WO2017038723A1

WO2017038723A1 - Xanthine oxidase inhibitor

Info

Publication number: WO2017038723A1
Application number: PCT/JP2016/075084
Authority: WO
Inventors: 周作松田; 周作岡
Original assignee: Ils株式会社
Priority date: 2015-08-28
Filing date: 2016-08-26
Publication date: 2017-03-09
Also published as: JPWO2017038723A1; JP6873904B2

Abstract

The present invention provides a novel xanthine oxidase inhibitor. More specifically, the present invention provides a xanthine oxidase inhibitor comprising a compound represented by formula (I) or an orally acceptable salt thereof. (wherein R represents OH or H).

Description

Xanthine oxidase inhibitor

Reference to related applications

This patent application is accompanied by a claim of priority based on Japanese Patent Application No. 2015-169753 filed on Aug. 28, 2015, and the entire disclosure in such earlier patent application is incorporated by reference. It is made a part of this specification.

The present invention relates to a novel xanthine oxidase inhibitor.

In recent years, there has been a concern that lifestyle-related diseases will increase along with the westernization of food and the increase in alcohol intake. Among them, a state in which the serum uric acid level is higher than usual is called hyperuricemia, but gout caused by this state causes uric acid to form stones in the joint fluid, causing inflammation, and the base of the thumb, ankle, foot It is known as a disease accompanied by severe pain in the upper, knee, and Achilles (Non-Patent Document 1).

According to the National Living Standards Survey (2004), the number of gout patients exceeds 870,000, which is a 3.4-fold increase compared to 1986. Hyperuricemia is a cause of urate deposition such as kidney damage and urolithiasis, and is defined as hyperuricemia when the serum uric acid level exceeds 7.0 mg / dL regardless of age and sex. . According to recent surveys, the frequency of hyperuricemia in adult males is estimated to be 21.5-26.2%, reaching 30% after 30 years of age, which is a problem. In the case of women, the frequency of hyperuricemia is 1.3% under 50 years old and 3.7% after 50 years old, which is lower than that of men, but in the case of women, the serum uric acid level is 7. Even if it is 0 mg / dL or less, it is said that the risk of lifestyle-related diseases increases as the serum uric acid level rises, and it has been pointed out that it may be accompanied by an increase in the relative risk of total death.

Hyperuricemia is classified into disease types, and is roughly classified into “uric acid production excess type”, “uric acid excretion reduced type”, and “mixed type”. In drug therapy, probenecid and benzbromarone are examples of uric acid excretion promoters, and allopurinol is representative of uric acid production inhibitors. Similarly, febuxostat can be prescribed from May 2011 as a uric acid production inhibitor. Drug selection according to the type of disease is in the basic principle, but if there is a history or complication of urinary calculi, it is necessary to select a uric acid production inhibitor even if it is a “uric acid excretion reduced type”. This uric acid production inhibitor is a substance that inhibits the activity of an enzyme that generates uric acid from xanthine, a metabolite of purine in vivo (xanthine oxidase (XOD), hereinafter also referred to as XOD). Serum uric acid level is reduced / suppressed by inhibiting the production of. However, these drugs can be expected to have a high serum uric acid level reducing effect, but side effects such as severe renal function and liver dysfunction must also be considered. In patients with asymptomatic hyperuricemia at a level that does not require drug treatment at the present time, treatment has been addressed by improving lifestyle, ingesting foods rich in purines, and avoiding excessive intake of alcohols and calories. (Non-Patent Document 2).

The state of hyperuricemia may develop into a serious disease in the future, and it goes without saying that efforts such as improving lifestyles are included, but functional substances having an action to reduce serum uric acid levels are included There is a demand for materials that can more actively control serum uric acid levels by ingesting foods and uric acid level reducing agents. Many of the substances that have been reported to have a serum uric acid level reducing action in patent documents and academic papers so far are derived from plants, and most are polyphenols. The action of reducing uric acid level is due to the effect of XOD inhibition by polyphenols. Exceptionally, animal-derived anserines are said to have a uric acid level-reducing action, but the mechanism of action is unclear unlike XOD inhibition. In particular, there is no report that exceeds the activity of pharmaceutical allopurinol with XOD inhibitors derived from natural products.

In addition, there is no report on the relationship between the degradation product of tetrapyrrole-containing product or its extract and the XOD inhibitory activity.

The object of the present invention is to provide a new technical means for effectively inhibiting the activity of XOD.

The present inventors have now found that XOD can be effectively inhibited using a tetrapyrrole-containing decomposition product or extract thereof or a specific compound derived therefrom.

According to the present invention, the following inventions are provided.
(1) A xanthine oxidase inhibitor comprising a compound represented by formula (I) or an orally acceptable salt thereof:

(Wherein R is OH or H).
(2) The xanthine oxidase inhibitor according to (1), which is obtained by containing a decomposition product of tetrapyrrole-containing product or an extract thereof.
(3) The agent according to (1) or (2), which is in the form of a single oral intake unit.
(4) The agent according to any one of (1) to (3), which is a blood uric acid lowering agent.
(5) The agent according to any one of (1) to (4), which is a therapeutic agent for a disease or condition caused by excessive production of uric acid or decreased excretion.
(6) The agent according to any one of (1) to (5), which is a therapeutic agent for hyperuricemia.
(7) The agent according to any one of (1) to (6), which is a composition.
(8) The agent according to any one of (1) to (7), which is a food or drink or a pharmaceutical product.
(9) Compound represented by formula (Ia) or an orally acceptable salt thereof:

(10) A method for producing a xanthine oxidase inhibitor, which comprises blending a tetrapyrrole-containing decomposition product or an extract thereof into the agent.
(11) The method according to (10), wherein the tetrapyrrole-containing material is at least one selected from the group consisting of erythrocytes, hemoglobin, hemin, protoporphyrin, hematoporphyrin, bilirubin and chlorophyllin.
(12) The method according to (10) or (11), wherein the decomposition of the tetrapyrrole-containing material is performed by at least one reaction selected from the group consisting of an oxidation reaction, a reduction reaction, and an enzyme reaction.
(13) The method according to (10), wherein the xanthine oxidase inhibitor comprises a compound represented by formula (I) or an orally acceptable salt thereof:
(Wherein R is OH or H).
(14) Use of a tetrapyrrole-containing decomposition product or an extract thereof in the production of a xanthine oxidase inhibitor.
(15) The use according to (14), wherein the xanthine oxidase inhibitor comprises a compound represented by formula (I) or an orally acceptable salt thereof:

(Wherein R is OH or H).
(16) The use according to (14) or (15), wherein the xanthine oxidase inhibitor is a therapeutic agent for a disease or condition caused by excessive production or decreased excretion of uric acid.
(17) A disease caused by excessive production or decreased excretion of uric acid, comprising ingesting an effective amount of a compound represented by formula (I) or an orally acceptable salt thereof into a subject in need thereof How to treat the condition:

(Wherein R is OH or H).
(18) A compound represented by formula (I) or an orally acceptable salt thereof for inhibiting xanthine oxidase or treating a disease or condition caused by excessive production or decreased excretion of uric acid:

(Wherein R is OH or H).

According to the present invention, XOD can be effectively inhibited. In addition, the present invention can be advantageously used to effectively reduce blood uric acid in subjects such as hyperuricemia. Furthermore, the present invention can be advantageously used to treat diseases or conditions resulting from excessive production of uric acid or decreased excretion.

It is a graph which shows the XOD inhibitory activity of the porcine erythrocyte enzyme degradation product adjusted to various pH. 2 is a chart showing an elution profile obtained by subjecting an erythrocyte enzyme degradation product to an ODS-A column of reverse phase resin and gradient elution with an acetonitrile-TFA system. The arrow indicates a peak having XOD inhibitory activity. 3 is a chart showing an elution profile obtained by subjecting the peak fraction having XOD inhibitory activity in FIG. 2 to a reverse phase ODS-120T and performing a gradient elution with a methanol-TFA system. The peak having the highest inhibitory activity was designated as P1, and the peak having the second highest inhibitory activity was designated as P2. 3 shows XOD inhibitory activity curves of P1 derived from hemin-hydrogen peroxide reactant, P1 obtained by organic synthesis (hereinafter also referred to as synthetic P1), and allopurinol. FIG. 4 is a chart in which P1 after reverse phase chromatography in FIG. 3 is adsorbed to an anion exchange resin GigaCap Q-650M (manufactured by Tosoh Corporation) under conditions of 50 mM Tris-HCl pH 9 and eluted with a sodium chloride gradient. The absorption spectra of P1 and P2 derived from hemin-hydrogen peroxide reactant are shown. The LC / MS analysis (negative mode) result of P1 derived from erythrocyte enzymatic degradation product and the LC / MS analysis (negative mode) result of P1 and P2 derived from hemin-hydrogen peroxide reaction are shown. The LC / MS analysis result of P2 derived from hemin-hydrogen peroxide reactant and the estimation of the structure of P2 are shown. The ¹ H-NMR and ¹³ C-NMR results of P1 derived from a hemin-hydrogen peroxide reactant are shown. The chart of reverse phase chromatography of P1 derived from erythrocyte enzyme degradation product, purified product of erythrocyte enzyme degradation product by activated carbon (activated carbon 1) or a mixture thereof is shown. The LC / MS analysis (negative mode) result of the synthesis P1 is shown. The result of ¹ H-NMR of Synthesis P1 is shown. It is a graph which shows the serum uric acid level change of the mouse | mouth in a potassium oxonate administration group, a potassium oxonate + synthetic P1 administration group, and a control group. It is a graph which shows the comparison of AUC (area under a curve) about the serum uric acid level until 6 hours after synthetic P1 administration.

DETAILED DESCRIPTION OF THE INVENTION

Agent The agent of the present invention is characterized in that it contains a tetrapyrrole-containing decomposition product or an extract thereof.

In the present invention, tetrapyrrole means a compound containing four pyrrole rings, and includes linear and cyclic compounds.

Examples of the linear tetrapyrrole include villin (bilirubin, biliverdin, urobilinogen, urobilin, stercobilin, phytochrome, phycobilin, etc.).

Examples of the cyclic tetrapyrrole include porphyrins (ethioporphyrin, mesoporphyrin, protoporphyrin, deuteroporphyrin, hematoporphyrin, coproporphyrin, uroporphyrin, pyroporphyrin, philoporphyrin, rhodoporphyrin, phytoporphyrin, etc.) and chlorins. .

The porphyrin includes an intramolecular metal complex into which a metal atom such as iron, copper, magnesium or cobalt is introduced. For example, heme, hematin, hemin, chlorophyll, vitamin B12, iron chlorophyllin, iron chlorophyllin sodium, copper chlorophyllin, copper chlorophyllin sodium, magnesium chlorophyllin and the like.

The tetrapyrrole-containing material of the present invention may be tetrapyrrole itself or a composition containing tetrapyrrole. Specifically, the tetrapyrrole-containing product is preferably red blood cells, hemoglobin, chloroplasts, hemin, protoporphyrin, hematoporphyrin, bilirubin, chlorophyll, chlorophyllin, and more preferably red blood cells, hemoglobin, hemin, hematoporphyrin or It is chlorophyllin.

The origin of the tetrapyrrole-containing material is not particularly limited as long as the effects of the present invention are not hindered. Animals having blood such as mammals, birds, reptiles, fish, amphibians and other vertebrates, or plants and algae having chloroplasts. Preferred are mammals, and more preferred are livestock animals such as pigs, cows, horses, sheep and goats.

Moreover, the tetrapyrrole-containing material may be obtained from a natural product, or a commercially available product may be used. Further, the tetrapyrrole-containing material may be subjected to a decomposition treatment as it is, or may be subjected to a decomposition treatment after performing a drying treatment such as spray drying, concentration drying, freeze drying and the like.

The decomposition of the tetrapyrrole-containing material is preferably performed by at least one reaction selected from the group consisting of an oxidation reaction, a reduction reaction and an enzyme reaction.

The oxidation reaction of the tetrapyrrole-containing material can be carried out by reacting the tetrapyrrole-containing material with an oxidizing agent such as hydrogen peroxide, hypochlorous acid or permanganate. More specifically, the oxidation reaction may be carried out at 45 to 60 ° C. for 1 to 16 hours by mixing a tetrapyrrole-containing material (in terms of dry matter) and an oxidizing agent in a molar ratio of 1:10 to 1: 500. preferable.

The tetrapyrrole-containing product is reduced by using a reducing agent such as cysteine (Cys), glutathione (GST), dithiothreitol (DTT), ascorbic acid (vitamin C), tocophenol (vitamin E) and the like. It can be carried out by reacting. More specifically, the reduction reaction may be carried out at 45 to 60 ° C. for 1 to 16 hours by mixing a tetrapyrrole-containing material (in terms of dry matter) and a reducing agent in a molar ratio of 1: 1 to 1: 100. preferable.

In addition, in the oxidation or reduction reaction of the tetrapyrrole-containing material, it is preferable to add iron ions such as iron (II) chloride and iron (III) chloride to the reaction system from the viewpoint of promoting the reaction.

The enzyme used in the enzyme reaction of the tetrapyrrole-containing material is not particularly limited as long as the effect of the present invention is not hindered, but a proteolytic enzyme (protease) is preferable. Such proteases include endo-type proteases or exo-type proteases, and such proteases may be alkaline proteases. Specific examples of the protease include Alcalase (trademark), Esperase (trademark), Neutrase (trademark) (manufactured by Novozymes), Orientase (trademark) 90N, Orientase (trademark) 22BF (manufactured by HIBI), Samoaase ( (Trademark) PC10F, protease P “Amano” 3SD (manufactured by Amano Pharmaceutical Co., Ltd.), Multifect (trademark) PR6L, Multifect (trademark) PR7L (manufactured by Danisco Japan Co., Ltd.), and the like, preferably alcalase and multifect PR6L . More specifically, the enzyme reaction is preferably carried out at 45 to 60 ° C. for 1 to 16 hours by mixing a tetrapyrrole-containing material (in terms of dry matter) and the enzyme at a weight ratio of 100: 1 to 100: 10.

Moreover, it is preferable that the tetrapyrrole containing material or extract thereof of the present invention contains a compound of the formula (I) or an orally acceptable salt thereof. Thus, according to a preferred embodiment, the agent of the present invention comprises a compound of formula (I) or an orally acceptable salt thereof.

(Wherein R is OH or H)

According to yet another preferred embodiment, the compound of the invention consists of a compound of formula (Ia) and / or a compound of formula (Ib).

In the present invention, the orally acceptable salt of the compound of formula (I) is not particularly limited, and examples thereof include inorganic acid salts, inorganic base salts, organic acid salts, and organic base salts.

In addition, the compound of formula (I) or an orally acceptable salt thereof absorbs moisture by attaching it to the atmosphere or recrystallizes, and adsorbs water or becomes a hydrate. In some cases, the present invention also includes such various hydrates, solvates and polymorphic compounds.

In addition, the compound of the formula (I) may be incorporated into the agent in the form of a prodrug, and the present invention includes such an embodiment. Suitable prodrugs include, for example, a nitrogen atom, a carboxyl group or an aldehyde group in formula (I), or a compound of formula (Ia) and / or formula (Ib), imine, nitrile, hydroxyl, amide, ester, carbamoyl. , Compounds converted or modified into acid anhydrides, and the like.

The extract of the present invention includes, for example, an extract of a decomposition product of tetrapyrrole-containing product using an aqueous medium (such as ethanol, water, or a mixture thereof), and is preferably an ethanol extract. As suitable extraction conditions, a tetrapyrrole-containing material (in terms of dry matter) and an aqueous medium are mixed at a weight ratio of 5: 1 to 1: 5, and extraction is performed at 20 to 65 ° C. for 0.5 to 3 hours. Is preferred.

In the production of the agent of the present invention, purification treatment, concentration treatment, sterilization treatment, etc. are performed as desired for the decomposition product of tetrapyrrole-containing product or extract thereof, or the compound of formula (I) or an orally acceptable salt thereof. May be. Examples of the purification treatment include adsorption treatment with a porous substance such as activated carbon, synthetic adsorption resin, silica gel, filtration treatment with a nanofilter (NF membrane), ultrafiltration membrane, etc., dialysis treatment (electrodialysis, etc.), and centrifugation. Processing and the like. Examples of the concentration treatment include ultrafiltration treatment, reduced pressure concentration treatment, lyophilization treatment, and the like.
The purification treatment or concentration treatment is preferable for separating, decomposing or removing inactive components and increasing the concentration of the compound of formula (I) or an orally acceptable salt thereof. Moreover, the said refinement | purification process, a concentration process, or a sterilization process etc. are preferable also when the pigment | dye or odor of a tetrapyrrole containing product decomposition product can be reduced significantly.
From this point of view, the activated carbon used for the purification treatment is preferably derived from wood flour, preferably in powder form, and most frequently having a pore diameter of 1 to 30 nm. The most frequent pore diameter of activated carbon can be measured by a nitrogen gas adsorption method. The amount of activated carbon used can be 0.1-20% of the decomposition product of tetrapyrrole-containing product or extract thereof, or the compound of formula (I) or an orally acceptable salt thereof, preferably 0. 4 to 1.25%. As a suitable condition for the purification treatment using activated carbon, activated carbon was added to a tetrapyrrole-containing decomposition product or extract thereof or a compound of formula (I) or an orally acceptable salt thereof adjusted to pH 2 to 5. It is recovered later, washed with 0.3 to 60 times the amount of tetrapyrrole-containing decomposition product or extract thereof or the compound of formula (I) or orally acceptable salt thereof, and then an alkaline buffer having a pH of 9 to 11 It is preferable to elute with a solution or 0.05 to 0.15 M sodium hydroxide solution. The amount of the alkaline buffer or sodium hydroxide solution should be 0.01 to 100 times the amount of tetrapyrrole-containing degradation product or extract thereof, or the compound of formula (I) or orally acceptable salt thereof. The amount is preferably 0.1 to 10 times. The synthetic adsorption resin is preferably a styrene-divinylbenzene system, and most preferably has a pore diameter of 5 to 100 nm. The most frequent pore diameter of the synthetic adsorption resin can be measured by a mercury intrusion method. The amount of the synthetic adsorption resin to be used can be 10 times to 1/50 of the tetrapyrrole-containing decomposition product or extract thereof, or the compound of formula (I) or an orally acceptable salt thereof. The amount is preferably equivalent to 1/10.
In addition, from the viewpoint of separation or removal of inactive components, or reduction of pigments or odors of tetrapyrrole-containing product decomposition products, the nanofilter used in the purification treatment can have a molecular weight cut-off of 150 to 800, preferably 150-500.
In the production of the agent of the present invention, the tetrapyrrole-containing product decomposition product or the extract thereof may be solidified or pulverized by, for example, freeze drying or spray drying.
Each treatment described above is carried out at any stage in the production of the tetrapyrrole-containing decomposition product or extract thereof, the compound of formula (I), or an orally acceptable salt thereof, as long as the effects of the present invention are not hindered. Also good.

Further, the agent of the present invention may be prepared using a decomposition product of tetrapyrrole-containing product or an extract thereof, a compound of formula (I) or an orally acceptable salt thereof as it is, and orally acceptable. You may mix | blend an additive further. Such orally acceptable additives are not particularly limited, but include solvents, solubilizers, lubricants, emulsifiers, isotonic agents, stabilizers, preservatives, preservatives, surfactants, regulators, Chelating agents, pH adjusters, buffers, excipients, binders, preservatives, thickeners, colorants, fragrances, fragrances, wetting agents, sweeteners, or antioxidants.
The pH of the agent of the present invention is not particularly limited, but can be pH 1 to 14, preferably pH 3 to 11, more preferably pH 4 to 10.

The agent of the present invention is not particularly limited as long as the effect of the present invention is not hindered. For example, it is solid, powder, granule, capsule, block, liquid (eg, solution, suspension, emulsion). , Any form of slurry, gel, paste, or paste. Specific dosage forms include solid preparations such as tablets, powders, fine granules, granules, capsules, pills, sustained release agents, drinks, and jelly preparations.

In addition, the agent of the present invention is preferably composed of a single oral intake unit form. The agent of the present invention may contain 1 to 5000 mg, more preferably 10 to 1000 mg, of a tetrapyrrole-containing decomposition product or an extract thereof in terms of dry mass as a single oral intake. In addition, the agent of the present invention is preferably 0.01 to 300 mg, more preferably 0. 0, in terms of dry mass of the compound of formula (I) or an orally acceptable salt thereof as a single oral intake. 1-100 mg may be contained.

Moreover, it is preferable to provide the agent of the present invention in a package form. The packaging form is not particularly limited, and examples thereof include a pack or a container. In addition, component display, dose / use display, etc. may be attached to the surface of the package. Suitable examples of such packaging forms include supplements, drinks or pharmaceutical preparations.

Further, the agent of the present invention can be advantageously used for lowering blood uric acid or reducing uric acid level, or suppressing increase in blood uric acid level or uric acid production. Therefore, according to one embodiment, the agent of the present invention is preferably provided as a blood uric acid lowering agent, more preferably a serum uric acid level lowering agent. According to another aspect, the agent of the present invention is provided as a therapeutic agent for a disease or condition caused by excessive production of uric acid or decreased excretion. According to another aspect, the agent of the present invention is provided as a therapeutic agent for hyperuricemia. In addition, the agent of the present invention reduces blood uric acid, thereby causing gout based on hyperuricemia, urolithiasis, renal dysfunction (preferably chronic kidney disease, chronic glomerulonephritis), cardiovascular disease It can be advantageously used for treatment of various diseases such as (preferably coronary artery disease, ischemic heart disease), hypertension, metabolic syndrome, malignant tumor, etc., or reduction or improvement of disease state. Therefore, the agent of the present invention is hyperuricemia, gout based on hyperuricemia, urolithiasis, renal dysfunction (preferably chronic kidney disease, chronic glomerulonephritis), cardiovascular disease (preferably, Coronary artery disease, ischemic heart disease), hypertension, metabolic syndrome, treatment of various diseases such as malignant tumor, or reduction or improvement of the disease state can be displayed and provided. The “treatment” of the present invention includes not only treating an established pathological condition but also preventing a pathological condition that may be established in the future. In addition, the “treatment” of the present invention includes not only a medical act but also an act of improving or preventing a disease state or a condition that has already occurred or that may occur in the future by causing a food or drink described below to be ingested. Preferably included.

Also, the agent of the present invention can be provided in the form of a composition in which a plurality of components are blended. Therefore, according to one embodiment of the present invention, blood uric acid for xanthine oxidase inhibition comprising a tetrapyrrole-containing degradation product or extract thereof or a compound of formula (I) or an orally acceptable salt thereof Compositions are provided for reducing, for treating a disease or condition resulting from overproduction or decreased excretion of uric acid, or for treating hyperuricemia.

The agent of the present invention may be a food or drink or a medicine as long as it contains a tetrapyrrole-containing decomposition product or extract thereof, or a compound of formula (I) or an orally acceptable salt thereof. Therefore, according to another aspect, the agent of the present invention is for inhibiting xanthine oxidase, comprising a tetrapyrrole-containing degradation product or extract thereof or a compound of formula (I) or an orally acceptable salt thereof, A food / beverage composition or a pharmaceutical composition for lowering blood uric acid, for treating a disease or condition caused by excessive production or decreased excretion of uric acid, or for treating hyperuricemia.

The food and drink of the present invention is not particularly limited, but for example, instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups and miso soups, freeze-dried foods; soft drinks, fruit juice drinks, vegetable drinks Beverages such as soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, alcoholic drinks; flour products such as bread, pasta, noodles, cake mixes, bread crumbs; rice cakes, caramel, chewing gum, chocolate, cookies, biscuits, Cakes, pies, snacks, crackers, Japanese confectionery, dessert confectionery, etc .; sauces, tomato processed seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew seasonings Oils and fats such as processed fats and oils, butter, margarine, mayonnaise; milk beverages, yogurts, lactic acid bacteria beverages, ice Reem class, cream, and the like of dairy products; agricultural canning, jam, marmalade class, agricultural processed products of serial and the like; ham, bacon, sausage, meat processed foods such as roast pork: frozen food, and the like.

The foods and drinks of the present invention include health foods, functional foods (for example, foods for specified health use, nutritional functional foods or functional labeling foods), nutritional supplements, foods for the sick, infant formulas, pregnant women, This includes milk powder for lactating women and food for those who have difficulty swallowing. In addition, the food and drink of the present invention is used for xanthine oxidase inhibition, blood uric acid reduction, uric acid overproduction or treatment of diseases or conditions caused by reduced excretion, or hyperuricemia treatment May be attached.

Further, the medicament of the present invention is also used for treatment of diseases or conditions caused by xanthine oxidase inhibition, blood uric acid reduction, excessive production or excretion of uric acid, or hyperuricemia. It may be a display. Such pharmaceuticals can be prepared according to the description regarding the above-mentioned agents.

Further, according to another aspect of the present invention, there is provided a method for inhibiting xanthine oxidase, comprising an effective amount of a tetrapyrrole-containing degradation product or extract thereof, a compound of formula (I) or an orally acceptable salt thereof, A method is provided comprising ingesting a subject in need thereof. According to another preferable aspect of the present invention, the above method is provided as a method for reducing blood uric acid. Moreover, according to another preferable aspect of the present invention, the above method is provided as a method for treating a disease or condition caused by excessive production of uric acid or decreased excretion. According to still another preferred aspect of the present invention, the above method is provided as a method for treating hyperuricemia. Moreover, according to another preferable aspect of this invention, it excludes medical practice, and this act is also called non-therapeutic act. The said method of this invention can be implemented according to description of the agent of this invention.

The ingestion method of the agent of the present invention is not particularly limited as long as the effect of the present invention is not hindered, but is preferably taken orally.

The subject in the present invention is a mammal, for example, a rodent, a dog, a cat, a cow, a primate, etc., preferably a human.
In addition, the serum uric acid level in the subject is not particularly limited, but it is preferably a high level in view of improvement of hyperuricemia due to a reduction in uric acid level. Such a serum uric acid level is preferably in a state exceeding 7.0 mg / dL.
According to another aspect of the present invention, the subject may be a healthy person.

In addition, the effective amount of tetrapyrrole-containing degradation product or extract thereof or the compound of formula (I) or orally acceptable amount thereof is not particularly limited as long as it does not interfere with the effect of the present invention. It is appropriately determined by those skilled in the art according to sex, age, symptoms, condition and the like. The agent of the present invention contains, as an effective amount, a tetrapyrrole-containing decomposition product or an extract thereof in terms of dry mass, 0.05 to 100 mg / kg body weight / day, preferably 0.5 to 25 mg / kg body weight / day. It can be. In addition, the agent of the present invention contains, for example, an effective amount of the compound of formula (I) or an orally acceptable salt thereof in the range of 0.01 to 15 mg / kg body weight / day, preferably 0. 1 to 5 mg / kg body weight / day.

The intake plan in the present invention can be appropriately set by those skilled in the art according to the species, age, sex, symptom, and state of the subject as long as the effects of the present invention are not hindered. In the present invention, the number of daily intakes is, for example, 1 to 6 times a day, and preferably 3 times a day.

According to another aspect of the present invention, there is provided use of a tetrapyrrole-containing degradation product or an extract thereof, a compound of formula (I), or an orally acceptable salt thereof in the manufacture of a xanthine oxidase inhibitor. The According to still another aspect of the present invention, there is provided use of a tetrapyrrole-containing degradation product or an extract thereof, a compound of formula (I) or an orally acceptable salt thereof in the manufacture of a blood uric acid lowering agent. Provided. According to still another aspect of the present invention, a tetrapyrrole-containing degradation product or an extract thereof or a compound of formula (I) in the manufacture of a therapeutic agent for a disease or condition caused by excessive production of uric acid or decreased excretion Alternatively, the use of an orally acceptable salt thereof is provided. According to still another aspect of the present invention, a tetrapyrrole-containing degradation product or an extract thereof, or a compound of formula (I) or an orally acceptable salt thereof in the manufacture of a therapeutic agent for hyperuricemia. Use is provided.

According to another aspect of the present invention, there is provided use of a tetrapyrrole-containing degradation product or extract thereof, or a compound of formula (I) or an orally acceptable salt thereof as a xanthine oxidase inhibitor. . According to still another aspect of the present invention, there is provided use of a tetrapyrrole-containing degradation product or an extract thereof or a compound of formula (I) or an orally acceptable salt thereof as a blood uric acid lowering agent. Is done. According to still another aspect of the present invention, a tetrapyrrole-containing decomposition product or an extract thereof, a compound of formula (I) or a compound as a therapeutic agent for a disease or condition caused by excessive production or decreased excretion of uric acid Use of the orally acceptable salt is provided. According to still another aspect of the present invention, use of a tetrapyrrole-containing degradation product or an extract thereof or a compound of formula (I) or an orally acceptable salt thereof as a therapeutic agent for hyperuricemia Is provided. According to one preferred alternative embodiment of the invention, the use of the invention is a non-therapeutic use.

According to another aspect of the present invention, the xanthine is characterized in that a tetrapyrrole-containing decomposition product or extract thereof, or a compound of formula (I) or an orally acceptable salt thereof is blended in the agent. A method for producing an oxidase inhibitor is provided. According to still another aspect of the present invention, the product comprising a decomposition product of tetrapyrrole or an extract thereof, or a compound of formula (I) or an orally acceptable salt thereof is characterized by comprising: A method for producing a blood uric acid lowering agent is provided. According to still another aspect of the present invention, the product comprising a decomposition product of tetrapyrrole or an extract thereof, or a compound of formula (I) or an orally acceptable salt thereof is characterized by comprising: A method for producing a therapeutic agent for a disease or condition resulting from excessive production of uric acid or decreased excretion is provided. According to still another aspect of the present invention, the product comprising a decomposition product of tetrapyrrole or an extract thereof, or a compound of formula (I) or an orally acceptable salt thereof is characterized by comprising: A method for producing a therapeutic agent for hyperuricemia is provided.

Also, according to another aspect of the present invention, there is provided a tetrapyrrole-containing degradation product or extract thereof, a compound of formula (I) or an orally acceptable salt thereof for xanthine oxidase inhibition. According to still another aspect of the present invention, there is provided a tetrapyrrole-containing degradation product or extract thereof or a compound of formula (I) or an orally acceptable salt thereof for reducing blood uric acid. The According to yet another aspect of the present invention, for the treatment of a disease or condition resulting from excessive production or decreased excretion of uric acid, or for reducing the risk of a disease or condition resulting from excessive production or decreased excretion of uric acid. , Tetrapyrrole-containing degradation products or extracts thereof, or compounds of formula (I) or orally acceptable salts thereof. According to still another aspect of the present invention, a tetrapyrrole-containing degradation product or an extract thereof, a compound of formula (I) or a compound for the treatment of hyperuricemia or reduction of the risk of hyperuricemia Its orally acceptable salt is provided. Any of these embodiments can be carried out in accordance with the description relating to the agent and method of the present invention.

Any of the above aspects of use and production method can be carried out in accordance with the description of the agent and method of the present invention.

Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples. Unless otherwise indicated, all percentages and ratios used in the present invention are by weight. Unless otherwise indicated, the units and measurement methods described in this specification are based on Japanese Industrial Standards (JIS).

Test Example 1: Red blood cell powder was used as a raw material for preparing a tetrapyrrole-containing product degradation product (porcine erythrocyte enzyme degradation product) . The erythrocyte powder was obtained by centrifuging pig blood to obtain erythrocytes and spray-drying the erythrocytes.
Preparation of the erythrocyte enzymatic degradation product was carried out as follows with reference to the method for producing heme iron complex (Japanese Patent Laid-Open No. 4-013680). First, about 7 times the amount of distilled water is added to red blood cell powder and dissolved, then sodium hydroxide is added as appropriate to adjust the pH to 9.5 ± 0.5, and a microorganism-derived endo-type protease (alkalase) (Novozymes) About 10% of red blood cell powder was added, and the enzyme reaction was carried out at 55 ± 1 ° C. for 1 hour or longer. Furthermore, the enzyme reaction solution was treated with an ultrafiltration membrane having a molecular weight cut-off of 10,000 or less, thereby removing high molecular weight substances and obtaining erythrocyte enzyme degradation products.

The erythrocyte enzymatic degradation product was adjusted to various pH by adding hydrochloric acid, and then the XOD inhibitory activity was measured in vitro.
In vitro XOD inhibitory activity was measured with reference to the method of Masuda et al. (Juniai Women's Junior College Bulletin, No. 41, 2008). Specifically, the inhibitory activity was determined from the amount of change in uric acid produced by adding xanthine as a substrate to xanthine oxidase (XOD).
First, 10 μL of the erythrocyte enzymatic degradation product was added to 240 μL of 0.1 M phosphate buffer (pH 7.4). As a control (no XOD inhibitor), 10 μL of 0.1 M phosphate buffer (pH 7.4) was added. Next, 50 μL of 0.24 U / mL bovine buttermilk-derived XOD (manufactured by Oriental Yeast Co., Ltd.) dissolved in 0.1 M phosphate buffer (pH 7.4) was added and stirred for 1 minute. Thereto was added 300 μL of 0.1 mM xanthine (manufactured by Wako Pure Chemical Industries) dissolved in 0.1 M phosphate buffer (pH 7.4), and a wavelength of 295 nm was immediately used using a spectrophotometer (DU7400, manufactured by Beckman). The absorbance values after addition of the substrate (after 0 minutes) and after 1 minute were measured. All the test solutions used for measurement and use were those at room temperature. ΔABS for 0 → 1 minute was determined from the obtained absorbance value, and the inhibitory activity of the sample was calculated by the following formula. All these reactions were carried out in the cell. The inhibitory activity of 0.7 mg / mL allopurinol (manufactured by Wako Pure Chemical Industries) (dissolved in 0.1 M phosphate buffer (pH 7.4)) in this measurement system was 99% or more.
Formula) A: Sample ΔABS (0-1 min)
B: Control ΔABS (0-1 min)
Inhibitory activity% = 100− {A / B × 100}

The measurement results of the XOD inhibitory activity of the erythrocyte enzyme degradation product of Test Example 1 are shown in Table 1 and FIG. As a result, the XOD inhibitory activity of the erythrocyte enzyme degradation product varied depending on the pH, and the inhibitory activity at pH 10 was 31.5%, but at pH 4, it was 62.4% (FIG. 1, Table 1).

Test Example 2: Preparation of tetrapyrrole-containing degradation product (bovine erythrocyte enzyme degradation product) Bovine blood-derived hemoglobin powder (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of porcine-derived erythrocyte powder in the same manner as in Test Example 1. An erythrocyte enzymatic degradation product was obtained.

Furthermore, after adding hydrochloric acid to the bovine erythrocyte enzymatic degradation product to adjust to pH 4, XOD inhibitory activity was measured in vitro by the same method as in Test Example 1.
As a result, 54.6% XOD inhibitory activity was observed (Table 1).
Therefore, it was speculated that the XOD inhibitory activity of the erythrocyte enzymatic degradation product is not unique to the species.

Test Example 3: Preparation of a decomposition product of tetrapyrrole-containing product (enzyme reaction product of a mixture of hemin and albumin) Although hemoglobin is composed of heme and globin, XOD can also be obtained by enzymatic reaction of a mixture of proteins other than heme and globin. It was confirmed whether it showed inhibitory activity.
Here, hemin was used as hem. After mixing hemin (manufactured by Kodak) and bovine-derived albumin (manufactured by Wako Pure Chemical Industries) at a ratio of about 1:10, protease was added and reacted in the same manner as in Test Example 1, and the mixture of hemin and albumin An enzyme reaction product was obtained.

The enzyme reaction product of the mixture of hemin and albumin was adjusted to pH 4, and then the XOD inhibitory activity was measured in vitro by the same method as in Test Example 1.
As a result, 23.7% XOD inhibitory activity was observed (Table 1).

Test Example 4: Preparation of tetrapyrrole-containing degradation product (reaction product of hemin and various reducing agents) The XOD inhibitor in the erythrocyte enzyme degradation product is a degradation product of heme (hemin) present in hemoglobin and myoglobin from the above examination. The possibility was suggested.
Therefore, cysteine (Cys) (manufactured by Wako Pure Chemical Industries, Ltd.), glutathione (GSH) (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 100 μmol / mL with respect to a hemin (manufactured by Kodak) solution having a final concentration of 10 μmol / mL. Alternatively, a reducing agent such as dithiothreitol (DTT) (manufactured by Wako Pure Chemical Industries, Ltd.) is added, adjusted to pH 10 with a 0.1 M sodium hydroxide (manufactured by Wako Pure Chemical Industries) solution, and reacted at 60 ° C. overnight. The reaction product was obtained.

The reaction product of hemin and various reducing agents was adjusted to pH 4 and then the XOD inhibitory activity was measured in vitro by the same method as in Test Example 1.
As a result, XOD inhibitory activity could be detected in all the reaction products (Table 1). Moreover, the peak which has XOD inhibitory activity on the reverse phase chromatography of these reaction products overlapped with the peak which has XOD inhibitory activity derived from the erythrocyte enzyme degradation product. Therefore, it was suggested that the substance showing XOD inhibitory activity in the reaction product is the same substance as the XOD inhibitor derived from the erythrocyte enzyme degradation product.

Test Example 5: Preparation of decomposition product of tetrapyrrole-containing product (reaction product of tetrapyrrole compound and cysteine) In addition to iron-free protoporphyrin (manufactured by Tokyo Kasei Kogyo) and heme (hemin), a different tetrapyrrole compound Cysteine was allowed to act on certain hematoporphyrin (manufactured by Sigma-Aldrich) and bilirubin (manufactured by Tokyo Kasei Kogyo Co., Ltd.), a heme degradation intermediate, to obtain a reaction product. Specifically, cysteine (Cys) is added to a final concentration of 10 μmol / mL protoporphyrin solution, final concentration of 10 μmol / mL hematoporphyrin or final concentration of 10 μmol / mL bilirubin to a final concentration of 100 μmol / mL. The pH was adjusted to 10 with 1 M sodium hydroxide solution and reacted at 60 ° C. overnight to obtain a reaction product.
Furthermore, iron (II) chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the reaction product of protoporphyrin or bilirubin and cysteine so as to have a final concentration of 10 μmol / mL, adjusted to pH 10, and then reacted at 60 ° C. overnight. Then, an iron (II) chloride addition reaction product was obtained.

The reaction product of the tetrapyrrole compound and cysteine was adjusted to pH 4 by adding hydrochloric acid, and then the XOD inhibitory activity was measured in vitro in the same manner as in Test Example 1.
As a result, XOD inhibitory activity could be detected in all the reaction products (Table 1).
In addition, with respect to the reaction product added with iron (II) chloride, after adjusting the pH to 4 by adding hydrochloric acid, the XOD inhibitory activity was measured in vitro in the same manner as in Test Example 1.
As a result, the reaction product added with iron chloride (II) was able to further increase the XOD inhibitory activity by about 10% as compared with the reaction product not added with iron chloride (II) (Table 1). This suggests that the presence of iron may increase the production rate of XOD inhibitors.

Test Example 6: Preparation of decomposition product of tetrapyrrole-containing product (reaction product of hemin and hydrogen peroxide) Reaction product of hemin and hydrogen peroxide instead of the reaction product of hemin and reducing agent (hereinafter referred to as hemin-peroxidation) It was investigated whether XOD inhibitory activity could be detected in the hydrogen reactant).
The reaction method of hemin and hydrogen peroxide was referred to the method of Iwata (Oka University Medical Bulletin, 4: 31-36, 1993). Specifically, first, 1/40 amount of 28% ammonia (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a hemin 10 mg / mL solution to dissolve hemin. While heating the solution at 60 ° C., a final concentration of 15% hydrogen peroxide solution (manufactured by Wako Pure Chemical Industries, Ltd.) that is half the amount of hemin solution (containing 1/33 amount of 28% ammonia) was added little by little to the solution. And reacted at 60 ° C. for 1 hour. After the reaction, the reaction product was cooled to room temperature.

The hemin-hydrogen peroxide reactant was adjusted to pH 4 by adding hydrochloric acid, and then the XOD inhibitory activity was measured in vitro by the same method as in Test Example 1.
As a result, the XOD inhibitory activity of the reaction product was 87.5% (Table 1).

Test Example 7: Preparation of tetrapyrrole-containing decomposition product (reaction product of chlorophyllin and hydrogen peroxide) between chlorophyllin, which is a tetrapyrrole compound involved in photosynthesis organisms such as plants, phytoplankton and algae, and hydrogen peroxide It was examined whether the reaction product could detect XOD inhibitory activity.
It was prepared by the same preparation method as the reaction product of hemin and hydrogen peroxide described in Test Example 6 except that iron chlorophyllin Na (iron chlorophyll) (manufactured by Japanese chlorophyll) was used instead of hemin.

The reaction product of chlorophyllin and hydrogen peroxide was adjusted to pH 4 by adding hydrochloric acid, and then the XOD inhibitory activity was measured in vitro in the same manner as in Test Example 1.
As a result, the XOD inhibitory activity of the reaction product was 24.2% (Table 1).

Test Example 8: Isolation of XOD inhibitor from tetrapyrrole-containing product (red blood cell enzyme degradation product or hemin-hydrogen peroxide reactant) XOD inhibitory activity was confirmed in the red blood cell enzyme degradation product. Was used to isolate an XOD inhibitor.
First, the erythrocyte enzymatic degradation product obtained in Test Example 1 was adjusted to pH 4, and 5-fold amount of 2-propanol was added to extract an XOD inhibitor. This 2-propanol-extracted fraction was applied to an ODS-A column (manufactured by YMC) with a reverse phase resin and was eluted with a gradient using an acetonitrile-trifluoroacetic acid (TFA) system (FIG. 2). As a result, as shown in FIG. 2, the peak fraction indicated by the arrow detected at 313 nm had a clear XOD inhibitory activity. In the other peaks, almost no XOD inhibitory activity was detected. Further, the peak fraction having the inhibitory activity was subjected to reverse phase ODS-120T (manufactured by Tosoh Corporation) and eluted with a gradient using methanol-TFA system (FIG. 3). As a result, three peaks were obtained at a detection wavelength of 313 nm. As a result of measuring the XOD inhibitory activity with respect to these peaks, the first and second peak fractions showed XOD inhibitory activity. The peak fraction in which the XOD inhibitory activity was confirmed was collected and used as a solution in which the absorbance value at 313 nm was adjusted to about 1. In the above solution, the peak showing 63% XOD inhibitory activity was taken as the P1 peak, and the peak showing 48% XOD inhibitory activity was taken as the P2 peak (FIG. 3). Fractions were collected for the P1 peak and repurified with ODS-120T to obtain XOD inhibitor P1 purified from the erythrocyte enzyme degradation product (hereinafter referred to as P1 derived from erythrocyte enzyme degradation product). Further, by the same isolation method, a substance equivalent to P1 and P2 derived from the erythrocyte enzyme degradation product was obtained from the hemin-hydrogen peroxide reactant of Test Example 6 (hereinafter referred to as hemin-hydrogen peroxide). Reactant-derived P1 and P2). The absorption spectra of hemin-hydrogen peroxide reactant-derived P1 and P2 were measured with a spectrophotometer (DU7400, manufactured by Beckman). As a result, the maximum absorption wavelengths were around 308 nm and 322 nm, respectively, and the spectra were similar (FIG. 6). -1).
The obtained hemin-hydrogen peroxide reactant-derived P1 was lyophilized and then dissolved in distilled water so that the P1 concentration was 0.7 mg / mL, and XOD was inhibited in vitro in the same manner as in Test Example 1. Activity was measured.
As a result, the 50% inhibitory concentration (IC ₅₀ value) of P1 derived from the hemin-hydrogen peroxide reactant was 2.45 μg / mL. (Fig. 4)
As described above, the XOD inhibitor can be separated using reverse phase chromatography, but can also be separated and purified using an ion exchange resin after reverse phase chromatography. P1 obtained after ODS-120T reverse phase chromatography was adsorbed on an anion exchange resin GigaCap Q-650M (manufactured by Tosoh Corporation) under the condition of 50 mM Tris-HCl pH9, and P1 was eluted with a sodium chloride gradient for fractionation. (Fig. 5).

Test Example 9: Molecular weight / structure analysis of XOD inhibitor derived from tetrapyrrole-containing product (red blood cell enzyme degradation product or hemin-hydrogen peroxide reaction product) The molecular weight and structure of the origin P1 and the hemin-hydrogen peroxide reactant-derived P1 and P2 were predicted by LC / MS (Synapt G2-S type, manufactured by Waters).
As a result, the molecular weights of P1 and P2 can be estimated, and the hemin-hydrogen peroxide reactant-derived P1 and the erythrocyte enzyme degradation product-derived P1 are eluted by the reverse phase ODS-120T (manufactured by Tosoh) shown in Test Example 8. In addition to the coincidence of time, it was confirmed that the signals of the molecular ion m / z 224 of P1 and other fragment ions almost coincided by LC / MS analysis (negative mode). From these, it was confirmed that P1 derived from erythrocyte enzymatic degradation product and P1 derived from hemin-hydrogen peroxide reactant are the same substance (FIG. 6-2).
Further, P1 derived from the hemin-hydrogen peroxide reactant was subjected to structural analysis by ¹ H-NMR, ¹³ C-NMR and differential NOE measurement (AVANCE 500 type, manufactured by BRUKER). The solvent used in ¹ H-NMR and ¹³ C-NMR was deuterated methanol. From the results of LC / MS (negative mode) and NMR, it was found that P1 has a molecular weight of 225 (FIG. 6-2) and has a structure having a methyl group, an aldehyde group, a carboxyl group, and propionic acid in the pyrrole ring. From the structure, it was confirmed that it was composed of a part of the porphyrin ring structure (FIG. 6-4). Since P2 is derived from the same hemin-hydrogen peroxide reactant as P1, and the absorption spectrum is similar, P2 was predicted to be an isomer similar to P1.
Therefore, the molecular weight and structure were estimated from the LC / MS data of P2. First, the molecular weight of P2 was estimated to be 209 from the LC / MS (negative mode) results (FIG. 6-2). From the LC / MS (positive mode) results, P2 was fragmented during ionization (a) to (f), and the molecular weight of each fragment ion coincided with the molecular weight of the predicted structure when fragmented. (Fig. 6-3). From these results, the P2 structure was estimated. The P1 and P2 structures are shown below.

As is clear from the above chemical formula, XOD inhibitors P1 and P2 do not have a purine skeleton. In contrast, allopurinol, a therapeutic drug for hyperuricemia, has a purine skeleton, which may cause side effects (such as nephrotoxicity) involved in purine metabolism. From this point of view, it is considered that the XOD inhibitor P1 and / or P2 is very useful in the reduction of blood uric acid and the treatment of hyperuricemia.

Test Example 10: Method for producing XOD inhibitor from tetrapyrrole-containing product degradation product (erythrocyte enzyme degradation product)
Test Example 10-1: Purification method of activated carbon for XOD inhibitor XOD inhibitor is efficiently purified from the erythrocyte enzyme degradation product using activated carbon for food production or pharmaceutical production used for decolorization or deodorization. It was possible to produce a raw material for the agent of the present invention in which the content of the inhibitory substance was increased.
First, about 1.2%, 0.6% or 0.48% amount of activated carbon was added to the erythrocyte enzyme degradation product of Test Example 1 adjusted to pH 4, and the XOD inhibitor was added. After adsorbing, the activated carbon was recovered. The recovered activated carbon is washed with an equal amount of water to the erythrocyte enzyme degradation product and then eluted with an alkaline buffer of pH 9 or pH 10.5 or 0.07M sodium hydroxide solution to effectively purify the XOD inhibitor. The concentration of P1 could be increased. The activated carbon used in the study was activated carbon 1 (Shirakaba P), activated carbon 2 (Shirakaba A) (above, most frequently around pore size 2.2 nm, wood-derived / steam activated carbon), activated carbon 3 (FP-3 (manufacturer)) (Most frequent pore diameter near 1.7 nm, derived from coconut shell / steam activated charcoal), activated carbon 4 (FP-6 (manufacturer temporary name)) (most frequent pore diameter near 3.3 nm, derived from wood / zinc chloride activated charcoal) , Nippon Enviro Chemicals Co., Ltd.) and activated carbon 5 (Dia Hope 6ED (derived from wood / zinc chloride activated charcoal) (Calgon Carbon Japan Co., Ltd.)). As a result of the examination, activated carbon 4 had the highest adsorption ability of the XOD inhibitor among them, and then activated carbon 5, and activated carbon 1 and activated carbon 2 had less dye elution. Among these activated carbons, activated carbon 1 can increase the specific activity of XOD inhibition 62.8 times by eluting the XOD inhibitor with 50 mM Tris-HCl buffer pH 9.0, which is equivalent to the erythrocyte enzyme degradation product. did it. Regarding activated carbon 4 and activated carbon 2, when eluted with 1/10 amount of 100 mM sodium carbonate buffer pH 10.5 of the erythrocyte enzyme degradation product, activated carbon 2 increased the specific activity of XOD inhibition by 67.7 times, and activated carbon 4 In the case of activated carbon 4 using 70 mM sodium hydroxide, the specific activity of inhibition could be increased about 110 times (Table 2). In Table 2, the inhibitory activity yield indicates the ratio of the XOD inhibition rate after purification, assuming that the XOD inhibition rate of the erythrocyte enzyme degradation product before purification is 100%. The purified product by activated carbon 4 was darker than the purified product by activated carbon 2, but the specific activity of XOD inhibition was high. This activated carbon purification method from erythrocyte enzymatic degradation products requires very little activated carbon to be used at 1.25% or less of the reaction volume and requires no solvent for elution, so it is very low cost and has low environmental impact. It was considered a manufacturing method. The liquid obtained by this purification method using activated carbon can be dried, powdered and solubilized, and can be used as a raw material for oral preparations.

In addition, it verified whether the XOD inhibitory activity in the refined | purified material by the said activated carbon originates in the XOD inhibitory substance in an erythrocyte enzyme degradation product. For the verification, the product derived from the erythrocyte enzyme degradation product P1 obtained in Test Example 8 and the purified product of the erythrocyte enzyme degradation product by activated carbon (activated carbon 1) were used. These samples were subjected to reverse phase chromatography alone or as a mixture to confirm peak overlap. As a result, P1 derived from the erythrocyte enzyme degradation product and one of the peaks in the purified product of the erythrocyte enzyme degradation product by activated carbon overlapped, and an increase in area value was observed (FIG. 7). Therefore, it was confirmed that the XOD inhibitory activity in the purified product of the erythrocyte enzymatic degradation product by activated carbon is derived from P1.

Test Example 10-2: Extraction Method of XOD Inhibitory Substance with Hot Ethanol By efficiently extracting XOD inhibitory substance from dry product of erythrocyte enzyme degradation product pH 4 with hot ethanol, its concentration could be increased.
First, after adding 2 to 5 times the amount of 85 to 90% ethanol to the dried product of the erythrocyte enzyme degradation product of Test Example 1, heating to room temperature or 65 ° C and returning this solution to room temperature is unnecessary. Solids were precipitated. The solid content was removed by centrifugation to obtain a hot ethanol extract.
The hot ethanol extract was dried to remove ethanol, and then the XOD inhibitory activity was measured. When extracted with 90% hot ethanol twice the amount of the erythrocyte enzymatic degradation product, the salt concentration was reduced to about 36%, and the XOD inhibition specific activity could be increased nearly 3 times (Table 3). In Table 3, the remaining amount of salt indicates the ratio of the salinity concentration in the solid content after extraction when the salinity concentration in the solid content before ethanol extraction is defined as 100%. The salt concentration was measured with a salinometer (LAQUATwin B-721, manufactured by Horiba, Ltd.) after dissolving the dried hot ethanol extract in water. With hot ethanol extraction, pigment and odor could be reduced at the same time. This hot ethanol extract can be powdered and solubilized, and can be used as a raw material for oral preparations.

Test Example 10-3: Purification method using XNF inhibitor NF filter membrane XOD inhibitors P1 and P2 are substances having molecular weights of 225 and 209, respectively. Therefore, it was possible to increase the concentration of the XOD inhibitor by efficiently purifying the XOD inhibitor by using a filtration membrane having an appropriate fractional molecular weight.
As the filtration membrane, four types of NF membrane (nanofilter) (Muromachi Chemical) were used. The NF membrane used was NF membrane 1 (NFX (fraction molecular weight 150-300)), NF membrane 2 (NFW (fraction molecular weight 300-500)), NF membrane 3 (NFG (fraction molecular weight 600-800)) NF membrane 4 (NF270 (fraction molecular weight 200-400)). Using the NF membrane, the porcine erythrocyte enzymatic degradation product of Test Example 1 was adjusted to pH 4 and permeated.
As a result, the XOD inhibition specific activity can be increased in any of the membranes, in particular, the solid content can be reduced to about 1% in the NF membrane 1, and the XOD inhibition specific activity can be increased by 58.7 times ( Table 4). Moreover, it was possible to significantly reduce the pigment and odor when using any film.
The liquid obtained by this NF filtration membrane method can be powdered and solubilized, and can be used as a raw material for oral preparations.

Test Example 10-4: Purification Method of XOD Inhibitory Substance with Synthetic Adsorption Resin The concentration of XOD inhibitor could be increased by efficiently purifying the XOD inhibitory substance from the erythrocyte enzyme degradation product using the synthetic adsorption resin.
As the synthetic adsorption resin, synthetic adsorption resin 1 (diaion HP20 (styrene-divinylbenzene system, most frequent pore diameter 58 nm)), synthetic adsorption resin 2 (HP20SS (styrene-divinylbenzene system, most frequent pore diameter 58 nm)), Synthetic adsorption resin 3 (SP850 (styrene-divinylbenzene system, most frequent pore diameter 9 nm)) and synthetic adsorption resin 4 (HP2MGL (aliphatic ester system, most frequent pore diameter 48 nm)) (manufactured by Mitsubishi Chemical) were used. First, 1/10 amount of resin of the erythrocyte enzyme degradation product of Test Example 1 was added to the erythrocyte enzyme degradation product of Test Example 1 adjusted to pH 4 to adsorb the XOD inhibitor. After this adsorption, the resin was washed with water at least 10 times the resin volume, and then the XOD inhibitor was eluted with a 40% ethanol solution containing 0.01 M hydrochloric acid 10 times the resin volume. The eluate was concentrated and dried to remove ethanol and then redissolved to measure the inhibitory activity. As a result, the XOD inhibition specific activity could be increased 9 to 14 times (Table 5).
Moreover, the XOD inhibitory substance could be eluted even using a buffer solution having a pH of 9 or higher. When purified using a synthetic adsorption resin in the same manner as above except that the 40% ethanol solution was changed to Tris-HCl buffer pH 9 for elution, the XOD inhibitory specific activity could be increased 13.6 times (Table 1). 5).
The purification method using synthetic adsorption resin was able to significantly reduce salt, pigment and odor.
The liquid purified by the synthetic adsorption resin can be powdered and solubilized and can be used as a raw material for oral preparations.

Test Example 11: Production Method of XOD Inhibitory Substance by Organic Synthesis The XOD inhibitory substance was not only obtained from the reaction product of biological organic matter such as the above-mentioned erythrocyte enzyme degradation product or heme, but also could be synthesized organically.
XOD inhibitor P1 was synthesized by the following synthesis schemes (A) to (F).
(A) First, carbonyldiimidazole (CDI, 9.7 g) was added to a tetrahydrofuran solution of 7.3 g of monoethylsuccinic acid (Compound 1), stirred for 1 hour, magnesium chloride (4.8 g), potassium monoethylmalonate (8.5 g) was added and reacted at 60 ° C. for 1 hour to obtain diethyl 3-oxohexanedionate (Compound 2). (B) After adding an aqueous solution (70 mL) of sodium nitrite (17.46 g) to a mixture of Compound 2 (52.25 g) and acetic acid (100 mL), tertiary butyl acetoacetate (45.37 g) and acetic acid (100 mL) ), Zinc dust (50 g) and ammonium acetate (50 g) were added and reacted at 50 to 70 ° C. for 30 minutes to give 4-tertiarybutyl 2-ethyl 3- (2- (ethoxycarbonyl) ethyl) -5- Methyl-1H-pyrrole-2,4-dicarboxylate (Compound 3) was obtained. (C) Compound 3 (10 g) was added to N-methylpyrrolidone (70 mL), 4-toluenesulfonic acid (p-TSA, 10 g) was added, and the mixture was reacted at 160 ° C. for 3 hours to give ethyl 3- (2- ( Ethoxycarbonyl) ethyl) -5-methyl-1H-pyrrole-2-carboxylate (compound 4) was obtained. (D) Paraformaldehyde (1.2 g) was added to a solution of hydroiodic acid (50 mL), acetic anhydride (50 mL) and hypophosphorous acid (10 mL) to compound 4 (5.06 g) and reacted for 25 minutes. 3- (2- (Ethoxycarbonyl) ethyl) -4,5-dimethyl-1H-pyrrole-2-carboxylate (Compound 5) was obtained. (E) A solution of compound 5 (15 g) in tetrahydrofuran (536 mL), acetic acid (135 mL), and water (536 mL) was added cerium (IV) ammonium nitrate (CAN, 120 g), reacted at room temperature for 2 hours, and ethyl 3- (2- (Ethoxycarbonyl) ethyl) -5-formyl-4-methyl-1H-pyrrole-2-carboxylate (Compound 6) was obtained. (F) Lithium hydroxide monohydrate (1.78 g) was added to a solution of compound 6 (3 g) in THF-H ₂ O (5: 1) (115 mL) and reacted at 70 ° C. for 4 hours in the presence of N 2. A synthetic XOD inhibitor P1 3- (2-carboxyethyl) -5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid was obtained.

Synthesis scheme of XOD inhibitor P1

The structure of P1 obtained by organic synthesis (hereinafter also referred to as synthesis P1) was confirmed by LC / MS 1200A (manufactured by Agilent Technologies) and ¹ H-NMR Mercury Plus 400 MHz (manufactured by Varian). In LC / MS analysis (negative mode), a signal of molecular ion m / z 224 of P1 was detected (FIG. 8), and a proton signal consistent with the P1 structure was obtained in ¹ H-NMR (FIG. 9). The solvent used in ¹ H-NMR was deuterated DMSO. From the above, it was confirmed that the synthesized P1 had the same structure as the hemin-hydrogen peroxide-derived P1.

Test Example 12: Measurement of inhibitory activity of XOD inhibitor obtained by organic synthesis When XOD inhibitory activity of synthetic P1 (purity 95% or more) obtained in Test Example 11 was measured, the inhibitory activity curve was hemin-peroxidation. The inhibitory activity behavior similar to that of hydrogen-derived P1 was shown (FIG. 4).
The IC ₅₀ value of synthetic P1 was 1.56 μg / mL, which was about 27-fold inhibitory activity compared to the IC ₅₀ value of allopurinol of 42.8 μg / mL (Table 6).

Test Example 13: In vivo evaluation of XOD inhibitor obtained by organic synthesis In vivo evaluation of synthetic P1 in a hyperuricemia model rat was performed.
Specifically, the effect of reducing blood uric acid was tested using hyperuricemia model rats in which blood uric acid levels were increased by intraperitoneal administration of potassium oxonate to rats. In the test, potassium oxonate was intraperitoneally administered to 5-week-old male rats three times 1 hour before the start of the test, 1 hour after the start, and 3 hours after the start so that the weight of the rat was 250 mg / kg of the body weight of the rat ( Potassium oxonate group). One hour after the first administration of potassium oxonate, the sample (synthetic P1) was orally administered at a dose of 10 mg / kg based on the body weight of the rat (potassium oxonate + synthetic P1 group). Blood was collected from the tail vein at 0, 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after administration of synthetic P1. Serum uric acid levels were measured using Uric Acid C Test Wako (manufactured by Wako Pure Chemical Industries). The results of potassium oxonate + synthetic P1 group are shown in FIGS. Data are mean ± standard error values.
As a result, accumulation of uric acid in the blood was observed in the potassium oxonate group compared to the untreated control group, but the serum uric acid level significantly decreased 0.5 hours after administration in the potassium oxonate + synthetic P1 group. However, there was almost no accumulation of uric acid until 4 hours after administration, and the effect was maintained until 8 hours after administration (FIG. 10). Regarding the serum uric acid level at each measurement time, the potassium oxonate + synthetic P1 group was significantly compared with the potassium oxonate group at 0.5 to 8 hours after administration (1, 4, 6, 8 hours after administration: P <0 .01, 0.5, 2, 3 hours after administration: P <0.05) was observed to suppress the accumulation of uric acid in the blood (FIG. 10). AUC (area under the curve) was compared for serum uric acid levels up to 6 hours after administration of synthetic P1 in this study (FIG. 11). In the potassium oxonate group, the accumulation of uric acid can be confirmed significantly ( P <0.01) compared to the control group, but the potassium oxonate + synthetic P1 group significantly suppresses the accumulation of uric acid ( P <0.01). (FIG. 11).
From these results, it was confirmed that the XOD inhibitor P1 is highly effective in vivo even through the digestive tract by oral administration.

Claims

A xanthine oxidase inhibitor comprising a compound represented by formula (I) or an orally acceptable salt thereof:

(Wherein R is OH or H).
The xanthine oxidase inhibitor according to claim 1, which contains a tetrapyrrole-containing decomposition product or an extract thereof.
The agent according to claim 1 or 2, which is in the form of a single oral intake unit.
The agent according to any one of claims 1 to 3, which is a blood uric acid lowering agent.
The agent according to any one of claims 1 to 4, which is a therapeutic agent for a disease or condition caused by excessive production of uric acid or decreased excretion.
The agent according to any one of claims 1 to 5, which is a therapeutic agent for hyperuricemia.
The agent according to any one of claims 1 to 6, which is a composition.
The agent according to any one of claims 1 to 7, which is a food or drink or a medicine.
Compound represented by formula (Ia) or orally acceptable salt thereof:
A method for producing a xanthine oxidase inhibitor, comprising blending a tetrapyrrole-containing product decomposition product or an extract thereof into the agent.
The method according to claim 10, wherein the tetrapyrrole-containing material is at least one selected from the group consisting of erythrocytes, hemoglobin, hemin, protoporphyrin, hematoporphyrin, bilirubin and chlorophyllin.
The method according to claim 10 or 11, wherein the decomposition of the tetrapyrrole-containing material is performed by at least one reaction selected from the group consisting of an oxidation reaction, a reduction reaction and an enzyme reaction.
The method according to claim 10, wherein the xanthine oxidase inhibitor comprises a compound represented by formula (I) or an orally acceptable salt thereof.

(Wherein R is OH or H).
Use of tetrapyrrole-containing decomposition products or extracts thereof in the production of xanthine oxidase inhibitors.
15. Use according to claim 14, wherein the xanthine oxidase inhibitor comprises a compound of formula (I) or an orally acceptable salt thereof.

(Wherein R is OH or H).
The use according to claim 14 or 15, wherein the xanthine oxidase inhibitor is a therapeutic agent for a disease or condition caused by excessive production of uric acid or decreased excretion.
Treatment of a disease or condition resulting from overproduction or decreased excretion of uric acid, comprising ingesting an effective amount of a compound represented by formula (I) or an orally acceptable salt thereof to a subject in need thereof Method:

(Wherein R is OH or H).
A compound of formula (I) or an orally acceptable salt thereof for the inhibition of xanthine oxidase or for the treatment of diseases or conditions resulting from excessive production or decreased excretion of uric acid:

(Wherein R is OH or H).