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WO2021124316A1 - High cbd cannabis sativa lines and extracts with anti-inflammatory potencies for oral and gut health - Google Patents

High cbd cannabis sativa lines and extracts with anti-inflammatory potencies for oral and gut health Download PDF

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Publication number
WO2021124316A1
WO2021124316A1 PCT/IL2020/051237 IL2020051237W WO2021124316A1 WO 2021124316 A1 WO2021124316 A1 WO 2021124316A1 IL 2020051237 W IL2020051237 W IL 2020051237W WO 2021124316 A1 WO2021124316 A1 WO 2021124316A1
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WO
WIPO (PCT)
Prior art keywords
oral
extract
disease
extracts
disorder
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Application number
PCT/IL2020/051237
Other languages
French (fr)
Inventor
Anna KOVALCHUK
Rocio DEL CARMEN RODRIGUEZ-JUAREZ
Igor Kovalchuk
Olga Kovalchuk
Original Assignee
Swysh Inc.
I. P. Israel Patents Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Swysh Inc., I. P. Israel Patents Ltd. filed Critical Swysh Inc.
Publication of WO2021124316A1 publication Critical patent/WO2021124316A1/en

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    • 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)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/28—Cannabaceae, e.g. cannabis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the present invention relates generally to products and methods for treating inflammation of the mouth cavity or alimentary canal, and more specifically to methods and products for treating oral tissue diseases or alimentary canal diseases and inflammation from cannabis and hemp plants, high in cannabidiol (CBD).
  • CBD cannabidiol
  • Inflammation is a fundamental physiological reaction of tissues to injury caused by heat, chemical or bacterial agents. Inflammation can be short and acute, or, if unresolved, can turn out to be prolonged and chronic, leading to an array of pathological conditions [Cekici, et al., 2014].
  • the oral cavity represents a very complex microenvironment containing numerous microorganisms that is very important for human health and disease.
  • gingivitis and periodontitis Two key inflammatory oral pathologies are gingivitis and periodontitis; their pathogenesis is facilitated by the inflammatory response to bacteria and bacterial components, such as lipopolysaccharides [Sudhakara et al., 2018] and has a substantial immune component.
  • bacteria and bacterial components such as lipopolysaccharides [Sudhakara et al., 2018] and has a substantial immune component.
  • lipopolysaccharides microbial etiologic factors mediate inflammatory events.
  • Gingivitis is inflammation of the gingiva, whereby disease is limited to the soft-tissue compartment of the gingival epithelium and connective tissue.
  • Periodontitis inflammation involves supporting tissues of the teeth and is characterized by progressive soft tissue and bone destruction.
  • Periodontal diseases are very common, 54% of US population exhibits signs of gingivitis, and 37% suffer from severe periodontitis [Delwel et al., 2018].
  • Stomatitis refers to for an inflamed and sore mouth, a condition that can disrupt a person's ability to eat, talk, and sleep.
  • Types of stomatitis include oral ulcers (canker sores) and mouth irritation. Oral ulcers are very painful and have various etiologies such as mouth injury, food sensitivities, lack of essential vitamins, bacterial, viral, or fungal infections, immunological diseases or cancers.
  • Glossitis refers to inflammation of the tongue. General mouth irritation can be caused by trauma, gingivitis, autoimmune diseases, infections, cancer chemotherapy, radiation therapy, antibiotics, and other exposures.
  • IBS Irritable Bowel Syndrome
  • IBD Inflammatory bowel disease
  • ulcerative colitis and Crohn's disease are characterized by prolonged inflammation of gastrointestinal tract and usually involve severe diarrhea, abdominal pain, fatigue and weight loss.
  • Inflammatory bowel disease symptoms vary, depending on the severity of inflammation and where it occurs. Symptoms may range from mild to severe, and periods of illness are followed by periods of remission. IBD can be debilitating and sometimes leads to life-threatening complications.
  • anti-inflammatory drugs both steroid and non-steroid
  • immune system suppressors such as azathioprine, mercaptopurine, cyclosporine and methotrexate, as well as anti- TNF biologies. While these treatments are effective, they cause very significant side effects.
  • compositions for improving wellness in a human or mammalian organism It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or mammalian organism.
  • compositions for improving wellness in a human or mammalian organism It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or mammalian organism.
  • the present invention provides new unique high CBD Cannabis Sativa (hemp and cannabis) lines, extracts and methods for their use to treat inflammation of oral tissues and gut tissues as novel anti-inflammatory therapies and modalities that can be used for improved health.
  • the method includes generation of unique lines, whole plant extract preparation, treating normal human tissues with bacterial lipopolysacharide (LPS) or tumor necrosis factor alpha (TNF) to induce inflammation, and then with the CBD extracts in amount sufficient to profoundly down-regulate inflammation and molecular pathways involved in inflammation in the tissues.
  • LPS bacterial lipopolysacharide
  • TNF tumor necrosis factor alpha
  • the modulation of these pathways is a key to treatment success in oral diseases, oral cancer and diseases of the alimentary canal.
  • the present invention provides new Cannabis sativa lines with high levels of cannabidiol (CBD)/low levels of THC and extracts and method of using them as a means to modulate gene expression in oral tissues having an active disorder such as gingivitis, periodontitis, stomatitis, glossitis, oral cancer, and others.
  • CBD cannabidiol
  • the disclosure also provides methods of modulating gene expression through the application cannabis extracts to the oral tissues affected by mouth diseases such as gingivitis, periodontitis, stomatitis, glossitis, oral cancer, and others
  • Extracts of novel CBD lines can be combined with anti-inflammatory 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 (e.g., in oral tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the oral epithelial cells or oral tissue.
  • a source of new Cannabis sativa hemp and cannabis
  • the present invention provides freshly prepared extracts of seven new C. Sativa lines ( #90, #115, #129, #131, #157, # 167, #169 ) (Fig. 1 A-G) and, using 3D EpiOral tissue models identified anti-inflammatory properties of the new lines based on the expression of genes that are associated with molecular etiology and pathogenesis of oral diseases (Fig. 2).
  • the present invention provides compositions having potent therapeutic effects against LPS-induced and TNF-induced inflammation of oral epithelial tissues, and thus for treatment and prevention of gingivitis, periodontitis, stomatitis, and other oral inflammatory disorders and oral cancers.
  • the present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in oral tissues having an active oral disorder and others.
  • the disclosure also provides methods of modulating gene expression through the application cannabis extracts to the mouth affected by oral diseases and others.
  • the present invention further provides extracts for treating an oral disease or disorder.
  • the extract efficacy can be further increased by adding Cannabidiol (CBD), Cannabigerol (CBG), Cannabinol (CBN), terpenes or combinations thereof.
  • CBD Cannabidiol
  • CBG Cannabigerol
  • CBN Cannabinol
  • 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 provides new unique cannabis lines, extracts and methods for oral improvement and healing and reduction in inflammation.
  • the method includes generation of unique lines, whole plant extract preparation, exposing human oral to the extracts in amount sufficient to modulate gene expression in the oral.
  • the modulation of gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the cannabis-treated oral tissues.
  • improved methods and products are provided for oral treatment.
  • a method and product is described for improving oral appearance.
  • a method and product is described for improving oral health.
  • 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 (e.g., in oral cells or in oral tissue) by providing a source of new extracts, exposing oral cells or oral tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the oral cells or oral tissue.
  • the disclosure 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 inflammatory oral disorders and others.
  • the method includes generation of unique lines, whole plant extract preparation, treating human 3D oral tissues with extracts in amount sufficient to modulate gene expression in the oral tissues. The modulation of gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the treated oral tissues.
  • 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 oral disorders.
  • the invention relates to methods and compositions for preventing other gastrointestinal tract diseases beyond the oral cavity that harbor inflammation component.
  • the invention relates to methods and compositions for preventing systemic diseases beyond the oral cavity that are related to periodontal disease.
  • the present invention is directed to compositions and methods for treating disorders, in general, and more particularly, oral diseases and disorders.
  • 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.
  • compositions and methods are provided for treating and/or preventing inflammatory and proliferative disorders.
  • compositions and methods are provided for treating and/or preventing cancer.
  • 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 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 at least one inflammatory pathway gene product.
  • compositions, compounds, formulations and extracts for treating an oral disorder or disease, or oral aging, in a human patient.
  • composition derived from at least one of hemp and cannabis for treating an oral disorder or disease, 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 treatment of an oral disease or disorder.
  • Some embodiments of the present invention are directed to a method for treating an oral 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 treating an oral disorder or disease in a human patient comprising administering to said patient the oral 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 cancerous organs 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.
  • 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 proliferative 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 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.
  • the present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in intestinal tissues having an active inflammatory disorder such as ulcerative colitis, Crohn’s disease, and other GI inflammatory diseases.
  • the disclosure provides a means for modulating gene expression (e.g., in intestinal tissues and cells) by providing a source of new extracts, exposing intestinal cells or intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the intestinal cells or intestinal tissue.
  • a means for modulating gene expression e.g., in intestinal tissues and cells
  • the present invention provides freshly prepared extracts of six new C. Sativa lines (#1, #7, #9, #45, #274, #130) and, using 3D Epilntestinal tissue models identified anti-inflammatory properties of the new lines based on the expression of genes that are associated with molecular etiology and pathogenesis of IBD and other GI inflammatory diseases (Fig. 3).
  • the present invention provides compositions having potent therapeutic effects against TNF/IFN-induced inflammation of intestinal epithelial tissues, and thus for treatment and prevention of IBD, IBS and other GI inflammatory diseases, and other inflammatory diseases and conditions
  • the present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in intestinal tissues having an active inflammatory disease and others.
  • the present invention provides new unique cannabis lines, extracts and methods for intestinal improvement and healing and reduction in inflammation.
  • EMBODIMENTS are new unique cannabis lines, extracts and methods for intestinal improvement and healing and reduction in inflammation.
  • a method for treating a mammalian oral 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; and c) treating said oral disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said oral disease or disorder.
  • 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.
  • a method according to embodiment 3, wherein said at least one line is selected from the group consisting of designated lines #90, #115, #129, #131, #157, #167 and #169 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 15, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation 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 oral disease or disorder.
  • a combination therapy according to embodiment 21, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation and combinations thereof.
  • a Cannabis extract for treating an oral 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 an oral 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 #90, #115, #129, #131, #157, #167 and #169.
  • FIGS. 1A-1G High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
  • EpiOral tissues and experimental set-up consist of normal, human-derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes.
  • FIG. 4 Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (LPS vs DMSO, LPS-#131 vs DMSO, and LPS-#90 vs DMSO).
  • Figure 5 Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts # 115, 129, 157, 167, 169).
  • FIGS 6A-6F High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
  • FIG. 1 Epilntestinal Tissues and experimental set-up.
  • A. A method using Epilntestinal tissues to detect anti-inflammatory properties of extracts.
  • B. Scheme of TNFa/IFNy-i induced inflammation experiment, in accordance with some embodiments of the present invention.
  • Figure 8 Summary effects of all the pro-inflammatory gene changes in Epilntestinal tissues treated with high-CBD cannabis extracts in a single bar for each condition.
  • FIGS. 1A-1G High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention:
  • FIG. 1A Chromatogram of #90 extract. Total THC equivalent 1.05% and CBD 4.58%.
  • FIG. 1B Chromatogram of #131 extract. Total THC equivalent 0.12% and CBD 2.24%.
  • FIG. 1C Chromatogram # 115 extract. Total THC equivalent 0.4% and CBD 9.54%.
  • Figure ID Chromatogram of #129 extract. Total THC equivalent 0.34% and CBD 6.75%.
  • Figure IE Chromatogram of #157 extract. Total THC equivalent 0.2% and CBD 3.78%.
  • Figure IF Chromatogram of #167 extract. Total THC equivalent 0.08% and CBD 2.2 %.
  • FIG. 1G Chromatogram of #169 extract. Total THC equivalent 0.21% and CBD 1.88%.
  • EpiOral tissues and experimental set-up consist of normal, human-derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes.
  • 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT-400 was replaced and incubated for another 24 h. Then, set of tissues was treated for 24 h with 10 pg/ml of E. coli LPS, or DMSO only for control.
  • an extract treatment step 206 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 was used. 24 h after LPS treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
  • RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
  • an equilibration step 210 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT- 400 was replaced and incubated for another 24 h. Then, a set of tissues was treated for 24 h with TNFa (40 ng/ml) and IFNy (5 ng/ml), or DMSO only as a control.
  • TNFa 40 ng/ml
  • IFNy 5 ng/ml
  • an extract treatment step 212 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 was used. 24 h after TNF/IFN treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
  • RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
  • FIG. 4 Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (LPS vs DMSO, LPS-#131 vs DMSO, and LPS-#90 vs DMSO).
  • Tissues were treated for 24 h with 10 pg/ml of E. coli LPS, after which the extracts were added and the samples were incubated for another 24h; DMSO alone was added to LPS sample.
  • Y axis shows cumulative arbitrary expression of all pro- inflammatory genes listed in Table 1.
  • Figure 5 Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts # 115, 129, 157, 167, 169).
  • Tissues were treated for 24 h with TNF/IFN, after which the extracts were added and the samples were incubated for another 24h; DMSO alone was added to LPS sample.
  • Y axis shows cumulative arbitrary expression of all pro-inflammatory genes listed in Table 1.
  • FIGS 6A-6F High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
  • FIG. 7 Epilntestinal Tissues and experimental set-up.
  • A A method using Epilntestinal tissues to detect anti-inflammatory properties of extracts.
  • Epilntestinal model has normal small intestine epithelial cell structure and is constructed from human-derived columnar epithelial and endothelial cells. It exhibits in vzvo-like growth and morphological characteristics whereby cells sustain differentiation and metabolic status similar to those of human intestinal epithelium.
  • B Scheme of TNFa/IFNy-i induced inflammation experiment, in accordance with some embodiments of the present invention.
  • Step 702- a cultivar growing step. Around 250 unique marijuana and around 120 unique hemp cultivars were used to generate approximately 1,200 marijuana/marijuana, hemp/hemp and hemp/marijuana hybrids.
  • Cultivars are typically grown in soil/vermiculite (2:1) mix.
  • plants are grown under 16h day, 8h night for approximately 6 weeks when they were moved to another grow room and grown at 12h day and 12h night for another 6-8 weeks until they developed mature flowers.
  • HPS high pressure sodium
  • Collected flowers were then tested for cannabinoids and terpenoids and those with most diversity in composition, or those that had highest amount of one or more cannabinoid or terpenoid or those that had the presence of unique terpenoids were used for breeding.
  • Step 704 an extraction step, such as organic solvent extraction.
  • Most solvents can be used.
  • ethyl acetate was used. This should not be deemed as limiting.
  • 3 g of the powdered flower tissue were used in 100 ml of ethyl acetate in a 250 mL Erlenmeyer flask. The flasks were then wrapped with tin foil and shaken continuously (120 rpm) in an incubator at 21°C overnight and in the dark. After overnight solvent extraction the extracts were filtered through cotton into a 100 ml round bottom flask. The extracts were concentrated to around 2-3 ml using a rotary vacuum evaporator.
  • the extracts were then transferred to a tared 3 dram vial. The leftover solvent was evaporated to dryness in an oven overnight at 50°C to eliminate the solvent completely. Mass of each extract was recorded, and the extracts were stored at -20°C.
  • 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) to reach 60 mg/mL final concentration and stored at - 20°C. Around 400 solvent-based crude extracts of flowers were thus generated.
  • DMSO Dimethyl sulfoxide anhydrous
  • extracts were applied to Epilntestinal tissues as per below.
  • data analysis step 708 data were obtained from harvested tissue and altered pathways were analyzed bioinformatically.
  • a treatment step 710 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT-400 was replaced and incubated for another 24 h. Then, set of tissues was treated for 24 h with TNFa (40 ng/ml) and IFNy (5 ng/ml), or DMSO only for control.
  • TNFa 40 ng/ml
  • IFNy 5 ng/ml
  • an extract treatment step 712 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 was used. 24 h after TNF/IFN treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
  • RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
  • Figure 8 Summary effects of all the pro-inflammatory gene changes in Epilntestinal tissues treated with high-CBD cannabis extracts in a single bar for each condition.
  • TD - samples treated with TNF/IFN and then with DMSO TD24 - TD sample collected 24 hours after addition of DMSO.
  • Extracts were selected based on their strong anti-inflammatory properties. Treatment and prevention of oral and gingival diseases, IBD, IBS and other inflammatory conditions can be done by using either extracts alone or in combination with other anti-inflammatory herbs or/and probiotics.
  • a hybrid flower extraction step 902 identification of novel anti- inflammatory extracts hybrids of different cannabis varieties are created and full flower extracts of various hybrids are prepared. These extracts are then applied to various 3D tissues treated with LPS or with TNF/IFN. The tissues are then harvested and used for mRNA-seq and protein analysis.
  • an inflammatory gene identification step 904 a set of genes involved in inflammation response are identified and extracts are stratified according to the capacity to downregulate such pro-inflammatory genes (see Table 1).
  • extracts are used alone or in combination with other herbs, probiotics, vitamins and other anti-inflammatory molecules to downregulate TNF, interleukins, cytokines and other pro -inflammatory genes (Table 1).
  • formulations are used for prevention and treatment of oral and gingival diseases, IBD, IBS and other inflammatory conditions.
  • 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 + 10% FBS or EMEM + 10% FBS was used to dilute the 60 mg/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 um syringe filter. These filtrates were ready to be applied to cells and tested.
  • EpiOral tissue model 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 tissue model (Mat Tek) was used as inflammation model.
  • Epilntestinal is a 3D reconstructed tissue model produced from primary, human cell- derived small intestine epithelial and endothelial cells and fibroblasts.
  • the highly differentiated tissue model is produced at the air-liquid-interface (ALI) in easy-to- handle tissue culture inserts.
  • Structural analysis of the tissue model demonstrates columnar shaped basal cells and Kerckring folds.
  • Epilntestinal exhibits brush borders, functional tight junctions and mucous secreting granules, similar to in vivo tissue. It exhibits in vivo-like growth and morphological characteristics whereby cells sustain differentiation and metabolic status similar to those of human intestinal epithelium.
  • Tissues were equilibrated for 24 h (overnight) then culture media was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to either to LPS (10 ug/ml LPS) or to TNFa (40 ng/ml) and IFNy (5 ng/ml) or to DMSO only, resulting in TNF/IFN” or “Ct” samples, respectively.
  • EpiOral tissues treated with LPS were then treated with two extracts - from lines #90 and #131, whereas EpiOral tissues treated with TNF/IFN were treated with cannabis extracts from lines #115, #129, #157, #167 and #169.
  • Epilntestinal tissues treated with TNF/IFN were then treated with cannabis extracts from lines #1, #7, #9, #45, #274, #130.
  • Tissues once treated were allowed to equilibrate at 37°C in an incubator with 6% CO2, for 24 hours. Then all tissues were frozen using liquid N2 and stored at - 80°C.
  • Described herein is a method for treating oral disorders including, but is not limited to the steps of: 1) preparation of new cannabis extracts, 2) exposing normal and disease skin or oral tissue models to novel extracts and 3) modulating the gene expression 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.
  • Extracts can be used alone or in combination with anti-inflammatory plants and/or probiotics to potentiate their anti-inflammatory effects on the GI inflammatory diseases and conditions (Figure 9).
  • a method for oral inflammatory diseases and conditions that includes the steps of exposing a patient to novel cannabis extracts, resulting in a reduction in the disease 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.
  • genes down-regulated by new extracts are 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, FCER
  • 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 is 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.
  • 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
  • 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 intradermally.
  • 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, intraarterial, 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.
  • 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. Each possibility represents a separate embodiment of the present invention.
  • 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).
  • Each possibility represents a separate embodiment of the present invention.
  • 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.
  • 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 al., 1981; Newmark et al., 1982; and Katre et al., 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.
  • 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.
  • 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. In another embodiment, “treating” refers to inducing remission. In another embodiment, “treating” refers to slowing the progression of a disease.

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Abstract

The present invention provides new unique high CBD Cannabis Sativa (hemp and cannabis) lines, extracts and methods for their use to treat inflammation of oral tissues and gut tissues as novel anti-inflammatory therapies and modalities that can be used for improved health. The method includes generation of unique lines, whole plant extract preparation, treating normal human tissues with bacterial lipopolysacharide (LPS) or tumor necrosis factor alpha (TNF) to induce inflammation, and then with the CBD extracts in amount sufficient to profoundly down-regulate inflammation and molecular pathways involved in inflammation in the tissues. The modulation of these pathways is a key to treatment success in oral diseases, oral cancer and diseases of the alimentary canal.

Description

HIGH CBD CANNABIS SATIVA LINES AND EXTRACTS WITH ANTIINFLAMMATORY POTENCIES FOR ORAL AND GUT HEALTH
FIELD OF THE INVENTION
The present invention relates generally to products and methods for treating inflammation of the mouth cavity or alimentary canal, and more specifically to methods and products for treating oral tissue diseases or alimentary canal diseases and inflammation from cannabis and hemp plants, high in cannabidiol (CBD).
BACKGROUND OF THE INVENTION
Inflammation is a fundamental physiological reaction of tissues to injury caused by heat, chemical or bacterial agents. Inflammation can be short and acute, or, if unresolved, can turn out to be prolonged and chronic, leading to an array of pathological conditions [Cekici, et al., 2014]. The oral cavity represents a very complex microenvironment containing numerous microorganisms that is very important for human health and disease.
Two key inflammatory oral pathologies are gingivitis and periodontitis; their pathogenesis is facilitated by the inflammatory response to bacteria and bacterial components, such as lipopolysaccharides [Sudhakara et al., 2018] and has a substantial immune component. In periodontal diseases microbial etiologic factors mediate inflammatory events. Gingivitis is inflammation of the gingiva, whereby disease is limited to the soft-tissue compartment of the gingival epithelium and connective tissue. In periodontitis inflammation involves supporting tissues of the teeth and is characterized by progressive soft tissue and bone destruction.
Periodontal diseases are very common, 54% of US population exhibits signs of gingivitis, and 37% suffer from severe periodontitis [Delwel et al., 2018].
Stomatitis refers to for an inflamed and sore mouth, a condition that can disrupt a person's ability to eat, talk, and sleep. Types of stomatitis include oral ulcers (canker sores) and mouth irritation. Oral ulcers are very painful and have various etiologies such as mouth injury, food sensitivities, lack of essential vitamins, bacterial, viral, or fungal infections, immunological diseases or cancers. Glossitis refers to inflammation of the tongue. General mouth irritation can be caused by trauma, gingivitis, autoimmune diseases, infections, cancer chemotherapy, radiation therapy, antibiotics, and other exposures.
Poor oral health is linked to various diseases, such as to brain inflammation, neurodegeneration, and Alzheimer's disease, cardiovascular and autoimmune disorders [Delwel et ah, 2018]. Treatment and prevention of mouth tissues inflammation is rather challenging, and new treatment modalities are urgently needed.
The problem of Irritable Bowel Syndrome (IBS) affects up to 21% of the general population and is connected to 40-60% of gastroenterology referrals [Hasturk et ah, 2012, Akbar etl ah, 2009]. It is a clinical phenomenon with complex psychosocial and biological pathophysiology and similar symptoms: abdominal pain, bloating and distorted bowel habit. On the basis of bowel movements we may differentiate following subtypes: constipation (C-IBS), diarrhoea (D-IBS), or mixed IBS [Chey et al, 2015 Farzaei, et ah, 2016]. The abdominal visceral pain of IBS is considered to be because of enhanced perception to colonic distension in about 70% of patients. While IBS does not pose an immediate hazard to life, the intense impact of IBS on the quality of patient’s life leads to great clinical significance [Farzaei, et al., 2016].
Inflammatory bowel disease (IBD) is a general term encompassing severe chronic inflammatory disorders of digestive tract. Types of IBD include ulcerative colitis and Crohn’s disease. Both ulcerative colitis and Crohn's disease are characterized by prolonged inflammation of gastrointestinal tract and usually involve severe diarrhea, abdominal pain, fatigue and weight loss. Inflammatory bowel disease symptoms vary, depending on the severity of inflammation and where it occurs. Symptoms may range from mild to severe, and periods of illness are followed by periods of remission. IBD can be debilitating and sometimes leads to life-threatening complications. Current treatments are aimed to reduce the inflammation and include anti-inflammatory drugs (both steroid and non-steroid), immune system suppressors such as azathioprine, mercaptopurine, cyclosporine and methotrexate, as well as anti- TNF biologies. While these treatments are effective, they cause very significant side effects.
Therefore, there is a need for further methods of treating GI diseases and conditions, including IBD disorders that are minimally invasive, easy to use, and have no systemic side effects. There is a need for further methods of treating oral tissue disorders that are minimally invasive, easy to use, and have no systemic side effects. SUMMARY OF THE INVENTION
It is an object of some aspects of the present invention, to provide improved methods and products for treating, improving and curing oral ailments.
It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or 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 mammalian organism.
It is an object of some aspects of the present invention, to provide improved methods and products for treating, improving and curing IBD and IBS.
It is an object of some aspects of the present invention, to provide improved methods and products for treating, improving and curing GI inflammatory disease.
It is an object of some aspects of the present invention, to provide improved methods and products for treating, improving and curing inflammatory disease.
It is an object of some aspects of the present invention to provide compositions for improving wellness in a human or 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 mammalian organism.
The present invention provides new unique high CBD Cannabis Sativa (hemp and cannabis) lines, extracts and methods for their use to treat inflammation of oral tissues and gut tissues as novel anti-inflammatory therapies and modalities that can be used for improved health. The method includes generation of unique lines, whole plant extract preparation, treating normal human tissues with bacterial lipopolysacharide (LPS) or tumor necrosis factor alpha (TNF) to induce inflammation, and then with the CBD extracts in amount sufficient to profoundly down-regulate inflammation and molecular pathways involved in inflammation in the tissues. The modulation of these pathways is a key to treatment success in oral diseases, oral cancer and diseases of the alimentary canal.
The present invention provides new Cannabis sativa lines with high levels of cannabidiol (CBD)/low levels of THC and extracts and method of using them as a means to modulate gene expression in oral tissues having an active disorder such as gingivitis, periodontitis, stomatitis, glossitis, oral cancer, and others. The disclosure also provides methods of modulating gene expression through the application cannabis extracts to the oral tissues affected by mouth diseases such as gingivitis, periodontitis, stomatitis, glossitis, oral cancer, and others
Extracts of novel CBD lines can be combined with anti-inflammatory 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 (e.g., in oral tissues and cells) by providing a source of new Cannabis sativa (hemp and cannabis) extracts, exposing oral cells or oral epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the oral epithelial cells or oral tissue.
The present invention provides freshly prepared extracts of seven new C. Sativa lines ( #90, #115, #129, #131, #157, # 167, #169 ) (Fig. 1 A-G) and, using 3D EpiOral tissue models identified anti-inflammatory properties of the new lines based on the expression of genes that are associated with molecular etiology and pathogenesis of oral diseases (Fig. 2).
The effect of two extracts of novel cannabis lines (#90, #131) were evaluated during inflammation, provoked by LPS treatment, in EpiOral tissues and confirmed the potent anti-inflammatory potencies of the extracts of new lines against LPS- induced inflammation in EpiOral tissues.
The effects of five extracts of novel cannabis lines (#115, #129, #157, #167 and #169) were evaluated during inflammation, provoked by TNF treatment, in EpiOral tissues and confirmed the potent anti-inflammatory potencies of the extracts of new lines against TNF-induced inflammation in EpiOral tissues.
The present invention provides compositions having potent therapeutic effects against LPS-induced and TNF-induced inflammation of oral epithelial tissues, and thus for treatment and prevention of gingivitis, periodontitis, stomatitis, and other oral inflammatory disorders and oral cancers.
The present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in oral tissues having an active oral disorder and others. The disclosure also provides methods of modulating gene expression through the application cannabis extracts to the mouth affected by oral diseases and others.
The present invention further provides extracts for treating an oral disease or disorder. The extract efficacy can be further increased by adding 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 provides new unique cannabis lines, extracts and methods for oral improvement and healing and reduction in inflammation. The method includes generation of unique lines, whole plant extract preparation, exposing human oral to the extracts in amount sufficient to modulate gene expression in the oral. The modulation of gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the cannabis-treated oral tissues.
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 appearance.
In other embodiments of the present invention, a method and product is described for improving oral 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 (e.g., in oral cells or in oral tissue) by providing a source of new extracts, exposing oral cells or oral tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the oral cells or oral tissue.
The disclosure 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 inflammatory oral disorders and others. The method includes generation of unique lines, whole plant extract preparation, treating human 3D oral tissues with extracts in amount sufficient to modulate gene expression in the oral tissues. The modulation of gene expression then results in a reduction of the disease state-associated changes or aspects thereof in the treated oral 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 oral disorders.
Further, the invention relates to methods and compositions for preventing other gastrointestinal tract diseases beyond the oral cavity that harbor inflammation component.
Further, the invention relates to methods and compositions for preventing systemic diseases beyond the oral cavity that are related to periodontal disease.
The present invention is directed to compositions and methods for treating disorders, in general, and more particularly, oral diseases and disorders. 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 inflammatory and proliferative disorders.
In additional embodiments of the present invention, compositions and methods are provided for treating and/or preventing cancer.
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 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 at least one inflammatory pathway gene product.
In particular, there are described methods for preparing compositions, compounds, formulations and extracts for treating an oral disorder or disease, or oral aging, in a human patient.
There is thus provided according to some embodiments of the present invention, a composition, derived from at least one of hemp and cannabis for treating an oral disorder or disease, 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 treatment of an oral disease or disorder.
Some embodiments of the present invention are directed to a method for treating an oral 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 treating an oral disorder or disease in a human patient comprising administering to said patient the oral 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 cancerous organs 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 proliferative 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 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.
The present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in intestinal tissues having an active inflammatory disorder such as ulcerative colitis, Crohn’s disease, and other GI inflammatory diseases.
Accordingly, the disclosure provides a means for modulating gene expression (e.g., in intestinal tissues and cells) by providing a source of new extracts, exposing intestinal cells or intestinal epithelial tissue to those in an amount sufficient to modulate gene expression where modulation of gene expression results in a reduction of a disease state in the intestinal cells or intestinal tissue.
The present invention provides freshly prepared extracts of six new C. Sativa lines (#1, #7, #9, #45, #274, #130) and, using 3D Epilntestinal tissue models identified anti-inflammatory properties of the new lines based on the expression of genes that are associated with molecular etiology and pathogenesis of IBD and other GI inflammatory diseases (Fig. 3).
The effect of six extracts of novel high-CBD cannabis lines (1, #7, #9, #45, #274, #130) were evaluated during inflammation provoked by a TNF/IFN co treatment in intestinal tissues, and proved the potent anti-inflammatory potencies of the extracts of new lines against TNF/IFN-induced inflammation in Epilntestinal tissues (Fig. 2).
The present invention provides compositions having potent therapeutic effects against TNF/IFN-induced inflammation of intestinal epithelial tissues, and thus for treatment and prevention of IBD, IBS and other GI inflammatory diseases, and other inflammatory diseases and conditions
The present invention provides new Cannabis sativa lines and extracts and method of using them as a means to modulate gene expression in intestinal tissues having an active inflammatory disease and others.
The present invention provides new unique cannabis lines, extracts and methods for intestinal improvement and healing and reduction in inflammation. EMBODIMENTS
1. A method for treating a mammalian oral 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; and c) treating said oral disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said oral disease or disorder.
2. A method according to embodiment 1, wherein said treating step induces modulation of gene expression in at least one of oral cells and oral tissue; and wherein said modulation of gene said expression results in a reduction of at least one of an oral disease state, caries, an inflammatory state, an oral disorder state and combinations thereof.
3. 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.
4. A method according to embodiment 3, wherein said at least one line is selected from the group consisting of designated lines #90, #115, #129, #131, #157, #167 and #169 and combinations thereof.
5. A method according to embodiment 1, wherein said extracting step comprises extracting flowers of said at least one Cannabis line.
6. A method according to embodiment 5, wherein said extracting step comprises extracting said at least one compound in at least one organic solvent.
7. A method according to embodiment 6, 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.
8. A method according to embodiment 2, 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, 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 and ZYX and combinations thereof. 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. A method according to embodiment 1, wherein said at least one compound is provided in a solvent extract and said solvent extract exhibits oral disease or oral disorder healing properties. 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. The method of embodiment 2, wherein the disease state is an inflammatory oral disease.
13. The method of embodiment 12, wherein the disease state is selected from the group consisting of an oral insult, caries, an oral disorder, an oral disease, environmental factor-induced inflammation and combinations thereof.
14. A method according to embodiment 1, wherein said Cannabis line is a Cannabis sativa line.
15. 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.
16. An organic extract according to embodiment 15, wherein said at least one plant line comprises a Cannabis sativa line.
17. An organic extract according to embodiment 15, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation and combinations thereof.
18. An organic extract according to embodiment 15, wherein said extract is effective against chemo-resistant cancer cells and is suitable to overcome chemo-resistance.
19. An organic extract according to embodiment 15, wherein said extract potentiates effects of cytotoxic chemotherapy and is an effective and safe adjuvant modality.
20. An organic extract according to embodiment 15, 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.
21. 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 oral disease or disorder.
22. A combination therapy according to embodiment 21, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation and combinations thereof.
23. A Cannabis extract for treating an oral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
24. A Cannabis extract for treating an oral 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.
25. 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 #90, #115, #129, #131, #157, #167 and #169.
26. A method for treating a disease state in oral epithelial cells or oral tissue employing new lines of Cannabis sativa and/or new extracts thereof, the method comprising the steps of: a) providing a source of unique extract; and b) treating the oral tissue with aforementioned extracts in an effective amount to induce modulation of gene expression in the oral cells or oral tissue; wherein the modulation of gene expression results in a reduction of the disease state in the oral cells or oral tissue.
27. The method of embodiment 26, wherein the extracts is extract #90.
28. The method of embodiment 26, wherein the extracts is extract #131.
29. The method of embodiment 26, wherein the extracts is extract #115.
30. The method of embodiment 26, wherein the extracts is extract #129
31. The method of embodiment 26, wherein the extracts is extract #157.
32. The method of embodiment 26, wherein the extracts is extract #167.
33. The method of embodiment 26, wherein the extracts is extract #169. 34. The method of embodiment 26, wherein the modulation of gene expression is a down-regulation of gene expression.
35. The method of embodiment 26, wherein the modulation of gene expression is an up-regulation of gene expression.
36. The method of embodiment 26, wherein the disease state is periodontitis.
37. The method of embodiment 26, wherein the disease state is gingivitis.
38. The method of embodiment 26, wherein the disease state is stomatitis.
39. The method of embodiment 26, wherein the disease state is post-cancer treatment oral inflammation.
40. The method of embodiment 26, wherein the disease state is oral cancer.
41. An extract according to embodiments 1-40, wherein said effects are further extended by addition of CBD, CBG, CBN, terpenes or combinations thereof.
42. An extract according to embodiments 1-40, wherein said effects are further extended by addition of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics or combinations thereof.
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:
Figures 1A-1G. High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
Figure 2. EpiOral tissues and experimental set-up. A. EpiOral tissues consist of normal, human-derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes. B. Scheme of LPS-induced inflammation experiment; C. Scheme of the TNF— induced inflammation experiment.
Figure 3. Effects of extracts #90 and 131 on LPS-induced inflammation.
Gene expression changes of each inflammatory gene in LPS vs DMSO (x-axis) and the same change in LPS extract co-treatment vs DMSO (y-axis). A. Extract #90; B. Extract #131.
Figure 4. Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (LPS vs DMSO, LPS-#131 vs DMSO, and LPS-#90 vs DMSO).
Figure 5. Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts # 115, 129, 157, 167, 169).
Figures 6A-6F. High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
Figure 7. Epilntestinal Tissues and experimental set-up. A. A method using Epilntestinal tissues to detect anti-inflammatory properties of extracts. B. Scheme of TNFa/IFNy-i induced inflammation experiment, in accordance with some embodiments of the present invention.
Figure 8. Summary effects of all the pro-inflammatory gene changes in Epilntestinal tissues treated with high-CBD cannabis extracts in a single bar for each condition.
TD - samples treated with TNF/IFN and then with DMSO; TD24 - TD sample collected 24 hours after addition of DMSO. “#1, #7, #9, #45, #130 and #274” - extracts added to samples treated with TNF/IFN. Figure 9. Key experimental outcomes and potential uses of novel high- CBD extracts.
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.
Figures 1A-1G. High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention:
Figure 1A. Chromatogram of #90 extract. Total THC equivalent 1.05% and CBD 4.58%.
Figure IB. Chromatogram of #131 extract. Total THC equivalent 0.12% and CBD 2.24%.
Figure 1C. Chromatogram # 115 extract. Total THC equivalent 0.4% and CBD 9.54%.
Figure ID. Chromatogram of #129 extract. Total THC equivalent 0.34% and CBD 6.75%.
Figure IE. Chromatogram of #157 extract. Total THC equivalent 0.2% and CBD 3.78%.
Figure IF. Chromatogram of #167 extract. Total THC equivalent 0.08% and CBD 2.2 %.
Figure 1G. Chromatogram of #169 extract. Total THC equivalent 0.21% and CBD 1.88%.
Figure 2. EpiOral tissues and experimental set-up. A. EpiOral tissues consist of normal, human-derived oral epithelial cells that have been cultured to form multilayered, highly differentiated models of the human buccal phenotypes. B. Scheme of LPS-induced inflammation experiment; C. Scheme of the TNF— induced inflammation experiment.
In a tissue preparation step 202 - 3D EpiOral tissues were used.
Further in an incubation step 204, 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT-400 was replaced and incubated for another 24 h. Then, set of tissues was treated for 24 h with 10 pg/ml of E. coli LPS, or DMSO only for control.
In an extract treatment step 206, 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 was used. 24 h after LPS treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
In an analysis of data step 208, tissues were collected for RNA and protein extraction. RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
In an equilibration step 210, 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT- 400 was replaced and incubated for another 24 h. Then, a set of tissues was treated for 24 h with TNFa (40 ng/ml) and IFNy (5 ng/ml), or DMSO only as a control.
In an extract treatment step 212, 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 was used. 24 h after TNF/IFN treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
In an analysis of data step 214, tissues were collected for RNA and protein extraction. RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
Figure 3. Effects of extracts #90 and 131 on LPS-induced inflammation.
Gene expression changes of each inflammatory gene in LPS vs DMSO (x-axis) and the same change in LPS extract co-treatment vs DMSO (y-axis). Tissues were treated for 24 h with 10 pg/ml of E. coli LPS, after which the extracts were added and the samples were incubated for another 24h; DMSO alone was added to LPS sample. A. Extract #90; B. Extract #131.
Figure 4. Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (LPS vs DMSO, LPS-#131 vs DMSO, and LPS-#90 vs DMSO).
Tissues were treated for 24 h with 10 pg/ml of E. coli LPS, after which the extracts were added and the samples were incubated for another 24h; DMSO alone was added to LPS sample. Y axis shows cumulative arbitrary expression of all pro- inflammatory genes listed in Table 1.
Figure 5. Summary effects of all the pro-inflammatory gene changes in a single bar for each condition (TNF vs DMSO, and extracts # 115, 129, 157, 167, 169).
Tissues were treated for 24 h with TNF/IFN, after which the extracts were added and the samples were incubated for another 24h; DMSO alone was added to LPS sample. Y axis shows cumulative arbitrary expression of all pro-inflammatory genes listed in Table 1.
Figures 6A-6F. High performance liquid chromatography (HPLC) profiles of tested lines, in accordance with some embodiments of the present invention.
A. Chromatogram of #1 - Total THC equivalent 0.25% and CBD 6.79%.
B. Chromatogram of #7 extract. Total THC equivalent 0.21% and CBD 7.2%.
C. Chromatogram of #9 extract. Total THC equivalent 0.22% and CBD 6.91%.
D. Chromatogram of #45 extract. Total THC equivalent 0.03% and CBD 1.61%.
E. Chromatogram of #130 extract. Total THC equivalent 0.86% and CBD
2.63%.
F. Chromatogram of #274 extract. Total THC equivalent 0.5% and CBD
10.3%.
Figure 7. Epilntestinal Tissues and experimental set-up. A. A method using Epilntestinal tissues to detect anti-inflammatory properties of extracts. Epilntestinal model has normal small intestine epithelial cell structure and is constructed from human-derived columnar epithelial and endothelial cells. It exhibits in vzvo-like growth and morphological characteristics whereby cells sustain differentiation and metabolic status similar to those of human intestinal epithelium. B. Scheme of TNFa/IFNy-i induced inflammation experiment, in accordance with some embodiments of the present invention. Step 702- a cultivar growing step. Around 250 unique marijuana and around 120 unique hemp cultivars were used to generate approximately 1,200 marijuana/marijuana, hemp/hemp and hemp/marijuana hybrids. Cultivars are typically grown in soil/vermiculite (2:1) mix. First, plants are grown under 16h day, 8h night for approximately 6 weeks when they were moved to another grow room and grown at 12h day and 12h night for another 6-8 weeks until they developed mature flowers. In both rooms, they were grown under the high pressure sodium (HPS) lights of -400 W/m2. Collected flowers were then tested for cannabinoids and terpenoids and those with most diversity in composition, or those that had highest amount of one or more cannabinoid or terpenoid or those that had the presence of unique terpenoids were used for breeding. The progeny of these crosses was then grown and further tested for cannabinoids/terpenoids as well as for growth parameters, such as height, response to nutrients, responses to pathogens, amongst others. In some cases, these plants were then crossed again using siblings with similar traits (cannabinoids/terpenoids for example). The seeds of these cultivars (resulting from crosses) are stored at +4 °C in the fridge in the locked cage. Approximately 600 strains with the best parameters, such as diversity of cannabinoids and terpenoids, plant growth vigor (germination rate, mutation time, yield of flowers, nutrients response, response to pathogens, size of flowers) and other features such as distinct smell for example were germinated and approximately 400 extracts were made.
Step 704, an extraction step, such as organic solvent extraction. Most solvents can be used. In one experiment ethyl acetate was used. This should not be deemed as limiting. For extract preparation, 3 g of the powdered flower tissue were used in 100 ml of ethyl acetate in a 250 mL Erlenmeyer flask. The flasks were then wrapped with tin foil and shaken continuously (120 rpm) in an incubator at 21°C overnight and in the dark. After overnight solvent extraction the extracts were filtered through cotton into a 100 ml round bottom flask. The extracts were concentrated to around 2-3 ml using a rotary vacuum evaporator. The extracts were then transferred to a tared 3 dram vial. The leftover solvent was evaporated to dryness in an oven overnight at 50°C to eliminate the solvent completely. Mass of each extract was recorded, and the extracts were stored at -20°C. 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) to reach 60 mg/mL final concentration and stored at - 20°C. Around 400 solvent-based crude extracts of flowers were thus generated.
In an extract biological assay step 706, extracts were applied to Epilntestinal tissues as per below.
In a, data analysis step 708, data were obtained from harvested tissue and altered pathways were analyzed bioinformatically.
In a treatment step 710, 3D tissues are inserted in a well with medium. Tissues were equilibrated in EFT-400 for 24 h (overnight) then culture media EFT-400 was replaced and incubated for another 24 h. Then, set of tissues was treated for 24 h with TNFa (40 ng/ml) and IFNy (5 ng/ml), or DMSO only for control.
In an extract treatment step 712, 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 was used. 24 h after TNF/IFN treatment, 15 uL of 0.01 ug/uL extract solution or control were applied to the tissue and incubated for 24 hours.
In an analysis of data step 714, tissues were collected for RNA and protein extraction. RNA was used for mRNA analysis by sequencing. Bioinformatics analysis of mRNA revealed changes in biological pathways associated with inflammation (see Table 1 for the detail listed of inflammation-related genes). Extracts with most pronounced changes were identified.
Figure 8. Summary effects of all the pro-inflammatory gene changes in Epilntestinal tissues treated with high-CBD cannabis extracts in a single bar for each condition.
TD - samples treated with TNF/IFN and then with DMSO; TD24 - TD sample collected 24 hours after addition of DMSO. “#1, #7, #9, #45, #130 and #274” - extracts added to samples treated with TNF/IFN.
Figure 9. Key experimental outcomes and potential uses of novel high- CBD extracts.
Extracts were selected based on their strong anti-inflammatory properties. Treatment and prevention of oral and gingival diseases, IBD, IBS and other inflammatory conditions can be done by using either extracts alone or in combination with other anti-inflammatory herbs or/and probiotics.
In a hybrid flower extraction step 902, identification of novel anti- inflammatory extracts hybrids of different cannabis varieties are created and full flower extracts of various hybrids are prepared. These extracts are then applied to various 3D tissues treated with LPS or with TNF/IFN. The tissues are then harvested and used for mRNA-seq and protein analysis.
In an inflammatory gene identification step step 904, a set of genes involved in inflammation response are identified and extracts are stratified according to the capacity to downregulate such pro-inflammatory genes (see Table 1).
In a downregulating inflammation testing step 906, extracts are used alone or in combination with other herbs, probiotics, vitamins and other anti-inflammatory molecules to downregulate TNF, interleukins, cytokines and other pro -inflammatory genes (Table 1).
In an inflammation treating step 908, formulations are used for prevention and treatment of oral and gingival diseases, IBD, IBS and other inflammatory conditions.
MATERIALS AND METHODS
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 left over solvent was evaporated to dryness in an oven overnight @ 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 + 10% FBS or EMEM + 10% FBS) was used to dilute the 60 mg/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 um syringe filter. These filtrates were ready to be applied to cells and tested.
MODELS:
EpiOral tissue model: 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 tissue model (Mat Tek) was used as inflammation model. Epilntestinal is a 3D reconstructed tissue model produced from primary, human cell- derived small intestine epithelial and endothelial cells and fibroblasts. The highly differentiated tissue model is produced at the air-liquid-interface (ALI) in easy-to- handle tissue culture inserts. Structural analysis of the tissue model demonstrates columnar shaped basal cells and Kerckring folds. Ultrastructurally, Epilntestinal exhibits brush borders, functional tight junctions and mucous secreting granules, similar to in vivo tissue. It exhibits in vivo-like growth and morphological characteristics whereby cells sustain differentiation and metabolic status similar to those of human intestinal epithelium.
Exposure and inflammation induction by LPS or TNFa/IFNy^
Tissues were equilibrated for 24 h (overnight) then culture media was replaced and incubated for another 24 h. Tissues were then exposed for 24 h to either to LPS (10 ug/ml LPS) or to TNFa (40 ng/ml) and IFNy (5 ng/ml) or to DMSO only, resulting in TNF/IFN” or “Ct” samples, respectively.
EpiOral tissues treated with LPS were then treated with two extracts - from lines #90 and #131, whereas EpiOral tissues treated with TNF/IFN were treated with cannabis extracts from lines #115, #129, #157, #167 and #169.
Epilntestinal tissues treated with TNF/IFN were then treated with cannabis extracts from lines #1, #7, #9, #45, #274, #130.
All crude extracts were diluted from a 60 mg/mL stock (the stock is prepared in DMSO). After LPS or TNF/IFN exposure, 15 pL of crude extract solution or control (media with DMSO only) were applied to the tissue after exposure, resulting in treatment samples labeled as “LPS” or “TNF” or treatment samples with extracts, labeled as “#1”, “#7” etc. or control samples, labeled as “DMSO”. The samples then were collected for the analysis in 24h after exposure.
Application volume of extracts: 15 pL of these solutions were applied on top of the tissues (inside the cup holding the tissue) to the final concentration of the extract of 0.015 mg/ml ensuring even coverage of the tissue surface.
Tissues once treated were allowed to equilibrate at 37°C in an incubator with 6% CO2, for 24 hours. Then all tissues were frozen using liquid N2 and stored at - 80°C.
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.
Results for EpiOral tissue experiments
Described herein is a method for treating oral disorders including, but is not limited to the steps of: 1) preparation of new cannabis extracts, 2) exposing normal and disease skin or oral tissue models to novel extracts and 3) modulating the gene expression 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.
The effect of two extracts of novel cannabis lines (#90, #131) on inflammation provoked by LPS treatment in EpiOral tissues and of five novel cannabis extracts (#115, #129, #157, #167, #169) on inflammation provoked by TNF treatment in EpiOral tissues were analyzed.
First, the reversion of the gene expression profile changes was observed after compound co-treatment with LPS. In order to do this, significant gene expression changes were detected (fold change above +1 or -1 and FDR<0.01*) between the LPS samples and the different compound co-treatments (LPS and extracts #90; LPS and #131).
These gene expression alterations led us to assess the biological pathways that were affected for specific inflammatory response (LPS) and each compound (#90, #131). It was observed that the LPS treatment produced significant gene expression changes compared to DMSO, indicating a powerful effect of LPS treatment. The new extract treatments significantly reverted these gene expression changes.
Another approach was performed to observe the anti-inflammatory effect of the extracts in the LPS induced model. To do so, the set of human genes that are annotated as participants for ‘inflammatory response’ by Gene Ontology (Table 1) was obtained.
Results for Epilntestinal tissue experiments
The effect of six extracts of novel cannabis lines (#1, #7, #9, #45, #274, #130) on inflammation provoked by TNFa/IFNy treatment in Epilntestinal tissues were analyzed.
First, the reversion of the gene expression profile changes was observed after compound co-treatment with TNFa/IFNy. In order to do this, significant gene expression changes were detected (fold change above +1 or -1 and FDR<0.01*) between the TNFa/IFNy samples and the different compound co-treatments.
The effect of all the pro-inflammatory gene changes was summed in a single bar for each condition (Figure 8). This figure shows how TNFa/IFNy treatment produced a significant over-expression of pro-inflammatory signature that was significantly decreased or completely abolished by the extract treatments. Treatment with #7 produced the strongest negative response that can be considered as a major reversion, and #9, #130, #274 reversed most of the TNFa/IFNy pro-inflammatory response (Figure 8).
In sum, treatments of intestinal tissues with TNFa/IRNg caused inflammation, and further treatment with extracts of new cannabis lines (#1, #7, #9, #45, #274, #130) significantly affected gene expression in the intestinal tissues, leading to down- regulation of genes and pathways involved inflammation, immunity and autoimmunity and related pathways.
Extracts can be used alone or in combination with anti-inflammatory plants and/or probiotics to potentiate their anti-inflammatory effects on the GI inflammatory diseases and conditions (Figure 9).
Table 1. List of genes used for calculation of level of gene expression in inflammation
Figure imgf000028_0001
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Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Then, the gene expression change of each inflammatory gene in LPS vs DMSO (x-axis) was plotted and the same change in LPS extracts co-treatment vs DMSO (y-axis). If the points are located toward the origin point (0, 0), that indicates a minor inflammatory response change in both LPS and extract co-treatments. If the points are located near the x-axis, this indicates an effect on LPS samples that disappears after extract co-treatment. Their location in the diagonal would indicate an effect on LPS that is independent of the compound (Figure 3).
This information was further combined in a single bar figure. To do so, the effect of all the pro-inflammatory gene changes was summed in a single bar for each condition (LPS vs DMSO, LPS-#131 vs DMSO, and LPS-#90 vs DMSO). Treatment with #90 produced a negative response that can be considered as a major reversion, and #131 reversed most of the LPS pro-inflammatory response (Figure 4).
Further, the anti-inflammatory effects of extracts were analysed in the TNF- induced model, 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 #115, 129, 157, 167, 169). This figure shows how TNF treatment produced a significant over-expression of pro-inflammatory signature that was completely abolished by extracts. Treatment with #115, #129, and #169 were the strongest of all, albeit all extracts reversed most of the TNF pro-inflammatory response (Figure 5).
Further, the anti-inflammatory effects of extracts were analysed in the TNF- induced model of Epilntestinal tissues, 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, #130 and #274). This figure (Figure 8) shows how TNF treatment produced a significant over-expression of pro-inflammatory signature that was completely abolished by extracts. Treatment with #7, and #274 were the strongest of all, albeit all extracts reversed most of the TNF pro- inflammatory response (Figure 8).
In sum, treatments of oral and intestinal tissues with LPS or TNF caused inflammation, and further treatment with extracts of new cannabis lines significantly affected gene expression in the oral tissues, leading to down-regulation of genes and pathways involved inflammation, immunity and autoimmunity and related pathways. Lines were identified with the strong anti-gingivitis, anti-periodontitis, anti-stomatitis, and anti -cancer properties based on the expression of genes that are associated with molecular etiology and pathogenesis of oral diseases.
In a more specific embodiment, there is provided a method for oral inflammatory diseases and conditions that includes the steps of exposing a patient to novel cannabis extracts, resulting in a reduction in the disease 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 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 (Table 1).
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.
The presented Examples (Figures 3,4) 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. 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 intradermally. 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, intraarterial, 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 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 al., 1981; Newmark et al., 1982; and Katre et al., 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.
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3. Chey, W.D., J. Kurlander, and S. Eswaran, Irritable bowel syndrome: a clinical review. JAMA, 2015. 313(9): p. 949-58.
4. Farzaei, M.H., et ah, The Role of Visceral Hypersensitivity in Irritable Bowel Syndrome: Pharmacological Targets and Novel Treatments. J Neurogastroenterol Motil, 2016. 22(4): p. 558-574.
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Claims

1. A method for treating a mammalian oral 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; and c) treating said oral disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said oral disease or disorder.
2. A method according to claim 1, wherein said treating step induces modulation of gene expression in at least one of oral cells and oral tissue; and wherein said modulation of gene said expression results in a reduction of at least one of an oral disease state, an inflammatory state, an oral disorder state and combinations thereof.
3. A method according to claim 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.
4. A method according to claim 3, wherein said at least one line is selected from the group consisting of designated lines #90, #115, #129, #131, #157, #167, #169 and combinations thereof.
5. A method according to claim 1, wherein said extracting step comprises extracting flowers of said at least one Cannabis line.
6. A method according to claim 5, wherein said extracting step comprises extracting said at least one compound in at least one organic solvent.
7. A method according to claim 6, 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.
8. A method according to claim 2, 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, GATA3, 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 and ZYX and combinations thereof.
9. A method according to claim 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.
10. A method according to claim 1, wherein said at least one compound is provided in a solvent extract and said solvent extract exhibits oral disease or oral disorder healing properties.
11. A method according to claim 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.
12. The method of claim 2, wherein the disease state is an inflammatory oral disease.
13. The method of claim 1, wherein the oral disease or oral disorder is selected from the group consisting of an oral insult, an oral disorder, an oral disease, environmental factor-induced inflammation and combinations thereof.
14. A method according to claim 1, wherein said Cannabis line is a Cannabis sativa line.
15. 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.
16. An organic extract according to claim 15, wherein said at least one plant line comprises a Cannabis sativa line.
17. An organic extract according to claim 15, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation and combinations thereof.
18. An organic extract according to claim 15, wherein said extract is effective against chemo-resistant cancer cells and is suitable to overcome chemo- resistance.
19. An organic extract according to claim 15, wherein said extract potentiates effects of cytotoxic chemotherapy and is an effective and safe adjuvant modality.
20. An organic extract according to claim 15, 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.
21. 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 oral disease or disorder.
22. A combination therapy according to claim 21, wherein said mammalian oral disease or disorder is selected from the group consisting of an oral cancer, an inflammatory oral disease, an oral burn, an oral cut, an oral scar, an oral insult, periodontitis, gingivitis, stomatitis, an environmental factor-induced oral inflammation and combinations thereof.
23. A Cannabis extract for treating an oral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
24. 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 #90, #115, #129, #131,
#157, #167, #169.
25. New lines of Cannabis sativa and new extracts as 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 a unique extract; and b) treating the oral tissue with aforementioned extracts in an effective amount to induce modulation of gene expression in the oral cells or oral tissue; wherein the modulation of gene expression results in a reduction of the disease state in the oral cells or oral tissue.
26. The method of claim 25, wherein the extracts is extract #90.
27. The method of claim 25, wherein the extracts is extract #131.
28. The method of claim 25, wherein the extracts is extract #115.
29. The method of claim 25, wherein the extracts is extract #129.
30. The method of claim 25, wherein the extracts is extract #157.
31. The method of claim 25, wherein the extracts is extract #167.
32. The method of claim 25, wherein the extracts is extract #169.
33. The method of claim 25, wherein the modulation of gene expression is a down-regulation of gene expression.
34. The method of claim 25, wherein the modulation of gene expression is an up- regulation of gene expression.
35. The method of claim 25, wherein the disease state is periodontitis.
36. The method of claim 25, wherein the disease state is gingivitis.
37. The method of claim 25, wherein the disease state is stomatitis.
38. The method of claim 25, wherein the disease state is post-cancer treatment oral inflammation.
39. The method of claim 25, wherein the disease state is oral cancer.
40. The method of any of claims 1-39, further comprising at least one of CBD, CBG, CBN, terpenes or combinations thereof to said extract to attenuate at least one effect of said treatment.
41. The method of any of claims 1-40, further comprising at least one of turmeric, chamomile, sage, fennel, ginger, rosehip, as well as probiotics combinations thereof to said extract to attenuate at least one effect of said treatment.
42. A method for treating a inflammatory GI disease or disorder, the method comprising: a) combining at least one marijuana or hemp cultivar and at least one another hemp or marijuana cultivar to form at least one Cannabis line; b) extracting at least one compound from said at least one Cannabis line to form an extract; and c) treating said GI disease or disorder with at least one of said extract and said at least one compound in an effective amount to treat said intestinal disease or disorder.
43. A method according to claim 42, wherein said treating step induces modulation of gene expression in at least one of intestinal cells and intestinal tissue; and wherein said modulation of gene said expression results in a reduction of at least one of an intestinal disease state, an inflammatory state, an intestinal disorder state and combinations thereof.
44. A method according to claim 42, 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.
45. A method according to claim 44, wherein said at least one line is selected from the group consisting of designated lines (#1, #7, #9, #45, #274, #130) and combinations thereof.
46. A method according to claim 42, wherein said extracting step comprises extracting flowers of said at least one Cannabis line.
47. A method according to claim 46, wherein said extracting step comprises extracting said at least one compound in at least one organic solvent.
48. A method according to claim 47, 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.
49. A method according to claim 42, 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, GATA3, 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 and ZYX and combinations thereof.
50. A method according to claim 42, 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.
51. A method according to claim 42, wherein said at least one compound is provided in a solvent extract and said solvent extract exhibits oral disease or oral disorder healing properties.
52. A method according to claim 51, 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.
53. The method of claim 43, wherein the disease state is an inflammatory GI disease.
54. The method of claim 42, wherein the GI disease or GI disorder is selected from the group consisting of a GI insult, a GI disorder, a GI disease, environmental factor-induced inflammation and combinations thereof.
55. A method according to claim 42, wherein said Cannabis line is a Cannabis sativa line.
56. 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 or hemp cultivar; and b) at least one other marijuana or hemp cultivar, wherein said organic extract comprises at least one compound suitable for treating a mammalian oral disease or disorder.
57. An organic extract according to claim 56, wherein said at least one plant line comprises a Cannabis sativa line.
58. An organic extract according to claim 56, wherein said mammalian GI disease or disorder is selected from the group consisting of an GI organ cancer, an inflammatory GI disease, IBS, IBD, gastritis, esophagitis, colitis, duodenitis, oral tissue inflammatory diseases, an environmental factor-induced oral inflammation and combinations thereof.
59. An organic extract according to claim 56, wherein said extract is effective against chemo-resistant GI cancer cells and is suitable to overcome chemo- resistance.
60. An organic extract according to claim 56, wherein said extract potentiates effects of cytotoxic chemotherapy and is an effective and safe adjuvant modality.
61. An organic extract according to claim 56, 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.
62. 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 or hemp cultivar; and b) at least one other marijuana or hemp cultivar; and wherein said organic extract comprises a plurality of compounds suitable for treating a mammalian oral disease or disorder.
63. A combination therapy according to claim 62, wherein said mammalian GI disease or disorder is selected from the group consisting of an GI cancer, an inflammatory GI disease, IBS, IBD, gastritis, esophagitis, colitis, duodenitis, oral tissue inflammatory diseases, an environmental factor-induced GI inflammation and combinations thereof.
64. A Cannabis extract for treating an oral disease or disorder, wherein said extract efficacy can be further increased by adding CBD, CBG, CBN, terpenes or combinations thereof.
65. A line of Cannabis sativa formed by combining at least one marijuana or hemp cultivar and at least one other marijuana or hemp cultivar, said line will be deposited at public culture collection, currently under designation numbers #1, #7, #9, #45, #274, #130.
66. New lines of Cannabis sativa and new extracts as a method for treating a disease state in GI epithelial cells or GI tissue, the method comprising the steps of: a) providing a source of a unique extract; and b) treating the oral tissue with aforementioned extracts in an effective amount to induce modulation of gene expression in the GI cells or GI tissue; wherein the modulation of gene expression results in a reduction of the disease state in the GI cells or GI tissue.
67. The method of claim 66, wherein the extracts is extract #1.
68. The method of claim 66, wherein the extracts is extract #7.
69. The method of claim 66, wherein the extracts is extract #9.
70. The method of claim 66, wherein the extracts is extract #45.
71. The method of claim 66, wherein the extracts is extract #274.
72. The method of claim 66, wherein the extracts is extract #130.
73. The method of claim 66, wherein the modulation of gene expression is a down-regulation of gene expression.
74. The method of claim 66, wherein the modulation of gene expression is an up- regulation of gene expression.
75. The method of claim 66, wherein the disease state is periodontitis.
76. The method of claim 66, wherein the disease state is gingivitis.
77. The method of claim 66, wherein the disease state is stomatitis.
78. The method of claim 66, wherein the disease state is gastritis.
79. The method of claim 66, wherein the disease state is esophagitis.
80. The method of claim 66, wherein the disease state is enteritis.
81. The method of claim 66, wherein the disease state is colitis.
82. The method of claim 66, wherein the disease state is duodenitis.
83. The method of claim 66, wherein the disease state is IBS.
84. The method of claim 66, wherein the disease state is ulcerative colitis.
85. The method of claim 66, wherein the disease state is Crohn’s disease.
86. The method of claim 66, wherein the disease state is proctitis.
87. The method of claim 66, wherein the disease state is sigmoiditis.
88. The method of claim 66, wherein the disease state is post-cancer treatment oral inflammation or post-cancer treatment GI inflammation.
89. The method of claim 66, wherein the disease state is oral cancer.
90. The method of claim 66, wherein the disease state is esophageal cancer
91. The method of claim 66, wherein the disease state is gastric cancer
92. The method of claim 66, wherein the disease state is colon cancer
93. The method of claim 66, wherein the disease state is rectal cancer
94. The method of any of claims 42-93, further comprising at least one of CBD, CBG, CBN, terpenes, turmeric, chamomile, sage, fennel, ginger, rosehip, probiotics or any combination thereof to said extract to attenuate at least one effect of said treatment.
PCT/IL2020/051237 2019-12-17 2020-12-01 High cbd cannabis sativa lines and extracts with anti-inflammatory potencies for oral and gut health WO2021124316A1 (en)

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