JP2005126361A - Matrix metalloprotease inhibitor containing polylactic acid mixture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new matrix metalloprotease inhibitor by evaluating inhibitory action on matrix metalloprotease exhibited by a cyclic and/or a chain polylactic acid mixture. <P>SOLUTION: The matrix metalloprotease inhibitor comprises a cyclic and/or a chain polylactic acid mixture having 3-20 condensation degree. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a matrix metalloprotease inhibitor, and more particularly to a matrix metalloprotease inhibitor comprising a cyclic and / or chain polylactic acid mixture. The matrix metalloproteinase inhibitor of the present invention is useful for the treatment and / or prevention of diseases associated with excessive production of matrix metalloprotease.

  Matrix metalloproteinases (hereinafter sometimes abbreviated as MMP) are proteins that exist in extracellular matrix (eg, joint lining, interstitial connective tissue, basement membrane or cartilage). A group of proteolytic enzymes related to the degradation and remodeling of certain collagens, laminins, elastins, fibronectins, or proteoglycans).

  Due to differences in primary structure and substrate specificity of MMP, collagenase group (MMP-1, MMP-8 and MMP-13), gelatinase group (MMP-2 and MMP-9), stromelysin group (MMP-3 and MMP-10), membrane-bound matrix metalloprotease group (MMP-14, MMP-15, MMP-16 and MMP-17), others (MMP-7, MMP-11, MMP-12) Has been. In normal tissues, the activity of MMP is (1) production of latent enzyme (pro-MMP), (2) activation of the latent enzyme, and (3) tissue inhibitors (Tissue Inhibitor of Metalloproteinases; TIMPs). Is controlled by the control of the activating enzyme by, so that the degradation of connective tissue by MMP and the synthesis of new matrix tissue are balanced.

  However, under pathological conditions in which MMP activity increases, TIMPs present in the body become uncontrollable, and the degradation of extracellular matrix increases, causing arteriosclerosis, arthritis (eg, rheumatoid arthritis and osteoarthritis), teeth Peripheral disease, ectopic angiogenesis, neoplastic invasion, neoplastic metastasis, tissue ulceration (eg corneal ulcer, gastric ulcer, epidermal ulcer), bone disease (eg osteoporosis, after joint replacement) Bone resorbable diseases such as loosening), vascular reocclusion, vascular restenosis, HIV infection and one of the main causes of delaying healing of intractable diseases such as diabetic complications. Therefore, MMP inhibitors are considered useful as preventive and therapeutic agents for these intractable diseases.

  Thus, controlling the increase in MMP activity under pathological conditions with an MMP inhibitor is useful for the treatment of various diseases, and various MMP inhibitors have been reported. Examples of the MMP inhibitor include marimastat (3R- (2,2-dimethyl-1S-methylcarbamoyl-propylcarbamoyl) -2S-hydroxy-5-methyl-hexano-hydroxamic acid) having a hydroxamic acid skeleton, flavones In addition, anthocyanidins, esculetin derivatives, sulfonylamino acid derivatives, and the like are known, but from the viewpoint of activity, absorption in the body, toxicity, etc., development of new MMPs inhibitors is desired.

  Previous studies have reported that cyclic and / or chain poly L-lactic acid mixtures having a condensation degree of 3 to 20 are useful as antineoplastic agents (Japanese Patent Application Laid-Open No. 9-227388 and special patents). (Kaihei 10-130153). However, the effect of cyclic and / or chain polylactic acid mixtures having a condensation degree of 3 to 20 on matrix metalloprotease production has not been reported.

  An object of the present invention is to provide a novel matrix metalloprotease inhibitor by evaluating the matrix metalloprotease inhibitory action exhibited by a cyclic and / or chain polylactic acid mixture. This invention also made it the subject which should be solved to provide the food-drinks using the said matrix metalloprotease inhibitor.

  As a result of intensive studies to solve the above problems, the present inventors have evaluated the matrix metalloprotease inhibitory action using polylactic acid mixture produced by polymerizing these using lactic acid or lactide as raw materials. It was demonstrated that a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 exhibits a matrix metalloprotease inhibitory action. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a matrix metalloprotease inhibitor comprising a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20.
Preferably, the matrix metalloprotease is matrix metalloprotease-3 (MMP-3).
Preferably, lactic acid which is a repeating unit in polylactic acid substantially consists of L-lactic acid.
Preferably, the matrix metalloprotease inhibitor of the present invention can be used for the treatment or prevention of diseases associated with excessive production of matrix metalloprotease.

  According to another aspect of the present invention, there is provided a food or drink containing the matrix metalloprotease inhibitor of the present invention described above.

  According to yet another aspect of the present invention, there is provided the use of a cyclic and / or chain polylactic acid mixture having a degree of condensation of 3-20 in the manufacture of a matrix metalloprotease inhibitor.

  According to still another aspect of the present invention, for inhibiting matrix metalloprotease comprising administering an effective amount of a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 to a mammal such as a human. A method is provided.

  According to the present invention, it has become possible to provide a novel matrix metalloprotease inhibitor and a food and drink using the same. In addition, since the polylactic acid mixture used as an active ingredient in the present invention is a low-condensate of lactic acid derived from biological components, it has high biocompatibility and few side effects.

Hereinafter, the implementation method and embodiment of this invention are demonstrated in detail.
The matrix metalloprotease inhibitor of the present invention contains a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 as an active ingredient, for example, treatment of diseases associated with excessive production of matrix metalloprotease or Can be used for prevention.

  The matrix metalloprotease inhibition referred to in this specification includes all cases where the activity of matrix metalloprotease in vivo is inhibited, but preferably means that the expression level of matrix metalloprotease is inhibited. In addition, diseases associated with excessive production of matrix metalloprotease include both diseases caused by excessive production of matrix metalloprotease and diseases associated with excessive production of matrix metalloprotease. Specific examples of diseases associated with excessive production of matrix metalloproteinases include arthritic diseases (eg, rheumatoid arthritis, osteoarthritis, osteoarthritis), bone resorption diseases (eg, osteoporosis), and collagen associated with diabetes. Increased disintegration, arteriosclerosis, aneurysm, cirrhosis, pulmonary fibrosis, periodontal disease, tissue ulceration (eg, corneal ulcer, gastric ulcer, epidermal ulcer), tumor invasion / metastasis, ectopic angiogenesis, Examples include skin aging, vascular re-occlusion, vascular restenosis, HIV infection, and diabetic complications.

In the matrix metalloprotease inhibitor of the present invention, a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 is used as an active ingredient.
The term “polylactic acid mixture” as used herein means a mixture in which cyclic and / or chain polylactic acid having a condensation degree of 3 to 20 is present in an arbitrary ratio. That is, the term “mixture” means a mixture of polylactic acids having any degree of condensation of 3 to 20, and at the same time, is used as a concept including a mixture of cyclic and chain polylactic acids. Such a “polylactic acid mixture” can be obtained by dehydrating and condensing lactic acid and purifying it by an appropriate method, as described hereinbelow. In the present specification, the term “polylactic acid mixture” is used for the sake of convenience. Among these, a single component such as cyclic polylactic acid having a certain degree of condensation or chain polylactic acid having a certain degree of condensation is used. Also included is polylactic acid.

  The degree of condensation means the number of lactic acid units that are repeating units in polylactic acid. For example, cyclic polylactic acid is presumed to have the following structural formula, where n represents the degree of condensation (ie, n = 3 to 20).

  When simply referred to herein as “lactic acid”, this lactic acid includes all of L-lactic acid, D-lactic acid or mixtures of these in any proportion. Preferably in the present invention, the lactic acid consists essentially of L-lactic acid. Here, “substantially” means that the ratio of L-lactic acid units in the polylactic acid mixture [ie, (L-lactic acid unit number / L-lactic acid unit number + D-lactic acid unit number) × 100] is 70, for example. % Or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and particularly preferably 95% or more. The ratio of L-lactic acid units in the polylactic acid mixture depends on the ratio of L-lactic acid and D-lactic acid present in lactic acid used as a starting material.

The method for producing a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 is not particularly limited. For example, JP-A-9-227388, JP-A-10-130153, It can be obtained by the production method described in Japanese Patent Application No. 11-39894 (all the contents described in these patent specifications are included as disclosure of the present specification by reference).
More specifically, for example, a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 can be obtained by the following method A.

Method A:
First, lactic acid (preferably lactic acid substantially consisting of L-lactic acid) is subjected to dehydration condensation in an inert atmosphere. Examples of the inert atmosphere include nitrogen gas and argon gas, and it is preferable to use nitrogen gas.

  The dehydration condensation reaction is carried out under a reduced pressure of about normal pressure to about 1 mmHg and at a temperature of 110 to 210 ° C., preferably 130 to 190 ° C., but it is particularly preferable to carry out by a stepwise reduced pressure and a stepwise temperature increase. Although reaction time can be set suitably, for example, reaction can be performed for 1 to 20 hours. When stepwise pressure reduction and stepwise temperature rise are used, the reaction time is divided into partial reaction times consisting of two or more, and the reaction is carried out by setting the pressure and temperature in each part. When using stepwise pressure reduction, for example, normal pressure → 150 mmHg → 3 mmHg can be reduced, and when using stepwise temperature increase, for example, 145 ° C. → 155 ° C. → 185 ° C. can be raised. In practice, for example, a combination of these can be performed at 145 ° C. for 3 hours at normal pressure, 145 ° C. for 3 hours at 150 mm Hg for 3 hours, 155 ° C. for 3 hours at 3 mm Hg, and 185 ° C. for 3 hours at 3 mm Hg for 1.5 hours. it can.

  Subsequently, ethanol and methanol are added to the reaction mixture obtained by this dehydration condensation reaction, and the filtrate is dried by filtration to obtain an ethanol- and methanol-soluble component. That is, the term “ethanol and methanol soluble fraction” as used herein means a fraction that is soluble in a mixed solution of ethanol and methanol. In addition, when obtaining ethanol and methanol-soluble components, the reaction mixture of the dehydration condensation reaction is mixed with ethanol and methanol, and the ratio of ethanol and methanol at that time can be appropriately set. For example, ethanol: methanol = 1: 9. The order and method of adding ethanol and methanol to the reaction mixture are not limited and can be appropriately selected. For example, ethanol can be added first to the reaction mixture of the dehydration condensation reaction, and then methanol can be added. .

The ethanol / methanol-soluble matter obtained above is subjected to reverse phase column chromatography, particularly chromatography using an octadecylsilane (ODS) column, and first eluted with 25 to 50% by weight acetonitrile aqueous solution at pH 2 to 3. When the fraction is removed, and then the fraction eluted with 90% by weight or higher acetonitrile aqueous solution of pH 2-3, preferably 99% by weight or higher acetonitrile aqueous solution is collected, cyclic and / or chain having a condensation degree of 3 to 20 A polylactic acid mixture is obtained.
The cyclic and / or chain polylactic acid mixture obtained as described above is neutralized with an alkali substance such as sodium hydroxide, dried under reduced pressure, and then formulated into a desired form as described below by a conventional method. can do.

  As another method for producing a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 used in the present invention, for example, the method described in Japanese Patent Application No. 11-265715 (Method B and Or the method described in Japanese Patent Application No. 11-265732 (referred to as Method C) (the contents described in these patent specifications are all incorporated herein by reference). ). Hereinafter, the method B and the method C will be specifically described.

Method B:
In this method, a lactic acid oligomer is polymerized by polymerizing lactide in the presence of a lithium compound represented by RYLi (wherein R represents an aliphatic group or an aromatic group, and Y represents an oxygen atom or a sulfur atom). It is a manufacturing method. When performing a polymerization reaction, the usage-amount of a lithium compound (RYLi) is 1-0.1 mol per mol of lactide, Preferably it is a ratio of 0.2-0.3 mol. The reaction temperature is -100 to 0 ° C, preferably -78 to -50 ° C. The reaction is preferably started at a temperature of −78 to −50 ° C. and gradually raised to room temperature. The reaction is preferably carried out in the presence of a reaction solvent. As the reaction solvent, in addition to cyclic ethers such as tetrahydrofuran, diethyl ether, dimethoxyethane and the like can be used. As the reaction atmosphere, an inert gas atmosphere such as nitrogen gas or argon is used. The reaction pressure is not particularly limited and is preferably normal pressure.

  Note that the composition of the lactic acid oligomer obtained as described above (that is, the mixing ratio of the cyclic lactic acid oligomer and the chain lactic acid oligomer) varies depending on the lithium compound used as a reaction aid.

Method C:
In this method, (i) lactic acid is heated to a temperature in the range of 120 to 140 ° C. under a pressure condition of 350 to 400 mmHg to cause a dehydration condensation reaction, and only by-product water is distilled off without distilling lactide. A first heating step,
(Ii) After completion of the first heating step, the reaction product is heated to a temperature of 150 to 160 ° C., and the reaction pressure is reduced to 15 to 20 mmHg at a pressure reduction rate of 0.5 to 1 mmHg / min. Then, only by-product water was distilled off while avoiding the distillation of lactide, and after the reaction pressure dropped to 15-20 mmHg, the reaction was further continued under the same pressure condition and reaction temperature of 150-160 ° C. A second heating step for producing a dehydration condensate mainly comprising an oligomer;
(Iii) a third heating step in which, after completion of the second heating step, the chain lactic acid oligomer is cyclized by heating at 150 to 160 ° C. under a pressure condition of 0.1 to 3 mmHg to generate a cyclic oligomer;
It is the method characterized by comprising.

  In this method, first, in the first heating step, lactic acid is heated under reduced pressure to cause a dehydration condensation reaction. The reaction time in this case is 3 to 12 hours, preferably 5 to 6 hours. In the reaction under the first heating, by-product water generated by dehydration condensation of lactic acid is distilled off in order to make the reaction proceed smoothly. In this case, lactide which is a dehydration condensate of two molecules of lactic acid is removed. Carry out not to distill off. For this purpose, the reaction pressure is kept at a reduced pressure, preferably 300 to 500 mmHg, more preferably 350 to 400 mmHg, and heating is performed in the range of 100 to 140 ° C., preferably 130 to 140 ° C. under this pressure condition. Good. By the reaction in the first heating step, a reaction product mainly containing a dehydration condensate of 3 to 23 molecules of lactic acid is generated.

  After the completion of the first heating step, a temperature that is higher than the reaction temperature in the first heating step, preferably 145 to 180 ° C., so that an oligomer having an increased average polymerization degree is obtained in the second heating step. More preferably, while heating to a temperature of 150 to 160 ° C., the reaction pressure is lowered to a pressure of 10 to 50 mmHg, preferably 15 to 20 mmHg, and the dehydration condensation reaction is continued.

  As in the case of the reaction in the first heating step, this reaction is also carried out under the condition that by-product water is distilled off in order to make the reaction proceed smoothly, but lactide is not distilled off. The rate at which the reaction pressure is lowered to the pressure in the above range (pressure reduction rate) is 0.25 to 5 mmHg / min, preferably 0.5 to 1 mmHg in order to avoid the distillation of lactide and increase the reaction efficiency. It is usually necessary to keep in the range of / min. A pressure reduction rate lower than the above range is not preferable because it takes a long time to reduce the pressure to the predetermined pressure. On the other hand, a pressure reduction rate higher than the above range is not preferable because lactide distills together with by-product water. Absent.

After the reaction pressure has dropped to a predetermined pressure, the reaction is further continued at this reaction pressure. The heating time in this case is 3 to 12 hours, preferably 5 to 6 hours.
The reaction in the second heating step yields a lactic acid oligomer having an average degree of polymerization of 3 to 30, preferably 3 to 23. In this case, the ratio of the cyclic oligomer in the oligomer is usually 70 to 80% by weight. Degree.

After completion of the second heating step, in the third heating step, the reaction pressure is maintained at 0.25 to 5 mmHg, preferably 0.5 to 1 mmHg, and further reacted at a temperature of 145 to 180 ° C, preferably 150 to 160 ° C. Continue. The reaction time is 3 to 12 hours, preferably 5 to 6 hours. By-product water generated in this case is also distilled off. In this case, it is preferable to avoid evaporation of lactide, but since the reaction product contains almost no lactide, it is not necessary to reduce the rate of pressure reduction.
The reaction in the third heating step produces a lactic acid oligomer having an average degree of polymerization of 3 to 30, preferably 3 to 23, and a cyclic oligomer ratio of 90% by weight or more, preferably 99% by weight or more.

Method D:
In a particularly preferred embodiment of the present invention, lactide is reacted in the presence of an alkali metal compound represented by the formula (3): Me-N (R ¹ ) (R ² ). Hereinafter, Formula (3): Me-N (R ¹ ) (R ² ) will be described.

In the formula (3), Me represents an alkali metal. R ¹ and R ² each independently represents an aliphatic group or an aromatic group.
Examples of the aliphatic group include a saturated or unsaturated aliphatic hydrocarbon group having 1 to 12 carbon atoms, preferably 1 to 6 linear, branched, cyclic, or combinations thereof. And alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, octyl and dodecyl, and cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclooctyl and cyclododecyl. Is mentioned. The aliphatic group may be an unsaturated hydrocarbon group having a double bond or a triple bond.

  Here, examples of the aromatic group include aryl groups and arylalkyl groups having 6 to 30, preferably 6 to 20, more preferably 6 to 12, and further preferably 6 to 10 carbon atoms. Examples of the aryl group include phenyl, tolyl, naphthyl, and the like. Examples of the arylalkyl group include benzyl, phenethyl, naphthylmethyl, and the like.

  The aliphatic group and the aromatic group may have one or more substituents. The type of the substituent is not particularly limited, and examples thereof include a linear or branched, chain or cyclic alkyl group, a linear or branched, chain or cyclic alkenyl group, a linear or branched, chain or cyclic alkynyl group. , Aryl group, acyloxy group, alkoxycarbonyloxy group, aryloxycarbonyloxy group, carbamoyloxy group, carbonamido group, sulfonamide group, carbamoyl group, sulfamoyl group, alkoxy group, aryloxy group, aryloxycarbonyl group, alkoxycarbonyl Group, N-acylsulfamoyl group, N-sulfamoylcarbamoyl group, alkylsulfonyl group, arylsulfonyl group, alkoxycarbonylamino group, aryloxycarbonylamino group, amino group, ammonio group, cyano group, nitro group, carbo group Sil group, hydroxyl group, sulfo group, mercapto group, alkylsulfinyl group, arylsulfinyl group, alkylthio group, arylthio group, ureido group, heterocyclic group (for example, containing at least one nitrogen, oxygen, sulfur, etc. 12-membered monocyclic ring, condensed ring), heterocyclic oxy group, heterocyclic thio group, acyl group, sulfamoylamino group, silyl group, halogen atom and the like. In the above, the alkyl, alkenyl, alkynyl and alkoxy generally have 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and the aryl generally has 6 to 20 carbon atoms, preferably 6 to 6 carbon atoms. 10.

In the formula (3), Me represents an alkali metal. Examples of the alkali metal include Li, Na, and K, and Li is preferable.
The compound represented by formula (3) having an asymmetric carbon may be any of (R) isomer, (S) isomer, (R), (S) isomer.

  The method for obtaining the alkali metal compound represented by the formula (3) is not particularly limited, and those skilled in the art can appropriately obtain it. It can be obtained by reacting a dialkylamine such as diisopropylamine with an alkylated alkali metal such as n-butyllithium. More specifically, this reaction is carried out, for example, under a condition inert to the reaction such as under a nitrogen atmosphere, and with a solution containing a dialkylamine in an inert solvent such as THF and an alkyl in an inert solvent such as hexane. It can carry out by mixing and stirring with the solution containing alkali metal halide. The reaction temperature is not particularly limited as long as the reaction proceeds, but is preferably −78 ° C. to room temperature. The reaction time can be set as appropriate.

When the lactide is polymerized in the presence of the compound of the formula (3), the amount of the compound of the formula (3) (Me-N (R ¹ ) (R ² )) is preferably 1 to 0.00 per mole of lactide. 1 mol, more preferably 0.2 to 0.3 mol.

  Although the reaction temperature at the time of performing the polymerization reaction of lactide is not particularly limited as long as the reaction proceeds, it is preferably −100 ° C. to room temperature, more preferably −78 ° C. to room temperature.

  The polymerization reaction of lactide is preferably carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited as long as it is inert to the reaction, but cyclic ethers such as tetrahydrofuran, diethyl ether, dimethoxyethane and the like can be preferably used. As the reaction atmosphere, an inert gas atmosphere such as nitrogen gas or argon gas can be used. The reaction pressure is not particularly limited and is preferably normal pressure.

  The composition of the mixture of the chain and cyclic lactic acid oligomers obtained by the above method varies depending on the kind of the compound of the formula (3) used as a reaction aid, reaction conditions, etc., but preferably the chain lactic acid rather than the cyclic lactic acid oligomer. High oligomer content.

  According to the method described above, the following formula (1) or (2):

(In the formula, m represents an integer of 1 to 18, and n represents an integer of 1 to 18)
A chain and cyclic lactic acid oligomer mixture represented by

  The above-mentioned methods A, B, C and D are only a part of specific examples of the method for producing the polylactic acid mixture used in the present invention, and in the present invention, the polylactic acid mixture produced by other methods. Can also be used.

  In addition to the essential components described above, the matrix metalloprotease inhibitor of the present invention may further contain components and additives used in preparations such as pharmaceuticals and quasi-drugs as long as the effects of the present invention are not impaired. An agent can be arbitrarily selected and used in combination. The matrix metalloprotease inhibitor of the present invention can be used in combination with pharmaceuticals and quasi-drugs in addition to being usable as a single pharmaceutical.

The form of the matrix metalloprotease inhibitor of the present invention is not particularly limited, and it is possible to select an appropriate form most suitable for the purpose from the preparation forms for oral administration or parenteral administration.
Examples of the dosage form suitable for oral administration include tablets, capsules, powders, drinks, granules, fine granules, syrups, solutions, emulsions, suspensions, chewables, etc. Examples of the dosage form suitable for parenteral administration include, but are not limited to, injections (subcutaneous injection, intramuscular injection, intravenous injection, etc.), external preparations, drops, inhalants, sprays, and the like. Never happen.

  Liquid preparations suitable for oral administration, for example, solutions, emulsions, syrups, etc., water, sugars such as sucrose, sorbit, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc. Oils, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. For the production of solid preparations such as capsules, tablets, powders or granules, excipients such as lactose, glucose, sucrose and mannitol, starch, disintegrants such as sodium alginate, magnesium stearate, talc, etc. Lubricants, binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin, and the like can be used.

  An injectable or infusion preparation suitable for parenteral administration preferably contains the above-mentioned substances as active ingredients dissolved or suspended in a sterile aqueous medium isotonic with the blood of the recipient. For example, in the case of an injection, a solution can be prepared using a salt solution, a glucose solution, or an aqueous medium composed of a mixture of salt water and a glucose solution. Formulations for enteral administration can be prepared using carriers such as cocoa butter, hydrogenated fat, or hydrogenated carboxylic acid and provided as suppositories. In the production of a spray, a carrier that can disperse the above-mentioned substance as an active ingredient as fine particles, does not irritate the recipient's oral cavity and airway mucosa, and facilitates the absorption of the active ingredient. Can be used. Specific examples of the carrier include lactose and glycerin. Depending on the substance that is the active ingredient and the nature of the carrier used, a preparation in the form of an aerosol or dry powder can be prepared. These preparations for parenteral administration include one or more selected from glycols, oils, flavors, preservatives, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, and the like. Two or more kinds of foods and drinks can also be added.

  The dose and frequency of administration of the matrix metalloprotease inhibitor of the present invention can be appropriately set depending on various factors including the purpose of administration, the dosage form, the age, weight or sex of the intake person, etc. Specifically, the dose of the active ingredient is 1 to 10,000 mg / kg, preferably 10 to 2000 mg / kg, more preferably 10 to 200 mg / kg per day. It is preferable to administer the above-mentioned dosage in about 1 to 4 times a day.

The present invention further relates to a food or drink for matrix metalloprotease inhibition comprising a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20. That is, the cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20 used in the present invention is used not only in the form of a single preparation as described above but also in a food or drink. Can do.
If the food-drinks of this invention can be mix | blended without decomposing | disassembling a polylactic acid mixture, there will be no restriction | limiting in particular in the compounding form.

  Specific examples of food and drink products of the present invention include soft drinks, drinks, health foods, foods for specified health use, functional foods, functionally active foods, dietary supplements, supplements, feeds, feed additives, etc. Mentioned health foods or supplements, including beverages.

Specific examples of food and drink include, for example, chewing gum, chocolate, candy, tablet confectionery, jelly, cookies, biscuits, yogurt and other confectionery, ice cream, ice confectionery and other frozen confectionery, tea, soft drinks (juice, coffee, cocoa Etc.), beverages such as nutritional drinks and beauty drinks, bread, ham, soup, jam, spaghetti, frozen foods and the like. Alternatively, the polylactic acid mixture used in the present invention can be added to seasonings or food additives. By ingesting the food or drink of the present invention, a safe food or drink that exhibits a matrix metalloprotease inhibitory effect and does not exhibit substantially harmful side effects can be provided.
The food / beverage products of the present invention can be obtained by directly mixing and dispersing a polylactic acid mixture in a general raw material used in foods and then processing it into a desired form by a known method.

  The food / beverage products of this invention include food / beverage products of all forms, The kind in particular is not restrict | limited, Various food / beverage products as described above, or various nutritional compositions, for example, various oral or enteral nutrition, It can be provided as a food or drink by blending the matrix metalloprotease inhibitor of the present invention into a beverage or the like. The composition of such foods and drinks can include proteins, lipids, carbohydrates, vitamins and / or minerals in addition to cyclic and / or chain polylactic acid mixtures having a condensation degree of 3 to 20. The form of the food or drink is not particularly limited, and may be any of solid, powder, liquid, gel, slurry and the like as long as it is easy to ingest.

Although content of the polylactic acid mixture in food-drinks is not specifically limited, Generally it is 0.1-20 weight%, More preferably, it is about 0.1-10 weight%.
The amount of the polylactic acid mixture contained in the food or drink is preferably included to such an extent that the matrix metalloprotease inhibitory effect of the present invention can be exerted, and preferably 0.1 to 10 g in one food or drink taken. The degree is more preferably about 0.5 to 3 g.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited in any way by the examples.

Synthesis Example 1: Production of polylactic acid mixture (hereinafter also referred to as X01) 500 ml of L-lactic acid (which also contains D-lactic acid) was placed in a separable flask contained in a mantle heater. Nitrogen gas was introduced and stirred at 300 ml / min, and the distilled water was heated at 145 ° C. for 3 hours while being led to a flask with a reflux condenser through a temperature-reducing descending connecting tube. The pressure was further reduced to 150 mmHg and heated at the same temperature for 3 hours, and then heated at 155 ° C. for 3 hours under a reduced pressure of 3 mmHg and finally heated at 185 ° C. for 3 hours under reduced pressure of 3 mmHg to obtain polylactic acid as a reaction product. It was.

  The obtained polylactic acid was kept at 100 ° C., and 100 ml of ethanol was added followed by 400 ml of methanol, followed by cooling. This was added to 500 ml of methanol, allowed to stand with sufficient stirring, and then purified by filtration. The filtrate was dried under reduced pressure and dissolved in acetonitrile to make a total volume of 200 ml (stock solution).

  This stock solution was applied to a pre-equilibrated reverse phase ODS column (TSK gel ODS-80 ™) and eluted stepwise with 30%, 50% and 100% acetonitrile (pH 2.0) containing 0.01 M hydrochloric acid. The polylactic acid (condensation degree 3-20) which is a% elution fraction was obtained. The mass spectrum of the obtained substance is shown in FIG. As is clear from the regular fragment ion peaks in FIG. 1, the obtained polylactic acid mixture is in a state where a cyclic condensate and a linear condensate are mixed.

Synthesis Example 2: Production of cyclic polylactic acid mixture (hereinafter also referred to as X02) 10.0 g of (s)-(+)-lactic acid was placed in a 100 ml eggplant-shaped flask, and this was set on a rotary evaporator. The pressure in the flask is adjusted to 350 to 400 mmHg, heated to 140 ° C., and the reaction is continued at the same pressure and the same temperature for 6 hours (first heating step). By-product water produced by this reaction was distilled off. Moreover, under the reaction conditions, lactide hardly distilled out of the system.
Next, the reaction temperature was raised to 150 to 160 ° C., and the reaction pressure was gradually lowered from 400 mmHg over about 6 hours and dropped to 15 to 20 mmHg (pressure reduction rate: 1 mmHg / min). Under this pressure reduction rate, byproduct water was distilled off, but lactide was hardly distilled off. Thereafter, the pressure was maintained at 15 to 20 mmHg, and the reaction was continued for 6 hours (second heating step).

Next, the pressure was reduced to 1 to 3 mmHg over 30 minutes, and the reaction was continued for 5 hours at a reaction temperature of 160 ° C. (third heating step).
As a result of analyzing the reaction product after completion of the reaction, 6.80 g (yield 85%) of a cyclic oligomer having an average degree of polymerization of 3 to 21 was obtained.
The MS spectrum of the reaction product obtained in Synthesis Example 2 is shown in FIG. 3 is an overall view of NMR of the reaction product obtained in Synthesis Example 2, and FIGS. 4 and 5 are enlarged views of a part of FIG.

Synthesis Example 3: Production of lactic acid oligomer mixture (hereinafter also referred to as X03) Under nitrogen atmosphere, 0.degree. C., 0.101 g (1 mmol) of diisopropylamine in 5 mL of THF solution, 0.63 mL of n-butyllithium (1.6 M hexane solution) (1 mmol) was added, and the mixture was stirred for 10 minutes to obtain lithium diisopropylamide (LDA). Then, a 4 mL THF solution of 0.577 g (4 mmol) of L-(−)-lactide was added and stirred for 15 minutes to react. To the reaction mixture, 20 mL of saturated aqueous ammonium chloride solution was added to treat the reaction, and 10 mL of water was further added. Extraction was performed 5 times with THF (50 mL), and the organic layer was dried over anhydrous sodium sulfate. After anhydrous sodium sulfate was filtered off, the organic solvent was concentrated under reduced pressure to obtain 0.53 g of a crude product. 6 mL of ether was added to the obtained crude product, immersed in an ultrasonic cleaner for 10 minutes, and filtered to obtain 0.39 g of a white solid product having a melting point of 125 to 129 ° C.

  The physical property data of the obtained product are shown in FIGS. From the FABMS and NMR data shown in FIG. 6 to FIG. 12, it was confirmed that a trimer to a 21-mer cyclic lactic acid oligomer and a trimer to a 27-mer chain lactic acid oligomer were present in the solid product. It was.

Test Example 1:
(1) Culture of mouse B16 melanoma cells The mouse B16 melanoma cell line (RCB 0557) was obtained from RIKEN Cell Bank (Tsukuba).
In a plate with a diameter of 10 cm, B16 melanoma cells (initial number of cells of 1 × 10 ⁵ ) were mixed with fetal bovine serum (10%), L-glutamine (0.3 mg / ml), penicillin (100 units / ml), and streptomycin (0.1 The culture was carried out at 37 ° C. for 48 hours using 20 ml of DMEM culture solution containing mg / ml). When the cells became Confluent after cell growth, the cells were washed twice with phosphate-buffered saline, and then further cultured at 37 ° C. for 48 hours with the above culture solution containing lactoalbumin hydrolysate instead of fetal bovine serum. . In the 48-hour culture in the final process, X01, X02, and X03 produced in Synthesis Examples 1 to 3 were added at concentrations of 100 μg / ml and 500 μg / ml, respectively. In control experiments, only solvents in which these drugs were dissolved were added. B16 melanoma cells cultured for 48 hours in the presence or absence of drug were collected.

(2) Preparation of total RNA Total RNA was prepared from mouse B16 melanoma cells obtained in (1) above using ISOGEN (RNA extraction reagent, Nippon Gene). That is, 1 ml of ISOGEN was added to B16 melanoma cells collected from a plate having a diameter of 10 cm, and the mixture was aspirated and lysed. After leaving at room temperature for 5 minutes, 0.2 ml of chloroform was added and shaken vigorously for 15 seconds, and then left at room temperature for 3 minutes. Centrifugation was performed at 12,000 rpm for 15 minutes at 4 ° C., and the aqueous phase was recovered. 0.5 ml of isopropanol was added to the collected sample and left at room temperature for 10 minutes, followed by centrifugation at 4 ° C. and 12,000 rpm for 15 minutes. The precipitate fraction produced here was vortexed with 70% ethanol, and then centrifuged at 7,500 rpm for 5 minutes at 4 ° C. The 70% ethanol layer was discarded so as not to lose the precipitate fraction, and the remaining 70% ethanol was completely removed by drying, and then the precipitate fraction was dissolved in 44 μl of Milli-Q water. In order to completely remove genomic DNA from the obtained sample, DNase treatment was performed as follows. That is, 1 μl of DNase (1 unit / μl, Promega) and 5 μl of DNase-attached buffer were added to the crude total RNA fraction dissolved in Milli-Q water (44 μl) and reacted at 37 ° C. for 1 hour. After completion of the reaction, 50 μl of phenol solution and 50 μl of CIAA solution were added and vigorously shaken, and centrifuged at 13,000 rpm for 5 minutes at room temperature. The aqueous phase was collected, 50 μl of CIAA solution was added thereto and shaken vigorously, and the aqueous phase was collected again by centrifugation at 13,000 rpm for 5 minutes at room temperature. Add 125 μl of 100% ethanol and 5 μl of 3 M sodium acetate buffer (pH 5.2) to the collected aqueous phase, leave it at −80 ° C. for 15 minutes, and then centrifuge at 4 ° C. and 13,000 rpm for 10 minutes to obtain a precipitate fraction. It was. To the obtained precipitate fraction, 400 μl of 70% ethanol at 4 ° C. was added and centrifuged (4 ° C., 13,000 rpm, 5 minutes). After discarding the ethanol in the supernatant and further completely removing the ethanol by drying, the precipitate fraction was dissolved in 300 μl of milli-Q water. Through this series of operations, high-quality total RNA containing no genomic DNA was obtained at a concentration of about 1 mg / ml.

(3) cDNA synthesis by reverse transcriptase Single-stranded DNA using Superscript First Strand Synthesis System for RT-PCR (Invitrogen) using the total RNA derived from mouse B16 melanoma cells obtained in (2) above as a template. Was synthesized. That is, an RNA / primer mixture was first prepared with the following composition.
Total RNA solution (1mg / ml) 2μl (2μg)
oligo (dT) _12-18 solution (0.5 μg / ml) 1 μl (0.5 μg)
DEPC-treated milli-Q water 9μl
Total volume 12μl

The mixture was warmed at 70 ° C. for 10 minutes. Placed on ice for 1 minute, the following reagents were added in order.
RNA / primer mixture 12 μl
10 x PCR buffer 2 μl
25 mM MgCl ₂ 2 μl
10 mM dNTP mix 1 μl
0.1 M DTT 2 μl
Total volume 19μl

  The mixture was preincubated for 5 minutes at 42 ° C. Next, 1 μl (200 units) of SUPERSCRIPT II (reverse transcriptase) was added and mixed well, followed by incubation at 42 ° C. for 60 minutes. The reaction was then stopped by incubation at 70 ° C. for 15 minutes. After cooling on ice, 1 μl (2 units) of RNaseH was added and incubated at 37 ° C. for 20 minutes to decompose the template RNA to obtain single-stranded cDNA.

(4) Synthesis of MMP-3 specific primer As a primer for specifically amplifying only the gene by PCR from the nucleotide sequence (Accession No. NM_010809) of mouse MMP-3 already published in a public database, The following nucleotides were synthesized. That is, 5′-TGGGACTCTACCACTCAGCCAAGG-3 ′ (24-mer) (SEQ ID NO: 1) was used as the 5 ′ sense primer, and 5′-CCAGGGTGTGAATGCTTTTAGG-3 ′ (22-mer) (SEQ ID NO: 2) was used as the 3 ′ antisense primer. ) Was synthesized. Synthetic oligonucleotides were chemically synthesized using a fully automatic DNA synthesizer (Applied Biosystems).

(5) PCR amplification of MMP-3 cDNA fragment PCR was carried out using the cDNA prepared in (3) above as a template and the primers prepared in (4) above. The composition of the PCR reaction solution is as follows.
cDNA solution 1μl
Milli-Q water 34.5μl
10 × PCR buffer (containing 15 mM MgC1 _2, TAKARA Co.) 5 [mu] l
10 mM dNTP mix 4 μl
10 μM primer (sense) 2.5 μl
10 μM primer (antisense) 2.5 μl
Ex taq polymerase (5 units / μl, Takara) 0.5 μl
Total volume 50μl

  After thoroughly mixing the above reaction solution, PCR reaction was performed. PCR was first heated at 94 ° C. for 3 minutes, followed by 30 cycles of heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 30 seconds. . After completion of the reaction, this PCR product was subjected to 1.5% agarose gel electric permanence. The PCR product was detected by UV irradiation after the electrophoresis gel was immersed in a buffer solution containing ethidium promide for 30 minutes. The amplified product was DNA consisting of about 470 nucleotides. In mouse B16 melanoma cell culture, the addition of X01, X02, and X03 to the culture medium at a concentration of 500 μg / ml significantly reduced the PCR product quality. (FIG. 13).

(6) Extraction of PCR product and determination of nucleotide sequence The PCR product amplified in (5) above was excised from an agarose gel and purified using a gene clean kit (Q-BIO gene). The PCR product was then ligated to T-Vector and the recombinant plasmid was transformed into E. coli (JM109). Single colonies were cultured in Terrific Broth medium, plasmids were purified, and ABI PRISM Big-Dye Terminator Cycle Sequence Kit Version 1.1 (Applied Biosystems) was used with a fluorescent automated DNA sequencer (Applied Biosystems, Model 373). Analyzed. This confirmed that the PCR product was a fragment of the mouse MMP-3 gene (468 bases, corresponding to SEQ ID NO: 753-1220 in Accession No. NM_010809).

FIG. 1 shows a mass spectrum of the polylactic acid mixture obtained in Synthesis Example 1. FIG. 2 shows the MS spectrum of the reaction product obtained in Synthesis Example 2. FIG. 3 shows an overall NMR view of the reaction product obtained in Synthesis Example 2. FIG. 4 shows an enlarged view of a portion of FIG. FIG. 5 shows an enlarged view of a portion of FIG. FIG. 6 shows an overall view of the positive mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 10.0000 ~ 1305.5900 FIG. 7 shows an overall view of the negative mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 10.0000 ~ 2000.0000 FIG. 8 shows an enlarged view of the negative mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 10.0000-501.9260 FIG. 9 shows an enlarged view of the negative mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 490.298-01003.7700 FIG. 10 shows an enlarged view of the negative mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 999.9500 ~ 1504.3400 FIG. 11 shows an enlarged view of the negative mode FABMS spectrum of the product obtained in Synthesis Example 3. Range: m / z 1484.5300 ~ 2000.0000 FIG. 12 shows an overall view of the NMR spectrum of the product obtained in Synthesis Example 3. FIG. 13 is a graph showing the effect of X01, X02 and X03 on MMP-3 expression in mouse B16 melanoma cells. (A) shows amplification of MMP-3 fragment by PCR. (B) shows quantified data of PCR products.

Claims (5)

A matrix metalloprotease inhibitor comprising a cyclic and / or chain polylactic acid mixture having a condensation degree of 3 to 20. The matrix metalloprotease inhibitor according to claim 1, wherein the matrix metalloprotease is matrix metalloproteinase-3 (MMP-3). The matrix metalloprotease inhibitor according to claim 1 or 2, wherein lactic acid, which is a repeating unit in polylactic acid, consists essentially of L-lactic acid. The matrix metalloprotease inhibitor according to any one of claims 1 to 3, which is used for treatment or prevention of a disease associated with overproduction of matrix metalloprotease. Food-drinks containing the matrix metalloproteinase inhibitor in any one of Claim 1 to 4.