KR19980076997A - Topical drug delivery for the treatment of periodontal disease - Google Patents

Abstract

The present invention relates to a topical drug delivery agent for the treatment of periodontal disease. More specifically, the present invention not only inhibits the production of superoxide, prostaglandin (PGE ₂ ), interleukin-1β (Interleukin-1β) for the treatment of periodontal disease, but also degrades collagen protein, which is a substrate of periodontal tissue. Contains one or two or more components selected from urodesoxycholine acid, kenodesoxycholine acid, and derivatives of taurosoledesoxycholine acid, which inhibit the activity of collagenase enzymes as active ingredients It relates to a topical drug delivery agent for the treatment of periodontal disease, such as ointment or membrane-like preparation.

Description

Topical drug delivery for the treatment of periodontal disease

The present invention relates to a topical drug delivery agent for the treatment of periodontal disease. More specifically, the present invention not only inhibits the production of superoxide, prostaglandin (PGE ₂ ), interleukin-1β (Interleukin-1β) for the treatment of periodontal disease, but also degrades collagen protein, which is a substrate of periodontal tissue. Contains one or two or more components selected from urodesoxycholine acid, kenodesoxycholine acid, and derivatives of taurosoledesoxycholine acid, which inhibit the activity of collagenase enzymes as active ingredients It relates to a topical drug delivery agent for the treatment of periodontal disease, such as ointment or membrane-like preparation.

Periodontal disease is clinically caused by loss of teeth due to gingival inflammation and bleeding, periodontal pocket formation and destruction of alveolar bone. The periodontal disease proceeds as a process of destruction of the periodontal tissue by bacterial colonization and periodontal tissue infiltration. That is, the saliva protein in the oral saliva is first adsorbed to the dentin and chalky surfaces to form a film. Bacteria, such as Streptococcus and Actinomyces, grow on the surface, forming plaques, and as time passes, the plaque moves in the direction of the apical roots, while at the same time, porphyromonas and Anaerobic Gram-negative bacteria, such as Actinobacillus, grow and penetrate into the gingival connective tissue through the gingival epithelium, forming periodontal pockets. As a result of the metabolism of these bacteria, hydrogen sulfide, ammonia, and toxic amines are toxic to the periodontal tissue. Lipopolysaccharide, a cell wall component, is secreted at the same time as toxins of cells such as tissues directly destroyed by endotoxins such as charide (LPS) or simultaneously stimulated bioimmune system and secreted extracellularly by proliferation of humoral and cellular immune systems, prostaglandins, rucotes Inflammation of the gums by various cytokines such as Leukotriens, Histamines, Interleukins, etc., and enzymes such as collagenase secreted from bacteria and white blood cells When collagen, a substrate, is broken down, gum retraction occurs, and if left unattended, it progresses to periodontal disease.

In an effort to prevent the occurrence of periodontal disease, as a component that can be killed by short-term exposure of periodontal disease bacteria, Chlorohexidine gluconate, Cetylpyridium chloride, Sanguinarin ( Antibacterial agents such as Sanguinarine and Triclosan and anti-inflammatory agents such as triamcinolone acetonide have been developed and applied to oral products such as dentifrice, toothpaste and ointment, but they have not yet prevented the occurrence of gingivitis. There is a situation.

Therefore, in recent years, efforts have been actively made to suppress the development of gingival inflammation using extracts of herbal medicines such as myrrh, baekbaekpi, horse riding, green tea, licorice, golden, pogongyoung, gold and silver, and corn. Substances that inhibit the production of prostaglandins that play a decisive role in causing gingivitis or inhibit collagenase activity that degrades periodontal tissues have not been identified. To prevent the occurrence of periodontal disease through the action, such as convergence, haemostasis, blood circulation.

The most widely used method to treat gingival inflammation is to use a drug that inhibits the activity of collagenase enzymes that break down the periodontal tissue or inhibits the production of prostaglandins, which cause gingival inflammation. For example, U. S. Patent No. 5230895 proposed a method for treating periodontal disease by inhibiting the enzyme activity of collagenase, which degrades periodontal tissues, using a sustained release drug delivery system containing Tetracycline. European Patent Publication No. 528468 A1 proposes a method of inhibiting periodontal disease by inhibiting the production of prostaglandin, a gingivitis-inducing substance, using a toothpaste containing triclosan and a mouthwash.

However, drugs as mentioned above generally do not have both effects of inhibiting the production of gingival-inflammatory substances and at the same time inhibiting the activity of periodontal tissue degrading enzymes in the treatment of gingivitis, and exhibit only one action. Therefore, there is a limit to the complete treatment of gingival inflammation, as well as a variety of side effects when used for a long time because of the synthetic material rather than natural substances. For example, a study on the long-term administration of tetracycline at low concentrations showed that tetracycline was administered at 1000 mg / day for 2 weeks, followed by 250 mg once a day for 48 weeks and clinically or bacteriologically. It was reported to improve to the normal state, and clinically, gingivitis was reduced, motility strains in subgingival plaque were lost, and periodontal pocket depth and loss of adhesion were reported. However, as described above, when long-term use of low concentrations of tetracycline occurs, resistance expression of Gram-negative bacteria frequently occurs, and the expression of such resistant strains occurs frequently even when the antibiotics are stopped. Pain in the gastrointestinal tract, diarrhea, tooth coloring and other side effects appear very often, pregnant women and children are limited to refrain.

Therefore, the inventors of the present invention used a method of measuring collagenase activity that degrades periodontal tissue, quantitative determination of interleukin-1β and prostaglandin (PGE ₂ ), and superoxide production to induce gingivitis. Extensive research was conducted to select drugs that are effective against periodontal disease among various herbal medicines that have been secured. As one of these studies, the medicinal ingredients of animal medicines woodam and ungdam were studied. As is already known, Ful tauri (gallbladder of Bos taurus demesticus Gmelin) and ungdam (gallbladder of Fel ursi, Selenarctos thibetanus G. Cuvier) are herbal medicines. It has been known to have the efficacy of growth and anti-inflammatory, and has been widely used, and Korean Patent Publication Nos. 93-11992 and 93-11993 discloses biliary salt and glycocholic acid (glycocholic) made by mixing at high temperature by mixing the salt with gallbladder juice. It was intended to provide plaque removal effect, prevention of periodontal disease, and treatment by applying oral hygiene prepared by mixing calcined wood and salt containing taurocholic acid and taurocholic acid. However, Woodam and Woongdam have various components as animal medicines, and thus have poor stability when manufactured as a product, and have a high possibility of deterioration of the raw materials themselves and storage of drug efficacy. Therefore, the present inventors have concentrated on the anti-inflammatory effect of various active ingredients of Ungdam in order to improve these problems, as a result Ursodesoxycholic acid (Urso- desoxycholic acid), Kenodesoxycholic acid (Chenodesoxycholic acid) And its derivative, Tauroursodesoxycholic acid, have excellent efficacy in inhibiting superoxide production, interleukin-1β production, PGE ₂ production, and collagenase activity, which cause periodontal disease. The use of these ingredients has led to the search for a treatment for periodontal disease.

However, the methods that have been generally applied up to now have been used in the form of toothpaste, mouthwash, etc. in the form of toothpaste, oral cleansers, etc., but most of them are used in the periodontal tissue because active ingredients are washed away by water or saliva, etc. There is a problem that the residual concentration is extremely low, and the desired effective drug concentration cannot be maintained continuously. Therefore, in case of severe periodontal disease, it was necessary to solve this problem by formulating and using an effective drug in a specific dosage form in the pharmaceutical field that can continuously supply an effective drug to maintain an effective drug concentration in the periodontal tissue. .

As a result of the research, the present inventors have applied an active ingredient such as urodesoxycholine acid, kenodesoxycholine acid or taurosoledecoxycholine acid as a topical drug delivery system that can maximize their efficacy. And a clinical trial performed on patients suffering from periodontal disease by formulating in the form of a membrane-type topical drug delivery agent, and confirmed that the active drug ingredient exhibited the desired excellent efficacy when using these formulations. To complete.

1 is an enlarged plan view of a trapezoidal membranous topical drug delivery agent according to the present invention.

Accordingly, the present invention is a topical drug for treating periodontal disease, containing one or two or more components selected from the group consisting of urodesoxycholic acid, kenodesoxycholic acid, and taurusodesoxycholic acid as an active ingredient. It is about a delivery system.

More specifically, the present invention is a periodontal in the form of an ointment containing one or two or more components selected from the group consisting of urethanesoxycholine acid, kenodesoxycholine acid and taurosoledesoxycholine acid as an active ingredient. It relates to a topical drug delivery agent for the treatment of diseases.

The present invention also relates to a membrane-type topical drug for treating periodontal disease, which contains one or two or more components selected from the group consisting of urodesoxycholic acid, kenodesoxycholic acid, and taurusodesoxycholic acid as an active ingredient. It is about a delivery system.

The invention is explained in more detail below.

As an active ingredient in the topical delivery agent of the present invention, any one component of urodesoxycholic acid, kenodesoxycholic acid, and its derivatives, taurusodesoxycholic acid, may be used alone or two or more components may be used. Use by mixing. The content of the active ingredient is suitably in a ratio of 0.0004 to 5% by weight, preferably 0.005 to 1% by weight based on the total composition. If the content of the active ingredient is less than 0.0004% by weight, it is not suitable because the desired level of efficacy and effect for gingival inflammation cannot be expected, and if it exceeds 5% by weight, the safety of the product may be lowered.

Ointment when the one or two or more components selected from the group consisting of urodesoxycholine acid, kenodesoxycholine acid and taurusodesoxycholine acid as an active ingredient according to the present invention to be formulated into an ointment Can be prepared in the following composition.

Ointments according to the present invention, in addition to the active ingredients as mentioned above, may also include wetting agents, drug delivery systems, stabilizers, solubilizers, preservatives, sweeteners, buffers, flavoring and coloring ingredients.

In detail, the ointment formulation of the composition of the present invention uses a humectant to maintain state and prevent drying. Such humectants include glycerin, sorbitol liquid, polyethylene glycol and propylene glycol, carboxymethylcellulose, and poloxamer (Poloxamer 407). And one component selected from monoglycerides (Myverol 18-99) or a mixture of two or more components is preferably used in a ratio of 5 to 40% by weight, based on the total weight of the ointment. In the case where polyethylene glycol is used as the wetting agent, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600 or polyethylene glycol 1000 can be used as polyethylene glycol. In addition, the drug delivery system that can effectively deliver the active ingredient to the oral soft tissue is preferably used in a ratio of 1 to 30% by weight, based on the total weight of the ointment alone or a mixture of gelatin or pectin, respectively. Solubilizers are also used in the ointments of the present invention, preferably for this purpose lower alcohols such as ethanol or isopropanol, respectively or in combination, in a ratio of 1 to 20% by weight, based on the total weight of the ointment. As a stabilizer for the state stability of the ointment, a pluronic derivative such as Pluronic F-127 or Pluronic F-180, which is a surfactant, may be used in a ratio of 5 to 30% by weight based on the total weight of the ointment. Buffers for pH adjustment include alkali metal salts of regular phosphoric acid, especially groups consisting of sodium phosphate dibasic, sodium phosphate dibasic, sodium triphosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide, sodium pyrophosphate and pyrophosphate One or two or more components selected from among them can be used. In the ointment of the present invention, particularly preferably, as a buffer, two kinds of components of the first sodium phosphate, the second sodium phosphate and the third sodium phosphate are properly mixed and used in an amount suitable to adjust the pH to 5.0 to 8.0. In addition, flavors and sweeteners are used to control the bitter or somewhat bitter taste of the ointment. The flavors are mainly used in natural flavors such as peppermint and spearmint oil. As the sweetening agent, synthetic or natural non-fermentable sugars are mainly used, and representative examples thereof include saccharin sodium, aspartame, lactose, maltose and ziitol. In particular, it is preferable to use saccharin sodium as a sweetener in a ratio of 0.05 to 1% by weight based on the total weight of the ointment. The ointment of the present invention is also added with a preservative for the purpose of preventing the contamination of microorganisms that may occur during the manufacture and use of the ointment, for this purpose methyl paraoxybenzoate, paraoxy generally licensed for use in foods and pharmaceuticals One or two or more components selected from the group consisting of propyl benzoate, benzoic acid, sodium benzoate, salicylic acid and the like are used in a ratio of 0.01 to 0.5% by weight of the total weight of the ointment. In addition, an appropriate food coloring is added to the ointment of the present invention for the purpose of improving the appearance of the ointment.

The ointment according to the present invention is mixed with ingredients such as pharmacologically active ingredients, wetting agents, drug delivery systems, stabilizers, solubilizers, preservatives, sweeteners, flavoring agents, pigments and the like according to conventional methods in the pharmaceutical field, and the mixture is buffered. It can be prepared by a method of dissolving in a gel and gelling.

Topical delivery agents according to the invention can also be prepared in the form of membrane-type topical drug delivery agents.

In the case of a membrane-type topical drug delivery agent, the active ingredient may be prepared in a membrane form by mixing with a drug release controlling ingredient that controls the release rate and sustained release of the active ingredient. A drug release controlling ingredient for this may be a polymer such as hydroxypropyl cellulose, ethyl cellulose, etc., preferably 5 to 95 weight based on the total weight of the membrane-type preparation of hydroxypropyl cellulose or ethyl cellulose or a mixture thereof It is suitable to contain in ratio of%. The membrane-type topical drug delivery agent of the present invention preferably contains 70% to 90% of hydroxypropylcellulose and 5% to 30% of ethyl cellulose in an appropriate ratio of dry hydroxypropylcellulose and ethylcellulose. After mixing uniformly in proportion, the active ingredient as defined above is added in the ratio of the predetermined range, homogeneously mixed, and pressed to prepare a film having a thickness of 100 to 500 µm. The membrane-type topical drug delivery agent is prepared to have a form that can be easily inserted into the periodontal disease site and attached for a long period of time, and is preferably formed into a trapezoidal shape with rounded corners as shown in FIG. 1.

The present invention will be described in detail by the following examples and comparative examples, but these examples are provided to aid the understanding of the present invention, and the technical scope of the present invention is not limited by them in any way.

Example 1-8 and Comparative Example 1-8

Using the ingredients as shown in Tables 1 to 4, the active ingredient is mixed with a humectant, a drug delivery system, a stabilizer, a solubilizer, a preservative, a sweetener, a flavoring agent, and a pigment according to a conventional ointment preparation method. The ointment was prepared by dissolving, adding residual amount of water and gelling.

TABLE 1

Ointment compositions of Examples 1-2 and Comparative Examples 1-2 (unit: wt%)

division ingredient Example 1 Example 2 Comparative Example 1 Comparative Example 2 Humectant glycerin 5 5 Polyethylene Glycol 200 5 5 Carboxymethylcellulose Poloxamer 407 0.1 0.1 Monoglycerides (Myverol 18-99) 10 15 10 15 Drug Delivery System gelatin 5 5 5 5 pectin 5 5 5 5 Stabilizer Pluronic F-127 20 20 Pluronic F-108 20 20 Solubilizer ethanol 5 5 Isopropanol 5 5 antiseptic Methyl paraoxybenzoate 0.1 0.1 0.1 0.1 Paraoxy Benzoate 0.05 0.05 0.05 0.05 Sweetener Zaccarin Sodium 0.1 0.2 0.1 0.2 Buffer Monosodium phosphate 0.1 0.2 0.1 0.2 Trisodium Phosphate 0.05 0.05 0.05 0.05 Active ingredient Ursodesoxycholic acid 0.0004 0.005 Kenodes oxycholine acid 0.0004 0.005 Taurusodesoxycholic acid 0.0004 0.005 Spices 0.75 0.75 0.75 0.75 Food coloring 0.0002 0.0002 Purified water Remainder Remainder Remainder Remainder

TABLE 2

Ointment compositions of Examples 3-4 and Comparative Example 3-4 (unit: wt%)

division ingredient Example 3 Example 4 Comparative Example 3 Comparative Example 4 Humectant glycerin 10 10 Polyethylene Glycol 400 5 5 Carboxymethylcellulose 0.5 0.1 0.5 0.1 Poloxamer 407 10 10 Monoglycerides (Myverol 18-99) 20 20 Drug Delivery System gelatin 3 2 3 2 pectin 3 2 3 2 Stabilizer Pluronic F-127 15 15 Pluronic F-108 15 15 Solubilizer ethanol 5 5 Isopropanol 5 5 antiseptic Methyl paraoxybenzoate 0.1 0.1 0.1 0.1 Paraoxy Benzoate 0.05 0.05 0.05 0.05 Sweetener Zaccarin Sodium 0.1 0.2 0.1 0.2 Buffer Monosodium phosphate 0.1 0.2 0.1 0.2 Trisodium Phosphate 0.05 0.05 0.05 0.05 Active ingredient Ursodesoxycholic acid 0.01 0.1 Kenodes oxycholine acid 0.01 Taurusodesoxycholic acid 0.1 Spices 0.75 0.75 0.75 0.75 Food coloring 0.0002 0.0002 Purified water Remainder Remainder Remainder Remainder

TABLE 3

Ointment compositions of Examples 5-6 and Comparative Examples 5-6 (unit: weight%)

division ingredient Example 5 Example 6 Comparative Example 5 Comparative Example 6 Humectant glycerin 15 15 Polyethylene Glycol 200 5 5 Carboxymethylcellulose 0.5 0.5 Poloxamer 407 5 One 5 One Monoglycerides (Myverol 18-99) 5 5 Drug Delivery System gelatin One 10 One 10 pectin One 5 One 5 Stabilizer Pluronic F-127 10 10 Pluronic F-108 10 10 Solubilizer ethanol 5 5 Isopropanol 5 5 antiseptic Methyl paraoxybenzoate 0.1 0.1 0.1 0.1 Paraoxy Benzoate 0.05 0.05 0.05 0.05 Sweetener Zaccarin Sodium 0.1 0.2 0.1 0.2 Buffer Monosodium phosphate 0.1 0.2 0.1 0.2 Trisodium Phosphate 0.05 0.05 0.05 0.05 Active ingredient Ursodesoxycholic acid 0.5 One Kenodes oxycholine acid One Taurus-Lesodes oxycholine acid 0.5 Spices 0.75 0.75 0.75 0.75 Food coloring 0.0002 0.0002 Purified water Remainder Remainder Remainder Remainder

TABLE 4

Ointment compositions of Examples 7-8 and Comparative Examples 7-8 (unit: weight%)

division ingredient Example 7 Example 8 Comparative Example 7 Comparative Example 8 Humectant glycerin 20 20 Polyethylene Glycol 400 5 5 Carboxymethylcellulose Poloxamer 407 10 10 Monoglycerides (Myverol 18-99) 20 20 Drug Delivery System gelatin 5 20 5 20 pectin 5 5 5 5 Stabilizer Pluronic F-127 5 5 Pluronic F-108 5 5 Solubilizer ethanol 10 10 Isopropyl Alcohol 10 10 antiseptic Methyl paraoxybenzoate 0.1 0.1 0.1 0.1 Paraoxy Benzoate 0.05 0.05 0.05 0.05 Sweetener Zaccarin Sodium 0.1 0.2 0.1 0.2 Buffer Monosodium phosphate 0.1 0.2 0.1 0.2 Trisodium Phosphate 0.05 0.05 0.05 0.05 Active ingredient Ursodesoxycholic acid 2 5 Kenodes oxycholine acid Taurusodesoxycholic acid 2 5 Spices 0.75 0.75 0.75 0.75 Food coloring 0.0002 0.0002 Purified water Remainder Remainder Remainder Remainder

Example 9-16 and Comparative Example 9-16

Using each component in the amount as described in Table 5 below, hydroxypropyl cellulose and ethyl cellulose are mixed, and the active ingredients are uniformly mixed therein and then compressed into a specified thickness using a compactor, as shown in FIG. The trapezoidal membrane-like preparation was prepared by cutting to the size as shown and rounding the four corners.

TABLE 5

Ointment compositions of Example 9-16 and Comparative Example 9-16 (unit: wt%)

division Ursodes oxycholine acid Kenodes oxycholine acid Taurusodesoxycholic acid Hydroxypropylcellulose Ethylcellulose Thickness (㎛) Example 9 0.0004 0.0004 0.0004 70 29.99 100 Example 10 0.005 0.005 0.005 75 29.95 200 Example 11 0.01 - 0.01 80 19.9 200 Example 12 0.1 0.1 0.1 85 14.5 200 Example 13 0.5 - 0.5 86 13 300 Example 14 One One One 87 11 300 Example 15 2 - 2 88 9 400 Example 16 5 - 5 90 5 500 Comparative Example 9 - - - 70 29.99 100 Comparative Example 10 - - - 75 29.95 200 Comparative Example 11 - - - 80 19.9 200 Comparative Example 12 - - - 85 14.5 200 Comparative Example 13 - - - 86 13 300 Comparative Example 14 - - - 87 11 300 Comparative Example 15 - - - 88 9 400 Comparative Example 16 - - - 90 5 500

Experimental Example 1 Inhibitory Effect on Superoxide Production

The venous blood collected from citric acid as an anticoagulant from a healthy adult without centrifugation was centrifuged at 1200 rpm for 10 minutes, and then the leukocyte concentrate of the middle layer was recovered, and the ratio was 1: 1 with RPMI 1640 medium. Diluted. 12 ml of Ficoll-Paque was added to a 50 ml centrifuge tube, and 30 ml of the diluted blood was carefully added to form a middle layer, followed by centrifugation at 1600 rpm for 30 minutes, and the upper layer containing serum. After removing the monolayer containing the mononuclear cells with RPMI 1640 medium carefully, add 3 times of RPMI 1640 medium, centrifuge at 800 rpm for 10 minutes, discard the supernatant and add 10 ml of RPMI 1640 medium and gently Pipetting was followed by centrifugation at 800 rpm for 10 minutes. Discard the supernatant and pipet with HBSS (HANKS 'BALANCED SALT SOLUTION) buffer, and then divide the human mononuclear leukocytes into 0.5-well wells at a concentration of 10 ⁶ cells / well in a 24-well plate as shown in Table 6. After aseptically incubated for 2 hours under% air, 5% CO ₂ , 100% humidity, 0.1 mL Cytochrome C to 80 μM, 0.1 mL Superoxide dismutase to 30 μg, 0.1 ml of the ointment composition of the Example and Comparative Example diluted three-fold with HBSS was added, HBSS was added so that the remaining total reaction solution was 0.9 ml, and then maintained at 37 ° C. for 10 minutes, and the stimulated phagocytized Zymosan ( Zymosan) A was added 0.1 ml so that the final concentration was 1.3 mg / ml, and the mixture was kept at 37 ° C. for 90 minutes while shaking. Then, the reaction was stopped for 10 minutes in a 4 ° C. refrigerator, and then stopped at 4 ° C. and 1500 rpm for 10 minutes. Higher at 550 nm after centrifugation Of the emission of the measured absorbance, and the superoxide anion was calculated by the food.

O ^-2 = 10 ^-3 (nanomol / 10 ^-6 cells, minute)

* △ O.D. = (B-D)-(A-C) = (B + C)-(D + A)

A: absorbance of A well

B: absorbance of the B well

C: absorbance of C wells

D: absorbance of the D well

TABLE 6

Overview of Experimental Methods for Superoxide Determination

division A well B well C well D well Mononuclear leukocytes 0.5 ml 0.5 ml 0.5 ml 0.5 ml Cytochrome C 0.1 ml 0.1 ml 0.1 ml 0.1 ml SOD - - 0.1 ml 0.1 ml Zymosan - 0.1 ml - 0.1 ml Experimental group - 0.1 ml - - HBSS 0.4 ml 0.2 ml 0.3 ml 0.2 ml total 1.00 ml 1.00 ml 1.00 ml 1.00 ml

TABLE 7

Experimental results of superoxide formation inhibition effect of ointment composition (unit: nanomole / 10 ⁶ cells / min)

Ointment composition Example Comparative Example One 7.57 8.47 2 5.42 8.47 3 1.52 8.47 4 0.75 8.51 5 0.78 8.50 6 0.77 8.47 7 0.76 8.45 8 0.76 8.48

As can be seen from the results described in Table 7, the superoxide production inhibitory effect was found to be increased as the concentration of the active ingredient is increased, the content of the active ingredient is 0.1% or more Examples 4, 5, 6 , 7, 8 ointment was excellent in inhibiting superoxide production.

Experimental Example 2: Inhibitory effect on collagenase enzyme activity

In order to determine whether the ointment prepared according to the present invention has an inhibitory effect on collagenase, which is a periodontal tissue degrading enzyme, the experiment was conducted according to the following method.

This study was performed to investigate the secretion of bacterioides gingivalis , polymorphonuclear leukocytes, and neutrophils, which are found in the saliva and gingival fluid of patients with periodontal disease. It is an experimental method that mimics the oral environment in which gum degeneration occurs due to the breakdown of collagen, which is the substrate of periodontal tissue, by the action of collagenase enzyme.

Add 100 μl of 2% Azocoll solution, a red collagen substrate, to 21 1.5 ml Eppendorf tubes, using one Eppendorf tube as blank and three tubes from Sigma. One collagenase type I standard enzyme solution (collagen degrading activity: 315 units / mg) was added to a concentration of 10, 100, 200 ppm, and each of the remaining tubes was crushed from saliva and gingival sac of patients with periodontal disease. 100 μl of collagenase enzyme purely separated by Acrylac S-200 chromatography was added, and another tube was used as a control, and each of the remaining tubes was treated with experimental ointment (Examples 1 to 8). Comparative ointment (Comparative Examples 1 to 8) was mixed with distilled water in a ratio of 1: 2, homogenized completely, and 10 µl of the supernatant obtained by centrifugation at 5000 g for 10 minutes was added, followed by buffer solution (0.05M Tris-HCl, 1 nM CaCl ₂ , pH 7.8) was added to 500 μl of the total reaction solution and reacted for 18 hours at 37 ° C. incubator. Centrifuge each Eppendorf tube at 10000 g for 5 minutes to precipitate undigested collagen, take supernatant containing digested collagen, measure absorbance at 540 nm, create a standard activity curve, and prepare enzymes from the resulting standard curve. By comparing the activity concentration of the enzyme activity of the experimental group and the control group in comparison to evaluate the results as shown in Table 8 below.

TABLE 8

Inhibitory effect of collagenase activity (unit:% = enzyme activity of experimental group ÷ enzyme activity of control group X100)

Ointment composition One 2 3 4 5 6 7 8 Comparative example 98 94 93 95 96 95 97 94 Example 88 69 61 51 53 3 2 2

As can be seen from the experimental results described in Table 8, the inhibitory effect on the collagenase activity by the ointment of Examples 1, 2, 3, 4, 5, 6, 7 and 8 is the concentration of the active ingredient It was shown to increase with increase, and the collagenase activity was completely inhibited by the ointment of Examples 6, 7, 8, wherein the content of the active ingredient is 1% or more of the total weight of the composition.

Experimental Example 3: Inhibitory effect on the production of interleukin (IL-1β) in mononuclear leukocytes

0.8 ml of blood mononuclear leukocytes isolated from healthy healthy blood were added to a 24-well plate and aliquoted to 10 ⁶ cells / well, and the wells to which 200 μl of RPMI 1640 medium were added were added to the control, E. coli. The wells to which 100 μl of E. Coli LPS (250ppm) was added and 100 μl of ointment diluted three-fold with 100 μl of LPS (250 ppm) and RPMI 1640 medium were incubated for 24 hours. 50 µl of arachidonic acid was added to incubate for 30 minutes. To a well of a 96-well plate to which an antibody of interleukin (IL-1β) is attached, 50 μl of IL-1β standard solution (0, 10.24, 25.6, 64, 160, 400 pg / well) was added to the well of the experimental group. 50 μl of cell culture medium was added, and 50 μl of biotinylated antibody reagent was added to all wells, and then maintained at 25 ° C. for 3 hours. M phosphate buffer, pH 7.5), and washed three times, streptavidin (Streptavidin) -HRP conjugate was added to all wells 100μL was maintained again at 25 ℃ for 30 minutes. Again wash three times with the same wash buffer solution and immediately add 100 [mu] l of enzyme substrate [TMB (3,3 ', 5,5'-tetramethylbenzidine) -hydrogen peroxide solution]. The solution was kept open for 30 minutes, and 100 μl of 0.18M sulfuric acid was added, and the absorbance was measured at 450 nm using a microplate reader. A standard curve was prepared using the absorbance value of the standard solution. Calculated.

TABLE 9

Inhibitory Effect of Ointment Composition on IL-1β Production (Unit: pg)

Ointment composition Example IL-1β Comparative Example IL-1β One 57 289 2 56 284 3 54 276 4 48 276 5 21 285 6 18 286 7 12 284 8 11 286

As can be seen from the results described in Table 9 above, The inhibitory effect of the ointment of Examples 1, 2, 3, 4. 5, 6, 7, 8 on the production of IL-1β of human mononuclear leukocytes stimulated with E. coli LPS was increased as the concentration of the active ingredient increased. appear.

Experimental Example 4: Inhibitory effect on prostaglandin (PGE ₂ ) production of monocytes

Using the ointment according to the present invention was tested the inhibitory effect on the production of prostaglandin, a gingival inflammation-inducing agent by the following method.

In this test method, prostaglandins (PGE ₂ ) induced by stimulating human mononuclear leukocytes with lipopolysaccharide (LPS), a cell wall component of Bacteroides gingivalis, a disease-causing bacterium during periodontal disease progression. The supernatant of the experimental group ointment (Examples 1 to 8) and the comparative group ointment (Comparative Examples 1 to 8) were mixed with distilled water in a ratio of 1: 2 and completely homogenized and centrifuged at 5000 g for 10 minutes. After treatment, comparison and evaluation were performed by measuring the inhibitory effect by immuno-diagnosis of the antigen-antibody. The specific experimental method is as follows.

50 µl of buffer solution (0.1 M phosphate buffer solution containing 0.9% NaCl, 0.1% bovine serum albumin, 0.5% Kathon) in a blank well of a 96-well plate to which 1 gG of chlorine anti-mouse was attached 50 μl of PGE ₂ standard solution of appropriate concentration (0, 2.5, 5, 10, 20, 40, 80, 160, 320 pg) was added to the standard well, and the experimental group ointment prepared as described above Examples 1 to 8) and 50 μl of the comparative group ointment (Comparative Examples 1 to 8) were added to the wells set as the example group and the comparative group, and 50 μl of the antibody against PGE ₂ was added to all the wells except the blankwell. 50 μl of PGE ₂ conjugate peroxidase was added to all wells except the blankwells. Cover the 96 well-plate and hold at 25 ° C. for 1 hour, wash four times with washing buffer solution (phosphate buffer solution containing 0.05% Tween 20: pH 7.5), and wash with enzyme substrate (20% dimethylformamide at room temperature). 150 μl of dissolved 3,3 ′, 5,5′-tetramethylbenzidine / hydrogen peroxide) were added immediately, held at 25 ° C. for 30 minutes, and 100 μl of 1M sulfuric acid was added, followed by absorbance at 450 nm with a microplate reader. Measured. A standard curve was prepared from the absorbance values of the standard solution, and the amount of prostaglandin produced in each experimental group was calculated from the standard curve. The measured results are shown in Table 10 below.

TABLE 10

Inhibitory Effect of Ointment Composition on PGE ₂ Production (Unit: pg)

Ointment composition One 2 3 4 5 6 7 8 Comparative Example 37 39 43 38 40 41 42 38 Example 10.9 8.7 8.5 8.4 8.4 4.8 2.7 2.7

As can be seen from the results shown in Table 10, all the ointments of the examples containing 0.0004% or more of the active ingredient. It showed an excellent inhibitory effect on PGE ₂ production of human mononuclear leukocytes stimulated with E. coli LPS.

Experimental Example 5: Clinical Experiment

Using the ointment of the present invention, the clinical effect on the treatment of periodontal disease was measured by the following experimental method.

The subjects were periodontal disease patients with even orthodontic teeth and no defects, and 160 patients were selected from the age group of 30 to 50 years old. Divided into two groups of people, one group was to use the experimental group ointment (ointments of Examples 1 to 8, 10 people for each ointment), the remaining one group is a comparative group ointment (comparative examples 1 to 8 ointments, 10 for each ointment) Persons). First, the subjects of each group were subjected to dentifrice scalp to score the initial gingivitis index, and the experimental group ointment and the comparison group ointment were fed twice a day (0.1 wt% / 1 times), that is, after breakfast and before sleeping. One week and one month later, oral examination was performed to check the gingivitis index. The gingivitis index is scored based on the criteria as shown in Table 11 by inserting a periodontal probe into the gingival sulcus and probing continuously around each tooth without applying force and measuring bleeding after 30 seconds. Was obtained by recording. The measured results are shown in Table 12 below.

TABLE 11

Gingivitis index

score Contents 0 points A bleeding state 1 point A bleeding condition 2 points Glandular bleeding 3 points Interdental triangle bleeding 4 points Total gingival bleeding

TABLE 12

Clinical trial results on treatment effect of periodontal disease (unit: gingivitis index)

Lead solid composition Early 1 week 2 weeks Example Comparative example Example Comparative example Example Comparative example One 1.04 1.05 1.72 2.42 2.02 3.29 2 1.03 1.07 1.62 2.38 1.80 3.35 3 1.05 1.05 1.54 2.41 1.74 3.42 4 1.06 1.06 1.47 2.39 1.61 3.45 5 1.07 1.06 1.24 2.34 1.34 3.38 6 1.04 1.05 1.30 2.36 1.32 3.43 7 1.06 1.04 1.25 2.37 1.32 3.45 8 1.05 1.07 1.21 2.41 1.32 3.47

As can be seen from the results shown in Table 12, the present invention can inhibit the production of IL-1β and prostaglandin and superoxide that cause gingivitis and inhibit the enzyme activity of collagenase that degrades periodontal tissues. In the ointment (Examples 1 to 8) according to the gingival inflammation treatment effect was shown to be much better than the ointment (1 to 8) of the comparative example from 1 week to 1 month. That is, in the comparative example, the gingivitis index increased continuously as time passed from 1 month, whereas in the case of using the ointment according to the present invention, the inhibitory effect of the periodontal disease increased as the content of the active ingredient increased, and gingivitis It was found that the index is significantly reduced over time, in particular, the effect of inhibiting periodontal disease of the ointment of Examples 2 to 8 containing 0.015% or more of the active ingredient was very excellent.

Experimental Example 6 In Vivo Release Test and Clinical Experiment of Membrane Topical Drug Delivery Agent

The release and treatment effect of periodontal disease of the membrane-type topical drug delivery agent according to the present invention was measured by the following in vivo release test and clinical experimental method.

Subjects were selected for 120 oral subjects from 30 to 50 years of age by age 30 years from 30 to 50 years of age. Initial gingivitis index by dividing 60 people into the membrane local drug delivery group (experimental group) of Examples 9 to 16 according to the invention and the membrane local drug delivery group (comparative group) of Comparative Examples 9 to 16, and then performing dentifrice Scored. Then, each group was supplied with experimental group membranous topical drug delivery agent and comparative group membranous topical drug delivery agent and inserted into the periodontal sac, and the content of the active ingredient released in the periodontal sac at intervals of 1, 6, 24 hours, 3, 7 days was measured. Derivatization was performed to quantitatively analyze by high performance liquid chromatography (HPLC). Meanwhile, each membrane-type topical drug delivery agent was used at three-day intervals, and oral examination was performed one week and one month later. The measurement method of gingivitis dimension is to insert the periorental probe into the gingival bulb and continuously probe each tooth without applying force and measure the bleeding state after 30 seconds and measure the score according to the criteria as shown in Table 11 above. Recorded. The measured results are shown in Tables 13 and 14, respectively.

TABLE 13

Test results of periodontal release of membrane-type topical drug delivery (unit: ㎍ / ㎖)

division 1 hours 6 hours 24 hours 3 days 7 days Example Comparative example Example Comparative example Example Comparative example Example Comparative example Example Comparative example 9 6.5 0 6.3 0 5.7 0 4.2 0 2.4 0 10 92 0 89 0 73 0 65 0 13 0 11 176 0 173 0 145 0 121 0 32 0 12 827 0 860 0 982 0 745 0 42 0 13 4524 0 4024 0 3024 0 2024 0 54 0 14 9576 0 8745 0 6840 0 5210 0 63 0 15 14800 0 12200 0 9390 0 6310 0 65 0 16 45240 0 43840 0 39020 0 12280 0 72 0

As can be seen from the results described in Table 13, the membrane-type topical drug delivery agent according to the present invention was inserted into the periodontal sac to measure the content of the active ingredient released in the periodontal sac, and as a result, the urine was inserted until 1, 6, 24 hours. Since the content of sodeoxycholine acid increases and then decreases on the third day and rapidly decreases on the seventh day, the membrane-type topical drug delivery agent of the present invention has been shown to be effective in treating periodontal disease by using a new one every three days.

TABLE 14

Treatment of Periodontal Disease with Membrane Topical Drug Delivery (Unit: Gingivitis Index)

Lead solid composition Early 1 week 1 month Example Comparative example Example Comparative example Example Comparative example 9 1.05 1.07 1.52 1.40 2.05 3.44 10 1.04 1.06 1.46 1.37 1.73 3.43 11 1.02 1.08 1.45 1.38 1.62 3.48 12 1.05 1.08 1.34 1.37 1.38 3.49 13 1.04 1.07 1.24 1.26 1.34 3.45 14 1.03 1.05 1.28 1.29 1.32 3.44 15 1.06 1.06 1.22 1.27 1.32 3.45 16 1.07 1.04 1.20 1.25 1.32 3.46

As can be seen from the results described in Table 14 above, urosin capable of inhibiting the production of IL-1β and prostaglandins and superoxide that causes gingivitis and inhibiting the enzymatic activity of collagenase that degrades periodontal tissues. Treatment effect of gingival inflammation of the membrane-type topical drug delivery agent (Examples 9 to 16) of the present invention containing an active ingredient such as desoxycholine acid did not contain ursodeoxycholic acid from one week to one month. The efficacy was shown to be superior to that of the membrane-type topical drug delivery agents (Comparative Examples 9-16). The gingivitis index of the comparative example is continuously increased with time after one month, whereas in the case of using the preparation according to the present invention, the inhibitory effect of periodontal disease increases as the content of the active ingredient in the preparation increases. Even after this, the gingivitis index was remarkably decreased, and in particular, the inhibitory effect of the periodontal disease of the formulations of Examples 10 to 16 containing 0.015% or more of the active ingredient was very excellent.

Claims (12)

Delivery of topical drug for the treatment of periodontal disease, characterized in that it contains one or two or more ingredients selected from the group consisting of urodesoxycholine acid, chinodesoxycholine acid, and taurosoledecoxycholine acid as active ingredients Formulation. The topical drug delivery agent for treating periodontal disease according to claim 1, wherein the active ingredient is contained in an amount of 0.0004 to 5% by weight based on the total weight of the preparation. The topical drug delivery agent according to claim 1, which is formulated in the form of an ointment. The topical drug delivery agent for treating periodontal disease according to claim 3, further comprising an ointment in which a humectant, a drug delivery system, a stabilizer, a solubilizer, a preservative, a sweetener, a buffer, a perfume, and a pigment are combined. The method according to claim 3, wherein one or two or more components selected from glycerin, sorbitol solution, polyethylene glycol, propylene glycol, carboxymethylcellulose, poloxamer, and monoglyceride as a humectant, based on the total weight of the formulation, is 5 to 40 weight. Topical drug delivery for the treatment of periodontal disease in the form of an ointment in the ratio of%. The topical drug delivery agent for treating periodontal disease according to claim 3, wherein the drug delivery system is in the form of an ointment containing gelatin and pectin or a mixture thereof in a ratio of 1 to 30% by weight based on the total weight of the preparation. The topical drug delivery agent for treating periodontal disease according to claim 3, which is in the form of an ointment containing a lower alcohol solvent as a solubilizer in a ratio of 1 to 20% by weight based on the total weight of the preparation. 4. The component according to claim 3, wherein the buffer is one or more selected from sodium sodium phosphate, sodium phosphate dibasic, sodium triphosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate. A topical drug delivery agent for treating periodontal disease in the form of an ointment in which the pH value is adjusted to about 5.0 to 8.0 by the compounding. The topical drug delivery agent for treatment of periodontal disease according to claim 1, which is formulated in the form of a membranous topical drug delivery agent. 10. The method for treating periodontal disease according to claim 9, wherein the drug release control component is hydroxypropyl cellulose or ethyl cellulose, or a mixture thereof in a ratio of 5 to 95% by weight based on the total weight of the preparation. Topical drug delivery agents. The topical drug delivery agent for treating periodontal disease according to claim 9, which is a membrane-type preparation having a film thickness of 100 to 500 µm. The topical drug delivery agent for treating periodontal disease according to claim 9, which is a membranous agent having a rounded trapezoidal shape.