JP5339708B2 - Matrix metalloproteinase-8 inhibitor containing cysteine as an active ingredient, and composition for preventing or treating periodontal disease for diabetic patients or persons with impaired glucose tolerance - Google Patents

Description

  The present invention relates to a technique utilizing an action of inhibiting the production or activity of matrix metalloproteinase-8 (MMP-8) of cysteines. More specifically, MMP-8 inhibitors containing cysteines, techniques for preventing or treating periodontal disease in diabetic patients or persons with impaired glucose tolerance, techniques for preventing or treating other diseases related to MMP-8 About.

  Matrix metalloprotease (hereinafter sometimes referred to as MMPs) is a general term for extracellular matrix degrading enzymes characterized by possessing zinc (II) ions in the active site, and the type of substance (substrate) that acts on them. According to the difference in the structure (domain structure), it is roughly classified into 5 groups, and about 27 types are currently confirmed.

  The main substrate of MMPs is the extracellular matrix, for example, biopolymers such as collagen, laminin, gelatin, fibronectin, proteoglycan, and elastin. MMPs are thought to function to maintain normal tissue by controlling angiogenesis and the construction of new tissues by decomposing unnecessary extracellular matrix in the process of tissue remodeling and wound healing in vivo. Yes. However, too much activity of MMPs causes structural abnormalities of the extracellular matrix and abnormal metabolic balance (imbalance in synthesis and degradation), tumor invasion and its metastasis, osteoarthritis and rheumatoid arthritis. It has been known to cause joint diseases such as vascular tissue and bone tissue metabolism, tissue destruction such as periodontal disease, and delayed healing of diabetic complications.

  MMP-8, a kind of MMPs, is classified into the collagenase group based on the type of substrate and the type of domain structure. Since MMP-8 is produced from neutrophils, it is also called neutrophil collagenase (see, for example, Non-Patent Document 1). The main substrates of MMP-8 are type I, type II, type III collagen and the like, which overlap with the substrate of MMP-1 (tissue collagenase) belonging to the same collagen group. MMP-1 is produced by fibroblasts derived from tissues such as skin and cornea, keratinocytes, osteoblasts, and other tissue cells, but these tissue cells do not migrate, so MMP-1 production is constant. Done at the site. In contrast, MMP-8 is produced by neutrophils as described above, but neutrophils migrate with cells of the immune system, so the production site of MMP-8 varies according to the movement of neutrophils. . MMP-1 and MMP-8 also differ in the activation mechanism. MMP-1 is activated by MMP-3 belonging to the same collagenase group, whereas MMP-8 is activated by MMP-10 belonging to the stromelysin group.

  MMP-8 is thought to play an important role in diseases caused by neutrophil adhesion, migration, and accumulation. Especially in diabetic patients with chronic inflammation, there is a strong correlation between the delayed healing of diabetes-related diseases, including diabetic complications, and the level of MMP-8 enzyme activity. It is considered to exist. That is, it is believed that the slower the cure of a disease associated with diabetes, the higher the activity level of MMP-8.

  It is well known that periodontal disease in diabetic patients has a high incidence and rapid progression. Because of the infectivity characteristic of diabetic patients, the inflammatory response to bacterial infection in periodontal tissues is significantly enhanced, the periodontal tissues are rapidly destroyed, and the responsiveness to treatment (therapeutic effect) is extremely poor Periodontal disease (periodontitis, gingivitis) in diabetic patients has been studied to be recognized as the sixth complication of diabetes (for example, see Non-Patent Document 2). As described above, periodontal disease in diabetic patients is refractory, and it is difficult to cure by normal periodontal disease treatment. Therefore, antibacterial agent is used in combination with blood glucose control and thorough plaque control considering susceptibility to infection. However, it is required to do it. In addition, those with impaired glucose tolerance also have a similar tendency, although milder than diabetic patients, and it is difficult to cure periodontal disease.

In periodontal disease in diabetic patients, it is reported that collagenase activity in gingival crevicular fluid (GCF) is high (see, for example, Non-Patent Document 3), and its origin is reported to be MMP-8 (eg, Non-Patent Document 4). In addition, collagenase activity and gelatinase activity in the gingiva of rats induced with streptozotocin in diabetes have been reported to be higher than those in non-diabetic rats that have developed periodontal disease with lipopolysaccharide (LPS) (eg, non-patented). Reference 5).
Yoshihara, Shinna, “Inflammation and Immunity”, 2, 177-185, 1994 Harald L., Diabetes Care, 16, Supplement 1, 329-334, 1993 Bedia S. et al., J. Periodontol., 77, 189-194, 2006 Sorsa T. et al., J. Clin. Periodontol., 19, 146-149, 1992 Kuang-min C. et al., Res. Commun. Mol. Pathol. Pharmacol., 91, 303-318, 1996

  An object of the present invention is to provide an inhibitor of MMP-8, improve various symptoms of a disease caused by MMP-8 production or increased activity, and prevent or treat the disease.

  The present inventors have found that the production or activity of MMP-8 is inhibited by oral ingestion or topical administration of cysteines, thereby completing the present invention.

That is, the present invention provides the following matrix metalloprotease-8 inhibitor and a composition for preventing or treating periodontal disease for diabetic patients or those having impaired glucose tolerance.
Item 1. A matrix metalloproteinase-8 inhibitor containing cysteines as an active ingredient.
Item 2. A composition for preventing or treating periodontal disease for a diabetic patient or a person having impaired glucose tolerance, comprising the matrix metalloproteinase-8 inhibitor according to Item 1.

  In the present invention, cysteines include cysteine, cysteine derivatives and salts thereof, and can be used alone or in combination of two or more. Examples of cysteine derivatives include cystine, cysteine methyl ester, cysteine ethyl ester, methyl cysteine, L-ethyl cysteine, N-acetyl cysteine and the like. The salt is not particularly limited as long as it is a salt acceptable for oral administration. For example, acid salts such as cysteine or inorganic acid salts such as hydrochlorides, nitrates and acetates of the above cysteine derivatives; alkali metal salts such as sodium salts and potassium salts of cysteine or the above cysteine derivatives, alkalis such as magnesium salts and calcium salts Examples include earth metal salts. Preferably, it is cysteine or hydrochloride of the above cysteine derivative, and specific examples include cysteine hydrochloride (particularly L-cysteine hydrochloride), methylcysteine hydrochloride, and L-ethylcysteine hydrochloride. Preferable cysteines are L-cysteine and cysteine hydrochloride, and more preferably L-cysteine.

  The matrix metalloprotease-8 (MMP-8) inhibitor of the present invention has an action of inhibiting the production or activity of MMP-8 and contains cysteines as an active ingredient. In addition, the preventive or therapeutic composition of the present invention has an action of preventing or treating periodontal disease for diabetic patients or persons with impaired glucose tolerance, and contains the inhibitor of the present invention as an active ingredient. The content of cysteine in the inhibitor and the preventive or therapeutic composition of the present invention is preferably 0.001 to 50% by weight, more preferably 0.01 to 20% by weight when converted to L-cysteine. .

  The intake amount of the inhibitor and the preventive or therapeutic composition of the present invention is an amount such that the intake amount of cysteine converted to L-cysteine is usually 100 to 2000 mg, preferably 200 to 1000 m per adult day. . In addition, the inhibitor and the preventive or therapeutic composition of the present invention may be taken once a day or divided into several times a day, and the time of eating is before meal (preferably within 5 minutes), between meals, after meal (preferably Within 5 minutes) or during a meal.

  The local dose of the inhibitor and the preventive or therapeutic composition of the present invention is usually 0.1 to 100 mg, preferably 1 to 10 mg, of cysteine converted to L-cysteine per adult day. This is the amount.

  In addition, preferred administration subjects of the inhibitor and the preventive or therapeutic composition of the present invention are diabetic patients and persons with impaired glucose tolerance, and more preferably diabetic patients. A person with impaired glucose tolerance is sometimes referred to as a boundary type, and the criterion is shown by WHO. That is, in the blood glucose level before loading obtained by the oral glucose tolerance test in which 75 g of glucose is orally loaded and the blood glucose level after 2 hours of loading, the blood glucose level before loading (mg / dL) is less than 110 and 2 hours after loading. Those whose blood glucose level (mg / dL) is less than 140 are normal, those whose blood glucose level before loading is 126 or higher, and those whose blood glucose level is 200 or higher after 2 hours of loading are diabetic, normal or diabetic It is a criterion for making the person who does not belong to the boundary type.

Moreover, the inhibitor and the preventive or therapeutic composition of the present invention can contain an orally acceptable carrier, other medicinal ingredients, additives and the like, in addition to cysteines, as necessary. The inhibitor and the preventive or therapeutic composition of the present invention are in a form suitable for oral ingestion or topical administration (preferably direct administration to the affected area (lesion)), for example, liquid, tablet, granule, depending on the purpose of use. In addition, it can be used in various forms such as capsules encapsulating solid agents or liquid agents or solid agents such as fine granules and powders, oral sprays, troches, ointments, gels, and gums. These forms of preparations can be prepared by conventional methods. Preferred dosage forms include tablets (sugar-coated tablets, coated tablets), granules, dry-coated tablets, multilayer tablets, capsules, gums, etc. for oral intake, and ointments, gels, pockets for topical administration. Examples include an irrigator liquid, an interdental brush gel, a form (temperature-sensitive gel, film, etc.) that adheres to oral tissues, and a periodontal pocket injection ointment. Examples of other medicinal components include antibacterial agents such as cetylpyridinium chloride (CPC), chlorhexidine, hinokitiol, triclosan, benzethonium chloride, lysozyme chloride, dipotassium glycyrrhizinate, glycyrrhetinic acid, tranexamic acid, allantoin, vitamin E, vitamin C, Anti-inflammatory agents such as phytonadione, bad breath suppressors such as sodium copper chlorophyllin and carbazochrome, and antioxidants. Moreover, as an orally acceptable carrier and additive, those used in the pharmaceutical field can be widely used. For example, the following materials may be mentioned, but not limited to these.
Sugar alcohols (such as maltitol, xylitol, sorbitol, erythritol), lactose, sucrose, sodium chloride, glucose, starch, carbonates (such as calcium carbonate), kaolin, crystalline cellulose, silicic acid, methylcellulose, glycerin, sodium alginate, Arabic gum, talc, phosphates (potassium hydrogen phosphate, calcium hydrogen phosphate, sodium hydrogen phosphate, dipotassium phosphate, disodium phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, dihydrogen phosphate Sodium), calcium sulfate, calcium lactate, cocoa butter and other excipients. Viscosity modifiers such as simple syrup, glucose solution, starch solution, gelatin solution. Polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxyvinyl polymer, crystalline cellulose, powdered cellulose, crystalline cellulose / carmellose sodium, carboxymethylcellulose, shellac, methylcellulose Binders such as ethyl cellulose, potassium phosphate, gum arabic powder, pullulan, pectin, dextrin, corn starch, pregelatinized starch, hydroxypropyl starch, gelatin, xanthan gum, carrageenan, tragacanth, tragacanth powder, polyethylene glycol and the like. Disintegrants such as dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose and the like. Disintegration inhibitors such as sucrose, stearic acid, cocoa butter and hydrogenated oil. Absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate. Adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid. Lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol. Emulsifiers such as sucrose fatty acid ester, sorbitan fatty acid ester, enzyme-treated lecithin, enzyme-degraded lecithin, saponin. Antioxidants such as ascorbic acid and tocopherol. Acidulants such as lactic acid, citric acid, gluconic acid and glutamic acid. Strengthening agents such as vitamins, amino acids, lactate, citrate, gluconate. Fluidizing agent such as silicon dioxide. Sweeteners such as sucralose, acesulfame potassium, aspartame, glycyrrhizin. Fragrances such as mint oil, eucalyptus oil, cinnamon oil, fennel oil, clove oil, orange oil, lemon oil, rose oil, fruit flavor, mint flavor, peppermint powder, dl-menthol and l-menthol. Oligosaccharides such as lactulose, raffinose, lactosucrose. Pharmaceutical preparations such as sodium acetate.

  Further, solid preparations such as tablets can be made into tablets with ordinary coatings if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets, multilayer tablets and the like. Liquid preparations may be aqueous or oily suspensions, solutions, syrups, elixirs, and can be prepared according to conventional methods using ordinary carriers, additives and the like.

  In addition, the present invention may also include embodiments as foods for preventing the above diseases and foods for improving the above symptoms, which contain cysteines as an active ingredient for MMP-8 inhibition. Foods include health foods, functional foods, foods for specified health use, functional foods for nutrition, foods for sick people, and the like. Foods can be prepared by conventional methods in combination with cysteines, if necessary, with edible carriers, food materials, food additives and the like according to the form of the food. For example, it can be used as liquid foods such as beverages, solid foods such as tablets, granules, chewable tablets, troches, and chewing gum.

  According to the study of the present inventor, when cysteines were orally ingested, production or activity of MMP-8 was inhibited in periodontal tissues. However, inhibition of the activity of other MMPs such as MMP-2 and MMP-9 was not observed. For this reason, cysteines are thought to selectively inhibit the production or activity of MMP-8. Thus, cysteines can prevent or treat periodontal disease in diabetic patients without interfering with normal tissue repair. Also, when periodontal tissue regeneration or dental implant impaction is performed, the tissue is required to form a new tissue at an early stage through normal remodeling. Since it is installed in the tissue, the initial effect may not be obtained by inducing the migration of neutrophils and causing tissue destruction due to the expression of MMP-8 activity. By using cysteines as a support agent for these treatments, it is possible to spontaneously heal periodontal tissues early while preventing recurrence of periodontal disease and peri-implantitis.

  Therefore, cysteines have the following preventive or ameliorating actions for diseases and symptom improving actions. Periodontal disease (gingivitis, periodontitis), diabetic complications (diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic macrovascular disorder, diabetes mellitus) Symptomatic cardiomyopathy), various diseases such as peri-implantitis, gingival retraction symptoms, periodontal pocket deepening symptoms, tooth swaying symptoms, periodontal tissue destruction symptoms after regenerative surgery. Therefore, the present invention may include a preventive or therapeutic composition for these diseases, a method for preventing or treating, an agent for improving these symptoms, a method for improving, and the like. Among these, the effect as a preventive or therapeutic composition of periodontal disease (gingivitis, periodontitis) in a diabetic patient or a person having impaired glucose tolerance is expected.

  The present invention can be used as an inhibitor of MMP-8, as well as the prevention or treatment of periodontal disease (gingivitis, periodontitis) in diseases involving MMP-8, such as diabetic patients or those with impaired glucose tolerance, It is useful for improving symptoms related to MMP-8.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not limited to these.

Test example 1
Test method <Test formulation>
A tablet containing 40 mg of L-cysteine in one tablet.

<Intake method>
Take 2 tablets of the test preparation 3 times a day with water or hot water after a meal. The daily intake of L-cysteine is 240 mg.

<Subject>
Prior to the start of the study, the doctor in charge conducted a preliminary examination and interview, and selected subjects who met all of the following conditions.
Subject conditions:
・ People who understand the purpose and content of this study and have consent to participate in the document ・ Men and women aged 25 to 65 ・ People who have not been treated for periodontal disease within 3 months before the start of this study ・ Pocket depth ( Exclusion criteria for subjects diagnosed as periodontal disease patients who have 1 or more sites with PD) of 4 mm or more, or 2 sites with bleeding even if PD is less than 4 mm:
・ People who have taken health foods and supplements containing L-cysteine preparations, reducing agents, cystine, and antioxidants within the past 2 weeks ・ Lactating women or pregnant women ・ Drugs that affect gingival conditions within the past 3 months Or antibiotics taken for more than 3 weeks

<Number of subjects>
19 (17 men, 2 women, average age 46.8 years)

<Examination design>
Open test only for the test group without the control group

<Test schedule>
Subjects selected by pre-examination performed a baseline examination approximately 2 weeks later. Test preparations were ingested for 4 weeks after baseline examination. Examination was conducted 2 weeks and 4 weeks after the start of ingestion.

<Combination drugs and foods>
Since health foods and supplements containing L-cysteine preparations, reducing agents, cystine, and antioxidants, glucocorticoids, drugs and antibiotics that affect the gingival state affect the evaluation, in principle, they are prohibited from being used together.

<Biochemical and clinical indicators>
Teeth with a pocket depth (PD) of 4 mm or more are mainly used as test teeth, and gingival crevicular fluid (GCF) is collected at baseline (at the start of the test), 2 weeks, and 4 weeks (at the end of the test) The activities of MMP-8 and MMP-9 in GCF were measured. PD and bleeding during probing (BOP) were also measured.

Test result <Pocket depth change over time>
Table 1 and FIG. 1 show the average values of PD at the second week of intake and the fourth week of intake for the subjects (16 subjects) who had PD of 4 mm or more at the baseline.

There was no improvement in PD from baseline to baseline, but it improved significantly (P <0.001) 2 weeks after ingestion and maintained that value from 2 weeks to 4 weeks .

<BOP change over time>
Table 2 and FIG. 2 show the average values of the ratio of bleeding (BOP) sites to the total probing sites at baseline, 2 weeks after intake, and 4 weeks after intake for all subjects.

There was no improvement in BOP from baseline to baseline, but it improved significantly (P <0.05) 2 weeks after ingestion and gradually improved from 2 weeks to 4 weeks.

<Change in GCF over time>
FIG. 3 shows the average value of the amount of GCF of 40 specimens that were within the measurable range of Periotron in the pockets with PD of 4 mm or more from which GCF (gingival crevicular fluid) was collected. There was almost no change in the amount of GCF from baseline to the fourth week after the end of the study.

<Changes in activity of MMP-8 and MMP-9 in GCF over time>
Twenty sites were selected at random from sites with a PD of 4 mm or more, and the total MMP-8 amount, active MMP-8 amount, total MMP-9 amount, and active MMP-9 amount contained in GCF were measured. The total amount of MMP is the total amount of active MMP and latent MMP.

  GCF sampling was performed using Periopaper, and the activities of MMP-8 and MMP-9 were measured using Biotrak Activity Assay System (Amersham Biosciences). Periopaper was placed in extraction solution (0.05% (w / w) Brij-35 / assay buffer), shaken on a rotary shaker for 30 minutes, and then centrifuged. The supernatant was collected and subjected to the test.

The MMP-8 antibody solution was placed in a 96-well plate and reacted at 37 ° C. for 2 hours to allow the antibody to adhere to the bottom of the plate. Thereafter, the antibody solution was removed and the wells were washed 4 times with wash buffer. In the MMP-9 test, the one having the MMP-9 antibody fixed on the bottom of the well was used. MMP standard solution and sample solution were placed in each well and allowed to stand overnight at 4 ° C. to bind the MMP in the sample to the antibody. The wells were washed 4 times with wash buffer, APMA was added only to the sample for which MMP standard and total amount of MMP were measured, and reacted at 37 ° C. for 1.5 hours to convert latent MMP into active form. At this time, an assay buffer was added to a sample for measuring active MMP. After completion of the reaction, a detection reagent was added to all the samples, and the absorbance at 405 nm was measured. Create a calibration curve using the concentration of the MMP standard solution and the MMP activity ((absorbance at the end of the reaction−initial absorbance value) / (reaction time) ² × 1000) determined from the change in absorbance from the initial value. The MMP activity in the medium was calculated in terms of the amount of MMP. The measurement results are shown in FIG. In MMP-8, the total amount of MMP-8 decreased and the amount of active MMP-8 significantly decreased during the 4 weeks of ingestion. In contrast, MMP-9 showed almost no change in total MMP-9 and active MMP-9 levels after 4 weeks of ingestion.

  Next, the total amount of MMP-9 and the amount of active MMP-9 were measured for all samples collected from 48 sites where PD was 4 mm or more. The measurement results are shown in FIG. Even after 4 weeks of ingestion, there was almost no change in the total amount of MMP-9 and the amount of active MMP-9.

Test example 2
Test method Human monocytic cell line U937 was cultured in the presence of 5 mM glucose (normal glucose: NG) (20 mM mannitol was also added for osmotic pressure adjustment) or 25 mM glucose (high glucose: HG) for 3 days. Each was assumed to be a normal blood glucose state and a hyperglycemic state. Thereafter, the sugar was removed, 0.01 μg / mL L-cysteine (L-cysteine hydrochloride monohydrate) was added, and the mixture was cultured for 20 hours. Furthermore, L-cysteine was removed, 100 ng / mL LPS, 10 ng / mL PMA (Phorbol 12-myristate 13-acetate) was added and cultured for 24 hours. Thereafter, total RNA was collected from the cells, cDNA was synthesized, and then the expression of MMP-8 was examined by Real-time PCR.
Test results The cultured U937 was assumed as follows depending on the additives during the culture.
NG + PMA: healthy person NG + PMA + LPS: periodontal disease affected person HG + PMA + LPS: diabetes + periodontal disease affected person in U937, with or without L-cysteine added, data obtained from each sample in FIG. Show. In particular, Table 3 shows how much the expression of MMP-8 was inhibited by the system in which 0.01 μg / mL L-cysteine was added to each of “NG + PMA + LPS” and “HG + PMA + LPS”.

  When cultured in a hyperglycemic state, compared to when cultured in a normoglycemic state, the increased expression of MMP-8 in response to LPS and PMA stimulation is significantly higher, and the enhanced MMP-8 expression is significantly suppressed by L-cysteine. It was. It was also found that the effect was higher when cultured in a hyperglycemic state.

  Therefore, MMP-8 expression is significantly higher in those with diabetes or impaired glucose tolerance and suffering from periodontal disease, and L-cysteine is more effective than MMP. -8 works to suppress production and is effective in preventing and treating periodontal disease.

  The present invention can be used in the fields of inhibitors of MMP-8, prevention or treatment of diseases associated with MMP-8, improvement of symptoms associated with MMP-8, and the like.

It is a graph showing a time-dependent change of PD (average value). The vertical axis shows PD (mm), and the horizontal axis shows the second week of intake and the fourth week of intake at the baseline from the left. It is a graph showing a time-dependent change of BOP (average value). The vertical axis shows the percentage (%) of BOP, and the horizontal axis shows the second week of intake and the fourth week of intake at the baseline from the left. It is a graph showing a time-dependent change of the amount of GCF. The vertical axis indicates the amount of GCF (μL), and the horizontal axis indicates the baseline from the left, the second week of intake, and the fourth week of intake. 2 is a graph showing a change with time in the amount of MMP-8 in GCF and a graph showing a change with time in the amount of MMP-9. The vertical axis indicates the total MMP amount or active MMP amount (ng / site), and the horizontal axis indicates the 4th week of intake at the baseline from the left. It is a graph showing the time-dependent change of the amount of MMP-9 in GCF. The vertical axis indicates the total amount of MMP-9 or active MMP-9 (ng / site), and the horizontal axis indicates the second week of intake and the fourth week of intake at the baseline from the left. It is a graph showing the result of having investigated the difference in the expression of MMP-8 by Real-time PCR when cultivating human monocytic cell line U937 by changing the conditions of additives.

Claims (2)

A composition for improving or treating periodontal disease for a person having diabetes or impaired glucose tolerance and suffering from periodontal disease, comprising a matrix metalloproteinase-8 production inhibitor containing L-cysteine as an active ingredient. The periodontal disease improving or treating composition according to claim 1, which is an oral composition or a composition directly administered to an affected area.

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