Composition for the prevention / treatment of advanced glycation end product-related disorders

Abstract

Provided is a composition having both an anti-aging effect by inhibiting the production of advanced glycation end products (AGEs) and an anti-aging and therapeutic effect by cleaving AGE crosslinks. 【Solution means】A compound represented by the following formula (I): 【Chemical formula 1】 TIFF2026073747000008.tif3475[wherein, R 1 , R 2 , R 3 , R 4 , R 5 are each independently hydrogen, halogen, or a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms which may contain an ether bond], or a pharmaceutically acceptable salt or solvate thereof, for preventing and / or treating a disorder associated with the accumulation of advanced glycation end products in a subject.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing and / or treating disorders (diseases) associated with the accumulation of target advanced glycation end products. [Background technology]

[0002] Advanced Glycation End Products (AGEs) are known as one of the substances that accelerate aging. AGEs are glycated proteins formed when excess carbohydrates in the body combine with proteins, and are known as one of the substances that contribute to aging. Non-patent document 1 reports that in a four-year follow-up study of approximately 70,000 people, the group with a high accumulation of AGEs in their bodies had a significantly higher risk of developing diabetes and cardiovascular diseases such as myocardial infarction and stroke.

[0003] Aminoguanidine is known as an AGEs formation inhibitor. In vitro studies have confirmed that aminoguanidine inhibits the formation of AGEs through glycation reactions and inhibits protein cross-linking and polymerization formation. Animal studies have confirmed that it has preventive and inhibitory effects on the progression of nephropathy, retinopathy, and neuropathy (Non-Patent Literature 2).

[0004] N-phenacylthiazolium bromide (PTB) and 3-phenacyl-4,5-dimethilthiazolium bromide (ALT-711) are known to have the effect of degrading AGEs crosslinks. PTB can suppress the increase in the amount of AGEs accumulated in blood vessels and reduce the amount of AGEs accumulated, suggesting the possibility of treating diabetic vascular disease (Non-Patent Literature 3). ALT-711 was confirmed to suppress vascular sclerosis and AGEs accumulation when administered to diabetic model rats (Non-Patent Literature 4). Furthermore, oral administration to humans was confirmed to have an improving effect on vascular sclerosis and poor systolic blood pressure (Non-Patent Literature 5-6).

[0005] Patent Document 1 reports that black galangal extract has the effect of inhibiting protein glycation, cleaving AGEs crosslinks, and enhancing oxidised protein hydrolase (OPH) activity. Patent Document 2 reports that extracts from plants of the genus Lindera are effective in suppressing AGEs formation and reducing AGEs. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2017-210452 [Patent Document 2] Japanese Patent Publication No. 2018-65769 [Non-patent literature]

[0007] [Non-Patent Document 1] Waateringe, et al., Diabetologia, Vol. 62, pp. 262-280 (2019) [Non-Patent Document 2] Bolton, et al., Am. J. Nephrol., Vol. 24, pp. 32-40 (2004) [Non-Patent Document 3] Copper, et al., Diabetologia. Vol. 43, pp. 660-664 (2000) [Non-Patent Document 4] Freidja, et al., Cardiovascular Diabetology, Vol. 13, pp. 55-71 (2014) [Non-Patent Document 5] Bakris, et al., Am. J. Hypertens., Vol. 17, pp. 23S-30S (2004) [Non-Patent Document 6] Kass, et al., Circulation, Vol. 104, pp. 1464-1470 (2001) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Aminoguanidine, a known AGEs formation inhibitor, and PTB and ALT-711, known AGEs crosslinking cleavage agents, respectively, have anti-aging effects through inhibition of AGEs formation and anti-aging / therapeutic effects through AGEs crosslinking cleavage, but it has not been reported that they possess both of these effects. The black galangal extract described in Patent Document 1 and the Kuromoji plant extract described in Patent Document 2 are presumed to have a crosslinking cleavage effect based solely on the result that they cleave the CC bond of the α-diketone structure, but it has not been demonstrated that they actually cleave AGEs crosslinks. Furthermore, none of these compounds have been put into practical use as pharmaceutical compositions for the prevention or treatment of AGEs-related disorders (diseases).

[0009] The present invention aims to provide a composition that has both an anti-aging effect by inhibiting AGEs formation and an anti-aging and therapeutic effect by cleaving AGEs crosslinks. [Means for solving the problem]

[0010] This specification provides the following invention. [1] An anti-glycation compound represented by the following formula (I): [Chemical formula] [wherein, R 1 , R 2 , R 3 , R 4 , R 5 are each independently hydrogen, halogen, or a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms which may contain an ether bond], or a composition for preventing and / or treating a disorder related to the accumulation of advanced glycation end products in a subject, which comprises a pharmaceutically acceptable salt or solvate thereof. [2] The composition according to [1], which prevents the disorder by inhibiting the generation of advanced glycation end products. [3] The composition according to [1], which prevents the disorder by inhibiting the cross-link formation of advanced glycation end products. [4] The composition according to [1], which prevents and / or treats the disorder by cleaving the cross-links formed in advanced glycation end products. [5] The composition according to any one of [1] to [4], wherein the disorder related to the accumulation of advanced glycation end products is a cell disorder. [6] The composition according to any one of [1] to [5], wherein the advanced glycation end products include those generated by the glycation of arginine in proteins. [7] The composition according to [6], wherein the advanced glycation end products include those generated by methylglyoxal. [8] The composition according to any one of [1] to [7], wherein the disease related to the accumulation of advanced glycation end products is an age-related disease. [9] The composition according to any one of [1] to [8], wherein the anti-glycation compound is taxifolin.

[10] The composition according to any one of [1] to [9], which is a composition for oral administration.

[11] The composition according to any one of [1] to

[10] , which is a pharmaceutical composition.

[12] The composition according to any one of [1] to

[10] , which is a food composition.

[13] An anti-glycation compound represented by the following formula (I): [Chemical formula] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 are each independently hydrogen, halogen, or a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms which may contain an ether bond], Or a method for preventing and / or treating a disorder related to the accumulation of advanced glycation end products in a subject, which comprises administering the pharmaceutically acceptable salt or solvate thereof.

[14] The method according to

[13] , wherein the administration is oral administration. [Advantages of the Invention]

[0011] According to the present invention, it is possible to provide a composition having both an anti-aging effect by inhibiting AGEs production and an anti-aging and therapeutic effect by cleaving AGEs crosslinks. [Brief Description of the Drawings]

[0012] [Figure 1] It is a schematic diagram showing the main biosynthetic pathway of advanced glycation end products (AGEs). [Figure 2] It is a graph showing the relationship between the concentration of methylglyoxal-hydroimidazolone (MG-H1) generated by the reaction of methylglyoxal (MGO) and bovine serum albumin (BSA) in Example 1 and the concentrations of added taxifolin and aminoguanidine. [Figure 3]This graph shows the relationship between taxifolin and aminoguanidine concentrations and the crosslinking rate between MGO-BSA and collagen in Example 2. Figure 3A shows the relationship between taxifolin concentration and crosslinking rate. Figure 3B shows the relationship between aminoguanidine concentration and crosslinking rate. The MGO-BSA crosslinking rate (%) was calculated from the absorbance of each solution, with the absorbance of the MGO-BSA solution without taxifolin or aminoguanidine set to 100%. [Figure 4] This graph shows the relationship between taxifolin concentration and crosslinking rate in Example 3. The crosslinking rate (%) here was calculated from the absorbance of each sample, with the absorbance of the negative control set to 100%. [Figure 5] This bar graph shows the relationship between the concentration of added taxifolin and taxifolin analogs and cell viability in Example 4. Here, cell viability (%) refers to the relative luminescence intensity, with the luminescence intensity in the control without MGO added set to 100%. [Figure 6] This graph shows the relationship between taxifolin concentration and cell viability in Example 5. The cell viability (%) here refers to the relative fluorescence intensity of each solution, with the fluorescence intensity of the control (MGO(-)) without MGO set to 100%. [Modes for carrying out the invention]

[0013] [1] Overview and definitions In this specification, each compound includes all geometric isomers and stereoisomers unless otherwise specified. Furthermore, unless otherwise specified, it includes both its salts and solvates.

[0014] In this specification, "pharmaceutically acceptable salt" means a pharmaceutically non-toxic salt of an active compound prepared using a base or acid based on a specific substituent (e.g., a hydroxyl group) of a compound. pharmaceutically acceptable salts can be classified into basic addition salts and acid addition salts depending on the base or acid used.

[0015] Examples of "basic addition salts" include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aliphatic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, and brocaine salt, aralkylamine salts such as N,N-dibenzylethylenediamine, heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, and isoquinoline salt, basic amino acid salts such as arginine salt and lysine salt, quaternary ammonium salts such as tetramethylammonium salt, tetraethylamonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt, and ammonium salts.

[0016] Examples of "acid addition salts" include inorganic salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, and perchlorate; organic salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, and ascorbate; sulfonates such as methanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate; and acidic amino acids such as aspartate and glutamate.

[0017] The solvents that can form a "pharmaceutically acceptable solvate" as used herein are not limited to, but may include, for example, water, or lower alcohols (e.g., alcohols having 1 to 6 carbon atoms, such as methanol, ethanol, or 2-propanol (isopropyl alcohol)), higher alcohols (e.g., alcohols having 7 or more carbon atoms, such as 1-heptanol or 1-octanol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine, or ethyl acetate.

[0018] In this specification, "Advanced Glycation End Products (AGEs)" refers to glycated proteins formed in the body when excess carbohydrates and proteins combine. There are multiple types of AGEs and biosynthetic pathways. Figure 1 shows the main biosynthetic pathways of AGEs. One of the biosynthetic pathways of AGEs is the Maillard reaction. The Maillard reaction is divided into an early stage in which reducing sugars such as glucose and fructose in the blood react non-enzymatically with the free amino groups of proteins to produce Amadori compounds from Schiff bases, and a later stage in which irreversible dehydration, condensation, oxidation, and reduction reactions are repeated to produce yellowish-brown AGEs with characteristic fluorescence. In the figure, the reaction of the early stage of the Maillard reaction is shown by a thick arrow. AGEs are produced not only through the Maillard reaction, but also from dicarbonyl compounds such as methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG) generated from the auto-oxidation and degradation products of glucose, as well as from Maillard reaction intermediates and carbohydrate metabolism intermediates. In this invention, AGEs encompass glycated proteins biosynthesized through all of these pathways. Non-limiting examples of AGEs include carboxymethyllysine (CML), glyoxal lysine dimer (GOLD), carboxymethylarginine (CMA), glycolaldehyde-pyridine (GA-Pyridine), carboxyethyllysine (CEL), methylglyoxalhydromidazolone (MG-H1), carboxyethylarginine (CEA), pyrraline, and 3-deoxyglucosone-imidazolonone (3DG-Imidazolone).

[0019] In this specification, "subject" refers to an animal, preferably a mammal. Mammals refer to animals belonging to the class Mammalia, subphylum Vertebrata, phylum Chordata, including humans and non-humans, such as humans, primates including chimpanzees, pet animals such as dogs and cats, domesticated animals such as cattle, pigs, horses, sheep and goats, rodents such as mice and rats, and mammals kept in zoos. In this specification, the subject is preferably a human.

[0020] In this specification, "disorders related to the accumulation of advanced glycation end products" (hereinafter also referred to as "AGEs-related disorders") encompass all disorders, including diseases and symptoms, that may result from the accumulation of AGEs. AGEs-related disorders include, in particular, cytotoxic disorders. Furthermore, age-related diseases are also included. More specifically, these include, but are not limited to, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, myocardial infarction, rheumatoid arthritis, chronic kidney disease, acute liver injury, cancer, arteriosclerosis, osteoporosis, ossification of the posterior longitudinal ligament, muscle atrophy, non-alcoholic steatohepatitis, neurodegenerative diseases, skin diseases, skin aging, age-related macular degeneration, retinopathy, cataracts and other eye diseases.

[0021] The pathways for suppressing (preventing and / or treating) AGEs-related disorders can be broadly categorized into the following four types: 1) Inhibit the formation of AGEs; 2) Inhibit cross-linking of AGEs; 3) To break the crosslinks of AGEs; and 4) It enhances the activity of oxidised protein hydrolase (OPH). The inventors of the present invention have discovered a compound (anti-glycation compound) related to at least one of the above pathways 1) to 3), and have completed the present invention.

[0022] In this specification, "anti-glycation compound" refers to a compound that tests "positive" in any of the following tests a) to c). Preferably, it refers to a compound that tests "positive" in all of the following tests a) to c): a) AGEs formation inhibition test; b) AGEs cross-linking inhibition test; and c) AGEs cross-linking cleavage test Examples of tests a) to c) are described below.

[0023] a) AGEs formation inhibition test MGO and an arbitrary protein (e.g., BSA) are coexisted in the presence or absence of the candidate compound, and the amount of MGO-hydroimidazolone (MG-H1) present in the resulting MGO-AGEs is measured. If the amount of MG-H1 in the presence of the candidate compound is significantly reduced compared to the amount of MG-H1 in the absence of the candidate compound, the inhibitory effect of the candidate compound on AGEs formation is determined to be "positive".

[0024] b) AGEs crosslinking inhibition test MGO is reacted with an arbitrary protein (e.g., BSA) to produce MGO-AGEs. A microwell plate is coated with another arbitrary protein (e.g., collagen), and the MGO-AGEs are added and reacted in the presence or absence of the candidate substance. The amount of MGO-AGEs remaining on the plate after washing is measured. If the amount of remaining MGO-AGEs in the presence of the candidate compound is significantly reduced compared to the amount of remaining MGO-AGEs in the absence of the candidate compound, the AGEs cross-linking inhibitory effect of the candidate compound is determined to be "positive".

[0025] c) AGEs cross-linking cleavage test MGO is reacted with an arbitrary protein (e.g., BSA) to produce MGO-AGEs. A microwell plate is coated with another arbitrary protein (e.g., collagen), and the MGO-AGEs are added to induce AGEs crosslinking. The candidate compound is added to the wells containing AGEs crosslinks and reacted. The amount of MGO-AGEs remaining on the plate after washing is measured. If the amount of remaining MGO-AGEs in the presence of the candidate compound is significantly reduced compared to the amount of remaining MGO-AGEs in the absence of the candidate compound, the AGEs crosslinking inhibitory effect of the candidate compound is determined to be "positive".

[0026] [2] Compositions for the prevention / treatment of AGEs-related disorders A first embodiment of the present invention is a composition for preventing and / or treating disorders associated with the accumulation of target AGEs. The composition of this embodiment is an anti-glycation compound represented by the following formula (I): [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 Each of these is independently a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, which may contain hydrogen, halogen, or ether bonds. It is characterized by containing either a pharmaceutically acceptable salt or solvate thereof.

[0027] A composition in one embodiment can inhibit the formation of AGEs in a subject when administered to that subject, thereby preventing AGEs-related disorders. More specifically, the composition in this embodiment can be used for the purpose of preventing the onset or progression of AGEs-related disorders.

[0028] Compositions of other embodiments can inhibit the cross-linking of AGEs in a subject when administered to that subject, thereby preventing AGEs-related disorders. More specifically, compositions of this embodiment can be used for the purpose of preventing the onset or progression of AGEs-related disorders.

[0029] Furthermore, compositions of other embodiments, when administered to a subject, can cleave crosslinks formed in AGEs in the subject, thereby preventing and / or treating AGEs-related disorders. More specifically, compositions of this embodiment can be used for the purpose of preventing the onset or progression of AGEs-related disorders. Alternatively, compositions of this embodiment can be used for the purpose of treating AGEs-related disorders.

[0030] In this context, the term AGEs preferably includes those produced by the glycation of arginine in proteins. Furthermore, in this case, it is preferable that the AGEs include those produced via MGO.

[0031] The anti-glycation compound represented by formula (I) in the composition of this embodiment is preferably taxifolin. Taxifolin refers to the compound represented by the following formula (II). [ka]

[0032] The composition of this embodiment can take various forms, including but are not limited to supplements, tablets, coated tablets, granules, powders, solutions, emulsions, capsules, injections or liquids, dry syrups, and syrups. In one embodiment, the composition of the present invention may be in an orally administered form (composition for oral administration). In another embodiment, the composition of this embodiment may be in an enteral administration form, a tube administration form, a gastrostomy administration form, an intravenous administration form, a transdermal administration form, an intraperitoneal administration form, or an intramuscular administration form.

[0033] In one embodiment, the composition of this embodiment may contain other components that are normally included in oral, enteral, tube, gastrostomy, intravenous, transdermal, intraperitoneal, or intramuscular compositions, such as excipients, disintegrants, binders, lubricants, colorants, flavoring agents, suspending agents, solubilizers, coating agents, auxiliaries, preservatives, flavoring agents, vitamins, pH adjusters, emulsifiers, thickeners, isotonic agents, antioxidants, chelating agents, sweeteners, flavoring agents, etc., but the additional components are not limited to these. In one embodiment, the composition of the present invention may be a pharmaceutical composition. In another embodiment, the composition of the present invention may be a food composition or a feed composition.

[0034] The composition of this embodiment may contain an anti-glycation compound in an amount of 0.1 mg / kg body weight or more, 0.2 mg / kg body weight or more, 0.5 mg / kg body weight or more, 1 mg / kg body weight or more, 2 mg / kg body weight or more, 5 mg / kg body weight or more, 10 mg / kg body weight or more, 20 mg / kg body weight or more, 50 mg / kg body weight or more, 100 mg / kg body weight or more, 200 mg / kg body weight or more, 500 mg / kg body weight or more, for example 1000 mg / kg body weight or more, for example 0.1 to 1000 mg / kg body weight, 0.1 to 500 mg / kg body weight, 0.1 to 100 mg / kg body weight, 0.1 to 50 mg / kg body weight, 0.1 to 20 mg / kg body weight, for example 0.1 to 10 mg / kg body weight. Alternatively, the composition of this embodiment may contain an anti-glycation compound in an amount of 0.01% or more by weight, 0.05% or more by weight, 0.10% or more by weight, 0.20% or more by weight, 0.5% or more by weight, 1% or more by weight, 2% or more by weight, 2.2% or more by weight, 3% or more by weight, 4% or more by weight, 5% or more by weight, 10% or more by weight, 20% or more by weight, 30% or more by weight, 40% or more by weight, 50% or more by weight, 60% or more by weight, 70% or more by weight, 80% or more by weight, 90% or more by weight, 90% or less by weight, 80% or less by weight, 70% or less by weight, 60% or less by weight, 50% or less by weight, 40% or less by weight, 30% or less by weight, 20% or less by weight, 10% or less by weight, 5% or less by weight, or 3% or less by weight.

[0035] When the composition of this embodiment is an orally administered composition, the amount of anti-glycation compound ingested may vary depending on the age, sex, symptoms, and method of administration when the subject is a human, but for an adult (weighing about 60 kg), it may be 1 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 100 mg or more, 150 mg or more, 200 mg or more, 500 mg or more, or 1000 mg or more per day.

[0036] The composition of this embodiment may be administered as a single dose or multiple doses. In the case of multiple doses, the frequency of administration is not particularly limited, but can be once every 3 days to once every 2 months, or more particularly once a week to once every 3 weeks. The administration period of the composition of this embodiment can be appropriately modified depending on the condition of the subject, the presence or absence of side effects, etc., and is not particularly limited, but can be, for example, 3 days to 6 months, more particularly 1 week to 3 months, 1 week to 1 month, etc.

[0037] If the composition of this embodiment is a food composition, it can be designated as a functional food or a food for specified health uses based on the above-mentioned functionality and beneficial effects on the body. Regarding the usefulness and functionality of the composition, the following may be indicated when the product is commercialized, but are not limited to these. For example, "functionality to prevent aging," "functionality to improve aging," "functionality to prevent age-related diseases," "functionality to suppress glycation," "functionality to improve glycation," "functionality to suppress the formation of advanced glycation end products (AGEs)," "functionality to suppress the crosslinking of advanced glycation end products (AGEs)," "functionality to break the crosslinking of advanced glycation end products (AGEs)," and similar indications. These indications may be indicated by affixing them to containers and packaging in known methods, displaying or distributing the above-mentioned explanations in advertisements, price lists or transaction documents related to the product, or providing information containing these by electromagnetic means (such as the internet).

[0038] [3] Methods to prevent / treat AGEs-related disorders A second embodiment of the present invention is a method for preventing and / or treating disorders associated with the accumulation of target AGEs. The method of this embodiment involves an anti-glycation compound represented by the following formula (I): [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 Each of these is independently a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, which may contain hydrogen, halogen, or ether bonds. The method is characterized by including the administration of a pharmaceutically acceptable salt or solvate thereof. More specifically, the method of this embodiment includes administering the compositions described in the section "[2] Compositions for the Prevention / Treatment of AGEs-Related Disorders" to a target.

[0039] The anti-glycation compound is preferably taxifolin.

[0040] In the method of this embodiment, the anti-glycation compound may be administered in any form, such as orally, enterally, via tube, gastrostomy, intravenously, transdermally, intraperitoneally, or intramuscularly, but it is particularly preferable to administer it orally.

[0041] In the method of this embodiment, the anti-glycation compound can be administered in amounts of 0.1 mg / kg body weight or more, 0.2 mg / kg body weight or more, 0.5 mg / kg body weight or more, 1 mg / kg body weight or more, 2 mg / kg body weight or more, 5 mg / kg body weight or more, 10 mg / kg body weight or more, 20 mg / kg body weight or more, 50 mg / kg body weight or more, 100 mg / kg body weight or more, 200 mg / kg body weight or more, 500 mg / kg body weight or more, for example 1000 mg / kg body weight or more, for example 0.1 to 1000 mg / kg body weight, 0.1 to 500 mg / kg body weight, 0.1 to 100 mg / kg body weight, 0.1 to 50 mg / kg body weight, 0.1 to 20 mg / kg body weight, for example 0.1 to 10 mg / kg body weight.

[0042] When administered orally, the amount of anti-glycation compounds ingested may vary depending on the age, sex, symptoms, and method of administration in humans, but for adults (weighing approximately 60 kg), it may be 1 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 100 mg or more, 150 mg or more, 200 mg or more, 500 mg or more, or 1000 mg or more per day.

[0043] In the method of this embodiment, the number of administrations may be a single dose or multiple doses. In the case of multiple doses, the frequency of administration is not particularly limited, but can be once every 3 days to once every 2 months, or more particularly once a week to once every 3 weeks. The administration period of the composition of this embodiment can be appropriately modified depending on the condition of the subject, the presence or absence of side effects, etc., and is not particularly limited, but can be, for example, 3 days to 6 months, more particularly 1 week to 3 months, 1 week to 1 month, etc.

[0044] [4] Screening method for anti-glycation compounds A third embodiment of the present invention is a screening method for selecting anti-glycation compounds from candidate compounds. The screening method of this embodiment includes confirming whether a candidate compound is "positive" in any of the following tests a) to c): a) AGEs formation inhibition test; b) AGEs cross-linking inhibition test; and c) AGEs cross-linking cleavage test Preferably, this includes confirming that all of tests a) to c) are "positive". The procedures for example tests a) to c) are described below.

[0045] a) AGEs formation inhibition test MGO and an arbitrary protein (e.g., BSA) are coexisted in the presence or absence of the candidate compound, and the amount of MGO-hydroimidazolon (MG-H1) present in the resulting MGO-AGEs is measured. If the amount of MG-H1 in the presence of the candidate compound is significantly reduced compared to the amount of MG-H1 in the absence of the candidate compound, the inhibitory effect of the candidate compound on AGEs formation is determined to be "positive". The amount of MG-H1 can be measured, for example, using a commercially available ELISA kit for measuring MG-H1 (Abcam, Inc.) (ab238543).

[0046] b) AGEs crosslinking inhibition test MGO is reacted with an arbitrary protein (e.g., BSA) to produce MGO-AGEs. A microwell plate is coated with another arbitrary protein (e.g., collagen), and the MGO-AGEs are added and reacted in the presence or absence of the candidate substance. The amount of MGO-AGEs remaining on the plate after washing is measured. If the amount of remaining MGO-AGEs in the presence of the candidate compound is significantly reduced compared to the amount of remaining MGO-AGEs in the absence of the candidate compound, the inhibitory effect of the candidate compound on AGEs cross-linking is determined to be "positive". For example, remaining MGO-AGEs can be easily detected by pre-labeling the MGO-AGEs with enzymes (horseradish peroxidase, alkaline phosphatase, etc.).

[0047] c) AGEs cross-linking cleavage test MGO is reacted with an arbitrary protein (e.g., BSA) to produce MGO-AGEs. A microwell plate is coated with another arbitrary protein (e.g., collagen), and the MGO-AGEs are added to induce AGEs crosslinking. The candidate compound is added to the wells containing AGEs crosslinks and reacted. The amount of MGO-AGEs remaining on the plate after washing is measured. If the amount of remaining MGO-AGEs in the presence of the candidate compound is significantly reduced compared to the amount of remaining MGO-AGEs in the absence of the candidate compound, the AGEs crosslinking inhibitory effect of the candidate compound is determined to be "positive". For example, remaining MGO-AGEs can be easily detected by pre-labeling the MGO-AGEs with enzymes (horseradish peroxidase, alkaline phosphatase, etc.).

[0048] In addition to the tests a) to c) above, it is preferable to perform a cytotoxicity test on the candidate compound. The cytotoxicity test can be performed, for example, by the following procedure: Cells such as human umbilical vein endothelial cells (HUVECs) are brought into contact with MGO in the presence or absence of the candidate compound, and the cell viability is measured. If the cell viability in the presence of the candidate compound is significantly higher than the cell viability in the absence of the candidate compound, it can be confirmed that the candidate compound has an inhibitory effect on the cytotoxicity of MGO, and that the candidate compound itself is not cytotoxic. [Examples]

[0049] The present invention will be further illustrated by the following embodiments, but these will not limit the scope of the present invention as described in the claims.

[0050] [Example 1] AGEs formation inhibition test Bovine serum albumin (BSA) was dissolved in 0.1 M phosphate buffer to prepare a 50 mg / mL BSA solution. Methylglyoxal (MGO) was prepared to a concentration of 20 mM in ultrapure water. 50 mg / mL BSA, 20 mM MGO, and taxifolin were mixed to prepare a 50 mM phosphate buffer solution containing a final concentration of 5.0 mg / mL BSA and 2 mM MGO. Taxifolin concentrations were adjusted to 250 μM, 500 μM, and 1000 μM. As a positive control, a similar solution was prepared using aminoguanidine instead of taxifolin. Each prepared solution was incubated overnight at 37°C. After incubation, each solution was collected, and the MG-H1 concentration was measured using a commercially available ELISA kit for MG-H1 (MGO-hydroimidazolon) (ab238543 (Abcam)).

[0051] Figure 2 shows the relationship between taxifolin, aminoguanidine concentrations and MG-H1 concentration. MG-H1 production was reduced in a taxifolin concentration-dependent manner. It was confirmed that it has an MGO-protein inhibitory effect almost equivalent to that of aminoguanidine (positive control), which is already known to have an AGE formation inhibitory effect.

[0052] [Example 2] AGE Crosslinking Inhibition Test Acetate buffer containing 25 μg / mL of collagen was prepared, and 200 μL / well was dispensed into a 96-well microplate. The plates were left standing overnight at 4°C. The wells were washed three times with PBS to obtain collagen plates.

[0053] BSA was dissolved in phosphate-buffered saline (PBS) to prepare a solution containing 10 mg / mL BSA and 1 mM MGO at final concentrations. The prepared solution was allowed to stand at 37°C for one week to prepare an MGO-BSA solution. MGO-BSA was labeled with HRP using a commercially available HRP labeling kit (Peroxidase Labeling Kit-NH2, Dojin Chemical Laboratories). Taxifolin was added to the MGO-BSA-HRP solution to concentrations of 250 μM, 500 μM, and 1000 μM, respectively. As a positive control, a similar solution was prepared using aminoguanidine instead of taxifolin. As a negative control, an MGO-BSA solution without taxifolin or aminoguanidine was used.

[0054] Each solution was dispensed into a collagen plate at a volume of 25 μL / well and incubated at 37°C for 4 hours. After incubation, the plate was washed three times with PBST, and 100 μL / well of TMB solution was added. The plate was allowed to develop color at room temperature for 10-20 minutes. The reaction was stopped by adding 100 μL / well of 1N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. A similar test was performed using PBS without taxifolin as a negative control.

[0055] Figure 3 shows the relationship between taxifolin and aminoguanidine concentrations and crosslinking rates. Figure 3A shows the relationship between taxifolin concentration and crosslinking rate. Figure 3B shows the relationship between aminoguanidine concentration and crosslinking rate. The crosslinking rate (%) here is calculated from the absorbance of each solution, with the absorbance of the negative control set to 100%. The MGO-BSA crosslinking rate (%) is calculated from the absorbance of each solution, with the absorbance of the MGO-BSA solution without taxifolin or aminoguanidine (negative control) set to 100%. It was confirmed that the amount of MGO-BSA remaining on the collagen plate, i.e., the amount of MGO crosslinking collagen and BSA, decreased in a taxifolin concentration-dependent manner. This result was equivalent to that of aminoguanidine (positive control), which is known to have an inhibitory effect on AGE crosslinking. This indicates that taxifolin has an inhibitory effect on protein crosslinking of AGEs.

[0056] [Example 3] Cross-linking cleavage test Collagen plates were prepared in the same manner as in Example 2. After preparing the MGO-BSA solution in the same manner as in Example 2, the MGO-BSA was labeled with HRP using a commercially available HRP labeling kit (Peroxidase Labeling Kit-NH2, manufactured by Dojin Chemical Laboratories).

[0057] A collagen-coated plate was inoculated with 100 μL / mL of 5 μg / mL MGO-BSA-HRP (PBS solution) and incubated at 37°C for 4 hours. After washing the plate three times with PBST, 100 μL / well of 100 μM, 500 μM, or 1000 μM taxifolin / PBS solution was added and incubated overnight at 37°C. After incubation, the plate was washed three times with PBST, 100 μL / well of TMB solution was added, and the plate was allowed to develop color at room temperature for 10-20 minutes. The reaction was stopped by adding 100 μL / well of 1N sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader. A similar test was performed using PBS without taxifolin as a negative control.

[0058] Figure 4 shows the relationship between taxifolin concentration and crosslinking rate. Here, crosslinking rate (%) refers to the absorbance of each component relative to the absorbance of the negative control, which is set to 100%. The amount of MGO-BSA remaining on the collagen plate and the amount of MGO crosslinking collagen and BSA decreased in a taxifolin concentration-dependent manner. This was consistent with the results of a similar test using ALT-711 (Cho, et al., Int. J. Biol. Macromol., Vol. 269, 131927 (2024)), which is known to have an effect on cleaving AGE crosslinks. This indicates that taxifolin can cleave already formed AGE crosslinks.

[0059] [Example 4] Cytotoxicity suppression test (1) Place 100 μL / well (1.0 × 10⁶) of human umbilical vein endothelial cells (HUVEC) (obtained from Lonza) into a 96-well microplate. 3Cells were seeded to a density of 1 / well. They were incubated statically in a CO2 incubator at 37°C for 24 hours. 50 μL of taxifolin solution at concentrations of 0 μM, 40 μM, 80 μM, 160 μM, or 320 μM (final concentrations of 0 μM, 10 μM, 20 μM, 40 μM, or 80 μM, respectively) was dispensed into each well. For comparative examples, solutions of myricetin, dihydromyricetin, aromadendrin, or fuscin, which have structures similar to taxifolin, were added in the same manner instead of taxifolin. Myricetin, dihydromyricetin, aromadendrin, and fuscin are compounds having structures of the following formulas (III) to (VI). [ka]

[0060] After culturing in a CO2 incubator for 1 hour, 50 μL of 1000 μM (final concentration 250 μM) MGO solution was dispensed. As a control, PBS was dispensed in the same manner instead of MGO solution. The cells were cultured in a CO2 incubator for 3 days, and after the cultured plates were left to stand at room temperature for 30 minutes, 100 μL of the solution was removed. 100 μL / well of cell viability assay reagent (CellTiter-Glo® Luminescent Cell Viability Assay (Promega)) was dispensed and reacted at room temperature for 10 minutes. The luminescence intensity of each well was measured using a microplate reader.

[0061] Figure 5 shows the relationship between the concentration of added taxifolin and taxifolin analogs and cell viability. Here, cell viability (%) is shown as relative luminescence intensity, with the luminescence intensity in the control without MGO added set to 100%. Under conditions without taxifolin, the addition of MGO reduced cell viability to approximately 50%. Under conditions with taxifolin added, cell viability increased in a concentration-dependent manner, recovering to approximately 80% at an addition amount of 40 μM. On the other hand, with other compounds, increasing the addition amount did not improve viability; in fact, it decreased. This indicates that taxifolin has an effect of suppressing MGO-induced HUVEC damage.

[0062] [Example 5] Cytotoxicity suppression test (2) Place 100 μL / well (1.0 × 10⁶) of cochlear cell culture line (HEI-OC1) (Oriental Yeast Co., Ltd.) into a 96-well microplate. 4 Cells were seeded to a density of 1 cell / well. They were incubated statically in a CO2 incubator at 37°C for 24 hours. The culture supernatant was removed by aspirator, and 100 μL / well of PBS solution containing 500 μM MGO and 0 μM, 1 μM, 10 μM, or 100 μM taxifolin was dispensed. As a control, PBS without MGO or taxifolin was dispensed similarly. They were incubated statically in a CO2 incubator at 37°C for 24 hours. After centrifugation, the culture supernatant was removed by aspirator. 100 μL / well of 5 μg / mL Hoechst 33342 solution was added. They were incubated statically in a CO2 incubator for 30 minutes. After centrifugation, the culture supernatant was removed by aspirator. 100 μL / well of D-PBS(-) was added, and fluorescence (incident light 340 nm, fluorescence 470 nm) was measured.

[0063] Figure 6 shows the relationship between taxifolin concentration and cell viability. Here, cell viability (%) is expressed as the relative fluorescence intensity of each solution, with the fluorescence intensity of the control (MGO(-)) without MGO set to 100%. It was shown that cell viability increased in a taxifolin concentration-dependent manner. This indicates that taxifolin has an effect of suppressing MGO-induced HEI-OC1 damage.

Claims

1. Compounds represented by the following formula (I): 【Chemistry 1】 [In the formula, R １ , R ２ , R ３ , R ４ , R ５ Each of these is independently a saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, which may contain hydrogen, halogen, or ether bonds. A composition comprising a pharmaceutically acceptable salt or solvate thereof, for preventing and / or treating disorders associated with the accumulation of an advanced glycation end product of a subject.

2. The composition according to claim 1, which prevents the aforementioned disorder by inhibiting the formation of advanced glycation end products.

3. The composition according to claim 1, which prevents the aforementioned impairment by inhibiting the cross-linking of advanced glycation end products.

4. The composition according to claim 1, which prevents and / or treats the aforementioned disorder by cleaving crosslinks formed in advanced glycation end products.

5. The composition according to claim 1, wherein the disorder associated with the accumulation of advanced glycation end products is cytotoxicity.

6. The composition according to claim 1, wherein the advanced glycation end products are produced by the glycation of arginine in a protein.

7. The composition according to claim 6, comprising a composition in which the advanced glycation end product is produced by methylglyoxal.

8. The composition according to claim 1, wherein the disease associated with the accumulation of advanced glycation end products is an age-related disease.

9. The composition according to claim 1, wherein the compound represented by formula (I) is taxifolin.

10. The composition according to claim 1, which is a composition for oral intake.

11. The composition according to claim 1, which is a pharmaceutical composition.

12. The composition according to claim 1, which is a food composition.

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