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WO2023182735A1 - Composition de prévention ou de traitement de la dégénérescence maculaire, comprenant un composé qui induit l'expression d'un gène klotho antivieillissement - Google Patents

Composition de prévention ou de traitement de la dégénérescence maculaire, comprenant un composé qui induit l'expression d'un gène klotho antivieillissement Download PDF

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Publication number
WO2023182735A1
WO2023182735A1 PCT/KR2023/003592 KR2023003592W WO2023182735A1 WO 2023182735 A1 WO2023182735 A1 WO 2023182735A1 KR 2023003592 W KR2023003592 W KR 2023003592W WO 2023182735 A1 WO2023182735 A1 WO 2023182735A1
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formula
macular degeneration
composition
compound represented
preventing
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Korean (ko)
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정동주
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Klotho Sciences Co Ltd
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Klotho Sciences Co Ltd
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Priority claimed from KR1020220187495A external-priority patent/KR102890732B1/ko
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the present invention relates to a composition for preventing or treating macular degeneration containing a compound that induces the expression of the anti-aging gene klotho.
  • the macula is located in the center of the retina at the back of the eye, where images of objects are formed, and is where visual cells are concentrated.
  • Age-related macular degeneration occurs when the macular area degenerates. Lack of oxygen is known to be the main cause of degeneration, and as a result, Vascular Endothelial Growth Factor (VEGF) increases and new blood vessels are formed in areas of the retina where there were no blood vessels, resulting in retinal degeneration.
  • VEGF Vascular Endothelial Growth Factor
  • the outer layer, where the retina's visual cells are gathered, is surrounded by polarized epithelial cells called retinal pigment epithelium (RPE).
  • RPE retinal pigment epithelium
  • RPE cells The apical side of these RPE cells is in direct contact with the visual cells, and the basal side is in contact with Burch's membrane located inside the choroid layer.
  • the function of RPE cells plays an important role in maintaining vision. Its function is to remove old visual cells and move waste materials discharged from visual cells to the choroid for cleaning.
  • Age-Related Macular Degeneration is a disease that occurs due to aging. It is a disease with a high incidence in the elderly, with one in three adults over the age of 70 suffering from symptoms. Age-related macular degeneration is a representative eye disease with a higher occurrence frequency than the combined incidence of other eye diseases, glaucoma and cataract. If the disease continues to progress, vision is lost.
  • Age-related macular degeneration occurs due to loss of RPE cells and visual cells in the macula.
  • the histological characteristic of age-related macular degeneration disease is the accumulation of crystals called drusen between the RPE cell layer and Burch's membrane. Most of the components of these crystals are formed through oxidative stress and inflammation. Therefore, oxidative stress or inflammation can be considered to be the cause of age-related macular degeneration.
  • the treatment for this mainly uses laser or drug administration to remove malignant blood vessels. However, this does not provide a fundamental treatment and is only a way to alleviate the worsening symptoms.
  • mice that later increased the expression of this gene had an increase in lifespan of 20.0 to 30.8% in males and 18.8 to 19.0% in females. This served as an opportunity to inform the world for the first time that the lifespan of mice can be increased or decreased depending on the expression of a single gene.
  • the base sequence of the Klotho gene is very similar between animals, and it has been reported that there is about 98% identity between mice and humans. This indicates that even in humans, lifespan can be regulated by the expression of the klotho gene.
  • the klotho gene is located on chromosome 13 and produces a membrane protein with a base sequence similar to that of ⁇ -glucosidase.
  • Klotho protein is mainly expressed in renal tubular epithelial cells and brain choroid plexus, and has been reported to be expressed in some parathyroid glands.
  • the Klotho gene is a gene involved in various aging phenotypes. In mice deficient in the klotho gene, the aging process includes reduced lifespan, reduced activity, growth retardation, atherosclerosis, arterial calcification, osteoporosis, reproductive immaturity, infertility, skin atrophy, and emphysema. A similar syndrome occurs.
  • Klotho mRNA expression is significantly higher in kidney tissue than in other tissues, but expression of klotho mRNA is reduced in the kidneys of mouse disease models for hypertension, type 2 diabetes, diabetic nephropathy, and chronic renal failure.
  • NO Nitric Oxide
  • OLETF Oleuka Long-Evans Tokushima fatty rats
  • the klotho gene also affects glucose and insulin metabolism in mice, and statins, a representative hypercholesterolemia treatment, increase the expression of klotho mRNA in kidney proximal tubule cells.
  • statins a representative hypercholesterolemia treatment
  • osteopenia with low bone turnover occurs due to impaired differentiation of both osteoblasts and osteoclasts, which is similar to the characteristics of age-related bone loss and senile osteoporosis in humans.
  • Klotho mutant mice abnormal elongation of trabecular bone in the epiphyseal region and abnormal trabecular bone tissue findings are observed in microcomputed tomography, which is due to a disruption in the bone resorption process.
  • KL-VS functional variant of klotho
  • klotho which has mutations in three regions of the Klotho gene exon2
  • lipid metabolism blood pressure, lifespan, cognitive function, coronary artery disease, and cerebrovascular disease
  • microsatellites of the Klotho gene polymorphisms and single nucleotide polymorphisms have been associated with bone mineral density, and it has also been reported that single nucleotide polymorphisms in the Klotho gene are associated with risk factors for cardiovascular disease and bone mineral density in healthy adult women.
  • Recently, the association between the Klotho gene and Alzheimer's has been reported in several papers.
  • the present inventor has continuously conducted research on compounds that induce klotho expression, and as a result, the new compound developed by the present inventor has excellent stability and has a protective effect against oxidative stress (oxidative toxicity) in retinal pigment epithelial cells (RPE). It was experimentally confirmed that it was excellent, and the present invention was completed.
  • RPE retinal pigment epithelial cells
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating macular degeneration containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Another object of the present invention is to provide a food composition for preventing or improving macular degeneration containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating macular degeneration, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a food composition or health functional food composition for preventing or improving macular degeneration, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 and R 2 are each -H, halogen or C 1-6 straight or branched alkyl
  • R 3 and R 4 may each be -H or halogen.
  • the compound represented by Formula 1 may be a compound represented by Formula 1-1 below.
  • R 1 and R 2 may each be -H, halogen, or C 1-6 straight or branched alkyl.
  • the composition can increase the expression of the Klotho gene.
  • the composition can protect retinal pigment epithelial cells from oxidative stress.
  • the present invention provides a method for preventing or treating macular degeneration, comprising administering to a subject a composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a use for preventing or treating macular degeneration of a composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound according to the present invention has the effect of improving the expression level of the Klotho gene, a gene related to aging, and has a protective effect against oxidative stress in retinal pigment epithelial cells (RPE), and is a pharmaceutical composition for preventing or treating macular degeneration. , or can be usefully used as a health functional food composition.
  • RPE retinal pigment epithelial cells
  • Figure 1 shows the results of confirming the stability of KS compounds according to Example 1 of the present invention by MS/MS analysis: KS101 (compound of Formula 1-1A), KS102 (compound of Formula 1-1B), KS103 (compound of Formula 1-1C) ), KS104 (Formula 1-1D compound).
  • Figure 2 shows the results of confirming the expression level of secreted klotho and membrane klotho genes by the KS compound according to Example 1 of the present invention by PCR.
  • Figure 3 shows the results of confirming changes in survival rate in cells (ARPE: retinal pigment epithelial cells) treated with the KS compound for a long time according to Example 1 of the present invention.
  • FIG 4 shows the results of confirming changes in reactive oxygen species (ROS) in cells (ARPE: retinal pigment epithelial cells) treated with the KS compound for a long time according to Example 1 of the present invention.
  • ROS reactive oxygen species
  • FIG. 5 shows the results of confirming changes in reactive oxygen species (ROS) in cells (ARPE: retinal pigment epithelial cells) treated with the KS compound for a short period of time (6 hours) according to Example 1 of the present invention.
  • ROS reactive oxygen species
  • Figure 6 shows the results of confirming the cytotoxicity of the KS compound according to Example 1 of the present invention.
  • Figure 7 shows the results confirming the cell protection effect of KS compounds from oxidative stress induced by BSO.
  • Figure 8 shows the results confirming the cell protection effect of KS compounds from oxidative stress induced by NaIO 3 .
  • the present invention provides a composition for preventing, improving or treating macular degeneration comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 and R 2 may each be -H, halogen, or C 1-6 straight or branched alkyl, and R 3 and R 4 may each be -H or halogen.
  • the compound represented by Formula 1 may be a compound represented by Formula 1-1 below.
  • R 1 and R 2 may each be -H, halogen, or C 1-6 straight or branched alkyl.
  • the compound represented by Formula 1-1 is:
  • the macular degeneration may be age-related macular degeneration.
  • composition of the present invention can increase the expression level of the Klotho gene.
  • composition of the present invention can protect retinal pigment epithelial cells from oxidative stress.
  • the composition may be a pharmaceutical composition, food composition, or health functional food composition.
  • the composition may contain 1 to 99% by weight, 5 to 95% by weight, or 10 to 90% by weight of the active ingredient based on the total weight of the composition, but is not limited thereto.
  • prevention refers to any action that delays the onset of macular degeneration by administration of the composition of the present invention
  • treatment and “improvement” refers to the improvement or beneficial change in symptoms of macular degeneration by administration of the composition of the present invention. It means all actions.
  • the compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt.
  • pharmaceutically acceptable salt refers to any organic or organic salt of the base compound of Formula 1 where side effects due to the salt do not reduce the beneficial effects of the base compound of Formula 1, at a concentration that is relatively non-toxic and harmless to the patient and has an effective effect. It means inorganic addition salt. For these salts, inorganic acids and organic acids can be used as free acids.
  • Hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarine can be used as organic acids.
  • Acids gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethane sulfuric acid Fonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid can be used.
  • these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).
  • acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, maleate, maleate , malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate.
  • stearate, succinate, tartrate, tosylate, trifluoroacetate aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, Sodium, tromethamine, zinc salt, etc. may be included, and among these, hydrochloride or trifluoroacetate is preferable.
  • the compound of the present invention includes not only pharmaceutically acceptable salts, but also all salts, isomers, hydrates and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by conventional methods.
  • the compound is dissolved in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and an excess amount of organic acid is added or an aqueous acid solution of an inorganic acid is added. It can be manufactured by adding and then precipitating or crystallizing. Then, the solvent or excess acid in this mixture is evaporated and dried to obtain an addition salt, or the precipitated salt can be prepared by suction filtration.
  • a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile
  • the compounds of the present invention can be usefully used as a preventive, ameliorating, or therapeutic agent for macular degeneration disease. .
  • compositions of the present invention can be prepared as pharmaceutical compositions.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-described compound of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to an amount sufficient to achieve the efficacy or activity of the above-described compound of the present invention or a pharmaceutically acceptable salt thereof.
  • Pharmaceutically acceptable carriers are those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Includes, but is not limited to, vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited and may follow methods commonly used in the art. More specifically, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It may be prepared using diluents or excipients.
  • the pharmaceutical composition may be formulated in any one dosage form selected from the group consisting of eye drops, injections, granules, tablets, troches, pills, capsules, gels, syrups, suspensions, emulsions, drops, and solutions.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include one or more compounds of the present invention and at least one excipient such as starch, calcium carbonate, water, etc. It is prepared by mixing sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used.
  • Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • injectable ester such as ethyl oleate.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
  • parenteral administration it can be administered by injection in the form of intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, ocular administration, or topical ocular administration.
  • Ocular topical administration for example, can be administered directly intraocularly, periocularly, retrobulbally, subretinal, central retinal, outside the fovea, subconjunctival, or intravitreous. ), intracameral, or suprachoroidal administration.
  • the effective dosage for the human body of the composition of the present invention may vary depending on the patient's age, weight, gender, dosage form, health condition, and degree of disease, and the amount of the active ingredient contained in the composition of the present invention is generally about 0.001-100 mg/kg/day, preferably 0.01-35 mg/kg/day. Based on an adult patient weighing 70 kg, the dose is generally 0.07-7000 mg/day, preferably 0.7-2500 mg/day, and is administered once a day at regular intervals depending on the judgment of the doctor or pharmacist. It may be administered in several divided doses.
  • composition of the present invention may be provided as a food composition or health functional food composition.
  • Examples of foods to which the active ingredient of the present invention can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, There are various soups, beverages, alcoholic beverages, vitamin complexes, dairy products and milk products, and it includes all foods, health foods and health functional foods in the conventional sense.
  • Food and health functional food compositions containing the active ingredients according to the present invention can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement).
  • the amount of the active ingredient of the present invention may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food (based on 100 parts by weight in total).
  • the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
  • the food and health functional food composition of the present invention has no particular restrictions on other ingredients other than containing the active ingredient of the present invention as an essential ingredient in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. can do.
  • natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the food or health functional food composition of the present invention.
  • food and health functional food compositions containing the active ingredients of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), It may contain pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.
  • the food and health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.
  • the ratio of these additives is not that important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the food and health functional food composition containing the active ingredient of the present invention.
  • Step 1 1-(3,4-difluorophenyl)-3-(2-hydroxyphenyl)thiourea (1-(3,4-difluorophenyl)-3-(2-hydroxyphenyl)thiourea ,FCCS-19025 -2-1) Manufacturing
  • reaction mixture was purified by silica-gel column chromatography (20% Acetone / n-hexane) to obtain 1-(3,4-difluorophenyl)-3-(2-hydroxyphenyl)thiourea (1 -(3,4-difluorophenyl)-3-(2-hydroxyphenyl)thiourea, FCCS-19025-2-1) was obtained as a pale yellow solid (354 mg, 92%).
  • Step 2 N-(3,4-difluorophenyl)benzo[d]oxazol-2-amine (FCCS-19025) manufacturing
  • FCCS-19025-2-1 (224 mg, 0.80 mmol) and potassium peroxide (KO 2 ) (284 mg, 4.00 mmol) obtained in step 1 were mixed with acetonitrile (MeCN) (25) under Ar gas atmosphere. mL) and stirred at room temperature for 14 hours. After confirming the completion of the reaction by TLC (thin layer chromatography), acetonitrile (MeCN) was removed under reduced pressure.
  • reaction mixture was purified by silica-gel column chromatography (10 ⁇ 20% Ethyl acetate / n-hexane) to obtain the target compound N-(3,4-difluorophenyl)benzo[d]oxazole-2- Amine (N-(3,4-difluorophenyl)benzo[d]oxazol-2-amine, FCCS-19025) was obtained as a white solid (160 mg, 82%).
  • 3,4-difluorophenyl isothiocyanate (210mg, 1.23mmol) and 2-amino-5-fluorophenol (2-amino-5) obtained in Example 1-2 -fluorophenol, 130 mg, 1.02 mmol) was reacted to produce KS103 compound ( N -(3,4-difluorophenyl)-6-fluorobenzo[ d ]oxazol-2-amine), represented by the following formula 1-1C, as an ivory solid (203). mg, 75%).
  • 3,4-difluorophenyl isothiocyanate (212 mg, 1.24 mmol) and 2-amino-4,5-fluorophenol (2- amino-4,5-difluorophenol, 150 mg, 1.03 mmol) was reacted to produce KS104 compound ( N -(3,4-difluorophenyl)-5,6-difluorobenzo[ d ]oxazol-2, represented by the following formula 1-1D -amine) was obtained as a light purple solid (187 mg, 64%).
  • KS compound in Example 1 was used dissolved in 10mM DMSO, and tolbutamide, used as a standard substance, was dissolved in methanol at a concentration of 10mM.
  • each KS compound solution in 50 ⁇ l to a final concentration of 1 ⁇ M, pre-incubate for 3 minutes in a shaking water bath (37°C, 40 rpm), and then add 25 ⁇ l of 10 mM NADPH. Incubation was performed for 30 minutes under conditions with or without addition.
  • Figure 1 shows quantitative confirmation of KS compounds cultured with human liver microsomes through MS/MS analysis. Microsome enzymes operate dependent on the coenzyme NADPH(+), so compound metabolism occurred only in reactions where NADPH(+) was added. KS compounds showed higher stability than tolbutamide used as a control, and in particular, KS104 was confirmed to have the best microsomal stability.
  • KS101, KS102, KS103, KS104 compounds of Example 1 was added to Retinal Pigment Epithelium (RPE cells), whose aging was accelerated by adding BSO (Buthionine sulfoximine) at a concentration of 1 ⁇ M to the medium during the culture process. ) were added at 2.5 ⁇ M each and cultured for 8 hours. All RNA expressed in the cells was collected, and changes in expression of only klotho mRNA were selectively confirmed through PCR. As a result of the experiment, all KS compounds induced an increase in the expression of secreted klotho and membrane klotho. In particular, KS104 had a higher ability to induce secreted klotho expression than other compounds ( Figure 2).
  • KS101, KS102, KS103, and KS104 compounds were added to the retinal pigment epithelial cells (AEPE) in culture, and the cells were subcultured for 10 times. Then, BSO was added at a concentration of 1 ⁇ M and cultured for an additional 20 passages. Cell viability was measured. The proportion of living cells among cultured cells was measured using a LUNA-IITM automatic cell counter. LUNA-IITM automatic cell counter uses a reagent called tryphan blue. Living cells do not accept this reagent, but only dead cells are stained, indicating the ratio of live cells among all cells. As a result, it was confirmed that the survival rate of retinal pigment epithelial cells grown in medium containing KS compound was statistically higher ( p ⁇ 0.05) than that of cells grown in control conditions containing DMSO ( Fig. 3 ).
  • each of the KS compounds of Example 1 (KS101, KS102, KS103, and KS104 compounds) were added to the retinal pigment epithelial cells in culture, and the cells were subcultured for 10 passages.
  • BSO was added at a concentration of 1 ⁇ M, and BSO was added at a concentration of 1 ⁇ M for an additional 20 passages.
  • Activity in the cells Oxygen (Reactive Oxygen Species, ROS) concentration was measured.
  • ROS detection reagents from Molecular Probes were used as measurement reagents, and the concentration was measured by measuring the absorbance of the pigment produced by reacting with active oxygen using a spectrophotometer according to the method required by the manufacturer.
  • KS101, KS102, KS103, KS104 compounds 2.5 ⁇ M each of the KS compounds (KS101, KS102, KS103, KS104 compounds) of Example 1 were added to the retinal pigment epithelial cells in culture, and the concentration of reactive oxygen species (ROS) in the cells was tested after culturing for an additional 6 hours. Measurements were made in the same way as in 4. Through the experiment, it was confirmed that the amount of intracellular reactive oxygen species in retinal pigment epithelial cells grown in medium supplemented with KS compound for a short period of time was statistically lower ( p ⁇ 0.05) than in cells grown in control conditions to which DMSO was added. Through this, it was confirmed that KS compounds can reduce the amount of reactive oxygen species in cells even with a short treatment time of 6 hours ( Figure 4).
  • KS101, KS102, KS103, and KS104 compounds were compared using compound H, which was the basis for the development of these compounds. Cytotoxicity was measured using a reagent called EZ-CYTOX, a product of Dugen Biotechnology Research Institute. Using this reagent, the formation of an orange-colored water-soluble substance through dehydrogenase, which is active only in living cells, was quantified by measuring the absorbance at 405 nm. As a result of the experiment, it was confirmed that compound H showed clear cytotoxicity even at a concentration of about 5 ⁇ M, while KS compounds did not show significant toxicity even at a concentration of 10 ⁇ M. In particular, in the case of KS104, it was confirmed that cytotoxicity was not high even at a concentration of 40 ⁇ M ( Figure 6).
  • BSO a substance that causes cellular oxidative stress, inhibits the production of glutathione (GSH), an intracellular oxygen radical scavenging substance.
  • GSH glutathione
  • BSO a substance that causes cellular oxidative stress, inhibits the production of glutathione (GSH), an intracellular oxygen radical scavenging substance.
  • KS101, KS102, KS103, KS104 compounds 2.5 ⁇ M each of the KS compounds (KS101, KS102, KS103, KS104 compounds) of Example 1 were added to retinal pigment epithelial cells in culture, and cultured for 3 hours. . These cells were additionally treated with BSO (50mM) at a concentration that induces cytotoxicity, which was confirmed through preliminary experiments. After 6 hours of incubation, cytotoxicity was measured using EZ-CYTOX reagent. As a result, it was confirmed that the cytoprotective effect of all KS compounds was statistically higher ( p ⁇ 0.05) than the DMSO-treated control group
  • NaIO 3 a substance that causes intracellular oxidative stress, is known to be a substance that causes age-related macular degeneration (AMD) in retinal pigment epithelial cells.
  • AMD age-related macular degeneration
  • KS compounds of Example 1 2.5 ⁇ M each of the KS compounds of Example 1 (KS101, KS102, KS103, and KS104 compounds) were added to the retinal pigment epithelial cells and cultured for 3 hours.
  • NaIO 3 was added at a concentration of 5 mM to the cells and incubated for 24 hours. Additional culture was performed. Afterwards, the toxicity protection effect in cells was measured using EZ-CYTOX reagent. As a result, it was confirmed that the cytoprotective effect of all KS compounds was statistically higher ( p ⁇ 0.05) than the DMSO-treated control group (FIG. 8).

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  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Ophthalmology & Optometry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une composition de prévention ou de traitement de la dégénérescence maculaire, comprenant un composé qui induit l'expression d'un gène Klotho antivieillissement. Le composé selon la présente invention présente un excellent effet d'amélioration du niveau d'expression du gène Klotho associé à l'âge, et également présente un effet protecteur contre le stress oxydatif dans des cellules d'épithélium pigmentaire de la rétine (RPE), et ainsi peut être avantageusement utilisé comme composition pharmaceutique ou composition alimentaire à fonction de santé pour prévenir ou traiter la dégénérescence maculaire.
PCT/KR2023/003592 2022-03-22 2023-03-17 Composition de prévention ou de traitement de la dégénérescence maculaire, comprenant un composé qui induit l'expression d'un gène klotho antivieillissement Ceased WO2023182735A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2022-0035142 2022-03-22
KR20220035142 2022-03-22
KR10-2022-0187495 2022-12-28
KR1020220187495A KR102890732B1 (ko) 2022-03-22 2022-12-28 항노화 유전자 klotho의 발현을 유도하는 화합물을 포함하는 황반변성의 예방 또는 치료용 조성물

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WO2023182735A1 true WO2023182735A1 (fr) 2023-09-28

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110049364A (ko) * 2009-11-05 2011-05-12 서울대학교산학협력단 벤조헤테로사이클 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 암 예방 및 치료용 조성물
KR20150079478A (ko) * 2013-12-30 2015-07-08 이화여자대학교 산학협력단 신경세포의 분화 또는 재생, 신경손상 또는 신경질환의 치료 효과가 있는 신규한 물질, 이의 제조방법 및 이를 포함하는 약학 조성물
KR20150091011A (ko) * 2014-01-29 2015-08-07 이화여자대학교 산학협력단 소양증 개선 활성을 갖는 벤즈옥사졸 유도체
KR20210039972A (ko) * 2019-10-02 2021-04-12 주식회사 클로소사이언스 항노화 유전자 klotho의 발현을 유도하는 화합물 및 이의 용도

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KR20110049364A (ko) * 2009-11-05 2011-05-12 서울대학교산학협력단 벤조헤테로사이클 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 암 예방 및 치료용 조성물
KR20150079478A (ko) * 2013-12-30 2015-07-08 이화여자대학교 산학협력단 신경세포의 분화 또는 재생, 신경손상 또는 신경질환의 치료 효과가 있는 신규한 물질, 이의 제조방법 및 이를 포함하는 약학 조성물
KR20150091011A (ko) * 2014-01-29 2015-08-07 이화여자대학교 산학협력단 소양증 개선 활성을 갖는 벤즈옥사졸 유도체
KR20210039972A (ko) * 2019-10-02 2021-04-12 주식회사 클로소사이언스 항노화 유전자 klotho의 발현을 유도하는 화합물 및 이의 용도

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MA ZHONGXU, LIU JINGJING, LI JING, JIANG HAO, KONG JUN: "Klotho Levels are Decreased and Associated with Enhanced Oxidative Stress and Inflammation in the Aqueous Humor in Patients with Exudative Age-related Macular Degeneration", OCULAR IMMUNOLOGY AND INFLAMMATION, AEOLUS PRESS, BUREN, NL, vol. 30, no. 3, 3 April 2022 (2022-04-03), NL , pages 630 - 637, XP009549109, ISSN: 0927-3948, DOI: 10.1080/09273948.2020.1828488 *

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