Fixed dose combination of cannabinoids and medicinal mushrooms for prevention and treatment of cancer, inflammatory or immune-mediated inflammatory diseases

Abstract

Disclosed is a composition that can be used to treat an immune-mediated inflammatory disease or cancer of a mammalian receptor, wherein the composition comprises a synergistic combination of a cannabis derivative compound and a pharmaceutically acceptable mushroom.

Description

[0001] Divisional application

[0002] This application is a divisional application. The application number of the original application is 202080038025.4, the application date is March 20, 2020, and the name of the invention is "Fixed-dose combination of cannabinoids and medicinal mushrooms for the prevention and treatment of cancer, inflammatory or immune-mediated inflammatory diseases." Technical Field

[0003] The present invention generally relates to novel therapeutic approaches and methods for cancer, inflammatory or immune-mediated inflammatory diseases.

[0004] More specifically, the present invention relates to synergistic interactions and fixed dose combinations of cannabinoids and medicinal mushrooms for the prevention and treatment of cancer, inflammatory or immune-mediated inflammatory diseases. Background Art

[0005] While cannabinoids (CBD, THC, and some others) are known to be potent anti-inflammatory agents with known anti-cancer activity, it has also been found that these compounds may impair, inhibit, or reduce immune-mediated processes. Reducing immune-mediated processes may have a positive impact in some clinical indications, but may have a negative impact on processes that require immune stimulation and anti-inflammatory effects (such as cancer).

[0006] Furthermore, most chemotherapeutic agents used to treat cancer may destroy tumors and prevent cancer progression as well as damage healthy cells and tissues during the course of treatment, furthermore, negatively affect cancer immunity which may reduce some of their beneficial anti-tumor effects.

[0007] Therefore, there is an unmet need for new more effective and safer agents for use as anti-cancer therapies and treatments of other immune-mediated inflammatory diseases (IMIDs). Summary of the invention

[0008] There is an unmet need for new more effective and safer agents for use as anticancer treatments and for the treatment of other immune-mediated inflammatory diseases (IMIDs) where a combination of anti-inflammatory and pro-immuno (mainly pro-Th1 type) or related cellular effectors (NK cell stimulation) may be considered beneficial.

[0009] While cannabinoids are anti-inflammatory and immunosuppressive, medicinal mushrooms are known to stimulate immunity while having some anti-inflammatory effects, so under certain conditions and in certain proportions, their combination will have a unique and unexpected synergistic anti-inflammatory activity spectrum as well as selective immunostimulatory properties.

[0010] An object of the present invention is to disclose a composition for treating immune-mediated inflammatory diseases or cancer and any combination thereof in a mammalian subject, wherein the composition comprises a synergistic combination of a. cannabis-derived compounds and b. medicinal mushrooms.

[0011] Another object of the present invention is to disclose a composition as defined in any one of the above items,

[0012] An object of the present invention is to disclose a synergistic composition useful for synergistically treating immune-mediated inflammatory diseases or cancers and any combination thereof, the composition comprising:

[0013] a. Cannabis-derived compounds that are agonists to CB1 or CBD receptors, or any combination thereof;

[0014] b. Medicinal mushrooms that have agonistic properties on pattern recognition receptors (PRRs);

[0015] wherein said cannabis-derived compounds are therapeutically anti-inflammatory and said medicinal mushrooms are immunostimulatory in mammalian receptors.

[0016] Another object of the present invention is to disclose a composition as defined in any one of the above items, wherein the immune-mediated inflammatory disease is selected from the group consisting of: asthma, chronic obstructive pulmonary disease (COPD); psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), ulcerative colitis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy; giant cell arteritis; GN, glomerulonephritis; juvenile idiopathic arthritis, polymyalgia rheumatica; SAPHO, synovitis, acne, impetigo, bone hyperplasia, osteitis, systemic lupus erythematosus; thrombotic / idiopathic thrombocytopenic purpura, Sjogren's syndrome, Crohn's disease, polymyositis / dermatomyositis, Wegener's granulomatosis, Wegener's vasculitis vasculitis, psoriasis, Behcet's disease, polyarteritis nodosa, Takayasu's arteritis, graft-versus-host disease, polyarteritis nodosa, sarcoidosis, adult-onset Still's disease, hydradenitis supprativa, cryoglobulinaemic vasculitis, pyoderma gangrenosum, Kawasaki disease, relapsing polychondritis, antiphospholipid syndrome, cryoglobulinaemic vasculitis, hidradenitis supprativa, idiopathic membranous, relapsing polychondritis, coeliac disease, chronic hepatitis C, myasthenia gravis, myelodysplastic syndrome pemphigus, refractory asthma, Graves' disease disease), pyoderma gangrenosum, erythema nodosum, SAPHO syndrome, multicentric reticulohistiocytosis, chronic hepatitis B, amyloidosis, and any combination thereof.

[0017] Another object of the present invention is to disclose the composition as defined in any one of the above items, wherein the pattern recognition receptor (PRR) is selected from the group consisting of To11-like receptor (TLR), mannose receptor, dectin1 and any combination thereof.

[0018] It is another object of the present invention to disclose the composition as defined in any one of the above items, wherein the medicinal mushroom is selected from the group consisting of Cordyseps, Maitake (Grifolafrondosa), Turkey Tail (Trametes versicolor, Coriolus versicolor), Reishi (Ganoderma lucidum), Chaga (Inonotus obliquus), Shiitake (Lentinus edodes), Agaricus Blazei Murill, and any combination thereof.

[0019] It is another object of the present invention to disclose the composition as defined in any of the above items, wherein said cannabis-derived compounds are selected from the group consisting of cannabinoids, terpenes, phenolic compounds and any combination thereof.

[0020] It is another object of the present invention to disclose a composition as defined in any of the above items, wherein the cannabinoid is at least one of the following: Tetra-hydro-cannabinoids (d9-THC), Tetrahydrocannabinol (d8-THC), Tetra-hydro-cannabinolic acid (THCA-d9), Tetra-hydro-cannabivarin (THCV / THC-C3), Cannabidiol (CBD), Cannabidiolic acid (CBDA), Cannabidivarin (CBDV), Cannabigerol (CBG), Cannabigerolic acid (CBGA), Cannabinol (CBN), Cannabidiolic acid (CBNA), Cannabichromene (CBC), Cannabichromenic acid (CBCA), and any combination thereof.

[0021] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the terpenes are monoterpenes or sesquiterpenes.

[0022] Another object of the present invention is to disclose a composition as defined in any one of the above items, wherein the phenolic compound is at least one of the following: O-glycoside Cannaflavin A, Cannaflavin B, Cannabisin D, and any combination thereof.

[0023] It is another object of the present invention to disclose the composition as defined in any one of the above items, wherein the composition is configured to be administered in a manner selected from the group consisting of an inhaler, a cigarette, a tablet, a capsule, a pill, a lyophilized powder, a powder, an emulsion, a granulated powder, a cream, an ointment, a paste, a lotion gel, a liquid, a solution, a patch, and any combination thereof.

[0024] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the composition is configured to be administered in a manner selected from the group consisting of fast release, slow release, sustained release, controlled release and any combination thereof.

[0025] Another object of the present invention is to disclose a composition as defined in any of the above items, wherein the composition further comprises an ingredient selected from the group consisting of a solubilizer, a stabilizer, a buffer, a tonicity adjuster, a filler, a viscosity enhancer / reducer, a surfactant, a chelating agent, an adjuvant and any combination thereof.

[0026] An object of the present invention is to disclose a method for treating immune-mediated inflammatory diseases or cancer and any combination thereof, comprising the following steps:

[0027] a. providing a composition, wherein the composition comprises a. a synergistic combination of a cannabis-derived compound and b. a medicinal mushroom; and

[0028] b. administering the composition to a mammalian recipient.

[0029] An object of the present invention is to disclose a method for treating immune-mediated inflammatory diseases or cancer and any combination thereof, comprising the following steps:

[0030] a. Providing a composition comprising: a cannabis-derived compound having agonism to CB1 or CBD receptors and any combination thereof, and a medicinal mushroom having agonism to a pattern recognition receptor (PRR); and,

[0031] b. administering the composition to a mammalian recipient;

[0032] wherein said cannabis-derived compound is therapeutically anti-inflammatory and said medicinal mushroom is immunostimulatory in said mammalian receptor.

[0033] An object of the present invention is to disclose a composition useful for treating immune-mediated inflammatory diseases or cancers and any combination thereof in a mammalian subject, the composition comprising

[0034] a. at least one medicinal mushroom that is agonistic to a pattern recognition receptor (PRR); and

[0035] b. At least a second medicinal mushroom.

[0036] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the composition for treating immune-mediated inflammatory diseases comprises Cordyceps M mushroom extract and THC.

[0037] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the treatment of immune-mediated inflammatory diseases comprises inhibiting nitric oxide secretion.

[0038] Another object of the present invention is to disclose the composition as defined in any one of the above, wherein the composition is useful for treating immune-mediated inflammatory diseases, the composition comprising Ganoderma lucidum mushroom extract and THC.

[0039] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the treatment of immune-mediated inflammatory diseases comprises reducing IL-6 secretion.

[0040] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the composition useful for treating immune-mediated inflammatory diseases comprises Cordyceps sinensis Cs-4 mushroom extract THC.

[0041] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the treatment of immune-mediated inflammatory diseases comprises reducing TNFα secretion.

[0042] It is another object of the present invention to disclose the composition as defined in any one of the above, wherein the treatment of immune-mediated cancer is provided by the composition comprising CBD and a mushroom extract selected from the group consisting of: Cordyceps (M) mushroom extract and Ganoderma lucidum mushroom extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Recommended plate formate for use in the nitric oxide standard reference curve;

[0044] Figure 2: Cordyceps sinensis Cs-4 (A) and Agaricus blazei (B) were incubated with 5000 PANC-1 cells for 2 hours, 6 hours, 24 hours, and 72 hours. Viability was measured using the XTT kit;

[0045] Figure 3: Cordyceps sinensis Cs-4 (A), Cordyceps sinensis M (B), Ganoderma lucidum (C), Agaricus blazei (D), Turkey tail RM (E), Shiitake mushroom (F), Turkey tail MS (G), Chaga (H), and Maitake (I) were cultured with 105 RAW cells for 24 hours. NO secretion in the supernatant was measured using the Greiss kit;

[0046] Figure 4: Cordyceps sinensis Cs-4 (A), Cordyceps sinensis M (B), Ganoderma lucidum (C), and Agaricus blazei (D) were incubated with 105 RAW cells in the presence and absence of CBD or THC for 24 hours. IL-6 and TNFα in the supernatant were measured by multiplexing;

[0047] Figure 5: CBD (A) and THC (B) were incubated with 105 RAW cells for 24 hours. NO secretion in the supernatant was measured using the Greiss kit;

[0048] Figure 6: Ganoderma lucidum (A+B), Agaricus blazei (C+D), Cordyceps sinensis Cs-4 (E+F), and Cordyceps sinensis M (G+H) were combined with DMSO, CBD, or THC with 105 RAW cells for 24 hours. N0 secretion in the supernatant was measured using the Greiss kit;

[0049] Figure 7: Ganoderma lucidum, Cordyceps sinensis M, Agaricus blazei, Cordyceps sinensis Cs-4, Turkey Tail (MS) (A), Chaga, Maitake, Turkey Tail (RM), and Shiitake (B) were cultured with 7500 PANC-1 cells for 72 hours. Viability was measured using the XTT kit;

[0050] Figure 8: Ganoderma lucidum, Cordyceps sinensis M, Agaricus blazei, Cordyceps sinensis Cs-4, Turkey Tail (MS) (A), Chaga, Maitake, Turkey Tail (RM), and Shiitake (B) were cultured with 5000 EGL-1 cells for 72 hours. Viability was measured using the XTT kit;

[0051] Figure 9: CBD and THC were incubated with 7500 Panc-1 (A) and 5000 EGL-1 (B) cells for 72 hours. Viability was measured using the XTT kit; and

[0052] Figure 10: Ganoderma lucidum (A), Cordyceps sinensis M (B), Cordyceps sinensis Cs-4 (C), and Turkey tail (D) were combined with DMSO, CBD, or THC with 5,000 PANC-1 cells for 72 hours. Viability was measured using the XTT kit. DETAILED DESCRIPTION

[0053] The following description and all sections of the present invention are provided to enable any person skilled in the art to utilize the invention and to illustrate the best mode of carrying out the invention contemplated by the inventors. However, various modifications will be apparent to those skilled in the art since the general principles of the invention have been specifically defined to provide means and methods for novel treatments of cancer, inflammatory or immune-mediated inflammatory diseases and medical conditions, prevention and treatment of these medical conditions through the interaction and fixed dose combination of cannabinoids and medicinal mushrooms.

[0054] Disclosed herein are means and methods for treating cancer, inflammatory or immune-mediated inflammatory diseases by compositions that synergistically combine cannabinoids with therapeutic mushrooms.

[0055] The present invention discloses such compositions, which exert a combined effect of anti-inflammatory and immunostimulatory effects, thereby being used as anti-inflammatory or anti-cancer drugs without inhibiting or reducing immune-mediated processes. In addition, the compositions disclosed in the present invention are derived from natural products and can therefore play an important role in developing more effective and safer agents for inhibiting the development and onset of cancer, inflammatory or immune-mediated inflammatory diseases.

[0056] Specifically, the present invention discloses a synergistic combination of cannabinoids and therapeutic mushrooms. Cannabinoids (CBD, THC and a few others) are potent anti-inflammatory agents with known anti-cancer activity that also weaken, inhibit or attenuate immune-mediated processes. Attenuating immune-mediated processes may negatively impact processes that require immune stimulation and anti-inflammatory effects (e.g., cancer).

[0057] Therapeutic mushrooms are known to be immunostimulants that primarily affect Th1, NK and macrophage polarization. These properties may be attributed to their known and claimed anticancer and regulatory effects in some immunoinflammatory processes.

[0058] Immunostimulation by medicinal mushrooms usually occurs via innate immunity and is usually mediated by phagocytes. These cells ingest invading pathogens or interact with pathogen components, which in either case further stimulate innate and adaptive immunity by secreting cytokines and chemokines. Pattern recognition receptors (PRRs) on the cell surface (such as Toll-like receptors (TLRs), mannose receptors and dectinl) trigger responses by recognizing conserved molecular patterns in microorganisms. There is increasing attention to immunomodulatory substances from mushrooms. For example, Lentinan has been widely used in Japan to treat cancer patients.

[0059] Fungal bioactive polysaccharides, mainly from the Basidiomycetes (and some from Ascomycetes) and medicinal mushrooms, are well known and widely used in Far Asia as part of traditional diets and medicines, and over the past few decades have been the focus of intensive research for understanding and exploiting their medicinal properties in natural medicines. In fact, some of these biopolymers, mainly β-glucans or heteropolysaccharides, have entered the market as anti-tumor, immunostimulatory or preventive drugs. Many of these biopolymers are produced by edible mushrooms, making them also very good candidates for the formulation of novel functional foods and nutraceuticals in order to exploit their immunomodulatory, anti-cancer, anti-microbial, cholesterol-lowering, blood sugar-lowering and health-promoting properties without any serious safety issues.

[0060] Combining cannabinoids with therapeutic mushrooms reveals a novel spectrum composition with unique and potent effects on clinical conditions such as immune-mediated inflammatory diseases (IMIDs) and medical disorders and cancer.

[0061] The therapeutic mushrooms are also combined to reveal a composition that has therapeutic effects on clinical conditions such as immune-mediated inflammatory diseases (IMIDs) and medical disorders and cancer.

[0062] As used herein hereinafter, the term immune-mediated inflammatory diseases (IMIDs) is a concept used to collectively describe a group of seemingly unrelated disorders that share common inflammatory pathways.

[0063] Immune-mediated inflammatory diseases (IMIDs) include, but are not limited to: asthma, chronic obstructive pulmonary disease (COPD); psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), ulcerative colitis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy; giant cell arteritis; GN, glomerulonephritis; juvenile idiopathic arthritis, polymyalgia rheumatica; SAPHO, synovitis, acne, impetigo, osteophyte, osteitis, systemic lupus erythematosus; thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, Crohn's disease, polymyositis / dermatomyositis, Wegener's granulomatosis, Wegener's vasculitis, psoriasis , Behcet's disease, polyarteritis nodosa, Takayasu's arteritis, graft-versus-host disease, polyarteritis nodosa, sarcoidosis, adult-onset Still's disease, hydrops suppurativa, cryoglobulinemic vasculitis, pyoderma gangrenosum, Kawasaki disease, relapsing polychondritis, antiphospholipid syndrome, cryoglobulinemic vasculitis, hydrops suppurativa, idiopathic membranous, relapsing polychondritis, celiac disease, chronic hepatitis C, myasthenia gravis, myelodysplastic syndrome pemphigus, refractory asthma, Graves' disease, pyoderma gangrenosum, erythema nodosum, SAPHO syndrome, multicentric reticulohistiocytosis, chronic hepatitis B, amyloidosis, and any combination thereof.

[0064] As used herein below, the term "cannabis-derived compounds" refers to compounds found in cannabis plants, including cannabinoids, terpenes and phenolic compounds. As used herein below, the term "cannabinoids" refers to a class of different chemical compounds, which are ligands of cannabinoid receptors in cells that change the release of neurotransmitters in the brain. Cannabinoids are mainly found in marijuana (marijuana flowers) and hashish (compressed cannabis resin) from the cannabis (cannabis sativa) plant. This plant contains more than 80 phytocannabinoids. The main active ingredient of cannabis is psychoactive Δ9-tetrahydrocannabinol (Δ9-THC), which acts as a partial agonist on cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptors. Other important natural cannabinoids present in cannabis are non-psychoactive cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THCV) and cannabichromene (CBC) [1-3]. Among them, CBD has attracted the greatest attention so far. It is shown to antagonize the effects of CB1 / CB2 receptor agonists to counteract the psychoactive and other negative effects of Δ9-THC, and several data indicate that it behaves as an inverse agonist of CB1 and CB2 receptors. Some of these plant-derived cannabinoids are used in medical practice, such as Δ9-THC (dronabinol) and its synthetic analogs, nabilone against chemotherapy-induced nausea and vomiting, and as an appetite stimulant (e.g. in AIDS patients). CBD is used in combination with Δ9-THC (nabiximols) to relieve neuropathic pain and spasticity in multiple sclerosis, and as an adjunctive analgesic treatment in advanced cancer pain.

[0065] As used hereinafter, the term "terpene" refers to a large and diverse class of organic compounds produced by various plants. Terpenes are biosynthesized from isoprene units having the molecular formula C5H8, the basic molecular formula of terpenes being a multiple of (C5H8)n, where n is the number of linked isoprene units. Terpenes are the aromatic oils that give cannabis its aromatic diversity.

[0066] As used herein below, the term cannabidiol (CBD) refers to a component of cannabis that is a pharmacologically broad-spectrum drug that has attracted increasing attention in recent years as a treatment for a range of conditions.

[0067] The cannabis-derived compounds include:

[0068] Cannabinoids: tetrahydrocannabinol (d9-THC), tetrahydrocannabinol (d8-THC), tetrahydrocannabinolic acid (THCA-d9), tetrahydrocannabinol (THCV / THC-C3), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidiol (CBDV), cannabigerol (CBG), cannabigeric acid (CBGA), cannabinol (CBN), cannabidiolic acid (CBNA), cannabichromene (CBC), cannabichromene acid (CBCA);

[0069] Terpenes: monoterpenes and sesquiterpenes; and

[0070] Phenolic compounds: 0-glycosides, cannaflavin A, cannaflavin B, cannabinamide D.

[0071] As used herein hereinafter, the term "about" means ±25% of a defined amount or measurement or value.

[0072] As used hereinafter, the term "therapeutic mushrooms" or "medicinal mushrooms" includes, but is not limited to, the following mushrooms: Cordyceps sinensis, Maitake (Grifola frondosa), Turkey Tail (Trametes versicolor, Trametes versicolor), Chaga (Inonotus obliquus), Reishi (Ganoderma lucidum), Shiitake (Lentinusedodes), Agaricus blazei Murill, and any combination thereof.

[0073] Cordyceps

[0074] Background: Cordyceps sinensis is the most diverse genus in the Clavicipitaceae family of the Order Hypocreales. Among the more than 400 species reported, the most documented species is Cordyceps sinensis.

[0075] Codyceps sinensis (C sinensis) is a traditional Chinese medicinal mushroom. It occurs naturally in the Himalayas at an altitude of more than 3,600 meters. In addition, Cordyceps sinensis can be produced from cultured mycelium extracts for commercial growth. Since 1964, the Chinese Pharmacopoeia has classified Cordyceps sinensis as a drug.

[0076] Uses in Traditional Medicine: In traditional Chinese medicine, Cordyceps sinensis has been used for fatigue, poor appetite, kidney deficiency, tuberculosis, chronic bronchitis, male impotence, and loss of libido (Chen PX et al., 2017). It is also used to treat health conditions related to the cardiovascular, immune, endocrine, and nervous systems (Zhu JS et al., 1998).

[0077] In addition, C. sinensis has been reported to possess immunomodulatory, anticancer, anti-aging, antioxidant, and anti-inflammatory activities (Tuli HS et al., 2014; Shashidhar MG et al., 2013; Nie S et al., 2013).

[0078] Extraction methods: Different compounds of Cordyceps sinensis can be extracted by using different media such as water, methanol, ethanol, trypsin, MeOH and EtOAc (Chen PX et al., 2013).

[0079] Bioactive compounds

[0080] The active compounds of Cordyceps sinensis include, but are not limited to, nucleosides (cordycepin and adenosine), polysaccharides (water-soluble polysaccharides isolated from Cordyceps sinensis (CPS1), water-soluble polysaccharides isolated from Cordyceps sinensis (CPS2), and acidic polysaccharides isolated from cultured Cordyceps sinensis (APC)), peptides (cyclodipeptides and cordycepin), and sterols (ergosterol and sitosterol).

[0081] Preclinical studies: Cordyceps sinensis extracts have been used in different preclinical studies for several indications, including antioxidant (Vasiljevic JD et al., 2016; Wang Jl et al., 2012; Ji, D.-B et al., 2009), reducing the formation of transplant arteriosclerosis (Zhang, Y et al., 2011) and anti-tumor (Chen, Y et al., 2009).

[0082] Pharmacological activity - inflammation and cancer: Many studies have shown that the active compounds of Cordyceps sinensis have anti-inflammatory and anti-cancer properties. The main pharmacological activities are mediated by attenuation of pro-inflammatory cytokines such as TNF-α and ICAM-1 and reduction of PCNA expression (Zhang et al., 2011), inhibition of IL-2 and TGF-β secretion, and enhancement of IL-4 secretion (Jeong et al., 2002). In addition, inhibition of COX-2 and NF-κB was shown (Wang J1 et al., 2012b).

[0083] Preclinical and clinical dosing

[0084]

[0085]

[0086] Maitake (Grifola frondosa)

[0087] Background: Grifola frondosa is a dark grey-brown fungus found primarily on the ground at or near the base of the trunk stumps of dead, dying or aged hardwood trees such as oak, elm, maple, black gum, beech and chestnut. Grifola frondosa is primarily distributed in northern temperate regions of Japan, European countries and the northeastern United States (Global Biodiversity Information Facility, www.gbif.or , 2017), and can also be found at high altitudes in subtropical temperate climates (SH Han et al., 1995). It is known worldwide by different names, including Maitake / Kumotake (Japan), Grifola frondosa (China), Signorina mushroom (Italy), and "Hen of the Woods" (North America).

[0088] Uses in traditional medicine: Grifola frondosa is considered a medicinal mushroom in traditional Chinese and Japanese "herbal medicine". It is used in different indications to protect the spleen (Zhonghua Bencao Editorial Committee, Zhonghua Bencao, Shanghai Science and Technology Press, Shanghai, 1999, pp. 548-549), stomach and liver (C. Zhou et al., 2013), and can also be used to calm the nerves and mind (T. Mizuno, 1995).

[0089] Extraction method: Polysaccharide: hot water 3% NH4 oxalate (100°C) 5% NaOH solution (30°C) (Mizuno T et al., 1995). Grifulan (GRN): Grifola frondosa was grown in 100 ml of a medium containing 2% glucose, 0.6% polypeptone, 2% sucrose and 0.1% soybean oil at pH 4.5, 25°C for 14 days. The mycelium was separated by filtration and cultured in 0.5% citrate buffer (pH 4.0, containing 5% glucose) at 25°C for 6 days. After 6 days of culture, the mixture was centrifuged, the supernatant was diluted with 1 volume of ethanol, and the precipitate was dissolved in 8M urea. The solution was further purified by DEADE-Sephadex A-25 to remove contaminating proteins, and the non-adsorbed fraction was dialyzed with water and precipitated with ethanol, followed by vacuum drying (Adachi Yoshiyuki et al., 1994)

[0090] Bioactive compounds: Grifola frondosa extracts contain a wide range of active compounds, such as glucans (e.g., NMF-5N, Grifolan LE, GFP2, GFPBW1) and heteropolymers (e.g., FII-3, FIII-1A, GFP1). Grifola frondosa is also a rich source of trehalose (SJ Huang, SY et al., 2011), which has been widely used as a food preservative, sweetener, and vaccine stabilizer.

[0091] Among the various maitake mushroom fractions prepared, the D fraction was found to be very promising for boosting the immune system, whether administered orally or injected. ∨ 1→6 branched main chain ∨ Many antitumor mushroom polysaccharides are composed of -1,3-glucan. The polysaccharide part of the D fraction is (1→3)-branched (1→6)- ∨ -dextran. Further purification of the D fraction produced the MID fraction (He, Xirui et al., 2017).

[0092] Preclinical studies: The biological activities of polysaccharides isolated from Grifola frondosa have been reported in many preclinical studies (He, Xirui et al., 2017) for different indications such as anti-tumor, dermatological treatment, immunomodulatory activity, and antioxidant activity.

[0093] Pharmacological activity - Inflammation and cancer: Several studies have shown that many anti-tumor polysaccharides act through host-mediated mechanisms rather than directly acting on tumor cells. Grifola frondosa polysaccharides not only act as biological response modifiers to help the host tolerate adverse biological stress and increase immunity against tumor cell development, but also directly induce tumor cell apoptosis (He, Xirui et al., 2017)o Another study has shown that Maitake D-fraction induces the initiation of immunity dominated by Th-1 cells converted from Th-0 cells, and thus stimulates cellular immunity and expresses anti-tumor effects (Inoue, Atsuyuki et al., 2002). A large number of studies have confirmed that Maitake has significant beneficial effects on immune function. It not only promotes the action of macrophages, but also promotes the action of a variety of other immune-related cells, such as natural killer (NK) cells and cytotoxic T cells (Tc) that can attack tumor cells. Maitake also increases the immune-related efficiency of these cells by increasing interleukin-1, interleukin-2 and lymphokines. In particular, the maitake fraction appears to have specific anti-tumor effects, potentially slowing the growth of tumors in the colon, lungs, stomach, liver, prostate, brain, and other organs. (It should be noted that any research references to the D-fraction also apply to the MD-fraction, as they are the same β1,6 / 1,3 glucans derived from Grifola frondosa) (Mayell, Mark, 2001).

[0094] Preclinical and clinical dosing

[0095]

[0096]

[0097] Turkey Tail (Trametes versicolor, Coriolus versicolor)

[0098] Background: Turkey tail is a mushroom that grows on dead logs around the world. It is named turkey tail because its brown and tan rings look like the tail feathers of a turkey. Its scientific name is Trametes versicolor or Trametes versicolor.

[0099] Turkey tail has been used in traditional Chinese medicine to treat lung disease for many years. In Japan, turkey tail has been used to boost the immune system when given standard cancer treatment.

[0100] Uses in Traditional Medicine: Strengthening the Immune System: Extraction Methods

[0101] Aqueous extracts obtained from Trametes versicolor have recently been shown to possess a wide range of biological activities, including regulatory and stimulatory effects on different immune cells, as well as inhibition of cancer cell growth. Among the different bioactive fractions derived from hot water and standardized ethanol-water extracts of Trametes versicolor, protein-bound polysaccharides (PSK, also known as Krestin) and polysaccharide peptides (PSP) possessed strong biological activities (Habibi, Emran et al., 2015).

[0102] Bioactive compounds: Polysaccharide peptides obtained from this mushroom have immunomodulatory, antitumor and hepatoprotective activities and have been used as immunomodulatory and antitumor drugs in cancer patients. A closely related polysaccharide, PSK (coriolus versicolor), has been isolated from T. versicolor by Japanese researchers and has been shown to have immunostimulatory activity.

[0103] Polysaccharide K (PSK) is the most famous active compound in turkey tail mushroom. In Japan, PSK is an approved mushroom product for the treatment of cancer (Hobbs C., 2004; Jeong, Sang-Chul et al., 2015).

[0104] Preclinical studies: PSK and PSP have demonstrated a wide range of immune effects in vitro and in vivo, especially reticuloendothelial system activation, cytokine regulation (IFN-γ production, IL-2 production), enhanced dendritic cell activity, T cell maturation, natural killer cell activity, antibody production, and anti-tumor and anti-cancer effects. The extracts can inhibit carcinogenesis and tumor cell growth by activating cancer cell apoptosis (Hobbs C., 2005).

[0105] Preclinical and clinical dosing

[0106]

[0107]

[0108] Shiitake (Lentinus edodes)

[0109] Background: Since ancient times in the East, shiitake mushrooms have been and remain one of the most edible mushrooms shown to have medicinal properties. Glucan components, particularly lentinan (a purified b-1,3-D-glucan with b-1,6 branches and a triple helical structure), have been shown to have significant anti-tumor activity. Lentinan has also been shown to be an immunopotentiator and appears to stimulate macrophage and T cell proliferation and has no direct cytotoxic effect on tumor cells (Ng, Mah-Lee, 2002).

[0110] Uses in Traditional Medicine: Shiitake is one of the well-known macrofungi used in several therapeutic applications. It is the source of several well-studied preparations with proven pharmacological properties. The medicinal properties of Shiitake have been studied since the Ming Dynasty (1369-1644). Elders from the Japanese Empire considered Shiitake to be the elixir of life, increasing vitality and energy. Antibiotic, anticancer, anticancer, antifungal, antibacterial and antiviral, antidiabetic, hypolipidemic compounds have been isolated from Shiitake both intracellularly (fruiting bodies and mycelium) and extracellularly (biculture). Some of these substances are lentinan, lectin, and eritadenine. Shiitake is used medically for diseases involving immunosuppression (including AIDv), cancer, diabetes, environmental allergies, fungal infections, frequent flu and colds, bronchial inflammation, and regulating urinary incontinence (Bisen, PS, et al., 2010).

[0111] Extraction method: Ethyl acetate frozen Lentinus edodes mushrooms (2000 g) were extracted with 80% methanol in water (3000 mL) and 50% methanol in water (3000 mL) at 5°C. The extracts were combined and concentrated on a rotary evaporator under reduced pressure and room temperature until the methanol was removed. The aqueous extract (200 mL) was partitioned twice with ethyl acetate (300 mL x 2). The combined ethyl acetate fractions were evaporated under reduced pressure and room temperature and then dried in a freeze dryer. The ethyl acetate fraction accounted for 2.12% of the frozen weight of Lentinus edodes mushrooms (Fang, Nianbai et al., 2006).

[0112] Ethanol precipitation and freeze drying in liquid nitrogen revealed that the β-D-glucan isolated by this method was Lentinan. Purity testing using a carbohydrate analysis column (chromatographic method) showed a significant Lentinan purity of 87.50% (Yap, Ann-Teck and Mah-Lee NG, 2001).

[0113] The powdered shiitake fruiting bodies (A) were extracted with a 1:1 mixture of EtOEt and EtOH at 42°C for 8 hours. The extraction was repeated 3 times. The extracts obtained were combined and evaporated under N2 gas. The extractable material (lipid) was taken as fraction B. The residue of the shiitake powder was extracted with a mixture of EtOEt and EtOH to provide fraction C. The extracted insoluble residue (lipid) was treated with hot water at 120°C for 2 hours to extract glucan. The residue was taken as fraction E. The shiitake powder (A) that had been treated with hot water was filtered to exclude soluble glucan, and the residue was taken as fraction D (Nanba Hiroaki et al., 1987).

[0114] Bioactive compounds: Scientific studies have isolated many compounds from shiitake mushrooms that have health-promoting activities. Shiitake fruiting bodies contain 88-92% water, proteins, lipids, carbohydrates, and vitamins and minerals. The mushroom is a good source of vitamins, especially provitamin D2 (ergosterol), which produces calciferol (vitamin D2) under ultraviolet (UV) light and heat. It also contains B vitamins, including B1 (thiamine), B2 (riboflavin), and B12 (niacin). Fatty acids account for 3.38% of the total lipids, with appreciable amounts of amino acids. In addition to glycogen-like polysaccharides, (1-4)-, (1-6)-D-glucans and antitumor polysaccharides, lentinan, (1-3)-, (1-6)-linked heteroglucans, heterogalactans, heteromannans, xyloglucans, etc. have been identified. Among the free sugars present are trehalose, glycerol, mannitol, arabitol, mannose, and arabinose. In shiitake mushrooms, dietary fiber consists of water-soluble materials (such as β-glucan and protein) and water-insoluble substances that can be extracted only with salt, acid and alkali, such as polysaccharide uronic acid glycosides (acidic polysaccharides), hemicellulose, β-glucan with heterosaccharide chains, lignin and chitin), which exist as cell wall components (Bisen, PS et al., 2010). Regarding the identification of anticancer components in shiitake mushrooms, an antitumor polysaccharide was isolated from the fruiting body and named shiitake polysaccharide. Its structure was confirmed to be β-(1 3)-glucan (C6H1005)n, with an average molecular weight of 5×105 kDa. Shiitake polysaccharide has been identified as the main antitumor active component in shiitake mushrooms. However, mammals lack the enzyme β-1 3-glucanase required for digestion of shiitake polysaccharide, and it is reported that decomposed shiitake polysaccharide and purified shiitake polysaccharide have no antitumor activity when orally administered (Fang, Nianbai et al., 2006).

[0115] Preclinical studies: Oral administration of shiitake mushroom fruiting bodies has been reported to have anticancer effects on some tumor types, and these effects appear to be strain-specific. A diet containing shiitake mushrooms significantly reduced the growth of sarcoma-180 cells in female ICR mice and MM-46 carcinoma in C3H mice, but had little effect on the growth of other tumor types such as B-16 melanoma, Lewis lung cancer, or Meth-A fibrosarcoma. In addition, the anticancer effects of the shiitake mushroom diet were found to be accompanied by the activation of macrophages and increased cytotoxic effects of NK and killer T cells, suggesting that one possible mechanism of this tumor suppressor activity may involve enhanced immune surveillance (Fang, Nianbai et al., 2006).

[0116] Pharmacological Activity - Inflammation and Cancer: Shiitake mushrooms have been described as having antiviral, antibiotic, anti-inflammatory, antihypertensive and anticancer properties and have been popular in the East for thousands of years and more recently in the West as a health food (Fang, Nianbai et al., 2006).

[0117] Lentinus edodes has attracted much attention due to its immunomodulatory effects. Lentinan is a well-known class of biological response modifiers (BRMs). It has been observed that administration of Lentinan enhances NK (natural killer cell), CTL (cytotoxic T lymphocyte), LAK (lymphokine-activated killer cell) activities and DTH (delayed type hypersensitivity) responses against tumor antigens. These activities are responsible for the anti-tumor effects of Lentinan (Bisen, PS et al., 2010).

[0118] Preclinical and clinical dosing

[0119]

[0120]

[0121] 1 Primary endpoint as not achieved

[0122]

[0123] Reishi (Ganoderma lucidum)

[0124] Background: Lingzhi (Reishi) is a Basidiomycetes, a non-lamellar fungus belonging to the family Polyporaceae. In nature, it grows in densely wooded hills with high humidity and dim light. It is rarely found as it grows mainly on the dry trunks of dead plum, kudzu or pasnia trees. Out of 10,000 of these aged trees, perhaps 2 or 3 will have Lingzhi growing, so it is very rare indeed. Lingzhi's place in the health food industry is unparalleled. It is the culmination of 5000 years of Eastern and Western knowledge and wisdom. More than 30 years of modern scientific research has proven its effectiveness as a health food and as a highly effective medicine. Of all people, Lingzhi can safely claim to be completely free of side effects.

[0125] Ganoderma lucidum is a natural medicine widely used and recommended by Asian doctors and natural pathologists for its supportive effects on the immune system. Laboratory studies and a small number of preclinical trials have shown that G. lucidum has promising anticancer and immunomodulatory properties. The prevalence of using G. lucidum as an alternative medicine is increasing among cancer patients (Jin, Xingzhong, et al., 2012).

[0126] Uses in Traditional Medicine: Ganoderma lucidum has been used as a medicinal remedy for centuries in China, Korea, and Japan. This edible mushroom is believed to protect human vitality and promote longevity. In addition, Ganoderma lucidum has been used to treat various human diseases such as allergies, arthritis, bronchitis, gastric ulcers, hyperglycemia, hypertension, chronic hepatitis, liver disease, insomnia, nephritis, neurasthenia, scleroderma, inflammation, and cancer (Daniel Sliva, 2003).

[0127] Extraction method: Ganoderma lucidum fruiting body fragments (1 kg) were extracted with ethanol (95%, 1 L) at room temperature for 24 hours to obtain 35 g of solid extract. The ethanol extract was suspended in chloroform and extracted with a saturated NaHCO3 solution. The NaHCO3 phase was then collected and adjusted to pH 3 with a cold HCl solution (6N). The resulting precipitate was extracted with chloroform, and 10.9 g of ethanol soluble and acidic components (ESAC) were obtained as yellow solids (Hu, Hongbo, et al., 2002).

[0128] According to the manufacturer's technical bulletin, Reishimax GLP TMThe active ingredients are standardized to 13.5% polysaccharides (β-1,3-glucan) and 6% triterpenes (ganoderic acid, etc.), which are the highest levels of extractable activity, while the remaining 80% consists of nucleosides, fatty acids and amino acids. The powdered extract was sonicated with 95% ethanol for 30 minutes, and the supernatant was further extracted by continuous sonication with anhydrous ethanol. A water-insoluble brown powder, i.e., GLE (passed through a 0.45 μm polypropylene filter) was recovered from the filtrate under reduced pressure (Yuen, John WM et al., 2007).

[0129] The fruiting bodies of G. lucidum collected from southern China were washed, disintegrated, and extracted twice with hot water at 70°C for 3 hours as described previously. 34 All hot water extracts were combined and the PS-rich fractions were precipitated by adding 75% (vol / vol) 160 GAO ETAL. ethanol. The PS-rich fractions were further purified by high-performance anion exchange and gel filtration chromatography using a 1.6-100-cm column filled with Sephadex G-25 (Gao, Yihuai, et al., 2005).

[0130] Bioactive compounds: The effectiveness of Reishi is attributed to the polysaccharide fraction, which is responsible for stimulating the immune system, or to the triterpenes, which show cytotoxic activity against various cancers (Suarez-Arroyo, Ivette J., et al., 2013).

[0131] The biologically active polysaccharides from Ganoderma lucidum exist mainly in the form of β-D-glucan, while the antitumor activity from Ganoderma lucidum exists mainly in the form of branched (1→3)-β-D-glucan.

[0132] Major bioactive components: More than 300 reports on the chemical composition of G. lucidum and related species have been published. The fruiting bodies, mycelium and spores of G. lucidum contain about 400 different bioactive compounds, which mainly include triterpenes, sterols, steroids, fatty acids, proteins, peptides and trace elements (Babu, P. Dinesh and RS Subhasree, 2008).

[0133] Preclinical studies: Ganoderma lucidum is a medicinal mushroom that has been reported to have antitumor activity in mice. Further studies have shown that the polysaccharide (PS) fraction of G. lucidum is involved in this antitumor effect. 12,13 G. lucidum PS is able to activate macrophages, T lymphocytes, and NK cells, and induce the production of cytokines such as tumor necrosis factor (TNF)-α, interleukins (IL), and interferons (IFN) in vitro using human immune cells and in vivo in mice.

[0134] Pharmacological activity - Inflammation and cancer: Ganoderma lucidum inhibits the constitutively active transcription factors nuclear factor KappaB (NF-κB) and AP-1, which results in the inhibition of the expression of urokinase-type plasminogen activator (uPA) and its receptor uPAR. Ganoderma lucidum also inhibits cell adhesion and cell migration of highly invasive breast and prostate cancer cells, indicating its efficacy in reducing tumor invasiveness. Therefore, Ganoderma lucidum clearly shows anticancer activity in experiments using cancer cells and has possible therapeutic potential as a dietary supplement for alternative therapies for breast and prostate cancer (Daniel Sliva, 2003). Reishi-treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G and p70S6K, as well as reduced activity of extracellular regulated kinases (ERK1 / 2). Reishi inhibits protein synthesis and tumor growth by affecting survival and proliferation signaling pathways acting on translation, suggesting that Reishi is a potential natural therapeutic agent for breast and other cancers (Suarez-Arroyo, Ivette J., et al., 2013).

[0135] The activity of polysaccharides from Ganoderma lucidum is believed to be mediated through complement receptor type 3 (CR3 receptor) binding to β-glucan. The activity of polysaccharides from mushrooms has been shown to indirectly prevent tumorigenesis and tumor metastasis by activating the immune response of the host organism through stimulation of natural killer cell, T cell, B cell and macrophage-dependent immune system responses. Polysaccharides (PS-G) isolated from fresh Ganoderma lucidum fruiting bodies stimulated the production of interleukin (IL)-1 β, tumor necrosis factor (TNF)-α, IL-6 from human monocyte-macrophages and interferon (IFN)-γ from T lymphocytes. In addition, these PS-G-induced cytokines inhibited the proliferation and clonogenicity of human leukemia cells. PS-G also enhanced the immune response of human neutrophils and caused antitumor effects by activating the phosphatidylinositol (PI) 3-kinase / Akt pathway to inhibit spontaneous and Fas-mediated apoptosis. In addition, PS-G enhances the phagocytic activity of neutrophils through the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. In addition, polysaccharides from Ganoderma lucidum exhibit chemopreventive effects, which are mediated by the induction of glutathione S-transferase (GST) activity (Daniel Sliva, 2003).

[0136] In addition, more than 100 highly oxidative and pharmacologically active lanostane-type triterpenes have been isolated from Ganoderma lucidum.

[0137] Preclinical and clinical dosing

[0138]

[0139]

[0140] Agaricus blazei Murill

[0141] Background: Agaricus Blazei Murill, a medicinal mushroom from the Brazilian rainforest, has been used in traditional medicine and as a health food for the prevention of a range of diseases, including infections, allergies, and cancer. Other scientists and we have examined whether there is scientific evidence behind this hypothesis. Agaricus Blazei Murill is rich in the immunomodulatory polysaccharide, β-glucan, and has been shown to have anti-tumor, anti-infective, and anti-allergy / asthma properties in mouse models, in addition to anti-inflammatory effects in patients with inflammatory bowel disease. These effects are mediated by the mushroom's stimulation of innate immune cells (such as monocytes, NK cells, and dendritic cells) as well as improvements in skewed Thl / Th2 balance and inflammation (Hetland, Geir, et al., 2011).

[0142] Uses in traditional medicine

[0143] Agaricus Blazei is a medically important mushroom that is widely consumed and prescribed in Japan.

[0144] Agaricus Blazei is traditionally used as a health food for preventing cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis.

[0145] According to legend, the elderly in the area suffer from fewer serious illnesses than those in neighboring communities, possibly due to the use of Agaricus Blazei as food. In addition to cancer and chronic hepatitis, this mushroom has been used in folk medicine to combat a variety of diseases, including diabetes, arteriosclerosis, and hyperlipidemia (Hetland, Geir et al., 2008).

[0146] Extraction method: Repeated extraction with hot water, cold NaOH, and then hot NaOH (Ohno, Naohito et al., 2001). Extract the hot water soluble fraction. The mycelial fraction (100 g) was extracted 3 times with hot water (500 ml) for 3 hours. The suspension was filtered to remove insoluble matter. After evaporation of the aqueous extract under reduced pressure, an equal volume of ethanol was added to the extract, and the precipitated polysaccharides were collected by centrifugation at 8000 rpm for 20 minutes. The crude polysaccharide was dissolved in water and dialyzed with distilled water. An equal volume of ethanol was added to the dialyzate again, and the suspension was centrifuged at 10000 rpm for 20 minutes. The polysaccharide fraction obtained was dissolved and lyophilized, and is hereinafter referred to as the hot water soluble (HWS) fraction.

[0147] Isolation of antitumor polysaccharides from HWS fraction: HWS fraction (100 mg) was dissolved in 100 ml of water. The solution was added to 100% saturated (NH4)2SO4 for salting out and then centrifuged at 120 xg for 15 minutes. The supernatant (FA) and precipitate (FB) were dialyzed against distilled water through cellulose tubes. The dialyzate was evaporated to a small volume and then lyophilized. The FA aqueous solution (5 mg) was subjected to ion exchange chromatography on a DEAE-Sepharose CL-6B column (5.2×38 cm), stepwise eluting with 2000 ml of distilled water containing 0.5 M and 1 M NaCl, and then eluting with 0.1N NaOH. A second anion exchange chromatography was performed on the same column with a gradient elution between 0 and 2 M NaCl in distilled water. The active fraction was further separated on a Sepharose 4B column (1.6×90 cm) using distilled water as the eluent (Mizuno, Masashi, et al., 1999).

[0148] 1 kg of dried thallus of A. Blazei was directly refluxed and extracted with chloroform / methanol (1:1, v / v) (2L 33) for 3 hours. The chloroform / methanol extract was concentrated under reduced pressure to obtain a brown extract (250 g). The chloroform / methanol extract (200 g) was separated into acetone soluble (160 g) and insoluble (40 g) fractions. The acetone soluble fraction (35 g) was further separated into a n-hexane insoluble fraction (16 g) and a n-hexane soluble fraction (14 g). The n-hexane insoluble fraction (15 g) was chromatographed on a silica gel column, and ergosterol (2.4 g) was isolated as an active substance (Takaku Takeshi et al., 2001).

[0149] The broken dry fruiting bodies (3kg) were extracted with 80% ethanol at 70-80°C under reflux for 22 hours. After centrifugation and removal of the insoluble fraction, the soluble fraction (~200g) was freeze-dried and designated as fraction 1. The insoluble material (about 2.5kg) was then extracted with water reflux at 80-90°C for 22 hours, and the water-soluble fraction (about 250g) was called fraction 2. By heating under reflux for 12 hours, the water-insoluble residue (~2kg) was extracted three times with 5% ammonium oxalate. About 20 liters of fractions soluble in 5% ammonium oxalate were concentrated to 4 liters. 2 liters of these concentrated fractions were ultrafiltered and freeze-dried on a Millipore filter (Pericon Minicassette) with a molecular weight cutoff of 5kDa. The freeze-dried fraction (final product, 711 g) was designated as fraction 3. The freeze-dried insoluble residue (309 g) was designated as fraction 4 (Oshiman, Ko-ichi et al., 2002).

[0150] Bioactive compounds

[0151] Agaricus blazei, originating from Brazil, is the source of several antitumor polysaccharides contained in its fruiting bodies. These polysaccharides include b-(1,6); b-(1,3) glucans, acidic b-(1,6); a-(1,3) glucans, and acidic b-(1,6); a-(1,4) glucans. In contrast to most mushroom glucans, the above glucans have a b-(1,6) pyranose glucose backbone rather than the more common b-(1,3) linked backbone. The fruiting bodies also contain a 380,000 Da antitumor water-soluble proteoglycan with a 4:1 ratio of a-(1,4) glucan backbone and b-(1,6) pyranose glucopyranoside branches, as well as two immunostimulatory heteroglucans containing glucose, galactose, and mannose, one composed of glucose and ribose, and xyloglucan. In submerged cultures in bioreactors, A. Blazei can also synthesize extracellular protein glucans with mannose, glucose, galactose and ribose groups and very high molecular weight (1000,000-10000,000 Da), which exhibit significant antitumor properties (Giavasis Ioannis, 2010).

[0152] Preclinical studies: β-glucan has known anti-tumor properties, as do proteoglycans and ergosterol, two other components of A. Blazei. The apparent anti-tumor effects of A. Blazei are due to β-1,3-glucan. In addition, β-1,6-glucan extracted from A. Blazei induced tumor regression in mice, and daily supplementation with β-glucan from A. Blazei reduced spontaneous metastasis of ovarian and lung cancer cells in a mouse model. In addition, β-glucan-protein complexes isolated from A. Blazei were shown to have an inhibitory effect on Meth A fibrosarcoma in a mouse model (Hetland, Geir et al., 2008).

[0153] Pharmacological activity-inflammation and cancer: Agaricus blazei is rich in immunomodulatory substances, such as highly branched β 1,3- / 1,6-glucan and proteoglycans. These are known ligands of CD11b / 18 (complement receptor 3, CR3), Dectin-1 and Toll-like receptor 2 (TLR2) on monocytes, dendritic cells (DC), granulocytes and NK cells of the innate immune system. Agaricus blazei also shows that it contains mushroom elements and ergosterol (provitamin D2) that induce leukemia cell apoptosis and inhibit tumor-induced angiogenesis, respectively, as well as isoflavone compounds with effective hypoglycemic effects, which can be used for anti-diabetes. It is reported that AbM has anti-tumor properties in mouse models of fibrosarcoma, myeloma, ovarian cancer, lung cancer and prostate cancer and in human studies for gynecological cancer (increased NK cell activity and quality of life) and leukemia (Hetland, Geir et al., 2011).

[0154] Preclinical and clinical dosing

[0155]

[0156]

[0157]

[0158] Chaga (Inonotus obliquus)

[0159] Background: Inonotus obliquus mushroom (Inonotus obliquus) is a white rot fungus belonging to the Hymenochaetaceae family of the genus Basidiomycetes. Inonotus obliquus mushrooms have been used in folk medicine for the treatment of cancer in Russia, western Siberia, Asia, and North America. Inonotus obliquus mushrooms have been shown to contain many polyphenolic compounds and exhibit various biological activities, including antibacterial, hepatoprotective, and antitumor properties (Ham, Seung-Shi et al., 2009).

[0160] Uses in Traditional Medicine: Inonotus obliquus mushrooms have been used as folk medicine in Russia and western Siberia for the prevention and treatment of gastric diseases and even cancer. Inonotus obliquus is said to have a variety of natural beneficial properties for human health based on its bioactive compounds, such as antibacterial, antiallergic, anti-inflammatory and antioxidant (Kang, Ju-Hee et al., 2015). It is also used to prevent and treat heart, liver and stomach diseases and tuberculosis. In addition, it has been traditionally used in Russia, Poland and many Baltic countries since the 16th century to treat gastrointestinal cancer, cardiovascular disease and diabetes (Kim, Yu Jin et al., 2011).

[0161] Extraction method: Inonotus obliquus samples were dried, crushed and stored at -20°C. The powdered sample (500 g) was extracted twice with 3 L methanol (99.8%) at 45-50°C for 3 hours. The methanol fraction (45 g) was then rotary evaporated, emulsified, dissolved in water, and extracted three times with ethyl acetate (H20: EtOAc, 5: 7, v / v). The ethyl acetate fraction (12 g) was obtained after rotary evaporation, and the dried extract was reconstituted in methanol. The stock solution was subjected to vacuum chromatography (N-2N, Eyela, Tokyo, Japan) using silica gel (60 g, 230-400 mesh) mixed with dichloromethane. The mobile phase used was dichloromethane and methanol with an increasing methanol (0-100%) gradient in dichloromethane, and the column was re-equilibrated with 100% dichloromethane for 3 minutes before the next run. The success of the fractionation separation was confirmed by TLC. Among the three ethyl acetate extract fractions, the most bioactive fraction, i.e., VCHG-fraction 1, was selected and subjected to reverse phase (ODS-C18) column chromatography using a reverse phase column (ODS-C18) containing Lichroprep RP-18 (25-40 μm) mixed with H2O:MeOH (1:30, v / v) with a gradient program ranging from 30% methanol to 100% methanol (mobile phase A: methanol; mobile phase B: ultrapure water). Four fractions were isolated, of which fraction 2 contained the greatest bioactivity. Fraction 2 was further fractionated by chromatography using a normal phase silica gel column (2.4 cm×15 cm) containing silica gel mixed with dichloromethane (200 g, 70-230 mesh; Merck Co.), eluted by a mobile phase gradient ranging from 100% dichloromethane to 50% methanol. The flow rate was 1.0 mL / min. This produced two subfractions, designated as subfractions 1 and 2, both of which showed biological activity. The compounds in subfractions 1 and 2 were crystallized and analyzed by measuring their MS (JEOL), 1H NMR and 13C NMR (Bruker DRX 500MHz) spectra (Ham, Seung-Shi et al., 2009).

[0162] The sclerotium (1.3 kg) of I. obliquus was extracted six times with methanol (MeOH) to obtain an extract (67.5 g), which was distributed with n-hexane, CH2Cl2 and ethyl acetate (EtOAc) in sequence. The CH2Cl2 fraction (24 g) was subjected to silica gel column chromatography and eluted with a CHCl3 / EtOAc gradient system (50:1→1:1) to obtain thirteen fractions (Fr.MCl~13). Compound 1 (105.2 mg) was recrystallized from fraction MC3 under CHCl3 and MeOH solvents. Fraction MC5 was subjected to repeated column chromatography with a gradient elution of n-hexane / EtOAc (10:1→1:1) to obtain 10 sub-fractions (Fr.MC5-1~MC5-10). Compound 2 (9.1 mg) was recrystallized from fraction MC5-5 under CHCl3 and MeOH solvents to obtain white crystals. Fraction MC5-6 (58 mg) was subjected to MPLC with n-hexane / EtOAc gradient (EtOAc in n-hexane 5-100% over 70 min, 100% over 10 min) to obtain compound 3 (7.1 mg), and fraction MC5-10 (13 mg) was subjected to semi-preparative HPLC with 0.1% trifluoroacetic acid aqueous solution / MeOH gradient (MeOH in water 50-80% over 5 min, 80-100% over 15 min, 100% over 25 min) to obtain compound 4 (2.2 mg). Compounds 5 (301.5 mg) and 6 (8.7 mg) were recrystallized from fractions MC7 and MC10, respectively, under CHCl3 and MeOH solvents. Fraction MC9 was subjected to silica gel column chromatography, eluted with n-hexane / EtOAc (15:1→1:1) gradient, to obtain 17 sub-fractions (Fr. MC9-1 to MC9-17). The fraction MC9-14 was subjected to semi-preparative high performance liquid chromatography (HPLC) using a 0.1% trifluoroacetic acid aqueous solution / MeOH gradient (MeOH aqueous solution 50-80% over 5 minutes, 80-100% over 15 minutes, 100% over 25 minutes) to obtain compounds 7 (2.7 mg) and 8 (3.2 mg). The Et0Ac fraction (12.4 g) was subjected to Sephadex LH-20 column chromatography and eluted with 100% MeOH to obtain ten fractions (Fr. E1-10). The fraction E4 (750 mg) was subjected to silica gel column chromatography and eluted with a gradient of n-hexane / EtOAc (8:5→1:2) to obtain 10 sub-fractions (FR. E4-1-E4-10). Compound 9 (2.4 mg) was isolated from fraction E4-3, and fraction E4-5 was subjected to semi-preparative HPLC using a water / MeOH gradient (aqueous MeOH 20-80% over 30 min, 80-100% over 5 min, 100% over 5 min) to give compound 10 (1.2 mg) (Kim, Yu Jin et al., 2011).

[0163] Dried inonotus birchii mushroom (8 kg) was extracted with 95% ethanol under reflux for 3 times, 4 hours each time. The extract was filtered and evaporated in vacuo to obtain an ethanol extract (212.95 g). The ethanol extract was then suspended in distilled water and partitioned with n-hexane, ethyl acetate and n-butanol in sequence. The n-hexane fraction (28 g) was chromatographed on a silica gel column using a hexane-EtOAc gradient system (30: 1-15: 1-8: 1-4: 1-1: 1, v / v, 2 L) to obtain 11 fractions (fractions 1-11). Fractions 1-10 were obtained by eluting with 1 L of solvent. Fraction 11 was eluted with hexane-EtOAc (0: 100, v / v, 2 L). Fraction 15 (360 mg, white crystals) was finally purified by recrystallization of fraction 10 (650 mg). Fraction 15 was identified by NMR analysis. The chemical structure of fraction 15 was confirmed by 1 H NMR (Kang, Ju-Hee et al., 2015). An aqueous extract from Inonotus obliquus (IOAE) was prepared by extracting powdered fruiting bodies (10 gm) in 200 ml warm water (40°C) (Mishra, SK et al., 2013).

[0164] Bioactive compounds: In previous studies on the chemical composition of this mushroom, lanostane-type triterpenoids such as inotodiol, trametenolic acid, and inonotsuoxides were reported to have antitumor and antifungal activities. Some phenolic compounds were also found in this mushroom (Kim, Yu Jin et al., 2011).

[0165] Inonotus obliquus contains excellent bioactive compounds. 3β-Hydroxy-lanosta-8,24-diene-21-aldehyde has been identified from Inonotus obliquus, and high molecular weight, water-soluble, lignin-like substances from edible mushrooms of Inonotus obliquus have been reported to inhibit the protease of human immunodeficiency virus type 1. Inotodiol isolated from sclerotia of Inonotus obliquus has been reported to have an inhibitory effect in a two-stage carcinogenicity test on mouse skin using 7,12-dimethyl-benz[α]anthracene. Hot water extracts of Inonotus obliquus exert inhibitory activity on the proliferation of human colon cancer cells (HT-29). Subfractions 1 and 2 are 3β-hydroxy-lanosterol 8,24-diene-21-aldehyde and inotodial, respectively, which have antimutagenic and antioxidant activities. In addition, subfraction 3 has antimutagenic and antioxidant activities (Chung, MiJA et al., 2010).

[0166] Preclinical studies: Chaga aqueous extract inhibits the proliferation of colorectal cancer cells in vitro and in vivo. It activates the mitochondrial intrinsic pathway of apoptosis, induces autophagy and S phase cell cycle arrest. Chaga aqueous extract can inhibit the expression of iN0S, COX-2 and proinflammatory cytokines, and improve intestinal epithelial cell inflammation. In addition, it downregulates β-catenin and NF-κ B signaling, which exerts anti-proliferative and anti-inflammatory activities in colorectal cancer cells (Mishra, SK, et al., 2013).

[0167] Pharmacological activity-inflammation and cancer: Ergosterol peroxide not only inhibits cell proliferation, but also inhibits clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines with different susceptibilities. The growth inhibition observed in these CRC cell lines is the result of apoptosis. Ergosterol peroxide inhibits the nuclear level of β-catenin, which ultimately leads to reduced transcription of c-myc, cyclin D1 and CDK-8. C-Myc, cyclin D1 and CDK-8 are important downstream targets of β-catenin, which regulate malignancies, including cell cycle and apoptosis in cancer (Kang, Ju-Hee et al., 2015). I0AE activates caspase-9 in human cancer cell lines in a dose-dependent manner, as observed by reduced levels of pro-caspase-9 and increased accumulation of cleaved caspase-9. IOAE subsequently activates caspase-3 and cleaves its nuclear substrate poly (ADP-ribose) polymerase (pARP) (Mishra, SK, et al., 2013).

[0168] Preclinical and clinical dosing

[0169]

[0170]

[0171] CBD

[0172] Background: Cannabis is the source of a unique group of at least 66 compounds collectively known as cannabinoids. Of these, much is known about the pharmacology of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component of cannabis, and about cannabidiol (CBD), which lacks psychoactive properties. Endogenous agonists of cannabinoid receptors have also been discovered. CB1 receptors are found at the terminals of central and peripheral neurons, where they regulate transmitter release. They are also present in some non-neuronal cells. CB2 receptors are mainly expressed by immune cells, one of their effects being to alter cytokine release. CBD has a much lower affinity for CB1 and CB2 receptors than Δ9-THC, and its pharmacological actions are not well characterized.

[0173] Both CB1 and CB2 receptors are coupled through Gi / o proteins, negatively coupled to adenylate cyclase and positively coupled to mitogen-activated protein kinase. CB1 receptors are also coupled to certain ion channels through Gi / o proteins, such as type A and inwardly rectified potassium 2 Colorectal adenoma model

[0174] Channel positive coupling, with N-type and P / Q-type calcium channels and D-type potassium channels negative coupling. CB1 receptors can also act through Gs protein to activate adenylate cyclase. Additional signal transduction mechanisms of cannabinoid CB1 and CB2 receptors have been proposed, and these descriptions can be found elsewhere (Pertwee, Roger G, 2004). Convincing evidence shows that the use of cannabinoids or the operation of cannabinoid receptors and endocannabinoid systems may be beneficial to many diseases, including pain, spasticity and tremor in multiple sclerosis, atherosclerosis, inflammatory bowel disease and cancer. Potential clinical applications of CBD and CBD-rich cannabis extracts include producing anti-inflammatory and neuroprotective effects, controlling epilepsy, anxiety, glaucoma and nausea, and regulating some effects of Δ9-THC.

[0175] CBD has been shown to antagonize some of the adverse effects of THC, including intoxication, sedation and tachycardia, while it itself has analgesic, antiemetic and anticancer properties. In modern clinical trials, this allows the use of higher doses of THC, providing evidence for the clinical efficacy and safety of cannabis-based extracts in the treatment of spasticity, central pain and lower urinary tract symptoms in multiple sclerosis, as well as sleep disorders, peripheral neuropathic pain, brachial plexus avulsion symptoms, rheumatoid arthritis and intractable cancer pain. Further examine the future application prospects of whole cannabis extracts in neuroprotection, drug dependence and tumor diseases. The combination of THC and CBD increases clinical efficacy while reducing adverse events, and is supported (Russo, Ethan and Geoffrey W. Guy, 2006).

[0176] Use in Traditional Medicine: Cannabis sativa L. (Cannabaceae) has a long history of use as a fiber and seed crop in China, and its achenes ("seeds") as well as other plant parts have been documented in Chinese medical texts for nearly 2,000 years. While the primary application of cannabis in traditional Chinese medicine revolves around the use of achenes, ancient indications for the female inflorescence and other plant parts include conditions such as pain and psychiatric disorders, which are the subject of current research on cannabinoids such as cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) (Brand, E. Joseph, and Zhongzhen Zhao, 2017).

[0177] Extraction method: The dried flowers and leaves of cannabis were extracted with CO2 at room temperature to obtain an extract, which was evaporated to dryness as a brown solid. A portion of the extract was dissolved in methanol and analyzed by HPLC (Agilent 1100) using a C18 column (150 mm × 4.6 mm, 1 mL / min flow rate) (Zhu JS et al., 1998).

[0178] Preclinical studies: CBD is considered an anti-tumor agent based on its in vitro and in vivo activity against tumor cells. However, the exact molecular mechanism by which CBD mediates this activity has not been elucidated. CBD induces cell death in breast cancer cells, independent of the activation of cannabinoids and valine receptors. Electron microscopy showed morphology consistent with the coexistence of autophagy and apoptosis. Western blot analysis confirmed these findings. CBD induces endoplasmic reticulum stress and subsequently inhibits Akt and mTOR signaling, as shown by decreased levels of phosphorylated mTOR and 4EBP1 and cyclin D1. Further analyzing the crosstalk between autophagy and apoptosis signaling pathways, we found that Beclin1 plays a central role in the induction of CBD-mediated apoptosis in MDA-MB231 breast cancer cells. Although CBD enhances the interaction between Beclinl and VPS34, it inhibits the association between Beclin1 and BCL-2. In addition, CBD reduces mitochondrial membrane potential, triggers the translocation of Bid to mitochondria, the release of cytochrome c to the cytosol, and ultimately activates the intrinsic apoptotic pathway in breast cancer cells. CBD increases reactive oxygen species (ROS) production, and ROS inhibition blocks the induction of apoptosis and autophagy. Our study reveals a complex interplay between apoptosis and autophagy in CBD-treated breast cancer cells and highlights the value of continuing to investigate the potential use of CBD as an anti-tumor agent.

[0179] Pharmacological activity - Inflammation and cancer: Cannabidiol (CBD), the main non-psychoactive component of cannabis, is considered an anti-tumor agent based on its in vitro and in vivo activity against tumor cells. Cannabinoids can modulate signaling pathways that are critical for cancer growth and spread. They inhibit cell cycle progression and chemotaxis, and block angiogenesis. CBD is cytotoxic to gliomas and inhibits tumor cell migration in vitro. In addition, CBD induces apoptosis in human leukemia cell lines by activating the classical caspase pathway and enhancing Nox4 and p22 (PHOX) function. Recent studies have reported that CBD inhibits breast cancer growth and downregulates ID1, a regulator of metastasis in breast cancer cell lines. In addition, CBD combined with THC induces programmed cell death (PCD) in glioma cells (Shrivastava, Ashutosh et al., 2011). Furthermore, cannabinoids induce inhibition of adenylate cyclase, affect ion channels, and stimulate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, suggesting that they may play a general role in cell survival or death decisions (Vaccani, Angelo et al., 2005).

[0180] Preclinical and clinical dosing

[0181]

[0182]

[0183] THC

[0184] Background: In recent years, cannabinoids (the active ingredients of marijuana, cannabis) and their derivatives have attracted intense attention as novel therapeutic agents for a range of common diseases. The identification of Δ9-tetrahydrocannabinol (THC), the major active ingredient of marijuana, led to the discovery that cannabinoids act by activating specific cell surface G protein-coupled cannabinoid receptors CB1 and CB2. CB1 receptors are particularly abundant in the brain, and CB2 receptors are highly expressed in cells of the immune system. Many of the effects of Δ9-THC are mediated by cannabinoid receptors, of which at least two types, CB1 and CB2, are present in mammalian tissues.

[0185] Cannabinoids have been proposed as new cancer therapeutics, primarily due to their ability to induce apoptosis in many different tumor cell types. THC is the psychoactive component of marijuana. In addition, THC exerts multiple biological effects by mimicking endogenous compounds, the endocannabinoids anandamide and 2-arachidonoylglycerol, which activate specific cannabinoid receptors.

[0186] Cannabinoids have been used successfully to treat some of the side effects, such as nausea and vomiting, weight loss, lack of appetite, and pain that often accompany cancer. Δ9-THC (dronabinol) and LY109514 (nabilone) are approved for the treatment of nausea and vomiting associated with cancer chemotherapy. Although cannabinoids are used in the palliative treatment of cancer, they have not yet been used as a treatment for tumor progression itself.

[0187] The main limitation of the possible future use of THC in oncology may be manifested in adverse reactions mainly at the level of the central nervous system, consisting mainly of perceptual abnormalities, occasional hallucinations, dysphoria, abnormal thinking, depersonalization and somnolence (Ligresti, Alessia et al., 2006).

[0188] Use in Traditional Medicine: Extracts from cannabis have been used for medicinal and recreational purposes for centuries.

[0189] Extraction method: Harvesting cannabis, which consists of seeds and husks; separating the husks from the seeds; extracting the husks with a solvent to produce an extract, and if desired, passing the extract through a chromatographic column configured to separate delta-9-THC from the extract (Webster, GR Barrie, 2002).

[0190] Δ9-THC was obtained from hashish by countercurrent distribution: automated assembly - 200 cells, 10 ml of either upper or lower phase, solvent system: petroleum ether / methanol / dimethylformamide / water, 10:8:2:1. After evaporation and extraction of the residue with petroleum ether, Δ9-THC was isolated as a mobile colorless oil [α] 25 D-141°(EtOH; c=3.16w / w). It is △ 9 - A mixture of THC and 7.90 / ow / w dimethylformamide, which is held by hydrogen bonding of the phenolic groups. The dimethylformamide content is measured by integration of the SMR signal and titration of THC with lithium isopropoxide in dimethylformamide. To remove the amide, the mixture is dissolved in 50-100 times the amount of CCl4 and shaken twice with water. The organic layer is separated and the solvent is removed under reduced pressure with cooling. The resulting viscous oil darkens shortly after exposure to air and light (Silva, M. Teresa A. et al., 1968).

[0191] Preclinical studies: Tumor cell growth and survival often depend on increased signaling through pathways that regulate cell survival and proliferation. Two of these include the MAPK signaling pathway (Ras / Raf-MAPK, extracellular signal-regulated kinase (ERK1 / 2)) and the PI3K / Akt pathway. Both CB1 and CB2 cannabinoid receptors are coupled to these pathways through heterotrimeric Gi / O-proteins. In Chinese hamster ovary (CHO) cells transfected with human CB1, CP 55940 (a highly potent non-selective CB1 and CB2 agonist) increased p42 / p44 MAPK activity in a time- and concentration-dependent manner, and this was reversed by the CB1 antagonist / inverse agonist SR141716A

[26] . Furthermore, activation of MAPK was blocked by pertussis toxin, thus indicating that signaling between CB1 and MAPK involves GTP-binding proteins. Similar results were observed when CHO cells were transfected with CB2, as CP 55940 increased p42 / p44 MAPK activity, and this effect was blocked by SR144528 (a CB2 selective antagonist) and pertussis toxin. Results from these studies suggest that activation of CB1 or CB2 receptors leads to increased signaling through the MAPK pathway (AlexanderAmy et al., 2009).

[0192] Pharmacological activity - Inflammation and cancer: THC has been shown to induce apoptosis in several tumor cell types. There is growing evidence that cannabinoids may be able to selectively target tumor cells, at least in part through differential regulation of signaling pathways that control cell survival and apoptosis. However, the mechanisms underlying the anti-tumor effects of cannabinoids are not fully understood, and evidence suggests that these effects may be cell type specific. Two signaling pathways reported to be positively or negatively regulated by cannabinoids (depending on the cell type and the cannabinoid studied) are the RAS-MAPK / ERK pathway and the PI3K-AKT pathway (Greenhough, Alexander et al., 2007).

[0193] Although the general consensus in the current literature indicates that cannabinoids have anti-cancer effects, there are some studies showing that D9-THC has a biphasic effect in cancer cells, where lower concentrations lead to increased cancer cell proliferation, while higher concentrations lead to decreased cell proliferation. For example, 100-300nM of D9-THC caused a 1.2-fold and 2-fold increase in the proliferation rate of NCL-H292 (lung cancer) and U373-MG (glioblastoma) cells, respectively. In contrast, higher concentrations of D9-THC (4-101M) were cytotoxic and increased the number of apoptotic cells (30-80%). It is well known that cannabinoids can cause suppression of the immune system through CB2 activation (Alexander Amy et al., 2009).

[0194] Preclinical and clinical dosing

[0195]

[0196]

[0197]

[0198]

[0199] The anti-inflammatory and anti-cancer effects of the composition of the present invention comprising mushroom extracts and cannabinoids were tested in vitro.

[0200] The objectives of this study were to evaluate:

[0201] A. Anti-inflammatory properties of different mushroom extracts, THC and CBD on RAW 264.7 cells.

[0202] B. Anticancer properties of different mushroom extracts, THC, and CBD on PANC-1 and EGL-1 cells.

[0203] The cell lines mentioned above in this article are:

[0204] a.RAW 264.7( TIB-71 TM ), Murine macrophage (American Type Culture Collection, Rockville, MD, USA).

[0205] b.PANC-1 CRL-1469 TM ), human pancreatic cancer.

[0206] c. EGL-1 human extrahepatic cholangiocarcinoma.

[0207] The mushroom extracts tested in the assay described herein below were:

[0208] a. Cordyceps sinensis C S -4 (Mushroom Science, batch number W0001576, experiment 11 / 2020). Hot water / alcohol extract.

[0209] b. Cordyceps sinensis-M (Real Mushrooms), batch number 18274, experiment 10 / 2020). Hot water extract.

[0210] c. Turkey Tail Colorius PSP (Mushroom Science, batch number 1820004, experiment 07 / 2020). Hot water / alcohol extract.

[0211] d. Turkey Tail Extract ((Real Mushrooms), Batch No. 18305, Experiment 11 / 2020). Hot water extract.

[0212] e. Shiitake (Mushroom Science, batch number 1800401, experiment 01 / 2020). Hot water / alcohol extract.

[0213] f. Agaricus Blazei - Full Spectrum Extract (Mushroom Science, Batch No. W0000008, Experiment 08 / 2020). Hot water / alcohol extract.

[0214] g. Chaga (Mushroom Science, batch number W0001580, experiment 11 / 2020). Hot water / alcohol extract.

[0215] h. Maitake (Mushroom Science, Lot #1802502, Exp 11 / 2020). Hot water / alcohol extract.

[0216] i. CBD (cannabidiol), Bol Pharma, batch number LG18086-36-2. CoA

[0217] j. Dexamethasone (West-Ward Pharmaceuticals Corp, catalog number 0641-0367-25, batch number 108381, experiment number 10 / 2020)

[0218] The mushroom extracts, cannabinoid solutions and growth media used in the assays described herein below were prepared as follows:

[0219] Mushroom Extracts:

[0220] a. Add 2 / 3 of the total final volume of warm water for injection (42°C) to the dried mushroom powder.

[0221] b. The suspension was vortexed and centrifuged (4100 rpm, RT, 5 min).

[0222] c. Separate the supernatant containing the water-soluble fraction into a clean tube.

[0223] d. Add 5% of the total final volume of EtOH to the pellet and vortex to extract the ethanol soluble fraction. Add appropriate amount of water to the final volume.

[0224] e. The suspension was vortexed and centrifuged. The resulting supernatants were combined and centrifuged again to remove debris, resulting in a final concentration of 50 mg / mL.

[0225] f. Pass the resulting solution through a cell strainer (40-70 microns).

[0226] g. Prepare two additional stocks in vehicle (5% EtOH in WFI): 10 mg / mL and 2 mg / mL.

[0227] h. All test articles / vehicles were added at 4 μL to cells in 96 μL of culture medium to give final working concentrations: 2 mg / mL, 0.4 mg / mL, and 0.08 mg / mL (in 0.2% EtOH, 4% WFI).

[0228] cannabinoid solution

[0229] a. THC-THC was diluted with vehicle (WFI containing 5% DMSO) to concentrations of 1875 μM, 500 μM, 100 μM, 25 μM, and 2.5 μM. All tested THC stocks / vehicles were diluted 1:15-1:5 with cells, depending on the assay.

[0230] b. CBD (314.46 g / mol) was dissolved in EtOH to a concentration of 10 mg / mL (31.8 mM). A 500 μM stock solution was prepared by combining a 31.8 mM ethanol solution with 5% DMSO and diluting with WFI to a final concentration of 500 μM. The resulting solution was further diluted with vehicle (WFI containing 5% DMSO) to concentrations of: 500 μM, 375 μM, 125 μM, 25 μM, and 2.5 μM. All tested CBD stock solutions / vehicles were diluted 1:15-1:5 with cells, depending on the assay.

[0231] c. Dexamethasone (392.46 g / mol) stock solution 10 mg / mL (25.47 mM). The stock solution was further diluted with vehicle (WFI containing 5% EtOH) to concentrations of 2.5 μM, 0.25 μM, and 0.025 μM. All dexamethasone stock solutions were added to cells in 96 μL of culture medium at 4 μL to produce final working concentrations of 100 nM, 10 nM, and 1 nM (in 0.2% EtOH and 4% WFI).

[0232] Cell culture medium preparation:

[0233] RAW growth medium: Dulbecco's Modified Eagle Medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin solution (PS).

[0234] Incubate cells with LPS and test items:

[0235] a. Harvest cells at 80% confluence, count and resuspend in growth medium to a final concentration of 1×106 / ml.

[0236] b. The cell suspension was seeded in a 96-well microtiter plate (1×105 cells / 100 μL / well). - Each plate was designed as shown in Table 1, Table 2 and Table 3.

[0237] c. Incubate the cells (37±10°C, 5% CO2) for 18-24 hours.

[0238] d. Remove growth medium; add 94 μL of medium and 4 μL of vehicle, test item, and control.

[0239] e. Mix the contents of all wells by pipetting up and down three times.

[0240] f. Incubate the plate for 2 hours (37±1°C, 5% CO2).

[0241] g. Add LPS (10 μL, final concentration 100 ng / mL LPS) to all wells.

[0242] h. Mix the contents of all wells by pipetting up and down three times.

[0243] i. Incubate the plate for 24±2h (37±1°C, 5% CO2).

[0244] j. Collect medium from 96-microtiter plates: 50 μL for NO secretion assay and the rest stored at -20°C for cytokine secretion assay.

[0245] Example 1

[0246] To evaluate the anti-inflammatory properties of the composition of the invention, assays of nitric oxide (NO) and cytokine secretion from RAW cells after incubation with mushroom extracts and cannabinoids, as well as viability assays were performed.

[0247] Table 1: *Suggested Assay Design

[0248]

[0249]

[0250] *The red one is the plate set up **Only in CBD / THC plate

[0251] Table 2: Plate setup for mushroom extracts (4 mushrooms / plate)

[0252]

[0253]

[0254] Table 3: Plate setup for cannabinoid extracts

[0255]

[0256]

[0257] Cell proliferation and viability (XTT assay)

[0258] a. Prepare XTT reaction solution.

[0259] b. Add 50 μL of XTT reaction solution to each well.

[0260] c. Incubate the plate in a humidified atmosphere (eg, 37° C., 5% CO 2 ).

[0261] d. Gently shake the plate to evenly distribute the dye in the wells.

[0262] e. Measure the absorbance of the samples at 450 nm using a spectrophotometer (ELISA reader) relative to a background control as a blank.

[0263] f. To measure a reference absorbance (to measure non-specific readings), use a wavelength of 620 nm to subtract from the 450 nm measurement.

[0264] g. The idle absorbance of cells in all wells is 0.5-1.5.

[0265] Nitric oxide (NO) test (Griess test)

[0266] a. Allow the sulfonamide solution and NED solution to equilibrate to room temperature (15-30 minutes).

[0267] b. Prepare 1 ml of a 100 μM nitrite solution by diluting the provided 0.1 M nitrite standard 1:1,000 in culture medium.

[0268] c. 2 columns (16 wells) are designated for the nitrite standard reference curve in a non-binding 96-well plate. Dispense 50 μL of assay medium into the wells of rows BH ( Figure 1 ).

[0269] d. Add 100 μL of 100 μM nitrite solution to the remaining 2 wells in row A.

[0270] Serial two-fold dilutions (50 μl / well) were immediately performed on the plate in duplicate to generate a nitrite standard reference curve (100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μM).

[0271] e. Dispense 50 μL of sulfanilamide solution into all experimental samples and into the wells containing the dilution series against the nitrite standard reference curve.

[0272] f. Incubate the plate at room temperature in the dark for 5-10 minutes.

[0273] g. Dispense 50 μL of NED solution into all wells.

[0274] h. Incubate the plate at room temperature in the dark for 5-10 minutes.

[0275] i. Measure the absorbance within 30 minutes in a plate reader with a filter between 520 nm and 550 nm.

[0276] j. Inhibition of NO secretion is considered relevant only at concentrations that have no effect on cell viability (viability greater than 95% compared to vehicle control). Representative results regarding viability are shown in FIG2 .

[0277] Results: Mushroom Extract

[0278] Nitric oxide (NO) secretion

[0279] a. In 2-3 independent experiments, Ganoderma lucidum, Agaricus blazei, Cordyceps sinensis CS-4 (2 mg / ml) and Cordyceps sinensis M (0.08 mg / ml) were the only effective mushrooms in inhibiting NO secretion (without any effect on cell viability).

[0280] b. Table 4 depicts NO secretion from RAW cells after treatment with vehicle control, mushroom extracts, and calculation of NO secretion inhibition rate (%) (100% - no mushroom / no vehicle * 100%). Cordyceps M was found to be the most effective in inhibiting NO secretion, which appeared to be dose-dependent. Significant inhibition was also observed after incubation with Ganoderma lucidum, however, inhibition was only observed at the highest concentration (2 mg / ml).

[0281] c. In vitro, there appears to be a very narrow range of activity (does not affect cell viability).

[0282] d. Turkey tail, maitake, shiitake and chaga had no effect on N0 secretion (see Table 4).

[0283] e. Figure 3 shows representative results of N0 secretion

[0284] Table 4: Summary of inhibition of N0 secretion*

[0285]

[0286]

[0287] *Mushrooms are arranged by most effective (top) and least effective (bottom).

[0288] Cytokine secretion

[0289] a. Ganoderma lucidum, Cordyceps sinensis, Agaricus blazei, and Cordyceps sinensis Cs-4 were incubated with 105 RAW cells / well at 0.08-2 mg / ml for 24 hours.

[0290] b. Culture medium from cells was collected in duplicate for multiplex analysis (CCL3 / MIP1α, IL-6, IL-1β, TNFα, IL-10, and IL-6).

[0291] c. IL-4, IL-10 and IL-1β were not secreted under these conditions (below the detection limit).

[0292] d. CCL3 / MIP-1α secretion was above the detection limit even when the sample was diluted 1:20.

[0293] e. IL-6 secretion Table 5 describes the IL-6 secretion of RAW cells after treatment with vehicle control, mushroom extract, and the calculation of the inhibition rate of IL-6 secretion (100%-IL-6 mushroom / IL-6 vector*100%) or the increase rate of IL-6 secretion (IL-6 mushroom / IL-6 vector*100%-100%).

[0294] f TNFαα secretion Table 6 describes the TNFα secretion of RAW cells after treatment with vehicle control, mushroom extracts, and the calculation of the secretion rate of TNFαα secretion (TNFα mushroom / TNFαα vector*100%) or the increase rate of TNFαα secretion (TNFαα mushroom / TNFα vector*100%-100%).

[0295] g. Results from the assay are also shown in FIG4 .

[0296] Table 5: Summary of IL-6 secretion

[0297]

[0298] Table 6: Summary of TNFα secretion

[0299]

[0300] Cannabinoid Extracts

[0301] Nitric Oxide Secretion: The results of NO secretion of the cannabinoid extracts are shown in Table 7 below.

[0302] a. CBD effectively inhibited NO secretion at 15-20 μM without affecting cell viability, while THC was effective at 20 μM.

[0303] b. Almost no dose response. Very narrow activity range (does not affect cell viability).

[0304] c. The results of the effect of cannabinoid extract on N0 secretion are also shown in Figure 5.

[0305] Table 7: Summary of inhibition of NO secretion*

[0306]

[0307] * Cannabinoids are listed by most potent (top) and least potent (bottom).

[0308] Cytokine secretion

[0309] a. CBD (15 μM) and THC (20 μM) were incubated with 105 RAW cells / well.

[0310] b. Culture medium from cells was collected in duplicate for multiplex analysis (CCL3 / MIP1α, IL-6, IL-1β, TNFα, IL-10, and IL-6).

[0311] c. IL-4, IL-10 and IL-1β were not secreted under these conditions (below the detection limit).

[0312] d. CCL3 / MIP-1α secretion was above the detection limit even when the sample was diluted 1:20.

[0313] e. IL-6 secretion:

[0314] i. CBD does not affect IL-6 secretion.

[0315] f. TNF α secretion:

[0316] i. CBD does not affect TNFα secretion.

[0317] ii.THC does not affect TNF α secretion.

[0318] The results are also shown in Figure 4.

[0319] Combination of mushroom extract and cannabinoid extract

[0320] Nitric Oxide Secretion: Table 8 depicts NO secretion from RAW cells after treatment with vehicle control, mushroom extracts, with / without cannabinoids, and calculation of % inhibition of NO secretion (100% - NO mushroom / NO vehicle * 100%).

[0321] a. DMSO, CBD (15 μM), or THC (20 μM) were combined with Ganoderma lucidum, Cordyceps sinensis M, Agaricus blazei, and Cordyceps sinensis Cs-4 (0.08-2 mg / mL) and incubated with 105 RAW cells / well for 24 hours.

[0322] b. The culture medium of the cells was collected and NO secretion was measured (Gteiss NO kit).

[0323] c. Lingzhi: THC significantly increased the inhibition of NO secretion by Lingzhi (2 mg / ml). CBD had only a slight additive effect. This combination appears to be the most effective combination in terms of NO secretion inhibition.

[0324] d. Cordyceps M (RM): THC enhanced the inhibitory effect of Cordyceps M (0.08 mg / mL) on NO secretion. CBD had no such effect.

[0325] e. Cordyceps sinensis C S -4(CS): Both THC and CBD increase the C S -4 (2 mg / mL) inhibits NO secretion.

[0326] f. Agaricus blazei: Both THC and CBD increased the inhibitory effect of Agaricus blazei (2 mg / ml) on NO secretion.

[0327] g. For the inhibition of N0 secretion, Cordyceps M 0.08 mg / mL and THC 20 μM were the most effective combination.

[0328] The results are also shown in Figure 6.

[0329] Table 8: Summary of inhibition of N0 secretion*

[0330]

[0331]

[0332] *Combinations are listed by most effective (top) and least effective (bottom)

[0333] Cytokine secretion

[0334] a. DMSO, CBD (15 μM) or THC (20 μM) were combined with Ganoderma lucidum, Cordyseps sinensis M, Agaricus Blazai, and Cordyceps sinensis Cs-4 (0.08-2 mg / mL) and incubated with 105 RAW cells / well.

[0335] b. Culture medium from cells was collected in duplicate for multiplex analysis (CCL3 / MIP1 a, IL-6, IL-1β, TNFα, IL-10, and IL-6).

[0336] c. IL-4, IL-10 and IL-1β were not secreted under these conditions (below the detection limit).

[0337] d. CCL3 / MIP-1α secretion was above the detection limit even when the sample was diluted 1:20.

[0338] e. IL-6 secretion (Table 9 depicts IL-6 secretion from RAW cells after treatment with vehicle control, mushroom extracts in the presence and absence of cannabinoids, and calculation of % inhibition of IL-6 secretion (100% - IL-6 mushroom / IL-6 vehicle * 100%) or % increase in IL-6 secretion.

[0339] f. Mushroom / IL-6 vector*100%-100%).

[0340] g. TNFα secretion: Table 10 describes the TNFα secretion of RAW cells after treatment with vehicle control, mushroom extract, with and without cannabinoids, as well as the calculation of the % secretion rate of TNFα secretion (TNFα mushroom / TNFα vector*100%) or the % increase in TNFα secretion (TNFα mushroom / TNFα vector*100%-100%).

[0341] i. CBD + Ganoderma lucidum (2mg / ml) reduced IL-6 secretion - a pro-inflammatory effect - compared to either extract alone.

[0342] ii. CBD + Cordyceps Cs-4 (0.08 mg / mL) reduced IL-6 secretion compared to either extract alone - pro-inflammatory effect. o CBD + Cordyceps Cs-4 (2 mg / ml) attenuated the inhibitory effect of Cordyceps Cs-4 (2 mg / ml) alone on IL-6 secretion. o CBD + Agaricus balzei (0.4 mg / mL) reduced the inhibitory effect of Agaricus balzei alone (0.4 mg / mL) on IL-6 secretion.

[0343] iii. THC + Ganoderma lucidum (0.08 mg / mL) reduced IL-6 secretion compared to either extract alone.

[0344] iv.THC + Cordyceps Cs-4 (2 mg / ml) increased IL-6 secretion compared to the two extracts alone. THC + Cordyceps Cs-4 (0.08 mg / mL) reduced IL-6 secretion compared to the two extracts alone. THC + Cordyceps M (0.08 mg / ml) reduced IL-6 secretion compared to the two extracts alone.

[0345] h.TNFα secretion:

[0346] i. CBD + Ganoderma lucidum (2 mg / ml) decreased TNFα secretion compared to either extract alone. THC + Ganoderma lucidum (0.4-2 mg / ml) decreased TNFα secretion compared to either extract alone. o CBD / THC + Cordyceps sinensis Cs-4 (2 mg / ml) increased TNFα secretion compared to either extract alone.

[0347] ii. THC + Cordyceps Cs-4 (0.08 mg / mL) reduced TNFα secretion compared to the two extracts alone.

[0348] iii. THC + Agaricus blazei (2 mg / mL) decreased TNFα secretion compared to either extract alone, whereas THC + Agaricus blazei (0.08 mg / mL) increased TNFα secretion compared to either extract alone.

[0349] iv.THC+Reishi (0.08 mg / mL) reduced IL-6 secretion the most.THC+Cordyceps Cs-4 (2 mg / mL) increased IL-6 secretion to the greatest extent.

[0350] v.THC+Cordyceps sinensis Cs-4 (0.08 mg / mL) reduced TNFα secretion to the greatest extent.

[0351] The results are shown in Figure 4.

[0352] Table 9: Summary of IL-6 secretion

[0353]

[0354]

[0355] Table 10: Summary of TNFα secretion

[0356]

[0357]

[0358] As can be seen from the data presented herein above, the anti-inflammatory properties of the combination of mushroom extract and THC or CBD are significantly different from the anti-inflammatory properties of each component alone, thus showing a synergistic anti-inflammatory effect of mushroom extract and THC or CBD.

[0359] Table 8 specifically shows that the combination of mushroom extract with THC or CBD resulted in a reduction in NO secretion relative to each component alone, indicating an increase in the anti-inflammatory properties of these combinations. The highest inhibition of NO secretion was observed for the combination of 0.08 mg / mL Cordyceps M with 20 M THC.

[0360] The effects of mushroom extracts in combination with THC or CBD on IL-6 secretion showed decreased IL-6 secretion THC + Ganoderma lucidum (0.08 mg / mL), as shown in Table 9, while THC + Cordyceps sinensis Cs-4 (2 mg / mL) resulted in the greatest increase in IL-6 secretion.

[0361] The effects of mushroom extracts in combination with THC or CBD on TNFα secretion are shown in Table 10: The highest reduction in TNFα secretion was observed for THC + Cordyceps Cs-4 (0.08 mg / mL), indicating anti-inflammatory activity.

[0362] Example 2

[0363] Viability assay of PANC-1 and GL-1 cells after treatment with various combinations of mushroom extracts and cannabinoids

[0364] Test system

[0365] a. PANC-1 cells (human pancreatic cancer cells) (American Type Culture Collection, Rockville, Maryland, USA).

[0366] b. EGL-1 cells (human extrahepatic cholangiocarcinoma)

[0367] Cell Growth:

[0368] Growth medium suitable for PANC-1 / EGL-1 cells: Dulbecco's Modified Eagle's Medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin solution (PS).

[0369] Growth medium suitable for EGL-1 cells: RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin solution (PS).

[0370] Assay medium for PANC-1: The assay medium contains only 1% FBS.

[0371] Assay medium suitable for EGL-1: Assay medium contains only 1% FBS in DMEM low glucose (1 g / L)

[0372] Experimental Design: Incubate cells with test items:

[0373] a. Harvest cells at 80% confluence, count and resuspend in growth medium to a final concentration of 75×103 / ml (PANC-1) or 50×103 (EGL-1).

[0374] b. PANC-1 cell suspension was seeded at 7500 cells / 100 μL / well in a 96-well microtiter plate, and EGL-1 cell suspension was seeded at 5000 cells / 100 μL / well in a 96-well microtiter plate.

[0375] c. Incubate the cells (37±10°C, 5% CO2) for 18-24 hours.

[0376] d. Remove growth medium; add 80 μL assay medium and 20 μL vehicle, test item, and control. Design each plate as shown in Table 11, Table 12, and Table 13.

[0377] e. Mix the contents of all wells by pipetting up and down three times.

[0378] f. Incubate the plate for 72±2 h (37±1° C., 5% CO 2 ).

[0379] g. Replace the medium on all plates with 100 μL of medium.

[0380] XTT assay

[0381] a. Prepare XTT reaction solution.

[0382] b. Add 50 μL of XTT reaction solution to each well.

[0383] c. Incubate the plate in a humidified atmosphere (eg, 37° C., 5% CO 2 ).

[0384] d. Gently shake the plate to evenly distribute the dye in the wells.

[0385] e. Measure the absorbance of the samples at 450 nm using a spectrophotometer (ELISA reader) relative to a background control as a blank.

[0386] f. To measure a reference absorbance (to measure non-specific readings), use a wavelength of 620 nm to subtract from the 450 nm measurement.

[0387] g. The idle absorbance of cells in all wells is 0.5-1.5.

[0388] Table 11: *Suggested Assay Design

[0389]

[0390]

[0391] Table 12: Plate setup for cannabinoid extracts

[0392]

[0393]

[0394] *The rows marked in red are the settings for each board

[0395] **Only in CBD / THC panels

[0396] Table 13: Plate setup for cannabinoid extracts

[0397] Example 3

[0398] Cancer Research Results: Mushroom Extracts

[0399]

[0400] a. Seven mushroom extracts were incubated with 0.5-10 mg / ml PANC-1 or EGL-1 for 72 hours.

[0401] b. Tables 12 and 13 describe the mushroom doses that resulted in maximum kill (lowest viability).

[0402] c. Test the viability of cells (XTT assay).

[0403] d. EGL-1 cells are much more resistant to cell death than PANC-1 cells. Cordyceps sinensis M (RM) was the most effective mushroom, followed by Ganoderma lucidum.

[0404] e. PANC-1 cells: Chaga was the most effective mushroom, followed by Ganoderma and Turkey Tail (MS).

[0405] f. The results are shown in Figures 7 and 8.

[0406] Table 12: Summary of PANC-1 cell viability*

[0407]

[0408]

[0409]

[0410] Table 13: Summary of viability in EGL-1 cells

[0411]

[0412]

[0413]

[0414] The results showed that both EGL-1 and PANC-1 cells incubated with Agaricus and Cordyseps Cs-4 (MS) showed % viability higher than 100%

[0415] To check whether mushrooms affect metabolic cell proliferation, both mushrooms were added to EGL-1 cells at the same concentration for 2 hours, 6 hours, 24 hours and 72 hours. As seen in Figure 2, in the case of Cordyseps Cs-4, higher viability was observed only after 72 hours, which implies a proliferative effect. However, in the case of Agaricus blazei, higher viability (compared to vehicle) was observed even after 2 hours, which implies a metabolic change.

[0416] Cannabinoid extracts

[0417] a. CBD (0.1-100 μM) or THC (0.5-375 μM) was incubated with 7,500 PANC-1 cells / well (pancreatic cancer) or EGL-1 cells / well (extrahepatic cancer) for 72 hours.

[0418] b. Test the viability of cells (XTT assay).

[0419] c.EGL-1:

[0420] i. CBD is effective at 40-100 μM (15%-30% activity).

[0421] ii. THC was effective at 100-375 μM (15% activity, no dose response).

[0422] d. PANC-1:

[0423] i. CBD was effective at 40-100 μM (35% activity, no dose response).

[0424] ii. THC was effective at 100-375 μM (35% activity, no dose response).

[0425] The results are shown in Figure 9.

[0426] Combination of mushroom extract and cannabinoid extract

[0427] a. DMSO, CBD (40 μM) or THC (50 μM) were combined with Ganoderma lucidum, Cordyceps sinensis M, Cordyceps sinensis Cs-4 and Turkey Tail (MS) extracts (0.5-6 mg / mL) and incubated with 7,500 PANC-1 cells / well.

[0428] b. Combination with THC did not improve the anticancer effects of mushrooms.

[0429] c. The combination with CBD reduced cell viability compared to all mushrooms alone. Cordyceps M (RM) and Ganoderma lucidum combined with CBD was the most effective combination in reducing cell viability.

[0430] As shown in FIG. 10 , the synergistic anticancer properties of the combination of mushroom extracts with and without THC or CBD were demonstrated by the viability of PANC-1 and EGL-1 cancer cell lines after incubation of cells with mushroom extracts with and without THC or CBD.

[0431] The combination of mushroom extracts with CBD showed increased anticancer effects relative to mushroom extracts alone, with the most significant effects observed for Cordyceps M (RM) and Ganoderma lucidum in combination with CBD.

[0432] Example 4

[0433] Route of Administration: The combination of cannabinoids and medicinal mushrooms is formulated for oral administration, intravenous administration, or topical administration.

[0434] The formulations of the present invention particularly include additional ingredients or pharmaceutical excipients in a non-limiting manner to further develop formulations with desired concentrations, effective doses, dosing regimens, and treatment durations. These ingredients particularly include solubilizers, stabilizers, buffers, tension regulators, fillers, viscosity enhancers / reducers, surfactants, chelating agents, and adjuvants.

[0435] The cannabis composition of the present invention is administered by smoking a plant-derived cigarette or by oral or intravenous administration. The smoking route is most commonly a pre-made cigarette or hand-rolled cigarette. The intravenous route provides precise control of dosage and time. The cannabinoid composition is also delivered via the oral mucosal route.

[0436] Nasal administration (through the nose) can be used for locally acting substances, as well as for insufflation of, for example, decongestant nasal sprays for absorption along the respiratory tract. Such substances are also called inhaled, for example, inhaled anesthetics.

[0437] Oral Administration Oral medications are taken in the form of tablets or capsules.

[0438] Tablets: The dissolution rate of tablets can be significantly affected by particle size and crystalline form. Dissolution time can be modified to obtain a rapid effect (fast dissolution) or sustained release (slow dissolution rate that prolongs duration of action or avoids initial high plasma levels).

[0439] Capsules: Capsules are gelatinous envelopes that enclose the active substance. Capsules can be designed to remain intact for several hours after ingestion to allow for delayed absorption. They can also contain a mixture of slow-release particles and fast-release particles to produce rapid and sustained absorption at the same dose.

[0440] Oral sustained release: Oral sustained release in capsules or tablets is achieved in non-limiting substances by embedding the active ingredient in an insoluble porous matrix so that the dissolved drug must leave the matrix before it can be absorbed, a sustained release formulation in which the matrix swells to form a gel through which the drug leaves, or by an osmotic controlled release oral delivery system in which the active compound is encapsulated in a water permeable membrane with laser-drilled holes at one end. As water passes through the membrane, the drug is pushed out through the holes and into the digestive tract where it can be absorbed.

[0441] Solutions: Pharmaceutical solutions are widely used as dosage forms for oral administration of therapeutic agents. Pharmaceutical solutions are defined as liquid preparations in which the therapeutic agent and various excipients are dissolved in a selected solvent system.

[0442] Drug solutions are homogeneous, i.e. the therapeutic agent and excipients are dissolved in the carrier

[0443] Parenteral Administration: Parenteral administration is carried out using intravenous, subcutaneous, intramuscular and intraarticular administration. The drug is stored as a liquid or, if unstable, in a lyophilized form.

[0444] Topical administration: Topical formulations include, inter alia, creams, ointments, pastes, lotions or gels.

[0445] Transdermal delivery: Transdermal delivery is achieved, for example, via transdermal patches.

[0446] Alternative routes of administration are suppository, intraventricular, intramuscular, inhalation, aerosol, and sublingual.

Claims

1. A composition for treating pancreatic cancer or bile duct cancer and any combination thereof in a mammalian recipient, wherein the composition comprises a. a cannabis-derived compound and b. a combination of a medicinal mushroom, wherein the cannabis-derived compound is CBD and the medicinal mushroom is Cordyceps sinensis, Ganoderma lucidum, or turkey tail, wherein the composition is characterized in that the cannabis-derived compound has a protective effect that counteracts the effect of the medicinal mushroom on increasing PANC-1 cell viability.

2. The composition according to claim 1, characterized in that The treatment of immune-mediated pancreatic cancer or bile duct cancer is provided by a composition comprising CBD and a mushroom extract selected from the group consisting of Cordyceps sinensis (M) mushroom extract, Ganoderma lucidum mushroom extract and turkey tail extract.

3. The composition according to claim 1, characterized in that The cannabis-derived compound has agonist properties at CB1 or CB2 receptors and any combination thereof; and the medicinal mushroom has agonist properties at pattern recognition receptors (PRRs).

4. The composition according to claim 1, characterized in that The composition is configured to be administered in a manner selected from the group consisting of an inhaler, a cigarette, a tablet, a capsule, a pill, a lyophilized powder, a powder, an emulsion, a granulated powder, a cream, an ointment, a paste, a lotion gel, a liquid, a solution, a patch, and any combination thereof.

5. The composition according to claim 1, characterized in that The composition is formulated to be administered in a manner selected from the group consisting of rapid release, slow release, sustained release, controlled release, and any combination thereof.

6. The composition according to claim 1, characterized in that The composition further comprises the following ingredients, which are selected from the group consisting of solubilizers, stabilizers, buffers, tonicity regulators, fillers, viscosity enhancers / reducers, surfactants, chelating agents, adjuvants, and any combination thereof.

7. A composition comprising a synergistic combination of a. a cannabis-derived compound and b. a medicinal mushroom, wherein the cannabis-derived compound is CBD and the medicinal mushroom is Cordyceps sinensis, Ganoderma lucidum, or turkey tail, for use in the preparation of a medicament for treating cancer of a mammalian receptor or bile duct cancer or any combination thereof.

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