Betulin as a hepatic X receptor agonist and its uses

Abstract

This invention relates to betulin as a hepatitis X receptor agonist and its use in treating hepatitis X receptor-mediated diseases. According to one aspect of the invention, betulin and its prodrug have specific agonist activity against hepatitis X receptor β (LXRβ). Therefore, compared with other hepatitis X receptor (LXR) agonists, it not only has significant stability but also has the effect of effectively treating hepatitis X receptor (LXR) related diseases, such as neurodegenerative diseases, autoimmune diseases, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), etc.

Description

Technical Field

[0001] This invention relates to betulin as a hepatic X receptor agonist and its use in treating hepatic X receptor-mediated diseases. Background Technology

[0002] Liver-X receptors (LXRs) are ligand-activated transcription factors that play a crucial role in regulating gene expression involved in lipid metabolism and cellular cholesterol homeostasis. LXR agonists have been shown to enhance reverse cholesterol transport (RCT), which involves the retrograde transport of cholesterol from the liver periphery back to the liver for processing and excretion. RCT occurs by upregulating cholesterol transport proteins (ATP-binding cassettes: ABCA1 and ABCG1) in peripheral phagocytes. Activation of RCT has been reported to inhibit the progression of atherosclerosis.

[0003] The liver X receptor (LXR) has two isoforms encoded by different genes: LXRα (NR1H3) and LXRβ (NR1H2). LXRβ is generally expressed in most tissues, while LXRα expression is tissue-selective and is expressed in the liver, small intestine, kidney, adipose tissue, and adrenal glands, which are important for lipid homeostasis. Both liver X receptors (LXRs) require a retinoid-X receptor (RXR) as a mandatory heterodimer partner for co-recognizing or binding liver X receptor response elements (LXREs), wherein the liver X receptor response elements (LXREs) consist of direct repetitions of two core hexameric sequences separated by four nucleotides (DR4). The ligand-binding domains of the two liver X receptors (LXRs) are remarkably well preserved (~78% amino acid homology) and bind to endogenous ligands, which are cholesterol oxidation derivatives (oxidized cholesterol; oxysterols) that act as intermediates in the synthesis of steroid hormones and bile acids. Among these, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol have been reported as the most potent ligands. These data suggest that liver X receptors (LXRs) may play an important role in maintaining cholesterol homeostasis, a role subsequently confirmed by gene knockout studies in mice. To date, non-steroidal ligands have been identified, and their use as chemical probes has led to the discovery of numerous genes regulating liver X receptors (LXRs). Genes containing multiple liver X receptor response elements (LXREs) are involved in cholesterol metabolism, reverse cholesterol transport (RCT), and lipogenesis. Other genes involved in inflammation and carbohydrate metabolism deficient liver X receptor response elements (LXREs) are inhibited by liver X receptors (LXRs) in a ligand-dependent manner.Based on these findings, the aforementioned liver X receptor has recently emerged as an excellent target for acting as an intracellular cholesterol sensor, providing a basis for the treatment of various diseases, including atherosclerosis, diabetes, Alzheimer's disease, skin diseases, reproductive diseases, and cancer (Viennoise et al., 2011, Expert Opin. Ther. Targets, 15(2):219-232). Furthermore, liver X receptor (LXR) agonists have been found to sequentially regulate intestinal and renal sodium phosphate (NaPi) transporters and serum phosphate concentrations (Caldas et al., 2011, Kidney International, 80:535-544). Therefore, liver X receptors (LXRs) could also serve as a target for the prevention of kidney diseases, particularly hyperphosphatemia and related cardiovascular complications. In addition, liver X receptors (LXRs) are considered to be targets for the treatment of osteoporosis and related diseases (Kleyer et al., 2012, J. Bone Miner. Res., 27(12): 24451).

[0004] Alzheimer's disease, the most common form of dementia, is characterized by the accumulation and deposition of amyloid-beta (Aβ) peptides in the brain, and by the perturbation of neural junctions and the loss of neurons in the brains of affected individuals. Neurons produce amyloid-beta (Aβ) peptides through the cleavage of amyloid precursor protein (APP) in the brain, and these peptides are typically eliminated through efflux into peripheral circulation and the action of proteolytic enzymes in the brain.

[0005] Apolipoprotein E (apoE) is associated with age-related risk in Alzheimer's disease and plays a crucial role in β-amyloid peptide (Aβ) homeostasis. Hepatic X receptors (LXRs) increase apoE expression and lipid binding. Decreases in β-amyloid peptide (Aβ), both intracellularly and extracellularly, are enhanced by lipid-bound apoE. LXR agonist treatment promotes reduced proteolysis of β-amyloid peptide (Aβ), decreases plaque disease, and enhances memory in mice transplanted with the amyloid precursor protein (APP) expression gene (Jiang et al., 2008, Neuron, 58:681-693).

[0006] Therefore, it is necessary to develop methods for treating liver X receptor (LXR)-mediated diseases such as arteriosclerosis, Alzheimer's disease, and metabolic diseases by developing liver X receptor (LXR) agonists. Summary of the Invention

[0007] Technical issues

[0008] One aspect of the present invention is to provide a liver-X receptor (LXR) agonist comprising betulin, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of liver-X receptor (LXR)-mediated diseases, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] Another aspect of the invention is the use of betulin, its prodrug, or a pharmaceutically acceptable salt thereof in the preparation of a liver-X receptor (LXR) agonist or a therapeutic agent for liver-X receptor-mediated diseases.

[0011] Another aspect of the invention is to provide a method for upregulating liver-X receptors (LXRs) and a method for treating liver-X receptor-mediated diseases, comprising administering betulin, its prodrug, or a pharmaceutically acceptable salt thereof to an individual in need of it.

[0012] Another aspect of the present invention is to provide a health product for the prevention or improvement of liver-X receptor (LXR) mediated diseases, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another aspect of the present invention is to provide a cosmetic composition for preventing or improving liver-X receptor (LXR) mediated skin conditions, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Technical solution

[0015] One aspect of the present invention provides a liver-X receptor (LXR) agonist comprising betulin, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of liver X receptor (LXR) mediated diseases, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] Another aspect of the invention provides the use of betulin, its prodrug, or a pharmaceutically acceptable salt thereof for the preparation of a liver X receptor (LXR) agonist or a therapeutic agent for liver X receptor-mediated diseases.

[0018] Another aspect of the invention provides a method for upregulating liver-X receptors (LXRs) and a method for treating liver-X receptor-mediated diseases, comprising administering betulin, its prodrug, or a pharmaceutically acceptable salt thereof to an individual in need of it.

[0019] As used in this article, the IUPAC name for "Inotodiol" is (3S,5R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopentadien[a]phenanthrene-3-ol ((3S,5R)-1H-cyclopentadien[a]phenanthrene-3-ol) R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol).

[0020] The betulin, as the main physiologically active component of Inonotus obliquus, can be chemically synthesized using conventional methods and can be prepared into a pharmaceutically acceptable salt, or can be isolated and purified from Inonotus obliquus extract.

[0021] The betulin or its precursor drug acts as a liver X receptor (LXR) agonist.

[0022] This document provides a method for treating subjects with diseases or disorders that can be treated with hepatic X receptor (LXR) agonists. In one embodiment, hepatic X receptors (LXRs) can be modulated by upregulating the activity of hepatic X receptors (LXRs). The method consists of administering an effective amount of betulin or a prodrug thereof.

[0023] The methods or pharmaceutical compositions described herein can be effective for disorders that can be treated by modulating hepatic X receptors (LXRs), particularly as hepatic X receptor (LXRs) agonists.

[0024] In one embodiment, betulin or its prodrug is effective in treating or preventing diseases or disorders related to altered cholesterol transport, fatty acid metabolism, cholesterol absorption, cholesterol reabsorption, cholesterol secretion, cholesterol excretion, or cholesterol metabolism. Representative diseases or disorders include lipid disorders; cancers, particularly hormone-dependent cancers including ovarian, breast, and prostate cancer; acne-prone skin conditions; inflammatory skin diseases; immune disorders; conditions characterized by disruption of the epidermal barrier function; disordered differentiation or excessive spread of the epidermis or mucous membranes; cardiovascular disorders; reproductive tract disorders; optic nerve and retinal abnormalities; neurodegenerative disorders caused by disease; damage to the central or peripheral nervous system; neuropathic diseases; or degenerative processes due to aging; kidney disease or disorders; and osteoporosis and related diseases, but not limited thereto.

[0025] In another embodiment, the disease or disorder is a neurodegenerative brain disease, an autoimmune disease, hyperlipidemia, hypercholesterolemia, hyperlipoproteinemia, hypertriglyceridemia, lipodystrophy, hepatosteosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), hyperglycemia, insulin resistance, diabetes mellitus, dyslipidemia, atherosclerosis, gallstone disease, acne vulgaris, or dermatitis (not limited to psoriasis or contact dermatitis). (dermatitis), hyperphosphatemia, cardiovascular complications of hyperphosphatemia, cancer, multiple sclerosis, or osteoporosis.

[0026] In another embodiment, the disease or disorder includes common acne; comedones; polymorphs; rosacea; nodulocystic acne; acne conglobate; senile acne; secondary acne, including but not limited to solar, medicinal, and occupational acne; ichthyosis; ichthyosiform conditions; Darier's disease; palmoplantar keratoderma; leukoplakia; leukoplakiform conditions; cutaneous or mucosal (oral) lichenification. Mucous (oral) lichen; including eczema, respiratory atopy, and gingival hypertrophy, with or without an inflammatory immunoallergic component and acellular proliferation disorder, including but not limited to cutaneous psoriasis, mucous psoriasis, ungual psoriasis, psoriatic rheumatism, and cutaneous atopy; benign or malignant skin or epidermal spreads caused by viruses or non-viral agents, including but not limited to common warts, flat warts, epidermal dysplasia verruciformis, and oral cauliflower-like papillomatosis. papillomatoses), and T-lymphoma or cutaneous T-cell lymphoma;The following conditions are considered to be caused by ultraviolet radiation: 1. Ultraviolet radiation-induced diffusion, including but not limited to basocellular epithelioma and spinocellular epithelioma; 2. Precancerous skin lesions, including but not limited to keratoacanthoma; 3. Immune dermatitis, including but not limited to lupus erythematosus; 4. Immune bullous diseases; 5. Collagen diseases, including but not limited to scleroderma; 6. Afflictions with an immune component; 7. Skin disorders caused by exposure to ultraviolet (UV) radiation; 8. Sun-induced skin aging or chronic skin aging; 9. Actinic pigmentations; 10. Keratosis; 11. Diseases related to age or photoaging, including but not limited to xerosis; 12. Sebaceous dysfunction. Disorders, including but not limited to seborrheic acne, simple seborrhea, and seborrheic dermatitis; cicatrization disorders, including but not limited to stretch marks; pigmentation disorders, including but not limited to hyperpigmentation, melasma, hypopigmentation, and vitiligo; and alopecia, including but not limited to chemotherapy-related alopecia and radiation-related alopecia.

[0027] In another embodiment, the disease or disorder is a neurodegenerative brain disease or an autoimmune disease.

[0028] The neurodegenerative diseases mentioned can be selected from dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloidosis, Dutch amyloidosis, Niemann-type amyloidosis, etc.

[0029] Pick's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy

[0030] The group consists of Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia.

[0031] The autoimmune diseases mentioned can be selected from the group consisting of rheumatoid arthritis, psoriasis, dermatitis, multiple sclerosis, and diabetes mellitus. The dermatitis mentioned can include atopic, acne-prone, and contact dermatitis, etc.

[0032] The inventors have confirmed that betulin can be effectively delivered to the brain of animals and have confirmed that it increases the expression of the genes of ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein E (APOE), which are known as factors for eliminating inflammation and β-amyloid in the brain. Therefore, it has been shown to be effective in preventing or treating neurodegenerative diseases.

[0033] Furthermore, betulin has been shown to alleviate arthritis in animal models of arthritis and has been shown to reduce the expression of tumor necrosis factor-α (TNF-α). Therefore, it has been confirmed that it can be effectively used to prevent or treat arthritis and autoimmune diseases, such as psoriasis and dermatitis.

[0034] In one specific embodiment, the prodrug of betulin can be a betulin ester derivative compound formed by the ester condensation reaction of the hydroxyl group of the betulin compound shown in Formula 1 with the carboxyl group of fatty acid, glucuronic acid, alkyl succinic anhydride or phenolic acid:

[0035] [Formula 1]

[0036]

[0037] Specifically, the prodrug can be a compound represented by Formula 2:

[0038] [Equation 2]

[0039]

[0040] In the formula, R1 and R2 are independently OH or -OC(O)-R3, and R3 is a straight-chain or branched alkyl, alkenyl, or alkynyl group having 1 to 30, 4 to 30, or 6 to 30 carbon atoms.

[0041] Furthermore, at least one of R1 and R2 is -OC(O)-R3.

[0042] In one specific embodiment, R3 has CH3(CH2) a -or CH3(CH2) b (CH=CH[CH2]) c (CH2) d - an unsubstituted straight-chain alkyl or alkenyl group, where a is an integer from 8 to 24, b is an integer from 1 to 5, c is an integer from 1 to 6, and d is an integer from 3 to 7.

[0043] As used herein, the term "prodrug" can refer to a drug that may itself lack biological activity, but exhibits effective pharmacological effects after undergoing a chemical / biochemical structural transformation during its residence in the body. In other words, although it is a useful drug, it can be chemically modified to address undesirable properties such as side effects, stability, solubility, absorption, and duration of action, thereby enabling clinical use. According to a specific embodiment, a betulinol derivative, as a compound formed by an ester bond between betulinol and a fatty acid, is not only improved in terms of solubility and stability in organic solvents, but is also readily absorbed by the small intestine due to its easy breakdown into betulinol and fatty acids in the small intestine. For example, the betulinol derivative can be a compound formed by an ester bond between betulinol and succinic acid. Specifically, the betulinol derivative can be synthesized by a condensation reaction of alkyl succinic anhydride and betulinol in the presence of p-toluenesulfonic acid as a catalyst. Adding an excess of alkyl succinic anhydride compared to betulinol induces the ester bond of succinic acid to all or at least one hydroxyl group of betulinol. After the reaction, sodium bicarbonate is used for neutralization to remove p-toluenesulfonic acid. In the betulinol derivative compound synthesized according to the method, all or at least one R1 and R2 in Formula 2 form a succinate bond, thereby improving water solubility and facilitating absorption by the small intestine due to the easy decomposition of the ester bond. Furthermore, according to another specific embodiment, the betulinol derivative not only increases the water solubility of betulinol through the ester bond between betulinol and fatty acids but also reduces hydrolysis and in vivo absorption in the small intestine, increasing the probability of the betulinol derivative reaching the large intestine. This allows betulinol to directly exert its pharmacological effects even when the betulinol derivative is decomposed by various microorganisms in the large intestine. For example, the betulinol derivative can be a compound formed by an ester bond between betulinol and phenolic acid. Specifically, the ester bond of the betulinol derivative is hydrolyzed in the large intestine to release betulinol and phenolic acid, thus allowing for the expectation of various physiological activities of both betulinol and phenolic acid. Therefore, the prodrug according to one aspect of the present invention can not only reduce the dosage of betulinol but also reduce the side effects that may occur when taken at high concentrations.

[0044] The betulinol prodrug derivative can be synthesized by chemical or biological methods. Specifically, the betulinol derivative can be synthesized based on the esterification reaction of betulinol and a fatty acid. The fatty acid can be C... 10 To C 30 Unsaturated or saturated fatty acids, and can be selected from C1 to C2. 10The group consisting of carboxylic acids or phenolic acids. The unsaturated fatty acids may be, for example, myristic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, trans-oleic acid, isoleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. The saturated fatty acids may be, for example, caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, arachidic acid, benzyl acid, ceric acid, waxy acid, and myristic acid. The saturated carboxylic acids may be, for example, formic acid, acetic acid, propionic acid, butyric acid, and succinic acid. The phenolic acid may be, for example, p-coumaric acid, cinnamic acid, ferulic acid, 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, sinapinica acids, etc. In one specific embodiment, the betulinol derivative may be prepared by mixing betulinol and selected unsaturated fatty acids, saturated fatty acids, and C1 to C2 fatty acids in a molar ratio of 1:10 to 30. 10 The betulinol derivative is synthesized from fatty acids in the group consisting of carboxylic acids. For example, the betulinol derivative can be prepared by mixing in molar ratios of 1:10 to 30, 1:10 to 25, 1:10 to 20, 1:10 to 15, 1:10 to 13, 5:10 to 30, or 5:10 to 15. In this case, when the mixing ratio of betulinol and fatty acids is less than or greater than the aforementioned range, the reaction proceeds incompletely, leading to a decrease in the efficiency of betulinol derivative formation or an increase in the reaction time. Furthermore, the esterification reaction can be carried out at 55°C to 65°C for 48 to 96 hours. In this case, when the esterification reaction conditions are less than or greater than the aforementioned range, the fatty acids used as the reaction substrate are difficult to completely dissolve, resulting in almost no reaction or reduced enzyme activity, leading to a decrease in esterification reaction efficiency. This results in the inability to synthesize the betulinol derivative as the final product in high yield. Furthermore, after the esterification reaction is completed, the purity of the esters contained in the product can be further improved by various known distillation or purification methods.

[0045] Furthermore, synthesis can be carried out by adding a bioenzyme to the mixture of betulinol and fatty acids, and the bioenzyme is suitable for preparing highly efficient betulinol derivatives, for example, it can be Candida Antarctica Lipase B (CalB). To improve the yield of the betulinol derivative as the final product, the Candida Antarctica Lipase B can be an immobilized enzyme instead of a conventional enzyme, and the immobilized enzyme can be a commercially available product or an immobilized enzyme prepared by conventional methods. Furthermore, based on 100 parts by weight of betulinol, 400 to 500 parts by weight of the Candida Antarctica Lipase B can be added to synthesize the betulinol derivative. However, when the content of Candida Antarctica Lipase B is less than the specified range, the esterification reaction is insufficient, resulting in low ester production efficiency; when it is greater than the specified range, the betulinol derivative as the final product cannot be synthesized in high yield, thus presenting an uneconomical problem.

[0046] According to one aspect of the present invention, the pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols by various conventional methods, or into external dosage forms, suppositories, and sterile injectable solutions. In order to formulate the pharmaceutical composition, suitable carriers, excipients, or diluents conventionally used in the preparation of the pharmaceutical composition may be included.

[0047] The carrier, excipient, or diluent may be a variety of compounds or mixtures including lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, tricalcium silicate, cellulose, lignocellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0048] When formulating dosage forms, commonly used fillers, weighting agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients can be used for preparation.

[0049] Another aspect of the invention provides a method for preventing or treating liver-X receptor (LXR) mediated diseases, comprising administering inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof to an individual in need of it.

[0050] Another aspect of the invention provides the use of inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof in the preparation of compositions for the prevention or treatment of liver-X receptor (LXR) mediated diseases.

[0051] The diseases mediated by the liver-X receptors (LXRs) are as described above.

[0052] This instruction manual provides methods for increasing cholesterol reverse transport and / or inhibiting the progression of atherosclerosis and promoting the relief of atherosclerosis.

[0053] This specification provides a method for treating a disease or disorder associated with the need to increase high-density lipoprotein (HDL) cholesterol levels, comprising administering an effective amount of betulin or a precursor thereof to a mammal (particularly a human) in need.

[0054] This specification provides a method for treating a disease or disorder associated with the need to lower low-density lipoprotein (LDL) cholesterol levels, comprising administering an effective amount of a compound, specifically betulin or a precursor thereof, to a mammal (particularly a human) in need.

[0055] Orally administered solid dosage forms can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the said legume extract. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0056] Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and mineral oil, which are commonly used simple diluents, they can also include various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc.

[0057] Parenteral preparations include sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, lyophilized preparations, and suppositories. Water-insoluble preparations and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, semi-synthetic fatty acid esters (Witepsol), polyethylene glycol, Tween 20, cocoa butter, lauryl acetate, and glycerin gelatin can be used.

[0058] The preferred dosage of the pharmaceutical composition according to one aspect of the invention can vary depending on the patient's condition, weight, disease severity, drug type, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. However, to obtain the desired effect, it can be administered daily at a dose of 0.0001 to 2000 mg / kg, preferably 0.001 to 2000 mg / kg. It can be administered once daily or divided into several doses. However, the scope of the invention is not limited to the stated dosage.

[0059] The pharmaceutical composition according to one aspect of the invention can be administered to mammals, such as rats, mice, livestock, and humans, via various routes. All routes of administration can include, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intraventricular injection.

[0060] Another aspect of the invention provides a cosmetic composition for preventing or improving liver-X receptor (LXR)-mediated skin conditions, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0061] The skin condition mediated by liver X receptors is as described above.

[0062] The cosmetic composition may have, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a serum, a mask, a gel, a concentrated serum, or a skin-adhesive cosmetic formulation.

[0063] In addition to the composition as an active ingredient, the cosmetic composition may also include ingredients conventionally used in cosmetic compositions, such as stabilizers, solubilizers, vitamins, pigments and flavorings, and other conventional adjuvants and carriers.

[0064] Another aspect of the present invention provides a health product for the prevention or improvement of liver X receptor-mediated diseases, comprising inotodiol, its prodrug, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0065] According to one aspect of the health product of the present invention, when the compound is used as an additive in the health product, it can be added directly or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to various purposes of use such as prevention, health care, or treatment.

[0066] Health supplements can be in any dosage form, including not only powders, granules, pills, tablets or capsules, but also general food or beverage dosage forms.

[0067] There are no particular restrictions on the types of food mentioned. Examples of foods to which the substance may be added include meat, sausage, bread, chocolate, candy, pastries, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, oral rehydration solutions, alcoholic beverages, and vitamin complexes, etc. It can include all foods in the conventional sense.

[0068] Typically, when preparing food or beverages, the amount of the compound added based on 100 parts by weight of raw materials can be less than or equal to 15 parts by weight, preferably less than or equal to 10 parts by weight. However, when consumed long-term for health and hygiene purposes or for health management purposes, the amount can be less than or equal to the range stated in the text, and since fractions of natural products are used, there are no safety concerns; therefore, amounts greater than or equal to the range can also be used.

[0069] The beverage in the health product according to one aspect of the present invention may include various flavorings or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as sematrandrine and stevia extract may be used, or synthetic sweeteners such as saccharin and aspartame may be used. Based on 100 mL of the beverage of the present invention, the proportion of the natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g.

[0070] In addition to the foregoing, health products according to one aspect of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the compositions of the present invention for improving sleep may include fruit pulp used in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used alone or in combination. The proportions of these additives are not limited, but are generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health product of the present invention.

[0071] Invention Effects

[0072] According to one aspect of the present invention, betulin and its prodrug have specific agonist activity against liver X receptor β (LXRβ). Therefore, compared with other liver X receptor (LXR) agonists, they not only have significant stability, but also have the effect of being effectively used to treat liver X receptor (LXR) related diseases, such as neurodegenerative diseases, autoimmune diseases, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), etc. Attached Figure Description

[0073] Figure 1 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in liver samples according to a specific embodiment.

[0074] Figure 2 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in spleen samples according to a specific embodiment.

[0075] Figure 3 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in peritoneal cell samples according to a specific embodiment.

[0076] Figure 4 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in lung samples according to a specific embodiment.

[0077] Figure 5 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in thymus samples according to a specific embodiment.

[0078] Figure 6 is a graph demonstrating that betulinol, according to a specific embodiment, increases gene expression associated with the inhibition of neurodegenerative diseases.

[0079] Figure 6a This is a chart demonstrating that, according to a specific embodiment, betulin increases the expression of a gene associated with the inhibition of neurodegenerative diseases, namely ATP-binding cassette transporter A1 (ABCA1). Figure 6b This is a graph demonstrating that, according to one specific embodiment, betulin increases the expression of apolipoprotein E (APOE), a gene associated with the inhibition of neurodegenerative diseases.

[0080] Figure 7 This is a graph showing the rheumatoid arthritic score of the efficacy of betulinol against rheumatoid arthritis according to a specific embodiment.

[0081] Figure 8 This is a graph showing the inhibition of tumor necrosis factor-α (TNF-α) expression by betulin according to a specific embodiment. Detailed Implementation

[0082] Preferred embodiments are presented below to aid in understanding the invention. However, the following embodiments are provided for ease of understanding, and the scope of the invention is not limited to these embodiments.

[0083] Example 1. Analysis of the effects of betulin on liver X receptor (LXRs)-related factors in vivo.

[0084] The effects of betulin on ATP-binding cassette transporter A1 (Abca1), sterol regulatory element-binding transcription factor 1c (Srebf1_1C), aquaporin 1 (AQP1), and apolipoprotein E1 (ApoE1), which are biomarkers associated with liver X receptors (LXRs), were analyzed.

[0085] First, a betulin emulsion was prepared for administration at a dose of 4 mg / kg. Specifically, the emulsion was prepared by mixing betulin with 10% olive oil (Monini, Classicoextra virgin olive oil), 0.5% Tween 80, and sterile water. Subsequently, the prepared emulsion was administered to BALB / cAnNTacSam (Sam Taco Bio Korea Co., Ltd.) daily at a dose of 0.15 mL for 5 consecutive days. After 5 days of administration, mice were euthanized, and spleen, lung, liver, and peritoneal cavity cell samples were isolated.

[0086] Next, in order to perform real-time quantitative PCR (qPCR) on the separated samples, Bead was used. TM TotalRNA Prep Kit (BioFACT) TM Total RNA was isolated from the sample. Primers for ATP-binding cassette transporter A1 (Abca1), sterol regulatory element-binding transcription factor 1c (Srebf1_1C), aquaporin 1 (AQP1), and apolipoprotein E1 (ApoE1) were then used, and the reaction reagent for real-time quantitative PCR (qPCR) was 2X Real-Time PCR Master Mix (BioFACT). TMThe instrument used for real-time quantitative PCR (qPCR) was an AriaMx Real-time PCR. The conditions for qPCR were: 50 cycles of 95℃ (15 min), 95℃ (20 s) - 55℃ (30 s) - 72℃ (0.5-1 min / kb), followed by one cycle of a melting curve of 95℃ (30 s) - 65℃ (30 s) - 95℃ (30 s). The relative expression level of mRNA was then measured. An untreated control group was used as a control, and the results were as follows: Figures 1 to 4 As shown.

[0087] Figure 1 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in liver samples according to a specific embodiment.

[0088] Figure 2 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in spleen samples according to a specific embodiment.

[0089] Figure 3 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in peritoneal cell samples according to a specific embodiment.

[0090] Figure 4 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in lung samples according to a specific embodiment.

[0091] Figure 5 This is a graph showing that betulin is a specific agonist of liver X receptor β (LXR-β) in thymus samples according to a specific embodiment.

[0092] like Figures 1 to 5 As shown, it can be observed that, according to a specific embodiment, betulin increases the expression of ATP-binding cassette transporter A1 (Abca1), sterol regulatory element-binding transcription factor 1c (Srebf1_1C), aquaporin 1 (AQP1), and apolipoprotein E1 (ApoE1) in various samples.

[0093] These results indicate that betulin activates the transcriptional regulatory functions of related genes such as ATP-binding cassette transporter A1 (Abca1), sterol regulatory element-binding transcription factor 1c (Srebf1_1C), aquaporin 1 (AQP1), and apolipoprotein E1 (ApoE1) by binding to liver X receptors (LXRs).

[0094] Furthermore, it is known that hepatic receptor X α (LXR-α) is typically expressed at high levels in the liver, while hepatic receptor X β (LXR-β) is expressed at low levels. Therefore, in liver samples, the expression of sterol regulatory element-binding transcription factor 1 (Srebf1), which is specifically regulated by hepatic receptor X α (LXR-α), was not significantly increased, while the expression of aquaporin 1 (AQP1), regulated by hepatic receptor X β (LXR-β), showed a significant increase. Thus, it can be concluded that betulin does not bind well to hepatic receptor X α (LXR-α), but rather acts as an agonist by more specifically binding to hepatic receptor X β (LXR-β).

[0095] Therefore, these results not only demonstrate that hepatic X receptors (LXRs) can be effectively used to treat LXR-mediated diseases, such as atherosclerosis, Alzheimer's disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic diseases, and dermatitis, but also indicate that due to their specificity for hepatic X receptor β (LXR-β), they are safer compared to other LXR agonists.

[0096] Example 2. Analysis of the safety of betulin in vivo.

[0097] Similar to Example 1, hepatotoxicity in mice was evaluated to confirm that betulin is a safe hepatic X receptor β (LXR-β) agonist. When betulin acts as an agonist of hepatic X receptor α (LXR-α), it induces hepatic steatosis in the liver by overexpressing the sterol regulatory element binding transcription factor 1 (Srebf1). The selectivity and safety of betulin for hepatic X receptors (LXRs) were evaluated in a 2-week mouse toxicity study.

[0098] Specifically, male and female ICR mice were orally administered the betulin emulsion once daily for two weeks at doses of 0.0 mg / kg (group 1), 5.0 mg / kg (group 2), and 20 mg / kg (group 3). For reference, the appropriate concentration of betulin for treating asthma and food allergies is expected to be 4.5 to 5.0 mg / kg. As a control group, 100% olive oil was administered. No signs of health abnormalities were observed in any of the subjects used in the experiment. All surviving animals were euthanized after 15 days of administration under isoflurane anesthesia, and liver tissue was then dissected for analysis of total fat content. The results are shown in Table 1 below. As summarized in Table 1, no significant changes were observed in the total liver fat content of either female or male mice at doses of 5 mg / kg or 20 mg / kg.

[0099] Table 1

[0100]

[0101]

[0102] Example 3. Analysis of the concentration of betulin in in vivo blood and brain tissue

[0103] The concentrations of betulin in the blood and brain tissue of mice when betulin was administered were analyzed.

[0104] Specifically, 7-week-old Balb / c mice were received and acclimatized for one week, then four mice were assigned to each group for the experiment. The mice were divided into four control groups (no betulin) and four groups (betulin administered). The betulin administered groups received oral betulin at a dose of 4.5 mg / kg for 10 consecutive days. On day 11, mice in both the control and betulin administered groups were sacrificed for blood and brain tissue samples.

[0105] First, to analyze the concentration of betulin in the blood, 20 μL of serum was transferred to a 1.5 mL microcentrifuge (Eppendorf) tube, and betulin was extracted twice in ethanol. In each extraction step, 200 μL of ethanol was mixed with serum, and the mixture was sonicated for 10 minutes and then centrifuged at 14000 x g for 15 minutes.

[0106] To analyze the concentration of betulin in brain tissue, the entire brain was weighed and mixed with 10 times (v / w) of 100% ethanol. The mixture was then sonicated and centrifuged.

[0107] After centrifugation, each effluent was reconstituted with 0.5 mL of 60% methanol for solid-phase extraction using a C18 column (Sep-Pak C18 3cc Vac Cartridge, 200 mg adsorbent, Waters). The sample was loaded onto the column and activated with methanol (6 mL), followed by conditioning with 60% methanol (6 mL). The column was then washed with 6 mL of 60% methanol. The column was then eluted in a glass tube with 100% methanol (3 mL) to collect betulin. After evaporation of the sample, the effluent was dissolved in methanol and analyzed by liquid chromatography-mass spectrometry (LC / MS).

[0108] The results are shown in Table 2. In the control group, betulin was not detected in brain tissue and blood. However, in the group that received betulin, an average of 151.7 ± 21.3 ng / g of betulin was detected in brain tissue and an average of 12.1 ± 1.3 ng / g of betulin was detected in blood. Therefore, it has been confirmed that the amount of betulin detected in the brain is about 12 times that in the blood.

[0109] These results indicate that betulin can be effectively delivered to the brain of animals.

[0110] Table 2

[0111]

[0112] Example 4. Analysis of gene expression associated with neurodegenerative diseases in in vivo brain tissue

[0113] The expression of ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein E (APOE), which are genes associated with neurodegenerative diseases, in brain tissue was analyzed when betulin was administered to mice.

[0114] Specifically, similar to Example 3, after preparing mice for the control group and the group administered betulin, 50 mg of brain tissue samples were first quantified, and then the cells were pulverized using a mortar and pestle to extract total RNA. 1 mL of Trizol was aliquoted into the pulverized sample and mixed by vortexing. Then, 0.2 mL of chloroform was aliquoted, and the mixture was reacted at room temperature for 3 minutes. After centrifugation at 12000 rpm and 4°C for 15 minutes, only the clear supernatant was transferred to a new tube, and then 0.5 mL of isopropyl alcohol was aliquoted, and the mixture was reacted at room temperature for 10 minutes. After centrifugation at 12000 rpm and 4°C for 10 minutes, the supernatant was completely removed, and then 1 mL of 75% ethanol (Ethanol) was used to remove impurities. After centrifugation at 7500 rpm and 4°C for 5 minutes, 75% ethanol (Ethanol) was added, followed by centrifugation, and then, after removing as much supernatant as possible, the mixture was dried on a heat block at 55°C for approximately 5 minutes. After dissolving the samples in DEPC water, total RNA was quantified to 1000 ng / uL using Nanodrop, followed by cDNA synthesis using PrimeScript RT Master Mix (Takara Korea). Real-time quantitative PCR was performed using Smartgene Sybr Green Q-PCR master mix (Samjung Bioscience) and primers specific to the apolipoprotein E (APOE) gene and the ATP-binding cassette transporter A1 (ABCA1) gene.

[0115] The results are shown in Figure 6. Compared with the control group (Naive) that did not receive betulin, the group that received betulin (Ino(10mpk)) showed high expression of the ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein E (APOE) genes in brain tissue. It is known that ATP-binding cassette transporter A1 (ABCA1) activates apolipoprotein E (APOE) and inhibits inflammation in the brain, and that activation of apolipoprotein E (APOE) eliminates β-amyloid.

[0116] Figure 6 is a graph demonstrating that betulinol, according to a specific embodiment, increases gene expression associated with the inhibition of neurodegenerative diseases.

[0117] Figure 6a This is a graph demonstrating that, according to a specific embodiment, betulin increases the expression of ATP-binding cassette transporter A1 (ABCA1), a gene associated with the inhibition of neurodegenerative diseases. Figure 6bThis is a graph demonstrating that betulinol, according to a specific embodiment, increases the expression of apolipoprotein E (APOE), a gene associated with the inhibition of neurodegenerative diseases.

[0118] Therefore, these results indicate that betulin promotes the excretion and breakdown of β-amyloid protein, reduces the inflammatory response of microglia, and suggests that betulin can be effectively used to treat neurodegenerative diseases.

[0119] Example 5. Confirmation of the in vivo therapeutic effects of betulin on rheumatoid arthritis and psoriasis.

[0120] To confirm whether betulinum toxin has a therapeutic effect on rheumatoid arthritis in mice, male DBA / 1J mice were given bovine type II collagen to induce arthritis, followed by oral administration of betulinum toxin.

[0121] Specifically, after a 6-day acclimatization period, an equal volume of complete Freund's adjuvant (Chondrex, Inc., USA) was slowly mixed with bovine type II collagen (2 mg / mL dissolved in 0.05 mol / L acetic acid; Chondrex, Inc., USA) while an emulsion was prepared using a homogenizer. Then, a secondary immunization was performed on mice by administering 0.05 mL per head subcutaneously to the tail using a disposable syringe to induce arthritis.

[0122] For animals without induced arthritis, six healthy animals with near-average weight were selected as a normal control group. For animals with induced arthritis, 18 healthy animals with near-average weight were selected, and then divided into three groups of six to ensure equal average weight. The animals with induced arthritis were divided into three groups: one without betulin, one receiving 4 mg / kg betulin, and one receiving 10 mg / kg betulin. Using a disposable syringe with a probe, the betulin was administered once daily for four weeks, for a total of 28 administrations.

[0123] Macroscopic observation of arthritis symptoms was conducted twice a week for 4 weeks starting from the date of administration. The knees, ankles, and dorsum of each leg of each animal were visually observed, and the arthritis index was recorded according to the macroscopic arthritic index evaluation criteria in Table 3 below. A macroscopic arthritic score (maximum score = 16) was calculated by summing the arthritis indices for each leg of each animal.

[0124] Table 3

[0125]

[0126]

[0127] The result is as follows Figure 7 As shown, it has been confirmed that arthritis animals treated with betulin had lower arthritis scores compared to those not treated with betulin.

[0128] Figure 7 This is a graph showing the rheumatoid arthritic score of the efficacy of betulinol against rheumatoid arthritis according to a specific embodiment.

[0129] Next, the expression of tumor necrosis factor-α (TNF-α), an activator of inflammation, was confirmed.

[0130] Specifically, the primary antibody (Biolegend) that specifically binds to mouse tumor necrosis factor-α (TNF-α) was diluted in coating buffer and aliquoted into plates at concentrations of 0.5 to 8 μg / mL in 100 μL increments, then stored at 4°C for 18 hours. After removing the coating buffer, the plate was washed three times with 200 μL of washing buffer (0.05% Tween 20 / PBS). To prevent non-specific binding, 200 μL of blocking solution (1% BSA / PBS) was reacted at room temperature for 1 hour. The plate was then washed three times with washing buffer. After diluting the standard reference material (Biolgend) and the sample with blocking solution, 100 μL increments were aliquoted and reacted at room temperature for 2 to 4 hours. The plate was then washed three times with washing buffer. Dilute the secondary antibody (biotin-labeled detection antibody, Biolegend) to 0.25–2 μg / mL using blocking solution, aliquot 100 μL, and incubate at room temperature for 1 hour. Wash three times with washing buffer. Dilute the avidin-horseradish peroxidase (Biolegend) to 1 / 1000 using blocking solution, aliquot 100 μL, and incubate at room temperature for 30 minutes. Wash five times with washing buffer. After aliquoting 100 μL of TMB (Biolegend), incubate for 4–30 minutes, and terminate the reaction by aliquoting 100 μL of 2N H₂SO₄ when the color changes. Verify the ELISA plate at 450 nm using an EPOCH microplate reader (Biotek).

[0131] The result is as follows Figure 8 As shown, lower expression of tumor necrosis factor-α (TNF-α) has been confirmed in arthritic animals treated with betulin.

[0132] Figure 8This is a graph showing the inhibition of tumor necrosis factor-α (TNF-α) expression by betulin according to a specific embodiment.

[0133] The results described above indicate that betulin can be effectively used not only to treat rheumatoid arthritis, but also to treat psoriasis.

[0134] The above description of the present invention is for illustrative purposes, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the invention. Therefore, the embodiments described above should be understood as exemplary in all respects and not restrictive.

Claims

1. The use of betulin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of hepatic receptor X-mediated diseases, wherein the hepatic receptor X-mediated diseases are Alzheimer's disease or rheumatoid arthritis.

2. The application according to claim 1, wherein the betulin acts as an agonist binding to liver X receptor β.

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