hemp strain, said line will be deposited at public culture collection, currently under designation numbers (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317, #CD10.
30. A method for treating a disease state in oral epithelial cells or oral tissue, the method comprising the steps of: a) providing a source of unique extract from at least one line of Cannabis sativa; and b) preventing and treating the COVID-19 and virus-caused disorders with aforementioned extract(s) in an effective amount to induce modulation of ACE 2 and inflammatory gene expression in the oral, airway and intestinal tissue; wherein the modulation of gene expression results in a reduction of the disease state in the aforementioned tissues.
31. The method of embodiment 30, wherein the extracts is extract #1.
32. The method of embodiment 30, wherein the extracts is extract #4.
33. The method of embodiment 30, wherein the extracts is extract #5.
34. The method of embodiment 30, wherein the extracts is extract #6.
35. The method of embodiment 30, wherein the extracts is extract #7.
36. The method of embodiment 30, wherein the extracts is extract #8.
37. The method of embodiment 30, wherein the extracts is extract #9.
38. The method of embodiment 30, wherein the extracts is extract #10.
39. The method of embodiment 30, wherein the extracts is extract #12.
40. The method of embodiment 30, wherein the extracts is extract #13.
41. The method of embodiment 30, wherein the extracts is extract #14.
42. The method of embodiment 30, wherein the extracts is extract #15.
43. The method of embodiment 30, wherein the extracts is extract #31
44. The method of embodiment 30, wherein the extracts is extract #45. 45. The method of embodiment 30, wherein the extracts is extract #49.
46. The method of embodiment 30, wherein the extracts is extract #81.
47. The method of embodiment 30, wherein the extracts is extract #90.
48. The method of embodiment 30, wherein the extracts is extract #98.
49. The method of embodiment 30, wherein the extracts is extract #114.
50. The method of embodiment 30, wherein the extracts is extract #115.
51. The method of embodiment 30, wherein the extracts is extract #129.
52. The method of embodiment 30, wherein the extracts is extract #130.
53. The method of embodiment 30, wherein the extracts is extract #131.
54. The method of embodiment 30, wherein the extracts is extract #132.
55. The method of embodiment 30, wherein the extracts is extract #155.
56. The method of embodiment 30, wherein the extracts is extract #157.
57. The method of embodiment 30, wherein the extracts is extract #166.
58. The method of embodiment 30, wherein the extracts is extract #167.
59. The method of embodiment 30, wherein the extracts is extract #169.
60. The method of embodiment 30, wherein the extracts is extract #207.
61. The method of embodiment 30, wherein the extracts is extract #274.
62. The method of embodiment 30, wherein the extracts is extract #317.
63. The method of embodiment 30, wherein the modulation of gene expression is a down-regulation of ACE2 gene expression.
64. The method of embodiment 30, wherein the modulation of gene expression is a down-regulation of proinflammatory gene expression and up-regulation of anti-inflammatory gene expression.
65. The method of embodiment 30, wherein the disease state is COVID-19.
66. The method of embodiment 30, wherein the disease state is corona virus disease.
67. The method of embodiment 30, wherein the disease state is viral disease.
68. The method of embodiment 30, wherein the disease state is Acute Respiratory Disease Syndrome
69. The method of embodiment 30, wherein the disease state is pneumonia.
70. Effects of extracts according to embodiments 19-25, wherein said effects are further extended by addition of CBD, CBG, CBN, terpenes or combinations thereof. 71. Effects of extracts in embodiments 19-25, wherein said effects are further extended by addition of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
72. A method according to embodiments 30-62, wherein said reduction of the disease state is further extended by addition of CBD, CBG, CBN, terpenes or combinations thereof to said unique extract.
73. A method according to embodiments 30-62, wherein said reduction of the disease state is further extended by addition of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
74. A method according to embodiment 1, wherein the cannabis extracts or components purified therefrom are adapted to inhibit processes critical for the vims life cycle.
The present invention will be more fully understood from the following detailed description of the preferred embodiments thereof, taken together with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figures so that it may be more fully understood.
With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:
Fig. 1A is a simplified image of an EpiOral tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention;
Fig. IB is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.lA, in accordance with an embodiment of the present invention;
Fig. 2A is a graph showing the effects of cannabis extract 81 on the levels of ACE2 gene expression and expression of inflammation-related genes in EpiOral tissues, in accordance with an embodiment of the present invention;
Fig. 2B is a graph showing the effects of cannabis extract 130 on the levels of ACE2 gene expression and expression of inflammation-related genes in EpiOral tissues, in accordance with an embodiment of the present invention;
Fig. 2C is a graph showing the effects of several pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts vs DMSO) for several different extracts, in accordance with an embodiment of the present invention;
Fig. 2D is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels in EpiOral tissues, in accordance with an embodiment of the present invention;
Fig. 2E is a graph showing the ratio of ACE2 protein to GAPDH protein after treatment with TNF/IFN and application of different cannabis extracts to EpiOral cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment; Fig. 3A is a simplified image of Epilntestinal tissues experimental set-up for testing candidate treatments for anti inflammatory properties, in accordance with an embodiment of the present invention;
Fig. 3B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-induccd inflammation using the set-up in Fig.3A, in accordance with an embodiment of the present invention;
Fig. 4 A is a graph showing the effects of different cannabis extracts on the levels of ACE2 gene expression in Epilntestinal tissues, in accordance with an embodiment of the present invention;
Fig. 4B is a graph showing the effects of extract of Cannabis Line 45 effectively decreasing levels of ACE2 gene expression in human 3D Epilntestinal tissues, in accordance with an embodiment of the present invention;
Fig. 5A is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels in Epilntestinal tissues, in accordance with an embodiment of the present invention;
Fig. 5B is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment;
Fig. 5C is a graph showing the effects of different cannabis extracts on down- regulation of inflammation-related PARP-1 and iNOS proteins in Epilntestinal cells, in accordance with an embodiment of the present invention;
Fig. 5D is a graph showing the ratio of PARP-1 to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention;
Fig. 5E is a graph showing the ratio of iNOS to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention;
Fig. 6A is a simplified image of an EpiAirway tissue model tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention; Fig. 6B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.6A, in accordance with an embodiment of the present invention.
Fig. 7 A is a graph showing the effects of cannabis extracts of novel cannabis lines in reducing levels of the ACE2 protein in human 3D EpiAirway tissue, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment;
Fig. 7B is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue, in accordance with an embodiment of the present invention;
Fig. 7C is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment;
Fig. 7D is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and Actin protein in human 3D EpiAirwayFT tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts, in accordance with an embodiment of the present invention;
Fig. 7E is a graph showing the ratio of ACE2 protein to Actin protein after induction of inflammation with TNF/IFN and after application of different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment;
Fig. 7F is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts, in accordance with an embodiment of the present invention;
Fig. 7G is a graph showing the ratio of ACE2 protein to GAPDH protein after induction of inflammation with TNF/IFN and after application of different cannabis extracts were applied to human 3D EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
Figs. 8A-8F are a set of graphs showing that cannabinoids affect processes involved in a viral life cycle - Figs. 8A-8C show that different cannabinoids applied at different concentrations to an in vitro viral experiment inhibit reverse transcription- namely Fig. 8A, CBD, Fig. 8B, THC, Fig. 8B THC and Fig. 8C, CB and Figs. 8D-8F show that different cannabinoids applied at different concentrations to an in vitro viral experiment inhibit DNA replication- namely Fig. 8D, CBD, Fig. 8E, THC and Fig. 8F THC, in accordance with some embodiments of the present invention;
Fig. 9 is a simplified pictorial illustration showing a schematic summary of the main effects of cannabis extracts on SARS-COV-2, in accordance with an embodiment of the present invention;
Fig. 10A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in HSIEC cells; asterisks indicated significant (p<0.01) difference;
Fig. 10B shows the dose-dependent increase in relative expression of COX2 protein as measured by western blot 24h after treatment with TNFa/IFNy in HSIEC cells;
Fig. IOC shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in HSIEC.
Fig. 10D is a graph showing ImageJ measured (from Fig. IOC) time-dependent increase in relative expression of COX2 in response to 10 ng/ml of TNFa/IFNy in HSIEC.
Fig. 11A shows the dose-dependent increase in relative expression of COX2 (as measured by qRT-PCR) in response to TNFa/IFNy in WI-38 human lung fibroblast cells;
Fig. 11B shows the dose-dependent increase in relative expression of COX2 (as measured by western blot) in response to TNFa/IFNy in WI-38 cells;
Fig. llC is a graph showing ImageJ measured dose-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells;
Fig. 11D shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to TNFa/IFNy in WI-38 cells;
Fig. 11E is a graph showing ImageJ measured time-dependent increase in relative expression of COX2 (done by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
Fig. 12A is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
Fig. 12B is a graph showing ImageJ measured expression of COX2 relative to GAPDH in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
Fig. 12C is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in HSIEC cells;
Fig. 12D is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in WI-38 cells;
Fig. 12E is a graph showing ImageJ measured expression of COX2 relative to GAPDH in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells;
Fig. 12F is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells;
Fig. 13A is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
Fig. 13B is a graph showing ImageJ measured expression of IL8 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
Fig. 13C is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
Fig. 13D is a graph showing ImageJ measured expression of IL8 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
Fig. 13E is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
Fig. 13F is a graph showing ImageJ measured expression of IL-6 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells;
Fig. 13G is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IRNg alone, or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
Fig. 13H is a graph showing ImageJ measured expression of IL-6 relative to GAPDH in response to treatment with 10 ng/ml TNFa/IRNg alone or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells;
Fig. 14A is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169;
Fig. 14B is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169;
Fig. 14C is a graph showing qRT-PCR analysis of COX-2 expression in WI- 38 in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169;
Fig. 14D is a graph showing qRT-PCR analysis of IL-6 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169;
Fig. 14E is a graph showing qRT-PCR analysis of IL-8 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IRNg alone, or followed by treatment with extracts #1, #5, #7 and #169;
Fig. 14F is a graph showing qRT-PCR analysis of COX-2 expression in HSIEC in response to treatment with 10 ng/ml TNFa/IRNg alone or followed by treatment with extracts #1, #5, #7 and #169.
Fig. 15A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 10 mM CBD or 0.015 pg of various extracts;
Fig. 15B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 10 pM CBD or 0.015 pg of various extracts;
Fig. 16A is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #1;
Fig. 16B is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #1;
Fig. 16C is a graph showing qRT-PCR analysis of IL-6 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #5;
Fig. 16D is a graph showing qRT-PCR analysis of IL-8 expression in WI-38 cells in response to 24, 48, 72 or 96 h of treatment with 10 ng/ml TNFa/IFNy alone or in combination with extract #5. Fig. 17A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of extracts #1 and #5;
Fig. 17B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to different concentrations of extracts #1 and #5;
Fig. 18A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
Fig. 18B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
Fig. 18C is a western blot image showing ACE2 and GAPDH protein levels in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
Fig. 18D is a graph showing the ratio of ACE2 protein to GAPDH protein in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC;
Fig. 19A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
Fig. 19B is a graph showing the ratio of ACE2 protein to GAPDH protein in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
Fig. 19C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC;
Fig. 20A shows graphs of the fold change in the expression of selected pro- inflammatory genes in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
Fig. 20B shows a graph of the fold change in the expression of PTGS2 (COX2) gene in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
Fig. 20C is a western blot image showing IL-6 and COX-2 protein levels in EpiDermFT human 3D tissues in response to UVC and treatment with extracts #4, #6, #8, #12, #13, #14 and #15;
Fig. 21 A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 2 IB is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 21C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 22A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 22B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 22C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of myrcene or CBD with myrcene;
Fig. 23A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 23B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 23C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 24A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 24B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 24C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of caryophyllene or CBD with caryophyllene;
Fig. 25A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 25B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 25C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 26A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 26B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 26C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of a-pinene or CBD with a-pinene;
Fig. 27A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 27B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 27C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 28A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 28B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 28C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of bisabolol or CBD with bisabolol;
Fig. 29A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 29B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 29C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 30A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 30B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 30C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of humulene or CBD with humulene;
Fig. 31A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
Fig. 3 IB is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
Fig. 31C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in WI-38 cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
Fig. 32A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene;
Fig. 32B is a graph showing the ratio of ACE2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene; and
Fig. 32C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein in HSIEC cells in response to 24 h treatment with 20 uM concentration of CBD and various concentrations of valencene or CBD with valencene. Fig. 33A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b-caryophyllene, a-bisabolol, a- humulene, a combination of all these components or to the extract #1 itself. Fig. 33B shows the same for COX-2, IL-6, IL-8 and GAPDH.
Fig. 33C and D show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 33A.
Fig. 34A shows the western blot in HSIEC cells exposed for 24h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #1 or its individual components.
Fig. 34B, C and D show ImageJ quantification of COX2, IL6 or IL8, respectively.
Fig. 35A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
Fig. 35B and C show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35A. Fig. 35D shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in Bj-5ta cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
Fig. 35E and F show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35D.
Fig. 36A shows the western blot image of IL6, COX2 and GAPDH proteins in WI-38 cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination. Fig. 36B and C show ImageJ quantification of IL6 and COX2 from Fig. 36A.
Fig. 36D shows the western blot image of IL8 and GAPDH proteins in HSIEC cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination. Fig. 36E shows ImageJ quantification of IL8 from Fig. 36D.
In all the figures similar reference numerals identify similar parts.
DETAILED DESCRIPTION OF THE EMBODIMENTS
In the detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that these are specific embodiments and that the present invention may be practiced also in different ways that embody the characterizing features of the invention as described and claimed herein.
Fig. 1A is a simplified image of an EpiOral tissue experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention. EpiOral tissues 110 consist of normal, human- derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes.
Fig. IB is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNY-induced inflammation using the set-up in Fig.lA, in accordance with an embodiment of the present invention;
Scheme of the TNF-induced inflammation experiment.
In an induction step 120, 3D EpiOral tissues 110 were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
In an extract treatment step 130, all crude extracts were diluted from a 60 mg/mL stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uL in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uL of 0.01 ug/uL extract solution or control (DMSO alone) were applied to the tissues.
In an analysis step 140, the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
Fig. 2 A and 2B represent the graphs showing the relative expression of ACE2 gene in EpiOral 3D tissues in response in response to treatment with cannabis extract #81 (2A) and #130 (2B), in accordance with an embodiment of the present invention; Extracts #81 and #130 down-regulate the levels of ACE2 in EpiOral tissues.
Fig. 2C is a graph showing the cumulative effect of several (as per Embodiment 11) pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts vs DMSO) in EpiOral tissues treated with TNF/IFN followed by treatment with several different extracts (as per Fig. 1), in accordance with an embodiment of the present invention.
Fig. 2D is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein levels using the same EpiOral tissues as per Fig. 1, in accordance with an embodiment of the present invention.
Fig. 2E is a graph showing the ImageJ-based measurement of the band intensity from Fig.2D, representing the ratio of ACE2 protein to GAPDH protein after treatment with TNF/IFN and application of different cannabis extracts to EpiOral cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
Fig. 3A is a simplified image of Epilntestinal tissues 310 experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention.
Epilntestinal tissues are 3D highly differentiated tissue models produced from normal, human cell-derived small intestine epithelial and endothelial cells and fibroblasts.
Fig. 3B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-induccd inflammation using the set-up in Fig.3A, in accordance with an embodiment of the present invention.
Fig. 3B is one simplified illustration of a scheme of a TNFa/IFNy-induccd inflammation experiment. 3D Epilntestinal tissues of oral epithelial cells 310 from Fig. 3A were used.
In an induction step 320, 3D tissues 310 were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
In an extract treatment step 330, all crude extracts were diluted from a 60 mg/mF stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uF in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uF of 0.01 ug/uF extract solution or control (DMSO alone) were applied to the tissues.
In an analysis step 340, the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
Fig. 4 A is a graph showing the relative expression of ACE2 gene in Epilntestinal tissues in response to treatment with different cannabis extracts, in accordance with an embodiment of the present invention.
Fig. 4B is a graph showing the effects of extract of Cannabis Line 45 effectively decreasing levels of ACE2 gene expression in human 3D Epilntestinal tissues, in accordance with an embodiment of the present invention.
Fig. 5A is an image showing the effects of different cannabis extracts on ACE2 protein and GAPDH protein in Epilntestinal tissues treated with TNF/IFN (as per Fig. 3), in accordance with an embodiment of the present invention.
Fig. 5B is a graph showing the ImageJ-based measurement of the band intensity from Fig. 5A showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
Fig. 5C is a graph showing the effects of different cannabis extracts on down- regulation of inflammation-related PARP-1 and iNOS proteins in Epilntestinal tissues treated with TNF/IFN, in accordance with an embodiment of the present invention.
Fig. 5D is a graph showing the ratio of PARP-1 to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
Fig. 5E is a graph showing the ratio of iNOS to actin after different cannabis extracts were applied to Epilntestinal cells, in accordance with an embodiment of the present invention.
Fig. 6A is a simplified image of an EpiAirway tissue model tissue 610 experimental set-up for testing candidate treatments for anti-inflammatory properties, in accordance with an embodiment of the present invention.
Fig. 6B is a simplified flowchart of a method for testing candidates for anti inflammatory properties after TNFa/IFNy-i induced inflammation using the set-up in Fig.6A, in accordance with an embodiment of the present invention. 3D EpiAirway tissues of oral epithelial cells 610 were obtained (per Fig. 6A).
In an induction step 620, 3D tissues were inserted in a well with medium. Tissues were equilibrated in the medium for 24 h (overnight) and then culture medium was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to TNFa (40 ng/ml) and IFNa (5 ng/ml) or to DMSO only.
In extract treatment step 630, all crude extracts were diluted from a 60 mg/mF stock (the stock is prepared in DMSO). For this experiment, a final concentration of 0.01 ug/uF in 30% glycerol-PBS was used. 24 h after TNF/IFN exposure, 15 uF of 0.01 ug/uF extract solution or control (DMSO alone) were applied to the tissues.
In an analysis step 640, the samples were harvested and used for the analysis of mRNA by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with changes in ACE2 expression or inflammation.
Fig. 7 A is a graph showing the effects of cannabis extracts of novel cannabis lines in reducing levels of the ACE2 protein in human 3D EpiAirway tissue (as described in Fig. 6), in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
Fig. 7B is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue (measured from Fig. 7A), in accordance with an embodiment of the present invention.
Fig. 7C is a graph showing the ratio of ACE2 protein to GAPDH protein after different cannabis extracts were applied to EpiAirway tissues, in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
Fig. 7D is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and Actin protein in human 3D EpiAirwayFT tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts (as described in Fig. 6), in accordance with an embodiment of the present invention; asterisks show significant difference from DMSO treatment.
Fig. 7E is a graph showing the ratio of ACE2 protein to Actin protein measured from the image in Fig. 7D, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
Fig. 7F is an image showing the effects of different cannabis extracts of novel cannabis lines on ACE2 protein and GAPDH protein in human 3D EpiAirway tissue after induction of inflammation with TNF/IFN and after application of cannabis extracts (as described in Fig. 6), in accordance with an embodiment of the present invention.
Fig. 7G is a graph showing the ratio of ACE2 protein to GAPDH protein measured from the image in Fig. 7F, in accordance with an embodiment of the present invention; asterisks show significant difference from TNF/IFN treatment.
Figs. 8A-8F are a set of graphs showing that cannabinoids affect processes involved in a viral life cycle - Figs. 8A-8C show that different cannabinoids applied at different concentrations inhibit reverse transcription in vitro - namely Fig. 8A, CBD, Fig. 8B, THC, and Fig. 8C, CBN and Figs. 8D-8F show that different cannabinoids applied at different concentrations inhibit DNA replication- namely Fig. 8D, CBD, Fig. 8E, THC and Fig. 8F CBN, in accordance with some embodiments of the present invention.
Fig. 9 is a simplified pictorial illustration showing a schematic summary 900 of the main effects of cannabis extracts on SARS-COV-2, in accordance with an embodiment of the present invention.
Step 910 illustrates mechanism of entry of SARS-COV-2 virus through the recognition of ACE2 receptor.
Step 920 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to block the viral entry by downregulating the expression of ACE2 receptor.
Step 930 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to block the virus life cycle, including but not limited to blocking reverse transcription and replication.
Step 940 illustrates mechanism of cytokine storm development in response to SARS-COV-2 vims.
Step 950 illustrates the use of cannabis extract or single cannabinoids or single terpene molecules or their combination to decrease the expression of pro- inflammatory molecules and prevent the development of cytokine storm.
Fig. 10A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in HSIEC cells; asterisks indicated significant (p<0.01) difference. Fig. 10B shows the dose-dependent increase in relative expression of COX2 protein as measured by western blot 24h after treatment with TNFa/IFNy in HSIEC cells.
Fig. IOC shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in HSIEC.
Fig. 10D is a graph showing ImageJ measured (from Fig. IOC) time-dependent increase in relative expression of COX2 in response to 10 ng/ml of TNFa/IFNy in HSIEC.
Fig. 11A shows the dose-dependent increase in relative expression of COX2 as measured by qRT-PCR 24h after treatment with TNFa/IFNy in WI-38 human lung fibroblast cells; asterisks indicated significant (p<0.01) difference.
Fig. 1 IB shows the dose-dependent increase in relative expression of COX2 as measured by western blot 24h after treatment with TNFa/IFNy in WI-38 cells.
Fig. llC is a graph showing ImageJ-measured dose-dependent increase in relative expression of COX2 (from Fig. 11B) in response to TNFa/IFNy in WI-38 cells.
Fig. 11D shows the time-dependent increase in relative expression of COX2 (as measured by western blot) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
Fig. 11E is a graph showing ImageJ measured time-dependent increase in relative expression of COX2 (from Fig. 11D) in response to 10 ng/ml of TNFa/IFNy in WI-38 cells.
Fig. 12A is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 5mM CBD or 15 uF of 0.01 ug/uF of various cannabis extracts in HSIEC cells.
Fig. 12B is a graph showing ImageJ measured expression of COX2 relative to GAPDH (from Fig. 12A) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in HSIEC cells; asterisks show significant difference.
Fig. 12C is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH (from Fig. 12 A) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in HSIEC cells; asterisks show significant difference. Fig. 12D is a western blot image showing the expression of COX2, SOCS3 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 5mM CBD or 15 uL of 0.01 ug/uL of various cannabis extracts in WI-38 cells.
Fig. 12E is a graph showing ImageJ measured expression of COX2 relative to GAPDH (from Fig.l2D) in response to treatment with TNFa/IFNy alone, or with addition of CBD or various cannabis extracts in WI-38 cells; asterisks show significant difference.
Fig. 12F is a graph showing ImageJ measured expression of SOCS3 relative to GAPDH (from Fig.l2D) in response to treatment with TNFa/IFNy alone or with addition of CBD or various cannabis extracts in WI-38 cells; asterisks show significant difference.
Fig. 13A is a western blot image showing the expression of IL8 and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by 24h treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells.
Fig. 13B is a graph showing ImageJ measured expression of IL8 relative to GAPDH (from Fig. 13A) in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells; asterisks show significant difference.
Fig. 13C is a western blot image showing the expression of IL8 and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells.
Fig. 13D is a graph showing ImageJ measured expression of IL8 relative to GAPDH (from Fig. 13C) in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells; asterisks show significant difference.
Fig. 13E is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to 24h treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in WI-38 cells.
Fig. 13F is a graph showing ImageJ measured expression of IL-6 relative to GAPDH (from Fig. 13E) in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in WI-38 cells; asterisks show significant difference. Fig. 13G is a western blot image showing the expression of IL-6, pAktl/2/3, Aktl and GAPDH in response to treatment with 10 ng/ml TNFa/IFNy alone, or followed by treatment with extracts #1, #5, #7 and #169 in HSIEC cells.
Fig. 13H is a graph showing ImageJ measured expression of IL-6 relative to GAPDH (from Fig. 13G) in response to 24h treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with 15 uL of 0.01 ug/uL of extracts #1, #5, #7 and #169 in HSIEC cells; asterisks show significant difference.
Fig. 14 shows qRT-PCR of IL-6, IL-8 and COX-2 genes in WI-38 and HSIEC cells in response to treatment with 10 ng/ml TNFa/IFNy alone or followed by treatment with extracts #1, #5, #7 and #169; asterisks show significant difference.
Specifically, Fig. 14A shows IL-6 expression in WI-38, Fig. 14B - IL-8 in WI-38, Fig. 14C - COX-2 in WI-38, Fig. 14D - IL-6 in HSIEC, Fig.l4E - IL-8 in HSIEC, Fig. 14F - COX-2 in HSIEC.
Fig. 15 A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 10 mM CBD or 0.015 pg of various extracts.
Fig. 15B is a graph showing the ratio of ACE2 protein to GAPDH protein measured from Fig. 15A using ImageJ; asterisks show significant difference.
Fig. 16 shows qRT-PCR of IL-6 and IL-8 genes in WI-38 cells treated for 24- 96h with 10 ng/ml TNFa/IFNy alone or in combination with 0.015 pg of extract #1 or #5; asterisks show significant difference. Specifically, Fig. 16A shows IL-6 expression in response to extract #1, Fig. 16B - IL-8 in response to extract #1, Fig.l6C - IL-6 in response to ext #5, Fig. 16D - IL-8 in response to ext #5.
Fig. 17 A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of extracts #1 and #5.
Fig. 17B is a graph showing the ratio of ACE2 protein to GAPDH protein in WI-38 cells (calculated from Fig. 17A) in response to different concentrations of extracts #1 and #5; asterisks show significant difference.
Fig. 18A is a western blot image showing ACE2 and GAPDH protein levels in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
Fig. 18B is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig. 18A) in WI-38 cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
Fig. 18C is a western blot image showing ACE2 and GAPDH protein levels in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
Fig. 18D is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig. 18A) in BJ-5ta immortalized foreskin fibroblast cells in response to 24 h treatment with different concentrations of CBD, CBN and THC.
Fig. 19A is a western blot image showing ACE2, TMPRSS2 and GAPDH protein levels in BJ-5ta cells in response to 24h treatment with 0.015 pg/pl concentration of CBD or various extracts.
Fig. 19B is a graph showing the ratio of ACE2 protein to GAPDH protein (calculated from Fig.l9A) in BJ-5ta cells in response to 24h treatment with 0.015 pg/pl concentration of CBD or various extracts.
Fig. 19C is a graph showing the ratio of TMPRSS2 protein to GAPDH protein (calculated from Fig.l9A) in BJ-5ta cells in response to 24 h treatment with 0.015 pg/pl concentration of CBD or various extracts CBN and THC.
Fig. 20A shows graphs of the fold change in the expression of selected pro- inflammatory genes in EpiDermFT human 3D tissues in response to UVC followed by 24h treatment with 0.015 pg/pl concentration of extracts #4, #6, #8, #12, #13, #14 and #15.
Fig. 20B shows a graph of the log2 fold change in the expression of PTGS2 (COX2) gene in EpiDermFT human 3D tissues in response to UVC f treatment with extracts #4, #6, #8, #12, #13, #14 and #15.
Fig. 20C is a western blot image showing IL-6 and COX-2 protein levels in EpiDermFT human 3D tissues in response to UVC or UVC followed by 24h treatment with 0.015 pg/pl concentration of extracts #4, #6, #8, #12, #13, #14 and #15; PBS and DMSO are controls; UV-PBS and UV-DMSO are samples treated with UV with PBS or DMSO added to them.
Fig. 21 -Fig.32 show the set of experiments to compare the effect of CBD and terpenes on the expression of ACE2 and TMPRSS2 proteins in WI-38 and HSIEC cells. Cells were treated for 24h with 20 uM CBD alone, with 10-100 pg/ml of an isolated terpene or with combination of CBD and terpenes. Asterisks show significant difference, either between control and any individual treatment, or between CBD and CBD + terpene, or terpene and CBD + terpene.
Specifically, Fig. 21 A shows the western blot image of ACE2, TMPRSS2 and GAPDH protein levels, while Fig. 2 IB and C show the ImageJ calculated expression of ACE2 and TMPRSS2 relative to GAPDH in response to CBD and myrcene in WI- 38 cells. Fig. 22 shows the same in HSIEC cells.
Correspondingly, Fig. 23 and 24 show the same for caryophyllene in WI-38 and HSIEC cells.
Fig. 25 and 26 show the same for a-pinene in WI-38 and HSIEC cells.
Fig. 27 and 28 show the same for bisabolol in WI-38 and HSIEC cells.
Fig. 29 and 30 show the same for humulene in WI-38 and HSIEC cells.
Fig. 31 and 32 show the same for valencene in WI-38 and HSIEC cells.
Since previous experiments hinted that CBD may be less efficient than whole extracts or isolated terpenes in downregulation of ACE2/TMPRSS2 and pro- inflammatory proteins COX, IL-6 and IL-8, it was attempted to check individual components of the extracts.
Fig. 33-36 show the results of exposure of three different cell types, WI-38, HSIEC and Bj-5ta for 24h or 48h to 15 pg/ml of two best working extracts, #1 and #7. To compare the effect of individual components with the extract, cells were exposed to the same concentrations found in the extract of three main cannabinoids, CBD, THC and CBN or 4-6 major terpenes or to a combination of all cannabinoids and major terpenes, attempting to reconstitute the effect of a whole extract. Asterisks show significant difference from DMSO control.
Specifically, Fig. 33A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-bisabolol, a-humulene, a combination of all these components or to the extract #1 itself. Fig. 33B shows the same for COX-2, IL-6, IL-8 and GAPDH.
Fig. 33C and D show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 33A.
Fig. 34A shows the western blot in HSIEC cells exposed for 24h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #1 or its individual components.
Fig. 34B, C and D show ImageJ quantification of COX2, IL6 or IL8, respectively. Fig. 35A shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in WI-38 cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
Fig. 35B and C show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35A.
Fig. 35D shows the western blot image of ACE2, TMPRSS2 and GAPDH proteins in Bj-5ta cells exposed for 24h to CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol, a combination of all these components or to the extract #7 itself.
Fig. 35E and F show ImageJ quantification of ACE2 and TMPRSS2 relative to GAPDH from Fig. 35D.
Fig. 36A shows the western blot image of IL6, COX2 and GAPDH proteins in WI-38 cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
Fig. 36B and C show ImageJ quantification of IL6 and COX2 from Fig. 36A.
Fig. 36D shows the western blot image of IL8 and GAPDH proteins in HSIEC cells exposed for 48h to 10 ng/ml of TNF/IFN followed by another 24h exposure to extract #7 or its individual components - CBD, THC, CBN, b-myrcene, b- caryophyllene, a-pinene, a-bisabolol, valencene, geraniol or their combination.
Fig. 36E shows ImageJ quantification of IL8 from Fig. 36D.
RESULTS
Described herein is a method for decreasing the expression of ACE2 and decreasing the levels of cytokine storm and inflammation, but is not limited to the steps of: 1) preparation of new cannabis extracts, 2) exposing normal and disease oral, lung and intestinal models to novel extracts and 3) modulating the gene expression and protein levels to cause a reduction of a disease state, or prevent an increase in the disease state in the body tissues, such as oral cavity tissues, lung tissues and intestinal tissues.
The present invention provides extracts of twenty nine new C. sativa lines (#1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317) ( see Table 1). The present invention further combines use of extracts of these lines, as well as combination of single cannabinoids and terpenes. The aforementioned extracts, single cannabinoids and terpenes have been tested for anti-inflammatory properties and/or ACE2 inhibition properties using human cells lines and 3D EpiOral, Epilntestinal and EpiAirway tissue models. This is performed by detecting the expression of genes that are associated with molecular etiology and pathogenesis of COVID-19 and ARDS and cytokine storms. Table 1. Level of single and total cannabinoids in the extracts of selected C. sativa cultivars.
Figure imgf000045_0001
Figure imgf000046_0001
The effects of thirteen extracts of novel cannabis lines (#81, #90, #130, and #131, #1, #7, #9, #45, #115, #129, #151, #167, and #169) on inflammation provoked by TNFa/IFNy treatment in EpiOral tissues were analyzed. It was observed that extracts of 3 lines (#81, #130, and #45) significantly decreased the levels of expression of ACE2 gene in EpiOral tissues (Figs. 2A-2B).
Also, the anti-inflammatory effects of extracts #1, #7, #9, #45, #115, #129, #151, #167, and #169 were analysed (Fig. 2C), whereby the effect of all the pro- inflammatory gene changes was summed in a single bar for each condition (TNF vs DMSO, and vs each of the extracts #1, #7, #9, #45, #115, #129, #151, #167, and #169). This figure shows how TNFa/IFNy treatment produced a significant over expression pro-inflammatory signature that was completely abolished by extracts (Fig. 2C). Figure 2D,E further demonstrate, in an independent experiment, that extracts #7, #9, #115, #157, #167 and #169 inhibit the expression of ACE2 in EpiOral tissues.
The effects of extracts #1, #7, #9, #45, #115, #129, #130, #169, and #274 on Epilntestinal tissues were also analysed. Global gene expression analysis revealed that extract #45 decreased the levels of ACE2 gene expression in Epilntestinal tissues. Further analysis of the levels of ACE2 protein revealed that extract #45, as well as extracts # 1, #7, #129, and #169 also suppressed the ACE2 levels in Epilntetsinal tissues. (Figs. 4A-4B). These data were supported by western blot analysis; extracts #1, #7, #45, #129 and #169 decreased the level of ACE2 protein (Figs. 5A-5B). Along with ACE2, some extracts also reduced the levels of PARP-1 and iNOS, as well as inhibited pro-inflammatory gene expression signatures induced by TNF (Figs. 5C- 5E).
The effects of extracts #81, #5, #31, #49, #114, #166, #10, #169, #155, and #207 on the levels of ACE2 in EpiAirway tissues were next evaluated. It was found that extracts #10, #31, #114, as well as #5 and #207 decreased the levels of ACE2 expression in EpiAirway tissues (Figs. 7A-7C). Induction of inflammation with TNF/IFN in EpiAirway and EpiAirwayFT 3D tissues, followed by treatment with extracts further revealed that extracts #1, #5, #7, #10, #45, #81 #129, #317 and #169 substantially decreased the expression of ACE2 (Figs. 7D-G).
In summary, treatments of oral tissues with extracts of new cannabis lines (#81, #130) significantly affected ACE2 gene expression in the oral tissues. ACE2- inhibitory potential of these lines in oral tissues may be used to develop modalities to prevent SARS-CoV2 entry via oral and nasopharyngeal epithelial cells.
Treatment of lung epithelial tissues with extracts of new cannabis lines (#10, #31, #114, as well as #5 and #207) significantly reduced the levels of ACE2 protein in the EpiAirway tissues (Figs. 7A-7C). ACE2-inhibitory potential of these lines in lung epithelial tissues may be used to develop modalities to prevent SARS-CoV2 entry via airway cells as well as to treat COVID-19.
Treatment of intestinal epithelial tissues with extracts of new cannabis lines (#45, # 1, #7, #129, and #169) significantly reduced the levels of ACE2 protein in the Epilntestinal tissues (Fig. 5A), and affected the levels of PARP-1 (Fig. 5C), iNOS, (Fig. 5C), as well as inhibited pro -inflammatory gene induction by TNFa/IFNy (Fig. 5B). ACE2-inhibitory and anti-inflammatory potential of these lines in intestinal epithelial tissues may be used to develop modalities to prevent SARS-CoV2 entry via intestinal cells as well as to treat COVID-19.
In a more specific embodiment, there is provided a method for COVID-19 prevention and treatment via affecting the levels of SARS-CoV2 receptor ACE2 in the oral and nasopharyngeal, lung and intestinal epithelial tissues that includes the steps of exposing a patient to novel cannabis extracts, resulting in a reduction in the disease incidence and manifestation in the patient.
In some embodiments, extracts of new cannabis lines are applied to a human patient. In other embodiments, they may be applied to human oral tissue, artificial human oral tissue, or even animal oral tissue to modulate gene expression leading to a reduction, or at least prevent an increase in a disease state.
Among those genes down-regulated by new extracts are ACE2, and pro- inflammatory genes ABR, ACP5, ACVR1, ADA, ADORA1, ADORA2A, ADORA2B, AGER, AGTR2, AHCY, AKT1, ALOX5, APOA2, ASH1L, ASS1, ATM, AXL, B4GALT1, BACE2, BAP1, BCR, BDKRB1, BMP6, BMPR1B, BTK, C1QTNF12, C3, C5orf30, C6, CALCA, CALCRL, CCL11, CCL24, CCR4, CD28, CD40, CD96, CDK19, CELA1, CLOCK, CNR1, CNR2, CXCR2, CYP19A1, CYP26B1, CYSLTR1, DROSHA, DUOXA1, DUOXA2, DUSP10, ECM1, EDNRB, EGFR, EIF2AK1, ELANE, EPHA2, EPO, ESR1, ETS1, FABP4, FANCA, FANCD2, FCER1G, FOXF1, FOXP3, GAL, GAT A3, GPR17, GPX1, GPX4, HAMP, HFE, HGF, HIST1H2BA, HSPD1, ICAM1, IDOl, IDOl, IGFBP4, IL10, IL12B, IL13, IL15, IL17B, IL17F, IL17RA, IL17RB, IL17RC, ILIA, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2, IL20RB, IL22RA2, IL25, IL2RA, IL31RA, IL33, IL5, IL5RA, ITGA2, ITGB2, ITGB6, JAK2, JAM3, JUN, KDM6B, KRT1, LBP, LDLR, LIPA, LRRK2, LTA, LYN, MAP2K3, MAS1, MCPH1, NAMPT, NFE2L2, NFKB1, NFKBIZ, NLRP6, NLRX1, NOTCH1, NPFF, NPPA, NPY5R, NT5E, NUPR1, OGGI, OPRM1, P2RX1, P2RX7, PBK, PGLYRP1, PGLYRP2, PIK3CG, PLAA, PLP1, POLB, PPARG, PRCP, PSMB4, PTAFR, PTGES, PTGS2, PYCARD, RASGRP1, RBPJ, RHBDD3, RICTOR, S100A8, S1PR3, SCN9A, SDC1, SEH1L, SELP, SERPINC1, SERPINF1, SGMS1, SLC7A2, SMAD1, SMAD3, SMO, SOCS3, SOCS5, SPHK1, SPP1, STAT3, STAT5B, STK39, SUCNR1, TAC1, TBC1D23, TFR2, TGM2, TIMP1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TNF, TNFAIP3, TNFRSF1B, TRPV1,TRPV4, TSPAN2, TUSC2, UCN, UGT1A1, UNC13D, VCAM1, WFDC1, ZYX.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or protein sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, 80%, 90%, or even at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C to about 20° C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. In certain embodiments, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Thus, the invention also provides nucleic acid molecules and peptides that are substantially identical to the nucleic acid molecules and peptides presented herein.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.
In addition, new extracts may modulate genes and proteins sharing a sequence identity or substantial sequence identity to those genes and proteins listed herein.
In addition, cannabinoids and new extracts may affect key processes of the viral life cycle - reverse transcription, replication, and other ones (Figs. 8A-8F).
Further analysis demonstrates that many cannabis extracts, including but not limited to #1, #5, #7, #10, #169 and #317, are more potent than CBD alone in downregulation of COX2 and upregulation of SOCS3 in two different models, intestinal epithelial cells (HSIEC) and lung fibroblast cells (WI-38) (Fig. 12).
Top performing extracts, such as #1, #5, #7 and #169 were further found to substantially downregulate the expression of IL-6, IL-8 and COX-2 on both mRNA and protein levels in both models, HSIEC and WI-38 (Fig. 13-14).
Extracts #317, #129 and #98 also more substantially inhibited ACE2 expression as compared to CBD alone (Fig. 15).
Extracts #1 and #5 demonstrated time-dependent decrease in TNF/IFN- induced expression of IL-6 and IL-8 (Fig. 16). Dose-dependent decrease in ACE2 expression in response to extracts #1 and #5 was also observed (Fig. 17).
Exposure to CBD, THC or CBN demonstrated that dose-dependent decrease in ACE2 expression is only observed for certain cannabinoids and only in certain cell type (Fig. 18).
Western blot analysis showed the extracts #1, #5, #7, #10 and #317 are substantially more potent in the decrease of ACE2 expression as compared to CBD and extracts #5, #7, #10, #129, #132, #169 and #98 are more potent in downregulation of TMPRSS2 as compared to CBD (Fig. 19). TMPRSS2 is a serine protease essential for activation of recognition of SARS-COV-2, namely by priming the viral spike protein to allow viral entry into the target cell.
Analysis of the KEGG pathway regulation demonstrated significant downregulation of key pro-inflammatory genes by extracts #4, #6, #8, #13 and #14, while showing upregulation by extract #12 and lack of effect by extract #15 (Fig. 20). These data were substantiated by western blot analysis of COX2 and IL-6 (Fig. 20D,E).
Top extracts (#1, #5 and #7) were common in the composition of cannabinoids (CBD-dominant) and terpenes and were enriched in caryophyllene, myrcene, pinene, bisabolol, humulene and valencene. Therefore, the contribution of each terpene on ACE2 and TMPRSS2 expression was tested (Fig. 23A-C). Caryophyllene demonstrated comparable to CBD effect on downregulation of ACE2 and TMPRSS2, with higher concentrations being more efficient. It was further found that combination of CBD with caryophyllene results in potentiation of the effect of CBD on ACE2 and TMPRSS2 in WI-38 cells (Fig. 23A-C) and HSIEC cells (Fig. 24A-C). Effect of a-pinene was less obvious, although synergistic effect of CBD and 10 pg/rnl pinene on ACE2 and TMPRSS2 expression was evident in WI-38 cells (Fig. 25A-C) and on TMPRSS2 in HSIEC cells (Fig. 26A-C). Bisabolol also downregulated ACE2 and TMPRSS2 expression in WI-38 cells, more prominent at lower concentration; no synergistic effect was observed (Fig. 27A-C). Bisbolol alone did not decrease the expression of ACE2 and TMPRSS2 expression in HSIEC cells, but synergistic effect on ACE2 expression was apparent (Fig. 28A-C). Humulene decreased the expression of ACE2 in WI-38 cells, but without synergistic effect; no effect on TMPRSS2 was observed (Fig. 29A-C). Humulene alone did not have any effect on ACE2 expression in HSIEC cells, but showed synergistic effect with CBD (Fig. 30A-C). Valencene decreased the expression of ACE2 in WI-38 cells, but without synergistic effect; no effect of valencene alone on TMPRSS2 was observed, but synergistic effect at low concentration was evident (Fig. 31A-C). In HSIEC cells, valencene decreased expression of ACE2 with synergistic effect with CBD (Fig. 32A-C).
While analyzing the effect of whole extracts and comparing it to the effect of CBD alone and seeing the potentiation effect some of the terpenes had on the expression of ACE2 and pro-inflammatory genes, it was decided to analyze the effect of single components present in the extracts #1 and #7. WI-38, HSIEC and Bj-5ta cells were used either directly exposing them to the extracts, their individual components or their combination or first treating cells with TNF/IFN and then exposing them to the extracts and their components. It was found that combined components were as effective as extract #1 in downregulation of ACE2, while CBD and myrcene alone were also effective (Fig. 33). In HSIEC cells, combined components were effective in reducing the expression of IF6 and IF8, although not as effective as extract #1; individual components were also effective, with humulene being the most effective (Fig. 34). Effect of combined components from extract #1 was more effective in reducing COX2 expression than extract #1 itself in HSIEC cells, and b-caryophyllene was also very effective (Fig. 34B).
Analysis of the components of extract #7 in Bj-5ta cells showed that several individual components, including b-caryophyllene, valencene and geraniol were more effective than extract #7 (Fig. 35E).
The effect of extract #7 on IL-6 in WI-38 revealed that it was the most effective, with combined components having statistically similar effect; individual components valencene, geraniol and bisabolol had very similar effect (Fig. 36B). The effect on COX2 was most prominent from combined components, followed by extract #7 and myrcene (Fig. 36C). Finally, similar analysis in HSIEC cells showed that extract #7 was the most efficient in downregulating IL8, while CBD, myrcene, pinene and geraniol were also very effective (Fig. 36E).
The presented Examples (Figures 2A-2C, 4A-4B, 5A-5E, 7A-7C, and 8A-8F, Fig. 12-36) are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.
MATERIALS AND METHODS PLANT GROWTH
All cannabis plants were grown in the licensed facility at the University of Lethbridge (license number LIC-62AHHG0R77-2019). C. sativa cultivars #1, #4, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #31, #45, #49, #81, #90, #98, #114, #115, #129, #130, #131, #132, #155, #157, #166, #167, #169, #207, #274, #317 were used for the experiments. Four plants per cultivar were grown at 22°C 18 h light 6 h dark for 4 weeks and then transferred to the chambers with 12 h light/ 12 h dark regime to promote flowering. Plants were grown to maturity and flowers were harvested and dried. Flower samples from four plants per cultivar were combined and used for extraction. PLANT CRUDE EXTRACT PREPARATION:
Solvent used: Ethyl acetate ACS grade from Fisher cat# E145-4 (99.9% pure)
Extract Preparation: 3 g of the powdered plant tissue were weighed using an analytical balance Plant material was placed inside a 250 mL Erlenmeyer flask (clean). 100 mL of Ethyl Acetate was poured into the flask containing the plant material. The flasks were then wrapped with tin foil and shaken continuously (120 rpm) in an incubator @ 21°C overnight and in the dark.
After overnight solvent extraction the extracts were filtered through cotton into a lOOmL round bottom flask. The extracts were concentrated to around 2-3mL using a rotary vacuum evaporator. The extracts were then transferred to a tared 3 dram vial (cat# 60975L Kimble obtained from Fisher Scientific). The leftover solvent was evaporated to dryness in an oven overnight at 50°C to eliminate the solvent completely. Mass of each extract was recoded.
BIOASSAY PREPARATION:
Preparation of 60 mg/mL stocks.
The stocks were prepared weighing a 3-6 mg of crude extract into a micro centrifuge tube. The crude extract was dissolved in DMSO (Dimethyl sulfoxide anhydrous from Life technologies cat # D 12345) to reach 60 mg/mL final concentration and stored at -20°C.
Preparation of Crude Extracts for Bioassay.
Appropriate cell culture media (in our experiments RPMI 1640 medium supplemented with 10% FBS or EMEM medium supplemented with 10% FBS) were used to dilute the 60mg/mL stock. The stocks are allowed to thaw then added to the cell culture media, mixed thoroughly to ensure they are in solution and filtered through a 0.22 pm syringe filter. These filtrates were ready to be applied to cells and tested.
Preparation of terpene stocks.
Terpenes were purchased from True Terpenes (www.trueterpenes.com) and were diluted to the assay concentrations using DMSO or media.
Preparation of CBD, THC or CBN stock.
CBD, THC and CBN were purchased from MilliporeSigma (Sigma- Aldrich) and were diluted to the assay concentrations using DMSO or media.
Analysis of cannabinoids Agilent Technologies 1200 Series HPLC system equipped with a G1315C DAD, G1316B column compartment, G1367D autosampler, and G1312B binary pump was used to analyse the acidic and neutral forms of phytocannabinoids. The separation was performed on a Phenomenex Kinetex EVO C18 column (5 pm, 100 x 2.1 mm id) with a Phenomenex SecurityGuard ULTRA guard column. Instrument control, data acquisition, and integration were done with ChemStation LC 3D Rev B.04.02 software (Agilent Technologies). A 2 pL injection volume was used for all calibration standards (THC, CBD, THC-A, CBD-A, CBG, CBG-A, all Sigma- Aldrich) and sample analysis. The compound peaks were detected for 230 nm and 280 nm. Mobile phases consisted of 50 mM ammonium formate (pH 5.19) (Sigma - Aldrich) in HPLC grade water (Lisher Chemical) on the A side and 100% methanol (Lisher Chemical) on the B side, with a flow rate 0.3 ml/min. Two samples per cultivar were analyzed, with two technical repeats per each sample.
Analysis of terpenes
Terpene analysis was performed on dry flowers using a 86 IOC GC coupled with a flame ionization detector (LID) from SRI Instruments at Canvas Labs (Vancouver, BC, Canada). Two samples per cultivar were analyzed.
MODELS:
EpiAirway (AIR-100) tissues: Mattek’s Epi Airway tissue model is a human 3D mucociliary tissue model that consists of normal, human-derived tracheal/bronchial epithelial cells, is cultured at the air-liquid interface and fully recapitulates the barrier, mucociliary responses, infection, toxicity responses of human airway tissues in vivo.
EpiAirwayFT (AFT-100) tissues: EpiAirway FT is a ready-to-use, 3D mucociliary tissue model consisting of normal, human-derived tracheal/bronchial epithelial cells and normal human stromal fibroblasts (Mattek Life Sciences, MA). H&E-stained section of EpiAirwayFT tissue (courtesy of Mattek) exhibits a pseudostratified epithelium with ciliated cells and an extracellular matrix containing fibroblasts on a microporous membrane.
EpiOral (ORL-20) tissues: MatTek’s EpiOral tissues consist of normal, human-derived oral epithelial cells. The cells have been cultured to form multilayered, highly differentiated models of the human buccal (EpiOral) phenotypes. The tissues are cultured on specially prepared cell culture inserts using serum free medium and attain levels of differentiation on the cutting edge of in vitro cell culture technology. The EpiOral tissue models exhibit in vivo-like morphological and growth characteristics which are uniform and highly reproducible.
Epilntestinal (SMI-100) tissues: Epilntestinal tissues are 3D highly differentiated tissue models produced from normal, human cell-derived small intestine epithelial and endothelial cells and fibroblasts. Grown at the air-liquid interface, Epilntestinal tissue models are similar to in vivo human epithelial tissues and exhibit columnar shaped basal cells and Kerckring folds, as well as brush borders, functional tight junctions and mucous secreting granules.
Cell cultures: Human normal foreskin fibroblasts (BJ-5ta), purchased from American Type Culture Collection (ATCC, Manassas, USA), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Human primary small intestinal epithelial cells (HSIEC), purchased from Cell Biologies, were cultured in Epithelial Cell Medium /w Kit supplemented with 1% P/S. Human lung fibroblasts (WI-38), purchased from ATCC, were cultured in EMEM supplemented with 10% FBS and 1% P/S. All cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2.
INDUCTION OF INFLAMMATION
To induce inflammation, tissues were either exposed to UVC (Figure 20) or TNF/IFN (all other experiments).
UVC was applied for 2 min, with tissues receiving 7000 erg. Distance from the light source was set to 10 cm. Upon exposure, tissues were treated with extracts. Specifically, right after the UVC treatment, the cannabis extracts (15 pi per sample) or vehicle (DMSO) were dissolved in media and applied to the media surrounding the tissues (n=3 for each condition). Control samples (PBS and DMSO) were sham treated - carried to the UVC source etc. but no UVC was given. The following experimental groups were set up: “UNT” - untreated tissues; “PBS” - 15 mΐ of 30% glycerol in PBS was applied to the tissues and no exposure was done; “DMSO” - 15 mΐ of DMSO (0.017% in 30% glycerolPBS) was applied to the tissues and no exposure was done; “UV-PBS” - tissues were exposed to UV and 15 mΐ of 30% glycerol-PBS was applied to them; “UV-DMSO” - tissues were exposed to UV and 15 mΐ of DMSO (0.017% in 30% glycerol-PBS) was applied to them; “#4” through “#15” - tissues were exposed to UV and 15 mΐ of extracts in DMSO was applied to them. Tissues were incubated with extracts for 24 h and flash frozen for RNA and protein analysis.
To induce inflammation using TNF/IFN, tissues or cells were equilibrated for 24 h, after equilibration tissues were treated with 10 ng/mL TNFa /IFNy alone or in combination with 5-30 mM CBD, 5-30 mM CBN, 5-30 mM THC, or different concentrations of extracts for various time. In yet another experiment, cells were treated either with CBD alone, terpenes alone or in combination of CBD with terpenes.
GENE EXPRESSION PROFILING:
Three tissues per group were used for the analysis of gene expression profiles. RNA was extracted from tissues using TRIzol® Reagent (Invitrogen, Carlsbad, CA), further purified using an RNAesy kit (Qiagen), and quantified using Nanodrop2000c (ThermoScientific). Afterwards, RNA integrity and concentration were established using 2100 BioAnalyzer (Agilent). Sequencing libraries were prepared using Illumina’s TruSeq RNA library preparation kits, and global gene expression profiles were determined using the Next 500 Illumina deep-sequencing platform at the University of Lethbridge Facility. Statistical comparisons between the control and treatment groups were performed using the DESeq Bioconductor package (version 1.8.3) and the baySeq Bioconductor package (version 1.10.0). Clustering of the samples was assessed with multidimensional scaling (MDS) plots built using the plotMDS function from the edgeR Bioconductor package. Features with a false discovery rate (FDR) < 0.1 (10% false positive rate) were considered differentially expressed between conditions.
The functional annotations of differentially expressed genes were performed using David, GO (Gene Ontology) Elite, and GO-TermFinder. Pathways were visualized using Pathview/KEGG and DAVID bioinformatics platforms DAVID Bioinformatics Resources 6.7 KEGG Pathway platforms. mRNA qRT-PCR
HSIEC and WI-38 cells grown to 70% confluency were exposed to either 10 ng/ml TNFa/IFNy or in combination with extracts #1 or #5 or #7 or #169 for 48 h, 1% DMSO served as a control; HSIEC and WI-38 cells grown to 80% confluency were treated with either 10 ng/ml TNFa/IFNy or in combination with extracts #1 or #5 for an indicated time-course, 1% DMSO served as a control; HSIEC cells grown to 70% confluency were exposed to either 10 ng/ml TNFa/IFNy or in combination with a range of doses of extracts #1 or #5 for 48 h, 1% DMSO served as a control; at the indicated time-point after treatment, total RNA was isolated using TRIzol RNA isolation reagent (ThermoFisher Scientific) and subjected to qRT-PCR analysis with iScript Select cDNA Synthesis kit (Bio-Rad) and SsoFast Evagreen SYBR Supermix (Bio-Rad) using primers specific for IL6, IL8, and COX2 according to the manufacturer’s instructions. Real-time quantitative RT-PCR analysis was carried out with CFX96 Real-Time System (Bio-Rad). Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) was used as the internal control to standardize the expression. All experiments for real-time RT-PCR were performed in triplicate and data was analysed using the comparative Ct method. Results are shown as fold induction of mRNA.
WESTERN BLOTTING
Tissues were harvested and solubilized in laboratory-prepared 1% sodium dodecyl sulfate (SDS) lysis buffer (BioUltraPure, Bioshop) by sonication using a Braunsonic model 1510 sonicator (B. Braun Germany) operating at 80% sonication intensity. Lysates were centrifuged at 15,000 rpm for 10 min and the supernatant was decanted for use. Protein concentrations were determined using the Bradford protein assay with bovine serum albumin as the standard protein using the Nanodrop 2000c spectrophotometer (v. 1.5). Total proteins were separated by electrophoresis on 10- 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). The protein was then electro-transferred onto activated polyvinyl difluoride (PVDF) membranes (Amersham Hybond P 0.45, GE Healthcare). The membranes were incubated for one hour in a blocking solution (5% dry skimmed milk in PBS, 0.5% Tween 20) at room temperature, and incubated with specific primary antibodies at 4°C overnight with 1:200 to 1:1,000 dilution of polyclonal/monoclonal antibodies against cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2) (all from Abeam, Cambridge, UK) or GPR18 (from LSBio, Seattle, USA) or CDK2, cyclin El, E2F1, ERK1/2, pERKl/2, PFKFB3 (all from Cell Signaling, Danvers, USA) or AKT1, p53, pAKTl/2/3 (all from Santa Cruz Biotechnology, Dallas, USA), IL-6, IL-8, COX-2, ACE2, TMPRSS2 (Abeam). Blots of the primary antibodies tested were developed with peroxidase- labelled secondary antibodies specific to the primaries. The membranes were washed (5 times of 5 min of washing with PBS-Tween) before and after adding secondary antibodies. The protein bands of interest were detected using an enhanced chemiluminescence (ECL) system by incubating for 5 min in ECL detection reagents (GE Healthcare, Amersham Biosciences) and visualized using the FluorChem HD2 ALC detection system (software v.3.2.2.0805, Cell Biosciences).
DOSAGE FORMS
The compositions of the present invention may be provided in any suitable dosage form. According to some embodiments, the dosage form is an oral dosage form. Oral dosage forms comprise liquids (solutions, suspensions, and emulsions), semi-solids (pastes), and solids (tablets, capsules, powders, granules, premixes, and medicated blocks).
Some examples of oral dosage forms in the art include, W090/04391, which discloses an oral dosage form of omega-3 polyunsaturated acids to overcome the problems of diseases. It is known to supply said acids in soft gelatine capsule shells.
EP 2 240 581 B1 discloses a gelatine capsule for pharmaceutical use with a controlled release of active ingredients and a process for the preparation of said gelatine capsules. During said process xylose is added to the liquid gelatine from which afterwards gelatine capsules are formed. Gelatine capsules manufactured according to the process provide retarded release of active ingredients.
US Patent No. 7,264,824 discloses and oral dosage form for food and food supplements, as well as dietetics comprising polyunsaturated acids in a xylose- hardened gelatine capsule with a retarded release time.
According to some embodiments of the present invention, the compositions described herein may be in a suspension or emulsion.
A suspension is a coarse dispersion of insoluble drug particles, generally with a diameter exceeding 1 pm, in a liquid (usually aqueous) medium. Suspensions are useful for administering insoluble or poorly soluble drugs/components or in situations when the presence of a finely divided form of the material in the GI tract is required. The taste of most drugs is less noticeable in suspension than in solution, due to the drug being less soluble in suspension. Particle size is an important determinant of the dissolution rate and bioavailability of drugs in suspension. In addition to the excipients described above for solutions, suspensions include surfactants and thickening agents. Surfactants wet the solid particles, thereby ensuring the particles disperse readily throughout the liquid. Thickening agents reduce the rate at which particles settle to the bottom of the container. Some settling is acceptable, provided the sediment can be readily dispersed when the container is shaken. Because hard masses of sediment do not satisfy this criterion, caking of suspensions is not acceptable.
An emulsion is a system consisting of 2 immiscible liquid phases, one of which is dispersed throughout the other in the form of fine droplets; droplet diameter generally ranges from 0.1-100 pm. The 2 phases of an emulsion are known as the dispersed phase and the continuous phase. Emulsions are inherently unstable and are stabilized through the use of an emulsifying agent, which prevents coalescence of the dispersed droplets. Creaming, as occurs with milk, also occurs with pharmaceutical emulsions. However, it is not a serious problem because a uniform dispersion returns upon shaking. Creaming is, nonetheless, undesirable because it is associated with an increased likelihood of the droplets coalescing and the emulsion breaking. Other additives include buffers, antioxidants, and preservatives. Emulsions for oral administration are usually oil (the active ingredient) in water, and facilitate the administration of oily substances such as castor oil or liquid paraffin in a more palatable form.
A paste is a 2-component semi-solid in which drug is dispersed as a powder in an aqueous or fatty base. The particle size of the active ingredient in pastes can be as large as 100 pm. The vehicle containing the drug may be water; a polyhydroxy liquid such as glycerin, propylene glycol, or polyethylene glycol; a vegetable oil; or a mineral oil. Other formulation excipients include thickening agents, co solvents, adsorbents, humectants, and preservatives. The thickening agent may be a naturally occurring material such as acacia or tragacanth, or a synthetic or chemically modified derivative such as xanthum gum or hydroxypropylmethyl cellulose. The degree of cohesiveness, plasticity, and syringeability of pastes is attributed to the thickening agent. It may be necessary to include a cosolvent to increase the solubility of the drug. Syneresis of pastes is a form of instability in which the solid and liquid components of the formulation separate over time; it is prevented by including an adsorbent such as microcrystalline cellulose. A humectant (eg, glycerin or propylene glycol) is used to prevent the paste that collects at the nozzle of the dispenser from forming a hard crust. Microbial growth in the formulation is inhibited using a preservative. It is critical that pastes have a pleasant taste or are tasteless.
A tablet consists of one or more active ingredients and numerous excipients and may be a conventional tablet that is swallowed whole, a chewable tablet, or a modified-release tablet (more commonly referred to as a modified-release bolus due to its large unit size). Conventional and chewable tablets are used to administer drugs to dogs and cats, whereas modified-release boluses are administered to cattle, sheep, and goats. The physical and chemical stability of tablets is generally better than that of liquid dosage forms. The main disadvantages of tablets are the bioavailability of poorly water-soluble drugs or poorly absorbed drugs, and the local irritation of the GI mucosa that some drugs may cause.
A capsule is an oral dosage form usually made from gelatin and filled with an active ingredient and excipients. Two common capsule types are available: hard gelatin capsules for solid-fill formulations, and soft gelatin capsules for liquid-fill or semi-solid-fill formulations. Soft gelatin capsules are suitable for formulating poorly water-soluble drugs because they afford good drug release and absorption by the GI tract. Gelatin capsules are frequently more expensive than tablets but have some advantages. For example, particle size is rarely altered during capsule manufacture, and capsules mask the taste and odor of the active ingredient and protect photolabile ingredients.
A powder is a formulation in which a drug powder is mixed with other powdered excipients to produce a final product for oral administration. Powders have better chemical stability than liquids and dissolve faster than tablets or capsules because disintegration is not an issue. This translates into faster absorption for those drugs characterized by dissolution rate-limited absorption. Unpleasant tastes can be more pronounced with powders than with other dosage forms and can be a particular concern with in-feed powders, in which it contributes to variable ingestion of the dose. Moreover, sick animals often eat less and are therefore not amenable to treatment with in-feed powder formulations. Drug powders are principally used prophylactically in feed, or formulated as a soluble powder for addition to drinking water or milk replacer. Powders have also been formulated with emulsifying agents to facilitate their administration as liquid drenches.
A granule is a dosage form consisting of powder particles that have been aggregated to form a larger mass, usually 2-4 mm in diameter. Granulation overcomes segregation of the different particle sizes during storage and/or dose administration, the latter being a potential source of inaccurate dosing. Granules and powders generally behave similarly; however, granules must deaggregate prior to dissolution and absorption.
A premix is a solid dosage form in which an active ingredient, such as a coccidiostat, production enhancer, or nutritional supplement, is formulated with excipients. Premix products are mixed homogeneously with feed at rates (when expressed on an active ingredient basis) that range from a few milligrams to -200 g/ton of food/beverage The density, particle size, and geometry of the premix particles should match as closely as possible those of the feed in which the premix will be incorporated to facilitate uniform mixing. Issues such as instability, electrostatic charge, and hygroscopicity must also be addressed. The excipients present in premix formulations include carriers, liquid binders, diluents, anti-caking agents, and anti dust agents. Carriers, such as wheat middlings, soybean mill run, and rice hulls, bind active ingredients to their surfaces and are important in attaining uniform mixing of the active ingredient. A liquid binding agent, such as a vegetable oil, should be included in the formulation whenever a carrier is used. Diluents increase the bulk of premix formulations, but unlike carriers, do not bind the active ingredients. Examples of diluents include ground limestone, dicalcium phosphate, dextrose, and kaolin. Caking in a premix formulation may be caused by hygroscopic ingredients and is addressed by adding small amounts of anti-caking agents such as calcium silicate, silicon dioxide, and hydrophobic starch. The dust associated with powdered premix formulations can have serious implications for both operator safety and economic losses, and is reduced by including a vegetable oil or light mineral oil in the formulation. An alternate approach to overcoming dust is to granulate the premix formulation.
A medicated block is a compressed feed material that contains an active ingredient, such as a drug, anthelmintic, surfactant (for bloat prevention), or a nutritional supplement, and is commonly packaged in a cardboard box. Ruminants typically have free access to the medicated block over several days, and variable consumption may be problematic. This concern is addressed by ensuring the active ingredient is nontoxic, stable, palatable, and preferably of low solubility. In addition, excipients in the formulation modulate consumption by altering the palatability and/or the hardness of the medicated block. For example, molasses increases palatability and sodium chloride decreases it. Additionally, the incorporation of a binder such as lignin sulfonate in blocks manufactured by compression or magnesium oxide in blocks manufactured by chemical reaction, increases hardness. The hygroscopic nature of molasses in a formulation may also impact the hardness of medicated blocks and is addressed by using appropriate packaging.
In another embodiment, the composition of the present invention is in a chewable oral dosage form. In another embodiment, the chewable oral dosage form is a chewable tablet. In another embodiment, the chewable tablet of the invention is taken slowly by chewing or sucking in the mouth. In another embodiment, the chewable tablet of the invention enables the dried cannabis extracts contained therein to be orally administered without drinking.
According to some embodiments of the present invention, the composition may comprise any suitable flavor or combination of flavors.
The composition may further comprise other additives, coloring, emulsifiers. The flavors and additives may be of a natural, semi-synthetic, synthetic source or combinations thereof.
In another embodiment of the present invention, the composition further comprises fructose, sorbitol, microcrystalline cellulose, magnesium stearate, or any combination thereof. In another embodiment, the composition further comprises chamomile. In another embodiment, the composition further comprises ginger. In another embodiment, the composition further comprises peppermint. In another embodiment, the composition further comprises anise. In another embodiment, the composition further comprises fennel. In another embodiment, the composition further comprises thyme. In another embodiment, the composition further comprises Arsenicum album. In another embodiment, the composition further comprises Carbo vegetabilis. In another embodiment, the composition further comprises Ignatia, homeopathic ipecac. In another embodiment, the composition further comprises Nux vomica. In another embodiment, the composition further comprises Zingiber officinale.
In another embodiment, the composition of the present invention is in the form of a chewing gum product. In another embodiment, chewing gum compositions contemplated by the present invention comprise all types of sugar and sugarless chewing gums and chewing gum formulations known to those skilled in the art, including regular and bubble gum types. In another embodiment, chewing gum compositions of the invention comprise a chewing gum base, a modifier, a bulking agent or sweetener, and one or more other additives such as, flavoring agents, colorants and antioxidants. In another embodiment, the modifying agents are used to soften, plasticize and/or compatibilize one or more of the components of the gum base and/or of the formulation as a whole.
In another embodiment, the present invention provides a soft, chewable dosage form which is pliable and chewy, yet dissolves quickly in the mouth, has a long shelf life, contains little moisture which improves stability and decreases the tendency for the dosage form to dry out, does not require cooking or heating as part of the manufacturing process. In another embodiment, the dosage form is used as a matrix for dried cannabis extracts.
In another embodiment, the chewable tablet of the invention comprises a metal salt such as calcium, magnesium, aluminum salt, or any mixture thereof. In another embodiment, the chewable tablet of the invention comprises hydroxyalkyl cellulose. In another embodiment, the chewable tablet of the invention comprises low viscosity hydroxyalkyl cellulose. In another embodiment, the chewable tablet of the invention comprises high viscosity hydroxyalkyl cellulose.
In another embodiment, the chewable tablet of the invention comprises various additives. In another embodiment, the chewable tablet of the invention comprises sweeteners. In another embodiment, the chewable tablet of the invention comprises acidic ingredients. In another embodiment, the chewable tablet of the invention comprises taste correctives. In another embodiment, the chewable tablet of the invention comprises polymeric compounds. In another embodiment, the chewable tablet of the invention comprises essential oils.
In another embodiment, the chewable tablet of the invention is a soft tablet. In another embodiment, the chewable tablet of the invention is made in a state of soft candy. In another embodiment, the chewable tablet of the invention is made in a state of jelly.
In another embodiment, the chewable tablet of the invention comprises a core comprising the vitamins of the invention. In another embodiment, the chewable tablet of the invention comprises an outer layer wrapping the core which is made up of chewable base such as a gum, a soft candy or a caramel.
In another embodiment, the compositions of the present invention may be provided in any suitable food of a solid, semi- solid or liquid form.
The preparation of pharmaceutical compositions that contain a dried cannabis extract, for example by mixing, granulating, or tablet-forming processes, is well understood in the art. The dried cannabis extracts are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active ingredients of compositions of the present invention are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.
In another embodiment, additional methods of administering the dried cannabis extracts, or compound(s) isolated therefrom, of the invention comprise injectable dosage forms. In another embodiment, the injectable is administered intraperitoneally. In another embodiment, the injectable is administered intramuscularly. In another embodiment, the injectable is administered intra-dermally. In another embodiment, the injectable is administered intravenously. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra arterial administration. In another embodiment, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra muscular administration. In another embodiment, additional methods of administering the dried cannabis extracts of the invention comprise dispersions, suspensions or emulsions. In another embodiment, the dispersion, suspension or emulsion is administered orally. In another embodiment, the solution is administered by infusion. In another embodiment, the solution is a solution for inhalation. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the pharmaceutical composition is administered as a suppository, for example a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides for controlled release of active compound agent over a period of time. Each possibility represents a separate embodiment of the present invention.
In other embodiments, pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs. Each possibility represents a separate embodiment of the present invention.
In another embodiment, parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs. Each possibility represents a separate embodiment of the present invention. In another embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the active compounds are released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate -release composition, i.e. a composition in which all the active compound is released immediately after administration. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the pharmaceutical composition is delivered in a controlled release system. In another embodiment, the agents are administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In another embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials are used; e.g. in microspheres in or an implant. In yet another embodiment, a controlled release system is placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990). Each possibility represents a separate embodiment of the present invention.
The compositions also include, in another embodiment, incorporation of the active materials into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Each possibility represents a separate embodiment of the present invention.
Also included in the present invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Each possibility represents a separate embodiment of the present invention.
Also comprehended by the invention are compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. The modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et ah, 1981; Newmark et ah, 1982; and Katre et ah, 1987). Such modifications also increase, in another embodiment, the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound. Each possibility represents a separate embodiment of the present invention.
The compositions of the present invention may comprise one or more additional components may further include an additional component selected from the group consisting of an anti-static agent, a buffering agent, a bulking agent, a chelating agent, a colorant, a diluent, a dye, an emollient, a fragrance, an occlusive agent, a pH- adjusting agent, a preservative, and a vitamin.
The compositions of the present invention may comprise one or more additional active agents, selected from the group consisting of active herbal extracts, analgesics, anti-allergic agents, anti-aging agents, anti-bacterials, antibiotic agents, anticancer agents, antidandruff agents, antidepressants, anti-dermatitis agents, anti- edemics, antihistamines, anti-helminths, anti-hyperkeratolyte agents, anti inflammatory agents, anti-irritants, anti-microbials, anti-mycotic s, anti-proliferative agents, antioxidants, anti-wrinkle agents, anti-pruritic s, antiseptic agents, antiviral agents, anti-yeast agents, astringents, topical cardiovascular agents, chemotherapeutic agents, corticosteroids, dicarboxylic acids, disinfectants, fungicides, hair growth regulators, hormones, hydroxy acids, immunosuppressants, immunoregulating agents, keratolytic agents, lactams, metals, metal oxides, mitocides, neuropeptides, non steroidal anti-inflammatory agents, oxidizing agents, photodynamic therapy agents, retinoids, sanatives, scabicides, self-tanning agents, skin whitening agents, vasoconstrictors, vasodilators, vitamins, vitamin D derivatives and wound healing agents. According to some embodiments, the composition may comprise one or more anti-oxidants/radical scavengers. The anti-oxidant/radical scavenger may be selected from butylated hydroxy benzoic acids and their salts, coenzyme Q10, coenzyme A, gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N,N- diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid and its salts, lycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts, grape skin/seed extracts, melanin, and rosemary extracts.
In one embodiment, the term “treating” refers to curing a disease. In another embodiment, “treating” refers to preventing a disease. In another embodiment, “treating” refers to reducing the incidence of a disease. In another embodiment,
“treating” refers to ameliorating symptoms of a disease. In another embodiment,
“treating” refers to inducing remission. In another embodiment, “treating” refers to slowing the progression of a disease.
The references cited herein teach many principles that are applicable to the present invention. Therefore, the full contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background.
It is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore described without departing from its scope, defined in and by the appended claims. REFERENCES 1. Zhao, S., et al., Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis, 2020. 92: p. 214- 217. . Li, G., et al., Coronavirus infections and immune responses. J Med Virol, 2020.
3. Channappanavar, R. and S. Perlman, Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. Semin Immunopathol, 2017. 39(5): p. 529-539. . Huang, C., et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, 2020. 395(10223): p. 497-506.
5. Guo, Y.R., et al., The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res, 2020. 7(1): p. 11.
6. Liu, Q., Y.H. Zhou, and Z.Q. Yang, The cytokine storm of severe influenza and development of immunomodulatory therapy. Cell Mol Immunol, 2016.
13(1): p. 3-10. . Tanaka, T., M. Narazaki, and T. Kishimoto, Immunotherapeutic implications ofIL-6 blockade for cytokine storm. Immunotherapy, 2016. 8(8): p. 959-70.
8. Tisoncik, J.R., et al., Into the eye of the cytokine storm. Microbiol Mol Biol Rev, 2012. 76(1): p. 16-32.

Claims

1. An organic extract of at least one plant line, said at least one plant line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain, wherein said organic extract comprises at least one compound suitable for treating a mammalian oral disease or disorder.
2. An organic extract according to claim 1, wherein said at least one plant line comprises a Cannabis sativa line.
3. An organic extract according to claim 1, wherein said mammalian oral disease or disorder is selected from the group consisting of a viral disease, an oral disease, a lung disease, an intestinal disease and combinations thereof.
4. An organic extract according to claim 1, wherein said extract is effective against corona virus disease.
5. An organic extract according to claim 1, wherein said extract is effective against other virus diseases.
6. An organic extract according to claim 1, wherein said extract is effective against ARDS and other cytokine-induced diseases and disorders.
7. An organic extract according to claim 1, wherein said organic extract is at least 2-20, 3-15, 4-12, 5-10 or 6-9 times as effective as at least one of THC and CBD, administered at the same concentration in treating said disease.
8. A combination therapy, isolated from an organic extract of at least one hybrid line, said at least one hybrid line formed from combining at least one of: a) at least one marijuana strain; and b) at least one hemp strain; and wherein said organic extract comprises a plurality of compounds suitable for treating a mammalian viral disease and disorder.
9. A Cannabis extract for treating and preventing a viral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
10. A Cannabis extract for treating and preventing viral disease or disorder, wherein said extract efficacy can be further increased by adding extracts of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
11. A line of Cannabis sativa formed by combining at least one marijuana strain and at least one hemp strain, said line will be deposited at public culture collection, currently under designation numbers #81, #130, #1, #7, #9, #45, #129, #169, #5, #10, #31, #49, #114, #207.
12. A pharmaceutical composition for treating a viral disease or disorder in a mammalian subject, the pharmaceutical composition comprising at least one of: a) an organic extract according to claim 1 ; b) at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD); and c) at least one terpene or terpene alcohol; wherein said pharmaceutical composition is suitable for treating said viral disease or said viral disorder in said mammalian subject.
13. A pharmaceutical composition according to claim 12, wherein said at least one terpene is selected from the group consisting of myrcene, humulene, pinene carophyllene and valencene.
14. A pharmaceutical composition according to claim 12, wherein said terpene alcohol comprises bisabolol.
15. A pharmaceutical composition according to claim 12, wherein said at least one of Cannabigerol (CBG), Cannabinol (CBN) and Cannabidiol (CBD) comprises only Cannabidiol (CBD).
16. A pharmaceutical composition according to claim 15, wherein said viral disease or disorder is caused by a corona virus, a mutant or variant thereof.
17. A pharmaceutical composition according to claim 16, wherein said viral disease is COVID 19.
18. A pharmaceutical composition according to claim 17, wherein said pharmaceutical composition is effective in treating a cytokine storm associated with said COVID 19.
19. A pharmaceutical composition according to claim 12, is suitable for reducing a level of an ACE2 protein in said mammalian subject.
PCT/IL2021/050265 2020-03-26 2021-03-10 Cannabis sativa (hemp and cannabis) products for viral disease prevention and management WO2021191888A1 (en)

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