Autophagy-promoting composition, food composition, pharmaceutical composition for cancer treatment, pharmaceutical composition for neurodegenerative disease treatment and cannabinoid-containing composition

Abstract

To provide an autophagy-promoting composition which has autophagy promotion or antagonistic effect by using a plurality of cannabinoids in combination.SOLUTION: An autophagy-promoting composition containing cannabidiol and cannabigerol is provided. Thus, the autophagy-promoting composition can be provided which exhibits promotion of autophagy or antagonistic effect as a novel effect which cannot be achieved by using each cannabinoid alone.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to an autophagy-promoting composition, a food composition, a pharmaceutical composition for treating cancer, a pharmaceutical composition for treating neurodegenerative diseases, and a cannabinoid-containing composition. More specifically, the present invention relates to an autophagy-promoting composition and a cannabinoid-containing composition that contain multiple specific cannabinoids, and further to a food composition, a pharmaceutical composition for treating cancer, or a pharmaceutical composition for treating neurodegenerative diseases that contains this autophagy-promoting composition. [Background technology]

[0002] Cannabinoids are compounds extracted from the cannabis plant (Cannabis sativa L.), an annual herb in the Cannabaceae family, and are known to function as physiologically active substances. One area of ​​applied research into cannabinoids is autophagy.

[0003] Autophagy is the process by which cells break down their own components, such as proteins, and is a key function present in all eukaryotic organisms, from yeast to humans. Autophagy promotes cellular metabolism and breaks down and eliminates accumulated waste and unnecessary proteins within the cell. Furthermore, it has become clear that autophagy suppresses the onset of serious diseases, including cancer, neurodegenerative diseases, immune diseases, respiratory diseases, kidney diseases, infectious diseases, lifestyle-related diseases such as type 2 diabetes, and various types of inflammation, and that it also has important physiological functions in development and differentiation, aging, and immunity. Autophagy is currently a hot topic in research.

[0004] As described above, regulating autophagy function can provide therapeutic and / or preventive effects against various diseases and aging, and therefore, there is a demand for novel compositions that can promote autophagy function.

[0005] For example, Patent Document 1 discloses an agent for promoting the uptake of nucleic acid molecules into cells or enhancing cellular autophagy, which uses cannabidiol (hereinafter also referred to simply as "CBD"), a type of cannabinoid, as a substance that activates specific transient receptor potential channels.

[0006] Furthermore, Patent Document 2 discloses a cannabinoid-containing complex mixture containing cannabidiol (CBD), cannabigerol (hereinafter also referred to simply as "CBG"), and terpenes, which is suitable for use as an active pharmaceutical ingredient for various treatments such as allergic diseases, as well as a method for producing the same and a method for using the pharmaceutical composition.

[0007] Furthermore, Patent Document 3 discloses a cannabinoid-containing composition suitable for use as an active pharmaceutical ingredient, i.e., a rapid-acting oral formulation comprising a major cannabinoid (a cannabinoid including tetrahydrocannabinol (THC) and / or CBD) and an entourage-restoring molecule (including a cannabinoid other than the major cannabinoid, such as CBG) that restores the entourage effect, in which the ratio of the major cannabinoid to the entourage-restoring molecule is 1000:1 to 0.1:1.

[0008] Meanwhile, Patent Document 4 discloses the use of a cannabinoid-containing composition for cholesterol management, using cannabidiol (CBD) alone or in combination with other cannabinoids, in the manufacture of a pharmaceutical or dietary supplement. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2022 / 118966 [Patent Document 2] Special Publication No. 2020-505417 [Patent Document 3] Special Publication No. 2020-512322 [Patent Document 4] Special Publication No. 2011-509989 Summary of the Invention [Problem to be solved by the invention]

[0010] It has already been found that CBD enhances autophagy, as described in Patent Document 1. Furthermore, as described in Patent Documents 2 to 4, it is also known that compositions containing CBD and other cannabinoids are suitable for use as active pharmaceutical ingredients. On the other hand, the effects of combining multiple cannabinoids (especially CBD and CBG) on autophagy have not been studied, and the effects of combining synthetic cannabinoids rather than cannabinoids extracted from cannabis (natural cannabinoids) have not been clarified.

[0011] An object of the present invention is to provide a composition for promoting autophagy that has the effect of promoting or antagonizing autophagy by using multiple cannabinoids in combination. Another object of the present invention is to provide a food composition, a pharmaceutical composition for treating cancer, and a pharmaceutical composition for treating neurodegenerative diseases, each containing the above-mentioned composition for promoting autophagy. A further object of the present invention is to provide a novel cannabinoid-containing composition using a combination of specific cannabinoids. [Means for solving the problem]

[0012] In order to solve the above problems, the present invention employs the following means.

[0013] That is, a composition for promoting autophagy according to one aspect of the present invention is characterized by containing cannabidiol and cannabigerol. As a result of extensive research, the inventors have discovered that when combining multiple cannabinoids, selecting a specific combination of cannabinoids can produce a new effect that is not achieved by using cannabinoids alone: ​​the promotion or antagonism of autophagy. In other words, the autophagy-promoting composition of the present invention makes it possible to exert an autophagy-promoting or antagonistic effect by using CBD and CBG in combination.

[0014] Furthermore, an autophagy-promoting composition according to one embodiment of the present invention is characterized in that the cannabigerol is synthetic cannabigerol (hereinafter also referred to simply as "CBG-syn"). Here, synthetic cannabigerol refers to cannabigerol that is biologically biosynthesized or chemically synthesized. Through extensive research, the inventors have discovered that when synthetic cannabigerol is used in combination with cannabidiol, it exhibits cell proliferation-promoting and autophagy-promoting effects that differ from those observed when it is used in combination with natural cannabigerol (hereinafter simply referred to as "CBG-ext"). In other words, the autophagy-promoting composition of the present invention can further enhance the autophagy-promoting or antagonizing effect of the combined use of CBD and CBG.

[0015] Furthermore, an autophagy-promoting composition according to one embodiment of the present invention is characterized in that the molecular mixing ratio of cannabidiol and cannabigerol is 10,000:1 to 0.01:1. This makes it possible to more effectively promote or antagonize autophagy by combining CBD and CBG.

[0016] Additionally, the autophagy-promoting composition according to one aspect of the present invention is characterized by further containing a functional component having physiological functionality. This means that in addition to its effectiveness as an autophagy-promoting composition, the combined use of CBD and CBG also provides other physiological functions.

[0017] A food composition according to one aspect of the present invention is characterized by comprising the above-described autophagy-promoting composition. The food composition of the present invention contains a composition that has an autophagy-promoting effect due to the combined use of CBD and CBG, and can be provided as a food composition that is expected to have therapeutic and / or preventive effects against various diseases and aging.

[0018] Additionally, a pharmaceutical composition for cancer treatment according to one aspect of the present invention is characterized by comprising the above-described composition for promoting autophagy. The pharmaceutical composition for cancer treatment of the present invention contains a composition that has the effect of promoting autophagy through the combined use of CBD and CBG, and can be provided as a pharmaceutical composition for cancer treatment that is expected to suppress the onset of disease.

[0019] Furthermore, a pharmaceutical composition for treating a neurodegenerative disease according to one aspect of the present invention is characterized by comprising the above-described composition for promoting autophagy. The pharmaceutical composition for treating neurodegenerative diseases of the present invention contains a composition that has an autophagy-promoting effect due to the combined use of CBD and CBG, and can be provided as a pharmaceutical composition for treating neurodegenerative diseases that is expected to suppress the onset of the disease.

[0020] Additionally, the cannabinoid-containing composition according to one aspect of the present invention is characterized by containing cannabidiol and synthetic cannabigerol. The cannabinoid-containing composition of the present invention can be provided as a novel cannabinoid-containing composition that exhibits particularly significant autophagy-promoting or antagonizing effects among compositions containing multiple cannabinoids. [Effects of the Invention]

[0021] According to the present invention, it is possible to provide a composition for promoting autophagy that has the effect of promoting or antagonizing autophagy by using multiple cannabinoids in combination. Furthermore, according to the present invention, it is possible to provide a food composition, a pharmaceutical composition for treating cancer, and a pharmaceutical composition for treating neurodegenerative diseases, each containing the above-mentioned composition for promoting autophagy. Furthermore, according to the present invention, it is possible to provide a novel cannabinoid-containing composition by using specific cannabinoids in combination. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a graph showing the results of an evaluation of the effect of the addition of CBD, CBG-syn, or CBG-ext alone on the viability of Caco-2 cells using a CCK-8 test. [Figure 2] 1 is a graph showing the results of an evaluation of the effect of combined use of CBD, CBG-syn, or CBG-ext on Caco-2 cell viability using a CCK-8 test. [Figure 3] 10 is a graph showing the effect of the addition of CBD, CBG-syn, or CBG-ext alone on autophagic flux in Caco-2 cells, calculated as an index of autophagic flux expressed as the GFP / RFP ratio. [Figure 4] 1 is a graph showing the results of a statistical analysis of the autophagy-promoting effect of combined use of CBD, CBG-syn, or CBG-ext in Caco-2 cells. DETAILED DESCRIPTION OF THE INVENTION

[0023] Hereinafter, embodiments of the autophagy-promoting composition, food composition, pharmaceutical composition for treating cancer, pharmaceutical composition for treating neurodegenerative disease, and cannabinoid-containing composition according to the present invention will be described in detail. The autophagy-promoting composition, food composition, pharmaceutical composition for cancer treatment, pharmaceutical composition for neurodegenerative disease treatment, and cannabinoid-containing composition described in the embodiments are merely examples used to explain the autophagy-promoting composition, food composition, pharmaceutical composition for cancer treatment, pharmaceutical composition for neurodegenerative disease treatment, and cannabinoid-containing composition according to the present invention, and are not limited to these.

[0024] First, the components contained in each composition according to the present invention will be described. Each of the compositions of the present invention (autophagy-promoting composition, food composition, pharmaceutical composition for treating cancer, pharmaceutical composition for treating neurodegenerative diseases, and cannabinoid-containing composition) contains multiple cannabinoids.

[0025] [Cannabinoids] First, cannabinoids will be explained. Cannabis sativa L. is an annual herb of the Cannabaceae family that has long been used as a folk remedy around the world to relieve symptoms such as pain, fever, anxiety, and diarrhea. To ensure efficacy and safety, compounds extracted from the cannabis plant are purified or synthesized, and compounds that function as physiologically active substances are collectively known as cannabinoids. Cannabis sativa contains over 120 cannabinoids, a unique compound found in cannabis plants. These include Δ9-tetrahydrocannabinol (THC), the main component of marijuana and the compound with the most psychoactive effect, as well as cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), and cannabicyclol (CBL), which are compounds with no psychoactive effect, and are primarily 21-carbon compounds.

[0026] In the present invention, cannabinoids are distinguished between natural cannabinoids and synthetic cannabinoids. More specifically, natural cannabinoids refer to cannabinoids obtained from at least one natural source (e.g., cannabis). On the other hand, synthetic cannabinoids refer to cannabinoids obtained through a chemical synthesis process, such as biosynthesis or chemical synthesis. Here, biosynthetic cannabinoids are chemically produced within living organisms, while chemically synthesized cannabinoids are chemically produced from reagents.

[0027] There are no particular limitations on the means of obtaining natural cannabinoids and synthetic cannabinoids. Specifically, they can be purchased commercially, or produced or obtained by known production methods. More specifically, natural cannabinoids can be produced by extraction methods such as supercritical CO2 extraction or high-temperature steam extraction of cannabis plants. Synthetic cannabinoids can be produced by known synthesis methods. An example of a synthesis method for producing synthetic cannabinoids will be described later.

[0028] (Application of cannabinoids) Currently, cannabinoids, including as dietary supplements, are used to relieve nausea caused by chemotherapy, alleviate cancer pain, prevent spasms caused by multiple sclerosis, and improve appetite in AIDS patients. Furthermore, research into its application as a therapeutic agent or nutritional supplement to prevent, reduce, or improve symptoms of inflammatory diseases such as neuroinflammation, pancreatitis, and periodontal disease, nicotine addiction, obesity, osteoporosis, osteopenia, pain, peripheral neuropathy, premenstrual syndrome (PMS), phantom limb pain, allergic diseases, proximal myotonic myopathy, and other mental disorders such as epilepsy, panic disorder, Parkinson's disease and syndrome, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), and Alzheimer's disease, as well as lifestyle-related diseases such as cancer, heart disease, cerebrovascular disease, diabetes, hypercholesterolemia, and hypertension, is actively being conducted.

[0029] The compositions of the present invention (autophagy-promoting composition, food composition, pharmaceutical composition for cancer treatment, pharmaceutical composition for neurodegenerative disease treatment, and cannabinoid-containing composition) focus on the above-mentioned cannabinoids, particularly cannabidiol (CBD) and cannabigerol (CBG), and are provided based on the inventors' findings that the combined use of CBD and CBG exerts an autophagy-promoting or antagonistic effect. The composition of each composition according to the present invention will be described in detail below with examples.

[0030] [Autophagy-promoting composition (cannabinoid-containing composition)] One embodiment of the present invention relates to a composition for promoting autophagy, which contains cannabidiol (CBD) and cannabigerol (CBG). In other words, the composition for promoting autophagy of the present invention can be considered a cannabinoid-containing composition containing multiple cannabinoids.

[0031] In one embodiment, the autophagy-promoting composition of the present invention contains cannabidiol (CBD) and synthetic cannabigerol (CBG-syn). Note that a composition containing cannabidiol and synthetic cannabigerol is also provided as a cannabinoid-containing composition, which is one embodiment of the present invention.

[0032] Furthermore, in one embodiment, the autophagy-promoting composition of the present invention comprises cannabidiol (CBD) and synthetic cannabigerol (CBG-syn), where the synthetic cannabigerol (CBG-syn) is chemically synthesized.

[0033] In one embodiment, the autophagy-promoting composition of the present invention comprises cannabidiol (CBD) and synthetic cannabigerol (CBG-syn), where the synthetic cannabigerol (CBG-syn) is biosynthesized.

[0034] (Cannabidiol (CBD)) Cannabidiol (CBD) is 2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-Benzenediol, which has the following chemical structural formula, and can be obtained by extraction from a natural source (cannabis) using known methods, or by synthesis such as biosynthesis or chemical synthesis. That is, the cannabidiol (CBD) of the present invention may be natural cannabidiol or synthetic cannabidiol. It is known that CBD is the predominant cannabinoid in certain cannabis strains. Furthermore, the CBD content in cannabis can range from trace amounts (<1%) to over 20%. Therefore, it is preferable to select a method for producing (obtaining) CBD appropriately, taking into consideration the efficiency of extraction from natural raw materials and the efficiency of synthesis. [ka]

[0035] (Cannabigerol (CBG)) The structural formula of cannabigerol (CBG) is 2-[(E)-3,7-Dimethyl-2,6-octadienyl]-5-pentyl-1,3-benzenediol, as shown in the chemical structural formula below, and can be obtained by known methods. [ka]

[0036] In one embodiment, the cannabigerol (CBG) of the present invention is preferably synthetically obtained. In this case, the combination of synthetic cannabidiol (CBG-syn) and cannabidiol (CBD) is also provided as a novel cannabinoid-containing composition that has the effect of modulating autophagy function.

[0037] For example, cannabigerol (CBG) used in the present invention may be produced by biosynthesis using yeast.

[0038] (Example of cannabigerol (CBG) biosynthesis) Cannabigerol (CBG) is biosynthesized using yeast according to the method described in Nature, 2019, Vol. 567, pp. 123-126. The method is shown below.

[0039] To produce olivetolic acid, an early intermediate in cannabinoid biosynthesis, we introduced the CsTKS and CsOAC expression cassettes into S. cerevisiae to generate strain yCAN01. We then introduced the cannabis-derived acryl-activating enzyme (AAE) into this strain. To optimize the conversion of hexanoic acid to hexanoyl-CoA, we introduced the cannabis-derived AAE, CsAAE1, into strain yCAN0112, resulting in strain yCAN02. To generate hexanoyl-CoA from galactose and complete the olivetolic acid pathway, we introduced the hexanoyl-CoA pathway into strain yCAN015, resulting in strain yCAN03, which efficiently produced olivetolic acid. Cannabigerolic acid (CBGA) is produced from olivetolic acid and geranyl pyrophosphate (GPP), an intermediate in the mevalonate pathway, by geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT). Therefore, we mined the publicly available cannabis transcriptome for GOT candidates. Using the Basic Local Alignment Search Tool (BLAST), we searched for full-length transcripts for CsPT1, HlPT1L, and HlPT2, and based on this, we selected six cannabis enzymes (CsPT2-CsPT7). For functional expression of six cannabis and two H. lupulus prenyltransferases in yeast, the predicted N-terminal plastid targeting sequences were removed, resulting in CsPT2-T through CsPT7-T, HlPT1L-T, and HlPT2-T, respectively. Next, each GOT candidate was introduced into yCAN10, and the resulting strains (yCAN12 through yCAN20) were transformed by converting malonyl-CoA and hexanoyl-CoA to olivetolic acid, yielding the desired cannabinoids.

[0040] Another example of cannabigerol (CBG) used in the present invention is chemically synthesized cannabigerol (CBG).

[0041] (Example of chemical synthesis of cannabigerol (CBG)) As an example of chemical synthesis of CBG, the method disclosed in Taura et al., The Journal of Biological Chemistry, 1996, Vol. 271, No. 21, pp. 17411-17416 is described below.

[0042] Geraniol (3 g, 0.0194 mol) and olivetol (2 g, 0.0111 mol) were dissolved in 400 mL of chloroform containing 80 mg of p-toluenesulfonic acid as a catalyst, and the reaction mixture was stirred in the dark at room temperature for 12 h. After 12 h, the reaction mixture was washed with saturated sodium bicarbonate (400 mL) followed by HO (400 mL). The chloroform layer was concentrated under reduced pressure at 40 °C, and the resulting residue was chromatographed on a 2.0 cm × 25 cm silica gel column using benzene (1000 mL) as the eluent to give 1.4 g (0.00442 mol) (39.9%) of CBG as product.

[0043] Alternatively, crude CBG was purified as follows: 7.25 g of crude CBG and 50 mL of benzene were added to a 250 mL beaker. The flask was swirled to dissolve the CBG, and 50 g of silica gel was added along with a stir bar. The solution was stirred overnight and then poured into a 44 cm x 2.75 cm column. The column was eluted with 300 mL of benzene. Approximately 70 mL fractions of the eluent were assayed for CBG. Fractions 1, 2, and 3 (approximately 230 mL) containing CBG were combined and the solvent removed under pressure, yielding 6.464 g of residue containing >80% CBG with a purity suitable for use in the next synthetic step.

[0044] (Ratio of components in the autophagy-promoting composition) In the autophagy-promoting composition of the present invention, the molecular mixing ratio of cannabidiol to cannabigerol ranges from 10,000:1 to 0.01:1, preferably from 1,000:1 to 10:1, and more preferably from 1,000:1 to 100:1. In other words, the autophagy-promoting composition of the present invention is prepared so that the concentration ratio of cannabidiol to cannabigerol ranges from 10,000:1 to 0.01:1, preferably from 1,000:1 to 10:1, and more preferably from 1,000:1 to 100:1. This makes it possible to more effectively promote or antagonize autophagy by using CBD and CBG in combination, as shown in the examples described below.

[0045] (Cannabidiol (CBD) content in autophagy-promoting compositions) In one embodiment, the content of cannabidiol (CBD) in the autophagy-promoting composition is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 10.0% by mass or more, more preferably 25.0% by mass or more, and particularly preferably 40.0% by mass or more. The upper limit of this content is preferably 99.0% by mass or less, more preferably 75.0% by mass or less, and particularly preferably 60.0% by mass or less.

[0046] (Cannabigerol (CBG) in autophagy-promoting compositions) In one embodiment, the content of cannabigerol (CBG) in the autophagy-promoting composition is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 10.0% by mass or more, more preferably 25.0% by mass or more, and particularly preferably 40.0% by mass or more. The upper limit of this content is preferably 99.0% by mass or less, more preferably 75.0% by mass or less, and particularly preferably 60.0% by mass or less. The cannabigerol (CBG) may be composed of natural cannabigerol (CBG-ext) and / or synthetic cannabigerol (CBG-syn), and there are no particular limitations on the content or ratio of each. For example, the cannabigerol (CBG) may preferably contain 50% or more, more preferably 80% or more, and particularly preferably 100% synthetic cannabigerol (CBG-syn).

[0047] (Total content of major cannabinoids in the autophagy-promoting composition) In one embodiment, the total content of cannabinoids (major cannabinoids) consisting of cannabidiol (CBD), natural cannabigerol (CBG-ext), and / or synthetic cannabigerol (CBG-syn) in the autophagy-promoting composition is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 10.0% by mass or more, more preferably 25.0% by mass or more, and particularly preferably 40.0% by mass or more. The upper limit of this content is preferably 99.0% by mass or less, more preferably 75.0% by mass or less, and particularly preferably 60.0% by mass or less.

[0048] (Functional ingredients with physiological functionality) In one embodiment, the autophagy-promoting composition of the present invention preferably contains a functional component having physiological functionality.

[0049] The physiologically functional component of the present invention is not particularly limited, and examples thereof include amino acids such as theanine, γ-aminobutyric acid, and taurine, hormones, neurotransmitters, pro-inflammatory cytokines such as G-CSF, IL-1, IL-6, IL-8, IL-11, IL-17, IL-18, IFN-α, IFN-β, IFN-γ, TNF-α, and TNF-β, anti-inflammatory cytokines such as IL-10, IL-12, IL-22, IL-37, IL-38, and TGF-β, GM-CSF, IL-2, IL-3, IL-4, IL-5, and IL- These include adaptive immune cytokines such as IL-9 and M-CSF, growth factors such as fibroblast growth factor (FGF), brain-derived neurotrophic factor, epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), vitamins and minerals, enzymes, nucleic acids, interferons, and physiologically active lipids such as platelet-activating factor (PAF), leukotrienes, steroids, sex hormones, inositol phospholipids, lysophosphatidic acid, and prostaglandins. As a result, the autophagy-promoting composition of the present invention has other physiological functionality in addition to the effect of an autophagy-promoting composition due to the combined use of CBD and CBG.

[0050] (The third cannabinoid component) In one embodiment, the autophagy-promoting composition of the present invention may additionally contain a third cannabinoid component in addition to CBD and CBG.

[0051] The third cannabinoid components include 3,4,5,6-tetrahydro-7-hydroxy-α-α-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocine-5-methanol (OH-iso-HHCV), 8,9-dihydroxy-delta-6a-tetrahydrocannabinol, 10-ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 10-oxo-delta-6a-tetrahydrocannabinol (OTHC), ethoxy-cannabidiol valine (CBTVE), cannabielson (CBE), and cannabinoids. Elsonic acid A (CBEA-A), cannabielsonic acid B (CBEA-B), cannabiolchol (CBN-C1), cannabichromanone (CBCN), cannabichromevalinic acid (CBCVA), cannabichromevarin (CBCV), cannabichromene (CBC), cannabigerol monomethyl ether (CBGM), cannabigerolic acid (CBGA), cannabigerolic acid monomethyl ether (CBGAM), cannabigerovalinic acid (CBGVA), cannabigerovarin (CBGV), cannabigeromenic acid (CBCA), cannabidiol mono Cannabidiol methyl ether (CBDM), cannabidiolic acid (CBDA), cannabidiolchol (CBD-C1), cannabicyclol (CBL), cannabicyclovaline (CBLV), cannabicyclolic acid (CBLA), cannabiditran (CBT), cannabidivalinic acid (CBDVA), cannabidivarin (CBDV), cannabiditriol (CBT), cannabiditriolvaline (CBTV), cannabinolic acid, cannabinol (CBN), cannabinol-C2 (CBN-C2), cannabinol-C4 (CBN-C4), cannabinol propionate lopil variant (CBNV), cannabinol methyl ether (CBNM), cannabinodivil (CBDL), cannabinodivarin (CBND), cannabinodivarin (CBVD), cannabinolic acid (CBNA), cannabivarin (CBV), cannabifuran (CBF), cannabilipsol (CBR), tetrahydrocannabinolic acid (THCA), tetrahydrocannabivarin (THCV), tetrahydrocannabivaric acid (THCVA), dehydrocannabifuran (DCBF), delta-11-tetrahydrocannabinol (Δ11-THC),Delta-7-cis-iso-tetrahydrocannabivarin, delta-8-tetrahydrocannabinol (Δ8-THC), delta-8-tetrahydrocannabinolic acid A (Δ8-THCA), delta-9-cis-tetrahydrocannabinol (Δ9-cis-THC), delta-9-tetrahydrocannabinol (Δ9-THC-C1), delta-9-tetrahydrocannabinol acid (Δ9-THCA-C1), delta-9-tetrahydrocannabinol (Δ9-THC), delta-9-tetrahydrocannabinol-C4( Δ9-THCA-C4), delta-9-tetrahydrocannabinolic acid-C4 (Δ9-THCA-C4), delta-9-tetrahydrocannabinolic acid A (Δ9-THCA-A), delta-9-tetrahydrocannabinolic acid B (Δ9-THCA-B), delta-9-tetrahydrocannabivarin (Δ9-THCV), delta-9-tetrahydrocannabivarinic acid (Δ9-THCVA), trihydroxy-delta-9-tetrahydroxycannabinol (triOH-Δ9-THC),

[0052] The content of the third cannabinoid component in the autophagy-promoting composition is, for example, 0.01 to 65.0% by mass. The lower limit of this content is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and particularly preferably 0.5% by mass or more. The upper limit of this content is preferably 65.0% by mass or less, more preferably 50.0% by mass or less, and particularly preferably 25.0% by mass or less.

[0053] (Other ingredients) The autophagy-promoting composition of the present invention may contain any other ingredients, such as pharmaceutically acceptable ingredients, cosmetically acceptable ingredients, or edible ingredients, as needed, depending on the dosage form, mode of use, etc., to the extent that the effects of the present invention are not impaired. Other ingredients include stabilizers, pH adjusters, nutritional ingredients, solubilizers, surfactants, nucleic acids, lubricants, sweeteners, buffers, diluents, absorption enhancers, adsorbents, flavoring agents, local anesthetic ingredients, binders, blood circulation promoting ingredients, suspending agents, glossing agents, antihistamines, anti-inflammatory ingredients, enzymes, fragrances, antioxidants, UV absorbers, humectants, fillers, sustained-release regulators, penetration enhancers, cooling agents, thickeners, extenders, colorants, coloring agents, antipruritic ingredients, alcohols such as lower alcohols (having 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms) and higher alcohols, animal and plant extracts, emulsifiers, film-forming agents, whitening ingredients, essential fatty acids, excipients, dispersants, moisturizing ingredients, preservatives, disintegrants, antiseptics, oils, solubilizers, amino acids, quinones, gelling agents, coating agents, steroids, vitamins, flavonoids, polyphenols, minerals, and water. The other components may be used alone or in combination of two or more, and the amounts of the other components to be added may be determined appropriately.

[0054] The autophagy-promoting composition of the present invention can be added to various compositions, which can be provided as compositions that are expected to have therapeutic and / or preventive effects against various diseases and aging, or to suppress the onset of diseases. Each composition containing the autophagy-promoting composition of the present invention will be described below.

[0055] [Food composition] A food composition according to one embodiment of the present invention is a food composition comprising the above-described autophagy-promoting composition.

[0056] The food composition of the present invention refers to a composition generally used for human consumption, including beverages, and may contain carriers, additives, or auxiliary substances that are generally accepted for use.

[0057] (Form of food composition) The form of the food composition is not particularly limited, and may be in any form such as solid, liquid, gel, or paste. The food composition may take the form of general processed foods, health foods, functional foods, dietary supplements, foods for specified health uses, foods with functional claims, foods for special dietary uses, beverages, and foods, or additives thereto. Specifically, it may be incorporated into supplements and beverages, but is not particularly limited thereto. Supplements may be in the same form as pharmaceuticals, such as liquids such as energy drinks, tablets, powders, granules, capsules, etc. The food composition of the present invention may also be animal feed or pet food. Furthermore, beverages, which are one form of food composition, may take the form of soft drinks, carbonated drinks, powdered drinks, coffee drinks, alcoholic beverages, energy drinks, juices, teas, liqueurs, soups, etc.

[0058] (Content of autophagy-promoting composition in food composition) In one embodiment, the content of the autophagy-promoting composition in the food composition of the present invention is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and particularly preferably 0.5% by mass or more. The upper limit of this content is preferably 99.95% by mass or less, more preferably 99.9% by mass or less, and particularly preferably 99.5% by mass or less.

[0059] [Pharmaceutical composition] Furthermore, the autophagy-promoting composition of the present invention exhibits both cell proliferation and autophagy-promoting effects due to the combined use of CBD and CBG. Therefore, the autophagy-promoting composition of the present invention is preferably added to a pharmaceutical composition. Pharmaceutical compositions containing the autophagy-promoting composition of the present invention are expected to be particularly useful for the prevention or treatment of diseases caused by the accumulation of abnormal proteins, such as cancer and neurodegenerative diseases.

[0060] (Content of autophagy-promoting composition as pharmaceutical composition) In one embodiment, the content of the autophagy-promoting composition in the pharmaceutical composition is, for example, 1.0 to 50.0 μg / m. The lower limit of this content is preferably 5.0 μg / m or more, more preferably 10.0 μg / m or more, and particularly preferably 25.0 μg / m or more. The upper limit of this content is preferably 50.0 μg / m or less, more preferably 40.0 μg / m or less, and particularly preferably 30.0 μg / m or less.

[0061] (Form of pharmaceutical composition) The pharmaceutical composition may be in any form suitable for human or animal medicine, such as liquid forms such as solutions, suspensions, oils, emulsions, gels, colloids, syrups, extracts, spirits, and elixirs; powders, granules, fine granules, tablets (including coated tablets such as sugar-coated tablets), solids, pills, granules, capsules (including hard capsules and soft capsules), troches, chewable tablets, semi-solid or solid forms such as creams, pastes, mousses, sheets, and liquid freeze-dried products; aerosols, patches, and poultices.

[0062] (Formulation of pharmaceutical compositions) In one embodiment, the pharmaceutical composition is formulated for administration by inhalation, a nebulizer, a vaporizer, or an aerosolizer.

[0063] (Method of manufacturing pharmaceutical composition) In one embodiment, the formulation of the pharmaceutical composition can be manufactured by a method that meets the standards set forth in the Japanese Pharmacopoeia or the standards required by other relevant foreign regulatory authorities or agencies.

[0064] (Dosage of pharmaceutical composition) In one embodiment, when a pharmaceutical composition containing the autophagy-promoting composition of the present invention is administered to a human, a useful dose of the autophagy-promoting composition per day, expressed as a weight per subject's body weight, can be in the range of 0.1 mg / kg to 100 mg / kg or 0.5 mg / kg to 50 mg / kg. When administered to a human, the composition is preferably administered once a day or in two or more divided doses. Furthermore, the daily dose may exceed the above amounts if necessary, at the discretion of a physician.

[0065] [Pharmaceutical composition for cancer treatment] In one embodiment of the present invention, the pharmaceutical composition for cancer treatment comprises the above-described composition for promoting autophagy.

[0066] The pharmaceutical composition for cancer treatment of the present invention can be used to treat cancer in mammals, particularly humans. The dosage and administration interval of the pharmaceutical composition for cancer treatment of the present invention can be appropriately selected at the discretion of a physician depending on the location of the disease and the patient's physical condition, i.e., height, weight, sex, medical history, etc. Furthermore, they can be adjusted as necessary to suit the observed effects of the treatment.

[0067] (Content of autophagy-promoting composition in pharmaceutical composition for cancer treatment) In one embodiment, the content of the autophagy-promoting composition in the pharmaceutical composition for cancer treatment is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and particularly preferably 0.5% by mass or more. The upper limit of this content is preferably 99.95% by mass or less, more preferably 99.9% by mass or less, and particularly preferably 99.5% by mass or less.

[0068] The type of cancer to which the pharmaceutical composition for cancer treatment of the present invention can be applied is not particularly limited, as long as sensitivity to the cancer has been confirmed, and examples include blood cancer, brain tumor, head and neck cancer, esophageal cancer, gastric cancer, appendix cancer, anal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, gastrointestinal stromal tumor, lung cancer, liver cancer, mesothelioma, thyroid cancer, kidney cancer, neuroendocrine tumor, melanoma, colon cancer, colorectal cancer, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, kidney cancer, bladder cancer, and testicular cancer.

[0069] [Pharmaceutical composition for treating neurodegenerative diseases] In one embodiment of the present invention, the pharmaceutical composition for treating a neurodegenerative disease comprises the above-described composition for promoting autophagy.

[0070] The pharmaceutical composition for treating neurodegenerative diseases of the present invention can be used to treat neurodegenerative diseases in mammals, particularly humans, similarly to the pharmaceutical composition for treating cancer described above. The dosage and administration interval of the pharmaceutical composition for treating neurodegenerative diseases of the present invention can be appropriately selected at the discretion of a physician depending on the location of the disease and the patient's physical condition, i.e., height, weight, sex, medical history, etc. Furthermore, they can be adjusted as necessary to suit the observed effects of the treatment.

[0071] (Content of autophagy-promoting composition in pharmaceutical composition for treating neurodegenerative diseases) In one embodiment, the content of the autophagy-promoting composition in the pharmaceutical composition for treating a neurodegenerative disease is, for example, 0.01 to 99.99% by mass. The lower limit of this content is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and particularly preferably 0.5% by mass or more. The upper limit of this content is preferably 99.95% by mass or less, more preferably 99.9% by mass or less, and particularly preferably 99.5% by mass or less.

[0072] Examples of neurodegenerative diseases to which the pharmaceutical composition for treating neurodegenerative diseases of the present invention can be applied include anxiety-related diseases, neuroses, convulsions, panic disorders, sleep disorders, depression, epilepsy, Parkinson's disease, Parkinsonian syndrome, Down's syndrome, schizophrenia, autonomic dysfunction, Huntington's disease, Alzheimer's disease-type dementia, affective disorders (including depressive or bipolar disorders), cognitive disorders, migraines, tension headaches, dissociative disorders, amyotrophic lateral sclerosis, acute disseminated (disseminated) encephalomyelitis, allergic encephalomyelitis, progressive multifocal leukoencephalopathy, postinfectious encephalitis, central pontine myelinolysis, adrenoleukodystrophy, multiple system atrophy, autism, insomnia, neurogenic cough, psychogenic convulsive seizures, and psychogenic syncope.

[0073] Furthermore, many diseases are caused by abnormalities in the autophagy system. In addition to the aforementioned pharmaceutical compositions for cancer treatment and pharmaceutical compositions for neurodegenerative diseases, compositions containing the autophagy-promoting composition of the present invention (pharmaceutical compositions) may be used. Examples of such diseases include metabolic disorders such as type II diabetes, nephropathy, metabolic syndrome, and fatty liver, as well as immune disorders such as Crohn's disease and autoimmune diseases. Furthermore, examples of infectious diseases include opportunistic infections. Other diseases include arteriosclerosis, muscle atrophy, myopathy, and anemia. [Example]

[0074] Hereinafter, one embodiment of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these.

[0075] [Reference example 1] First, as Reference Example 1 for the present invention, the effect of adding each cannabinoid (CBD, CBG-syn, or CBG-ext) alone on cell viability was evaluated by the following procedure.

[0076] (1)Cell culture Caco-2 cells (a human colon carcinoma-derived cell line) were cultured in a high-sugar DMEM (A1896701) supplemented with 20% fetal bovine serum (FBS; 10270-106), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin at 37°C, 5% CO2, and 95% humidity.

[0077] (2) Sample and drug matrix design Synthetic CBG (CBG-syn) ※ Naturally extracted CBG (CBG-ext), and naturally extracted CBD were obtained from Sophis Co., Ltd. These three cannabinoids were dissolved in DMSO. Various concentrations of CBD (concentrations: 0.01, 0.1, 1, 10, and 100 μM), CBG-syn (concentrations: 0.01, 0.1, 1, 10, and 100 μM), and CBG-ext (concentrations: 0.01, 0.1, 1, 10, and 100 μM) were used alone or in combination for 24 hours. *Manufactured by Willow Biosciences (Company website URL: https: / / willowbio.com / )

[0078] (3) Cell viability assessment Caco-2 cells (1 x 10 5 Cells / ml) were seeded into 96-well plates in a final volume of 110 μl / well. After one day of culture, each of the cannabinoids or solvent alone was added (n ≥ 4). Cell viability was measured using the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide], CCK-8 (Cell Counting Kit-8; Sigma Aldrich) method at 570 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).

[0079] The evaluation was carried out according to the above procedures (1) to (3). In Reference Example 1, each cannabinoid was used alone in procedure (2). More specifically, Caco-2 cells were treated with three cannabinoids (CBD, CBG-syn, or CBG-ext) at various concentrations (0.01, 0.1, 1, 10, and 100 μM) for 24 hours, and cell viability was measured using the CCK-8 assay. The results are shown in Figure 1. As shown in Figure 1, the addition of CBD, CBG-syn, or CBG-ext to Caco-2 cells resulted in a proliferation effect at cannabinoid concentrations ranging from 0.01 to 10 μM, and a cell death induction effect at a concentration of 100 μM. In other words, when the three cannabinoids were added alone at relatively low concentrations, they exhibited cell proliferation effects, but at high concentrations, all of them induced cell death.

[0080] [Example 1] As an example (Example 1) of the autophagy-promoting composition of the present invention, the effect of combined use of CBD with CBG-syn or CBG-ext on cell viability was evaluated. The procedures for evaluation in Example 1 are the same as (1) to (3) in Reference Example 1, and detailed explanations of each procedure will be omitted.

[0081] Here, Example 1 uses a combination of cannabinoids in step (2). More specifically, Caco-2 cells were treated with CBD and CBG-syn or CBD and CBG-ext at various concentrations (0.01, 0.1, 1, 10, and 100 μM) for 24 hours, after which cell viability was measured using the CCK-8 assay. The results are shown in Figure 2. As shown in Figure 2, when CBD and CBG-syn were used together, there was a tendency to promote cell proliferation compared to the addition of either alone in the range of 0.01 to 100 μM. On the other hand, when CBD and CBG-ext were used together, a cell proliferation effect was observed in the range of 0.01 to 10 μM, but at high concentrations (100 μM vs. 100 μM), a cell death-inducing effect was observed. Furthermore, high concentrations of CBD (100 μM) and low concentrations of CBG (0.01–10 μM) showed a reciprocal relationship (synergy score <1), indicating that the presence of low concentrations of CBG-syn inhibited the cell death induced by high concentrations of CBD alone. In other words, when CBD and CBG-ext were used together, they promoted cell proliferation at relatively low concentrations and induced cell death at high concentrations, similar to the results when either compound was added alone. On the other hand, when CBD and CBG-syn were used together, unlike the results obtained when either compound was added alone, cell proliferation was promoted at all concentrations and cell death was not induced. These results suggest that the combination of CBD and CBG-syn produces different effects than the combination of CBD and CBG-ext in assessing cell viability.

[0082] [Reference example 2] Next, as Reference Example 2 of the present invention, the effect of adding each cannabinoid (CBD, CBG-syn, or CBG-ext) alone on autophagic flux was evaluated by the following procedure.

[0083] Cell culture and sample preparation are carried out according to the procedures (1) and (2) in Reference Example 1 described above. Next, instead of the procedure (3) in Reference Example 1, the following procedures (4) and (5) are carried out.

[0084] (4) Preparation of GFP-LC3-RFP-expressing Caco-2 cells and measurement of cellular autophagic flux HEK293FT cells (a human colon carcinoma cell line) were transfected with GFP-LC3-RFP-LC3ΔG, pCG-VSV-G, and pCG-gag / pol. After 72 hours of incubation, the virus-containing medium was collected and incubated with Caco-2 cells for 48 hours. 1 μM Torin (an autophagy inducer) served as a positive control, and 200 μM Bafilomycin (an autophagy inhibitor) served as a negative control. After cannabinoid or vehicle treatment, cells were harvested and their fluorescence intensity was measured using a Cellometer® Vision (Nexcelom Bioscience LLC, Lawrence, MA, USA). The GFP / RFP fluorescence intensity ratio (GFP, excitation wavelength: 460-490 nm, emission wavelength: 500-550 nm; RFP, excitation wavelength: 530-560 nm, emission wavelength: 570-650 nm) of the intracellular GFP-LC3-RFP-LC3ΔG probe was used as an index of autophagic flux. Here, a lower GFP / RFP ratio indicates higher autophagic activity.

[0085] (5) Statistical analysis for evaluating the effect of promoting autophagy Data are expressed as mean ± SEM and were statistically analyzed using unpaired Student's t-test for comparisons between two independent groups, and one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test for multiple comparisons between each experimental and control group. The threshold for statistical significance was set at p < 0.05. Synergy was calculated using Synergyfinder and the Zero Interaction Potency (ZIP) method. ZIP scores were used to represent synergistic (ZIP score <1), additive (ZIP score = 1), and antagonistic (ZIP score >1) effects and were calculated according to the standard isobologram equation. Delta scores based on the ZIP model are expressed as a delta score of 0. A delta score greater than 0 indicates a greater effect (synergistic) than the expected effect, whereas a delta score less than 0 indicates a smaller effect (antagonistic).

[0086] The evaluation was carried out based on the above procedures (1), (2), (4), and (5). In Reference Example 2, each cannabinoid was used alone in procedure (2). More specifically, Caco-2 cells were treated with three cannabinoids (CBD, CBG-syn, or CBG-ext) at various concentrations (0.01, 0.1, 1, 10, and 100 μM) for 24 hours, and the autophagic flux index was calculated from the fluorescence intensity measurements. The results are shown in Figure 3. As shown in Figure 3, when added alone, the addition of CBD or CBG-syn to Caco-2 cells significantly promoted or inhibited autophagy at the concentrations shown below. Effect of CBD addition: Promotion: 10 and 100 μM Effect of CBG-syn addition; Inhibition: 0.01 and 10 μM On the other hand, the addition of CBG-ext did not have any inhibitory effect at any concentration, and promoted autophagy at a concentration of 100 μM. In other words, in the autophagic flux assessment, the three cannabinoids showed different effects when added alone. CBD showed pro-autophagy effects at relatively high concentrations, while CBG-ext showed pro-autophagy effects only at the highest concentrations. On the other hand, CBG-syn showed an autophagy inhibitory effect at both low and high concentrations.

[0087] [Example 2] As an example (Example 2) of the autophagy-promoting composition of the present invention, the effect of combined use of CBD with CBG-syn or CBG-ext on autophagic flux was evaluated. The procedures for evaluation in Example 2 are the same as (1), (2), (4), and (5) in Reference Example 2, and detailed explanations of each procedure will be omitted.

[0088] Here, Example 2 uses a combination of cannabinoids in step (2). More specifically, Caco-2 cells were treated with CBD and CBG-syn or CBD and CBG-ext at various concentrations (0.01, 0.1, 1, 10, and 100 μM) for 24 hours, and the autophagic flux index was calculated from the fluorescence intensity measurements. The results are shown in Figure 4. As shown in Figure 4, when CBD and CBG-syn were used in combination, a synergistic effect was observed, with a CBD concentration in the range of 10 to 100 μM and a CBG-syn concentration in the range of 1 to 100 μM, compared to the use of either alone (Figure 4(B) bottom). These results, unlike those obtained when each cannabinoid was added alone in Reference Example 2, demonstrated that the autophagy-promoting effect of the combined use of CBD and CBG-syn was significantly demonstrated within a specific cannabinoid concentration range. On the other hand, when CBD and CBG-ext were used in combination, an autophagy-promoting effect was observed compared to the addition of either alone, at CBD concentrations in the range of 1 to 100 μM and at CBG-ext concentrations in the range of 0.01 to 100 μM, and an antagonistic effect was observed (Figure 4(A)). This result differs from the result in Reference Example 2, in which an autophagy-promoting effect was observed only when CBG-ext was added alone at a concentration of 100 μM, and also differs from the result in this Example 2, in which CBD and CBG-syn were used in combination, demonstrating an antagonistic effect. Furthermore, as shown in Figure 4(C)C, when CBG-syn and CBD were used together, a significantly greater autophagy-promoting effect was observed than when CBG-ext and CBD were used together over the range of 0.01 to 100 μM.

[0089] When CBD and CBG-syn were used together, they promoted cell proliferation within a specific concentration range and simultaneously promoted autophagy, demonstrating a synergistic effect. In contrast, when CBD and CBG-ext were used together, they showed cell proliferation effects within a certain concentration range and also promoted autophagy, but these were antagonistic effects. These results suggest that the combined use of CBD and CBG may have a promoting or antagonistic effect on autophagy. In other words, it was shown that when CBD and CBG-syn or CBD and CBG-ext are used together, new effects are observed that do not occur when either is used alone. Furthermore, when preparing an autophagy-promoting composition (cannabinoid-containing composition), it was shown that combining multiple cannabinoids (CBD and CBG) within a specific concentration range further enhanced the cell proliferation-promoting effect of the combination, as well as the autophagy-promoting effect.

[0090] Furthermore, by adjusting these conditions, the autophagy-promoting composition of the present invention can be added to other compositions as an ingredient that enables cell proliferation and autophagy regulation. For example, it is expected to be used as an active pharmaceutical ingredient for functional foods and disease treatment, such as the food composition and pharmaceutical composition of the present invention (pharmaceutical composition for cancer treatment, pharmaceutical composition for neurodegenerative disease treatment). [Industrial Applicability]

[0091] The autophagy-promoting composition and cannabinoid-containing composition of the present invention can be used as compositions that exhibit an autophagy-promoting or antagonistic effect. Furthermore, autophagy is an important process for maintaining cellular homeostasis, and abnormalities in autophagy are associated with many diseases, particularly cancer and neurodegenerative diseases. Therefore, the autophagy-promoting composition and cannabinoid-containing composition of the present invention can be added to various compositions and used. In particular, when added to food compositions or pharmaceutical compositions, they can be suitably used in technical fields such as functional foods and pharmaceuticals.

[0092] Furthermore, the food composition, pharmaceutical composition for treating cancer, and pharmaceutical composition for treating neurodegenerative diseases of the present invention can be suitably used as foods and pharmaceuticals that can regulate autophagy function.

Claims

1. Cannabidiol and A composition for promoting autophagy, comprising cannabigerol.

2. 2. The autophagy-promoting composition according to claim 1, wherein the cannabigerol is synthetic cannabigerol.

3. The autophagy-promoting composition according to claim 1 or 2, characterized in that the molecular mixing ratio of the cannabidiol and the cannabigerol is 10,000:1 to 0.01:

1.

4. The autophagy-promoting composition according to claim 1 or 2, further comprising a functional component having physiological functionality.

5. A food composition comprising the autophagy-promoting composition according to claim 1 or 2.

6. A pharmaceutical composition for cancer treatment, comprising the autophagy-promoting composition according to claim 1 or 2.

7. A pharmaceutical composition for treating a neurodegenerative disease, comprising the autophagy-promoting composition according to claim 1 or 2.

8. A cannabinoid-containing composition comprising cannabidiol and synthetic cannabigerol.

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