JP7376889B2 - URAT1 inhibitor and food and drink composition for URAT1 inhibition - Google Patents

Description

The present invention relates to a URAT1 inhibitor and a food/beverage composition for inhibiting URAT1.

Hyperuricemia refers to a state in which the concentration of uric acid in the blood is high, and is a cause of the onset of gout. As methods for lowering blood uric acid concentration for the purpose of treating and preventing hyperuricemia, methods for suppressing uric acid synthesis and methods for promoting uric acid excretion are known. For example, Patent Document 1 discloses a composition for promoting uric acid excretion containing ampelopsin.

Japanese Patent Application Publication No. 2018-043985

By the way, in order to promote uric acid excretion, it is known that inhibiting URAT1, a uric acid transporter expressed in the kidney, is effective.

The present inventors have newly discovered that isoflavonoids, flavones, flavanones, and chalcone have a URAT1 inhibitory effect. The present invention is based on this novel finding and aims to provide a novel URAT1 inhibitor.

The URAT1 inhibitor of the present invention contains as an active ingredient at least one member selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof. The present invention has a URAT1 inhibitory effect because it contains at least one selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof.

The URAT1 inhibitor may be a uric acid excretion promoter.

The present invention also provides a food and drink composition for inhibiting URAT1, which contains as an active ingredient at least one member selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof. .

The present invention provides a novel URAT1 inhibitor.

Hereinafter, preferred embodiments of the present invention will be described in detail.

The URAT1 inhibitor of the present embodiment contains as an active ingredient at least one selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof.

"URAT1" is a transporter expressed in the kidney, and includes transporters with other names such as RST, OAT4L, and SLC22A12. "URAT1" is responsible for uric acid reabsorption on the luminal side. A typical nucleotide sequence and amino acid sequence of human-derived URAT1 is disclosed in GenBank (AB071863).

The URAT1 inhibitor of this embodiment has a URAT1 inhibitory effect. As mentioned above, since URAT1 is responsible for uric acid reabsorption on the luminal side, uric acid excretion can be promoted by inhibiting URAT1. In this case, the URAT1 inhibitor of this embodiment can also be called a uric acid excretion promoter.

In addition, by inhibiting URAT1, it is possible to lower the uric acid level in the blood, prevent uric acid crystal-dependent kidney damage, or reduce the risk of developing gout. Prolonged high blood uric acid levels can cause gout, but gout can be prevented by keeping blood uric acid levels low. Therefore, the URAT1 inhibitor of the present invention can be used to prevent gout onset and recurrence.

Isoflavonoids, flavones, flavanones and chalcone may be included in the URAT1 inhibitor as pharmaceutically acceptable salts. Such pharmaceutically acceptable salts are those formed by isoflavonoids, flavones, flavanones or chalcones and bases that form non-toxic salts. Specific examples include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and ammonium salts.

In this specification, isoflavonoid refers to a compound having an isoflavonoid skeleton. Isoflavonoids only need to have an isoflavonoid skeleton, and may exist as isoflavonoid glycosides. Examples of isoflavonoid glycosides include puerarin and daidzin. The isoflavonoid may be, for example, a compound represented by the following formula (1). In general formula (1), R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R 15 , R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are each independently a hydrogen atom, a ^hydroxyl group (-OH ) or a methoxy group (-OCH ₃ ). Examples of isoflavonoids include genistein (R ¹² , R ¹⁵ and R ¹⁷ : hydroxyl group, R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁴ , R ¹⁶ , R ¹⁸ and R ¹⁹ : hydrogen atom), daidzein (R ¹² and R ¹⁷ : hydroxyl group, R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁸ and R ¹⁹ : hydrogen atom), and the like.

Isoflavonoids may be derived from natural products (plants, microorganisms, etc.) or may be artificially synthesized. Moreover, if there is a commercially available product, it may be used. Isoflavonoids may be obtained by extracting, for example, seeds, leaves, branches, roots, flowers, etc. of leguminous plants with water, hot water, ethanol, methanol, etc.

As used herein, flavone refers to a compound having a flavone skeleton, a double bond between the 2-position and the 3-position, and no hydroxyl group at the 3-position. The flavone skeleton refers to the carbon skeleton that flavone has. The flavone may be, for example, a compound represented by the following formula (2). In general formula (2), R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ and R ²⁸ are each independently a hydrogen atom, a hydroxyl group (-OH) or a methoxy Indicates a group (-OCH ₃ ). Examples of flavones include luteolin (R ²¹ , R ²² , R ²⁵ and R ²⁷ : hydroxyl group, R ²⁰ , R ²³ , R ²⁴ , R ²⁶ and R ²⁸ : hydrogen atom), apigenin (R ²² , R ²⁵ and R ²⁷ : hydroxyl group, R ²⁰ , R ²¹ , R ²³ , R ²⁴ , R ²⁶ and R ²⁸ : hydrogen atom), baicalein (R ²⁵ , R ²⁶ and R ²⁷ : hydroxyl group, R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁸ : hydrogen atom), scutellarein (R ²² , R ²⁵ , R ²⁶ and R ²⁷ : hydroxyl group, R ²⁰ , R ²¹ , R ²³ , R ²⁴ and R ²⁸ : hydrogen atom), Tricetin (R ²¹ , R ²² , R ²³ , R ²⁵ and R ²⁷ : hydroxyl group, R ²⁰ , R ²⁴ , R ²⁶ and R ²⁸ : hydrogen atom), diosmetin (R ²¹ , R ²⁵ and R ²⁷ : hydroxyl group, R ²² : methoxy group, R ²⁰ , R ²³ , R ²⁴ , R ²⁶ and R ²⁸ : hydrogen atom), nobiletin (R ²² , R ²³ , R ²⁵ , R ²⁶ , R ²⁷ and R ²⁸ : methoxy group, R ²⁰ , R ²¹ and R ²⁴ ), and the like. The flavones are preferably luteolin, apigenin and nobiletin.

Flavones may be derived from natural products (plants, microorganisms, etc.) or may be artificially synthesized. Moreover, if there is a commercially available product, it may be used. Flavones may be obtained by extracting, for example, edible plants such as Apiaceae and Brassicaceae with water, hot water, ethanol, methanol, etc.

As used herein, flavanone refers to a compound having a flavanone skeleton and a single bond between the 2nd and 3rd positions. The flavanone may be a flavanonol in which the 3-position is a hydroxyl group (-OH). Further, the flavanone may exist as a flavanone glycoside. Examples of flavanone glycosides include hesperidin, naringin, sacranin, and poncillin.

The flavanone may be, for example, a compound represented by the following formula (3). In general formula (3), R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R 35 , R ³⁶ , R ^{37 ,} R ³⁸ and R ³⁹ are each independently a hydrogen atom, a hydroxyl group (-OH ) or a methoxy group (-OCH ₃ ). Examples of flavanones include butyne (R ³² , R ³³ and R ³⁸ : hydroxyl group, R ³⁰ , R ³¹ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ and R ³⁹ : hydrogen atom), eriodictyol (R ³¹ , R ³² , R ³⁶ and R ³⁸ : hydroxyl group, R ³⁰ , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), hesperetin (R ³³ , R ³⁶ and R ³⁸ : hydroxyl group, R ³² : methoxy group, R ³⁰ , R ³¹ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), homoeriodictyol (R ³² , R ³⁶ and R ³⁸ : hydroxyl group, R ³¹ : methoxy group) , R ³⁰ , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), isosacranetin (R ³⁶ and R ³⁸ : hydroxyl group, R ³² : methoxy group, R ³⁰ , R ³¹ , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), pinocembrin (R ³⁶ and R ³⁸ : hydroxyl group, R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), Sakuranetin (R ³² and R ³⁶ : hydroxyl group, R ³⁸ : methoxy group, R ³⁰ , R ³¹ , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), Sterubin (R ³¹ , R ³² and R ³⁶ : hydroxyl group, R ³⁸ : methoxy group, R ³⁰ , R ³³ , R ³⁴ , R ³⁵ , R ³⁷ and R ³⁹ : hydrogen atom), and the like. The flavanones are preferably naringenin and hesperetin.

Flavanones may be derived from natural products (plants, microorganisms, etc.) or may be artificially synthesized. Moreover, if there is a commercially available product, it may be used. Flavanones may be obtained by extracting, for example, fruits, leaves, branches, roots, flowers, etc. of plants such as mandarin oranges, grapefruits, oranges, and tomatoes with water, hot water, ethanol, methanol, and the like.

In this specification, chalcone refers to a compound having a chalcone skeleton. The chalcone only needs to have a chalcone skeleton. The chalcone may be a dihydrochalcone such as phloretin, and is preferably a compound represented by the following formula (4). In general formula (4), R ⁴⁰ , R ⁴¹ , R ⁴² , R ⁴³ , R ⁴⁴ , R 45 , R ⁴⁶ , R ^{47 ,} R ⁴⁸ and R ⁴⁹ are each independently a hydrogen atom or a hydroxyl group (-OH ) is shown. Examples of chalcone include naringenin chalcone (R ⁴² , R ⁴⁵ , R ⁴⁷ and R ⁴⁹ : hydroxyl group, R ⁴⁰ , R ⁴¹ , R ⁴³ , R ⁴⁴ , R ⁴⁶ and R ⁴⁸ : hydrogen atom).

Chalcone may be derived from natural products (plants, microorganisms, etc.) or may be artificially synthesized. Moreover, if there is a commercially available product, it may be used. Chalcone may be obtained by extracting, for example, fruits, leaves, branches, roots, flowers, etc. of plants such as morning leaves and apples with water, hot water, ethanol, methanol, etc.

The URAT1 inhibitor of this embodiment may be in any form such as solid (for example, powder), liquid (water-soluble or fat-soluble solution or suspension), paste, etc., and may also be in the form of powder, granule, tablet, or capsule. It may take any dosage form such as a drug, solution, suspension, emulsion, ointment, or plaster. It can also be in the form of a controlled release preparation. Alternatively, it may consist only of isoflavonoids, flavones, flavanones, chalcone, or pharmaceutically acceptable salts thereof.

The various formulations mentioned above contain isoflavonoids, flavones, flavanones, or chalcones, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable additives (excipients, binders, lubricants, disintegrants, emulsifiers). , a surfactant, a base, a solubilizing agent, a suspending agent, etc.).

For example, excipients include lactose, sucrose, starch, dextrin, and the like. Examples of the binder include polyvinyl alcohol, gum arabic, tragacanth, gelatin, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. Examples of lubricants include magnesium stearate, calcium stearate, and talc. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium hydrogen carbonate, and dextrin. Examples of the emulsifier or surfactant include Tween 60, Tween 80, Span 80, glyceryl monostearate, and the like. Examples of the base include cetostearyl alcohol, lanolin, polyethylene glycol, rice bran oil, fish oil (DHA, EPA, etc.), olive oil, and the like. Examples of the solubilizing agent include polyethylene glycol, propylene glycol, sodium carbonate, sodium citrate, Tween 80, and the like. Examples of the suspending agent include Tween 60, Tween 80, Span 80, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxymethylcellulose, sodium alginate, and the like.

The URAT1 inhibitor of this embodiment can be used as a component of products such as pharmaceuticals, quasi-drugs, food and drink compositions, feeds, and feed additives. Food and beverage compositions include foods and beverages (beverages, foods), food and beverage additives, health foods, foods with functional claims, foods for special uses, nutritional supplements, supplements, foods for specified health uses, and the like. Examples of beverages include water, soft drinks, fruit juice drinks, milk drinks, alcoholic drinks, sports drinks, and nutritional drinks. Examples of foods include breads, noodles, rice, tofu, dairy products, soy sauce, miso, and confectionery.

The above product consisting of or containing the URAT1 inhibitor of the present embodiment may be used for lowering blood uric acid levels, promoting uric acid excretion, or inhibiting URAT1. The above products include "improving blood uric acid levels", "controlling blood uric acid levels", "suppressing the increase in blood uric acid levels", "lowering blood uric acid levels", and "blood uric acid levels". Labels such as "Optimize uric acid level", "For those who are concerned about dietary purines", "For those who like alcohol and delicious food", etc. may be attached.

The method for producing the above products (medicines, quasi-drugs, food and drink compositions, feeds, feed additives, etc.) using the URAT1 inhibitor of the present embodiment is not particularly limited, and any known method can be followed as appropriate. For example, the URAT1 inhibitor of this embodiment can be mixed with an intermediate product or a final product in the manufacturing process of the above product to obtain the above product used for URAT1 inhibition.

As mentioned above, a URAT1 inhibitor containing as an active ingredient at least one member selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof can be used as a food or drink or a food or drink additive. It can be used as a component of Therefore, as an embodiment of the present invention, there is provided a food/drink composition for inhibiting URAT1, which contains as an active ingredient at least one member selected from the group consisting of isoflavonoids, flavones, flavanones, chalcone, and pharmaceutically acceptable salts thereof. things are provided.

The URAT1 inhibitor of this embodiment may be ingested by humans or non-human mammals. The dosage (intake amount) of the URAT1 inhibitor of this embodiment may be, for example, 0.1 mg to 1 g, preferably 1 to 500 mg, and more preferably 1 to 500 mg per kg of body weight per day for an adult, as an active ingredient. , 5 to 200 mg. The dosage can be appropriately determined depending on the condition, age, etc. of the individual.

The URAT1 inhibitor of this embodiment may be administered orally (ingested) or parenterally, but is preferably administered orally. The URAT1 inhibitor may be administered once a day, or may be administered in divided doses multiple times a day, as long as the daily amount of the active ingredient is within the above range.

Hereinafter, the present invention will be explained more specifically based on Examples. However, the present invention is not limited to the following examples.

[Test Example 1: Uric acid transport inhibition test using human URAT1-expressing cells]
(1) Preparation of test sample solution Genistein as an isoflavonoid, luteolin, apigenin, and nobiletin as flavones, naringenin and hesperetin as flavanones, naringenin chalcone as a chalcone, and benzbromarone (Wako), which is known as a URAT1 inhibitor, as a positive control. ) was dissolved in DMSO and diluted with DMSO to a concentration of 100 to 1000 times the target concentration. ¹⁴ C-labeled uric acid ([8- ¹⁴ C]-Uric Acid: ARC0513, manufactured by American Radiolabeled Chemicals, Inc.) was prepared using 125mM sodium gluconate, 4.8mM potassium gluconate, 1.2mM magnesium sulfate, and 1.3mM. Cl-free Hanks balanced salt solution (hereinafter referred to as HBSS Cl(-)) containing calcium gluconate, 1.2mM potassium dihydrogen phosphate, 25mM Hepes and 5mM glucose (pH 7.4) as final concentrations, respectively. The final concentration was adjusted to 5 μM. A test sample was diluted 100 to 1000 times using this ¹⁴ C-labeled uric acid solution to obtain a target concentration, which was then used as a sample solution. Note that a 0.1 to 1% DMSO solution containing no test sample was used as a control.

(2) Preparation of human URAT1 transiently expressing cells Human fetal kidney cell-derived 293A cells (manufactured by Invitrogen) 3.5 x 10 ⁵ cells/mL were added to DMEM medium containing 10% fetal bovine serum (manufactured by Nacalai Tesque). After suspending and inoculating 1 mL/well on a 12-well plate, the cells were cultured for 1 day in a 5% CO ₂ incubator at 37°C. 3.75 μL of polyethyleneimine "MAX" (manufactured by Polysciences) prepared at 1 mg/mL using sterilized MilliQ was taken per well, diluted with 50 μL of OptiMEM (manufactured by Invitrogen), and then allowed to stand at room temperature for 5 minutes (hereinafter, (referred to as “PEI-OptiMEM”). Dilute 0.75 μg per well (for example, 3.75 μL of a plasmid solution prepared to 0.2 μg/μL) of the human URAT1 expression vector and a control vector in which no human URAT1 has been introduced with 50 μL of OptiMEM, and incubate at room temperature for 5 minutes. (hereinafter referred to as "Plasmid-OptiMEM"). Plasmid-OptiMEM and PEI-OptiMEM were mixed, left at room temperature for 20 minutes or more, and then added to 293A cells in an amount of 107.5 μL per well. The 293A cells were cultured for 2 days in a 5% CO ₂ incubator at 37° C. and subjected to a uric acid uptake inhibitory activity test.

(3) Uric acid uptake inhibitory activity test The following tests were conducted on a hot plate heated to 37°C. After aspirating and removing the medium from each well containing the cells prepared in (2), each well was washed with HBSS Cl(-), replaced with 500 μL of HBSS Cl(-), and incubated at 37°C with 5% Pre-incubation was carried out for approximately 10 minutes in a CO ₂ incubator. After HBSS Cl(-) was removed by suction, 500 μL of the sample solution of (1) previously warmed to 37° C. was added, and an uptake reaction was performed for 20 seconds. After the reaction was completed, the sample solution was immediately removed by suction and washed with ice-cold HBSS Cl(-). Cells were lysed on ice using 500 μL of 0.2N sodium hydroxide aqueous solution per well, neutralized with 100 μL of 1N hydrochloric acid, and then 400 μL was transferred to a vial for liquid scintillation measurement (manufactured by Tokyo Glass Co., Ltd.). After mixing with 5 mL of Clearsol II (manufactured by Nacalai), radioactivity (disintegration number) was measured using a liquid scintillation counter (manufactured by PerkinElmer). Furthermore, the protein concentration was determined using 20 μL of the cell lysate using a PIERCE BCA Protein assay kit (manufactured by Thermo Fisher). The URAT1-specific uric acid transport activity rate in the presence of the test compound was calculated as follows using the measured radioactivity (disintegration number) value.
URAT1-specific uric acid transport activity rate (%) = [(A-C)/(B-C)] x 100
A: Transport activity when sample solution is added to human URAT1 transiently expressing cells B: Transport activity when 0.1-1% DMSO solution is added to human URAT1 transiently expressing cells C: Control vector is introduced Transport activity when 0.1-1% DMSO solution was added to the cells. Here, each transport activity of A, B, and C was calculated as follows.
Each transport activity of A, B and C [μL/mg/min] = X/(Y×Z)
X: Disintegration rate of ¹⁴ C-labeled uric acid taken up into cells in 600 μL cell lysis neutralization solution [DPM/600 μL/min]
Y: Protein amount in cells [mg/600μL]
Z: Disintegration number of ¹⁴ C-labeled uric acid per unit volume of the reaction mixture used in the uptake experiment [DPM/1 μL]

Table 1 shows the URAT1-specific uric acid transport activity rate in the presence of genistein, luteolin, apigenin, nobiletin, naringenin, hesperetin, and naringenin chalcone, and the IC ₅₀ value of each test compound for the uric acid transport activity by URAT1, obtained through the above study. show. Note that the IC ₅₀ value indicates the concentration of each test compound in the reaction mixture when inhibiting URAT1 activity by 50%. The IC ₅₀ value was calculated by plotting the URAT1-specific uric acid transport activity rate when each test compound was added at each concentration, and drawing an inhibition curve using the least squares method.

As shown in Table 1, isoflavonoids, flavones, flavanones, and chalcone had a URAT1 inhibitory effect.

Claims (3)

A URAT1 inhibitor containing as an active ingredient at least one member selected from the group consisting of genistein, apigenin, nobiletin, hesperetin, naringenin chalcone, and pharmaceutically acceptable salts thereof. The URAT1 inhibitor according to claim 1, which is a uric acid excretion promoter. A food or drink composition for inhibiting URAT1, which contains as an active ingredient at least one member selected from the group consisting of genistein, apigenin, nobiletin, hesperetin, naringenin chalcone, and pharmaceutically acceptable salts thereof.