KR100205968B1 - Thermal composition comprising lysozyme and method for preparation thereof - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a skin solvent containing lysozyme as a main component and a method for producing the same, wherein the lysozyme or salt thereof, an aliphatic base, an emulsifier (HLB 8.0-13.5), a co-emulsifier (HLB 10-13), a chelating agent, a humectant And a method of producing an ointment or cream such as a cream or ointment containing 0.1-10% by weight of a water-in-oil emulsified (W / O) lysozyme by adding a matrix agent and maintaining a pH of 5.0 ± 2 in a buffer solution. According to the invention, the lysozyme is stabilized, so that the preservation of the product is good, the skin may be moisturized and moisturized, and the effect of anti-inflammatory is maintained as it is.

Description

Outer sheath containing lysoli and its manufacturing method

FIELD OF THE INVENTION The present invention relates to a skin preparation containing lysosomes as a main component and a method for manufacturing the same, and particularly to a skin preparation having good penetration and no skin irritation, such as ointments, warnings, gels and patches, etc. And a method for preparing the same, wherein the pharmaceutical composition is stable.

Lysozyme is a polymer (molecular weight: 14,000 ± 100) that is obtained from chicken egg white and is a muco-enzyme that is distributed in vivo to promote the hydrolysis of polysaccharides. Known as saccharides or peptides, it has mainly the characteristics of basic polypeptides, and has non-polar amino acid side chains and SS bonds in its molecule, and its isoelectric point is 11.0, which is unstable at pH 9.5 and above, so it is relatively stable in acid salts. As a result, HEW lysozyme is preserved as a lysozyme chloride compound, but the formulation is lysozyme depending on the medium used, its liquid and the type and amount of the surfactant, and the nature of the environmental substance for the lysozyme. Depends on its activity. Therefore, in order to have the proper efficacy, it is necessary to fully consider the stability of the biological improvement of the skin, the redox system and the application site according to the environmental substances, and the interference of the hydrolase of the polypeptides.

The pharmacological action of lysozyme and its salts as antimicrobial agent is lysine, anti-inflammatory, hemostatic action, etc. It is known to repair damaged tissues. Especially in the latter study, the expectation of redox effect by cysteine is recently Research is ongoing.

However, it is known that the main formulation of pharmaceutical products containing lysozyme as a single component is mainly powder, granule, tablet, capsule, etc. Cosmetics include cream, jelly and rinse. Therefore, despite the feasibility of the development of a skin coating formulation applied directly to the skin in anticipation of the efficacy of the lysozyme for the purpose of treatment by bacterial diseases and tissue repair action of the skin described above, the skin of the lysozyme single component No formulation has been found so far. It is believed that this is due to the stability of the semi-solid formulation, and to date, reports on the stability of lysozyme have been reported as stabilization of the lysozyme itself as hydrochloride, carbonic acid, phosphoric acid, hexametaphosphate, sulfuric acid, glutamic acid or gluconic acid as its salt type. (JPA₂ small 63-174935), pH 4.0-7.0 (eyedropper pH 6.0) in liquid phase is known as the stabilization method of the formulation, although phytic acid for the purpose of maintaining the inclusion state as a stabilizer of aqueous formulations (phytic acid), there is a method of forming a complex by the polyoxyethylene fatty acid ether (emulsion) as an emulsifier (JPA₂ 평 4-26630), glycine (glycin) for the formation of chelate (chelate) , Amino acids such as alanine, disodium EDTA and sugar-phosphate have been reported (JP A₂ 평 1-283206), but glycine as a desiccant for moisture preservation to prevent drying of the product There is a drawback that serine, sudang, etc. are additionally used, and no semi-solids have been studied as a therapeutic agent such as a special stabilization method or an ointment for the skin preparation.

It is an object of the present invention to compensate for the deficiencies as described above, and to meet the development and physiology of the skin, while the ointment for the skin having the effect of enabling transdermal absorption of lysozyme and promoting the repair of damaged tissue and its The present invention aims to provide a new skin preparation that is stable in lysozyme and maintains its efficacy as a skin external agent by using a creamy emulsion ((O / W and W / O) as a preferred W / O type. In addition to stabilization, we have developed a product that maintains pH 5.0 ± 2 in consideration of the preferred liquidity (pH 3.5 to 6.5) for maintaining the skin's improvement, and in addition several improvements to the additives for stabilizing the formulation. Research and development of new formulations for which stability has been established and filed as patents.

The skin solvent of the present invention contains 0.1-10% by weight of a water-in-oil emulsified lysozyme containing a lysozyme or a salt thereof, an aliphatic base, an emulsifier, an auxiliary emulsifier, a chelating agent, a humectant, and a matrix agent at a pH of 5.0 ± 2. As the outer solvent, the preparation method is characterized by adding a lysozyme or a salt thereof, an aliphatic base, an emulsifier, a co-emulsifier, a chelating agent, a moisturizing agent and a matrix agent to maintain a pH of 5.0 ± 2 with a buffer and emulsify at 10 ~ 80 ℃ The outer skin solvent which contains 0.1-10 weight% of lysozyme of the water-in-oil emulsified type (W / O) which is pharmaceutical form stable is prepared.

The present invention provides a particularly strong chelating agent, alpha-gettogluramine, soy protein-based chelalte, lysine and cysteine for the stabilization of lysozyme. The stability of lysozyme chloride was increased with polyamihooacid suitable for the structure of lysozyme, not Sibgle amibno acid such as glutathione and glutathione.

On the other hand, the use of hyaluronic acid, kojic acid, or derivatives thereof, which is an inorganic salt-free salt as an acid salt of lysozyme, is not only stabilized by acidification than inorganic salts such as hydrochloride, but also moisturizes the skin. And it is effective in improving the shelf life of a product, and it is preferable at the point of water absorption.

At this time, the rapid transdermal absorption of lysozyme is expected to occur when the drug comes into contact with the skin, so it is necessary for the antiviral effect or safety. To do this, the selection of the emulsifier and the matrix agent is necessary. It must be designed.

In the present invention, the fatty base refers to conventional fats and fats, and may include all of the substances disclosed in the contract or the Cosmetic Toilet Fragrance Association (CTFA), and examples thereof include white petrolatum, liquid paraffin, cetyl alcohol, and stearyl alcohol. , Stearic acid, and the like can be used, and scylene and the like can be added.

In the present invention, the emulsifier belongs to natural lipids and synthetic emulsifiers, and natural lipids include ceramides, sphingolipids, phospholipids, etc., and synthetic emulsifiers are isopropyl adipate or lipophilic glycerin mono C _12-18 dissipation ester _{(glycerin mono-C 12-18 farry acid} esters) or its alkyl ether (alkul ethers), sucrose mono C _12-18 fatty acid ester _{(sucrose mono-C 12-18 fatty acid} esters) and PEG Or known nonionic surfactants such as fatty acid esters of PPG, but in addition to the preparation of emulsions, in the present invention, the stabilization of the lysozyme, interaction with the living body and absorption or improvement should be maintained. In the alkyl ether, HLB (hydrophilic lipophilic balance) value is 2.5 to 18.0, and the preferred one is selected in the range of 8.0 to 13.5. Ethel type is more preferable than stell type structure.

Specific examples of the compound usable as the emulsifier of the present invention include glycerol mono or distearate, propylene glycol mono or distearate, sorbitan mono or dioleate, propylene glycol mono or dilaurate and sorbitan stearate. , Diethylene glycol monostearate, diethylene glycol monolaurate. Sorbitan palmitate, tetraethylene glycol mono or distearate, tetraethylene glycol monooleate, sorbitan laurate, tetraethylene glycol monolaurate, polyoxyethylene sterate, hexane ethylene to lyc monostearate, Polyoxyethylene sorbitan oleate, polyoxyethylene oleate, polyoxyethylene palmitate, polyoxyethylene laurate, polyoxyethylene sorbitan laurate or polyoxyethylene sorbitan palmitate It selects from a group, Preferably it selects 1 or more types containing a monobody.

On the other hand, as the co-emulsifier composition, beta which is an emulsifier having a nonionic HLB value of 10-13 or an alkyl cationic fatty acid, a diamine alkyl sulfonate or an oxazoline derivative or a zwitterionic emulsifier Betaine, imidazloe and tego compounds are used. In addition, the HLB value of 10-13 was used for the wetting, moisturizing and antibacterial action and the promotion of absorption.

Specific examples of the compound usable as the moisturizing agent of the present invention include hyaluronic acid or salts thereof, kojic acid and derivatives thereof, chondroitin or salts thereof, collagen, lecithin, 1,3-furophilene glycol, polyglycerine, chitosan and chitins. , At least one selected from the group consisting of sorbitol, PEG, maltose, squalene or squalane.

As the matrix agent, gelatin, polyoxymethylcellulose, sodium polyacrylate, carboxyvinyl polymer and the like and a film forming agent are used. Examples of the polymo (polymer) include polyvinyl-based alcohols, synthetic resins, and ethylene vinyl acetate; Polyalkyl acrylate, ethylene acrylate based on acrylate; Polyisobutylene system; Polybutane system; PEG system; PPG-based and polyurethane, styrene-butadiene, silicone polymer or polypentene polymer and the like, lipophilic polymer was selected for the stability detection.

In particular, the polymer for the hydrogel (hudrogel) to use polycarbophils (polycarbophils), polymethacrylic acid cloth linked vinylbenzene polymethmethylacrylate or polyhydroxy ethyl methacrylate or amino-polyamide polymer good.

The skin preparation of the present invention and a method for producing the same are prepared according to the manufacturing method of the ointment and the cream, with lysozyme, lysozyme salt, or lysozyme chloride as an aqueous phase and other components as oil. Formulations containing base and lysozyme can be prepared by softening method at one time or separately. At this time, the preparation temperature can be 10-80 ℃ but more preferably 10-40 ℃ It is characteristic. And the roughness of the product can be adjusted to the type or amount of the composition to be 7-110 dyne / cm², more preferred roughness is to manufacture in the range of 80-100 dyne / cm². The outer solvent of the present invention contains 0.1-10% by weight of lysozyme (titer) as a main component. The outer preparation prepared in this way is applied to a variety of skin ulcers such as burns or bedsores once a day or several times depending on the symptoms and lesion size.

Hereinafter, examples and experimental examples of the present invention will be described.

Example 1 (W / O Mold Ointment)

B) ointment preparation

The remaining components except for lysozyme, carboxyvinyl polymer and kojic acid are emulsified at 60 ° C. or higher, and carboxy vinyl polymer is added while stirring. After cooling, the remaining aqueous phase components such as lysozyme and kojic acid are slowly added to be homogeneous while stirring and then left for maturation.

Example 2 (fatty ointment)

Preparation method: It is a formulation for a fat-soluble base (white vaseline, liquid parffin), and it is manufactured according to Example 1 according to the conventional method of fat-soluble ointment.

Example 3 (W / O Type, Absorbent Ointment)

Preparation: W / O absorption ointment is prepared according to the same method as in Example 1.

Example 4 (gel-type ointment)

Preparation method: It is prepared according to the general method of water-soluble ointment by mixing at a temperature of 60 ℃ or less.

Example 5 Hydrophilic Ointment, O / W Type

Preparation method: The same procedure as in Example 1 was carried out to obtain an oil-in-water (O / W) ointment.

EXAMPLE 6 (Number of g in 100 g of lysine chloride ointment)

Preparation Method: According to the preparation method of Examples 1 to 5, the fatty acid was warmed to 70 ° C., the aqueous phase component was warmed to 75 ° C., the water phase was cooled to 50 ° C. or lower, and a portion of the aqueous phase was lysozyme chloride stabilizer. It is prepared by emulsifying with softening while pre-emulsifying with softening and adding to the residual water phase and injecting it into oil phase (fatty phase).

Experimental Example 1 [Long-term preservation test]

In order to examine the stability of the samples prepared according to Examples 1 to 5, three batches were prepared and expanded tests were carried out by the disclosure method for two years.

[Experimental example 2 [lithography confirmation and quantitative test method]]

Exactly 1 g of the ointment prepared according to Examples 1 to 5 are treated with 20 ml of chloroform (or ethylenedichloride or other hydrophobic solvent) and 20 ml of 2% NaCl (pH 3.0) containing hydrochloric acid to give an oil layer. The aqueous layer was separated and repeated three times, and the obtained aqueous layer was confirmed to be consistent with the lysozyme pure band by electrophoresis method.

Experimental Example 3 Formulation of Quantitative Test Method

Introduction

After quantitative determination of lysozyme chloride ointment by the calibration curve of lysozyme chloride by HPLC analysis, the experiment was conducted by HPLC analysis, non-turbidity, cylindrical plate method for the purpose of securing stability in the preparation. Since the accuracy of inferiority was confirmed as in the disclosure method, the stability test method of the lysozyme in the ointment was shown to be significant, so it was adopted and applied to the expansion test.

2. Quantitative review by calibration curve

NO. Cone area

1 1.26mg / ml 0.030480

2 2.48mg / ml 0.060892

3 4.93mg / ml 0.118185

4 7.412 g / ml 0.185528

5 8.642 g / ml 0.218828

As shown in the attached graph (not shown), the formation of an almost linear calibration curve shows that lysozyme has sufficient quantification in the H. P. L. C. assay.

3. Data of content and titer in preparation (110% prescription)

4. Conclusion

As can be seen from the above test results, the chloride content in this formulation can be obtained from the HPLC analysis, non-takt method, and cylindrical plate method in the actual product, and it is possible to obtain a constant test value within ± 2.0% of the actual prescription of 110.0%. It is thought that the stability of lysozyme enzyme can be secured.

[Non-tax method]

1.Preparation of Standard Solution: Take 50 mg of lysozyme chloride standard product accurately, mark 50ml with pH 6.2 ginseng buffer solution, and use it as standard solution. (1MG / ML)

2. Preparation of test solution: Aliquot of lysine Chloride is exactly 50 mg equivalent, and put into 250 ml aliquots. Add 100 ml of benzene and shake for 40 minutes. After shaking, add 50 ml of PH6.2 ginseng buffer solution, and mix it for 30 minutes. Discard the benzene layer and filter the pH 6.2 ginseng buffer solution to make the sample solution. (1MG / ML)

3. Operation: Add 3ml of substrate solution to each prepared test tube and leave for 3 minutes at 35 °. Separately, the standard solution and the sample solution PH6.2 ginseng buffer solution are left at 35 degrees for 3 minutes. Add 3 ml of standard solution, sample solution, and buffer solution to each test tube containing the substrate solution for 3 minutes, and leave at 35 ° C for 10 minutes.

After standing for 10 minutes, the water was measured for absorbance at a wavelength of 640NM with a control solution.

※ U.V. Spectrometer: Shimadzu

Model: UV-160A

※ Substrate solution: After dissolving about 50 mg of microorganism LYSODEILCTICUS cells in PH 6.2 ginseng buffer solution 60ml, adjust the pH 6.2 ginseng buffer solution to 10% at 640NM wavelength as a control solution.

Reference: Notice No. 85-9, Food and Drug Safety Headquarters

[H. P. L. C. Assay]

1.Preparation of Standard Solution: Take 50 mg of lysozyme chloride standard accurately and dissolve in 50 ml of distilled water and filter with 0.4 μMFILTERF to make standard solution.

2. Quarantine preparation: As lysozyme chloride, ointment of about 50 MG is precisely taken, put into aliquots, put into 50 ml of benzene, shaken for 30 minutes, dissolved, and added twice with 20 ml of distilled water. Filter by adjusting the sample solution.

3. Analysis condition: ① Column: ODS-2

② Mobile phase: 95% Methanol

③ Fliw Rate: 0.8ml / min

④ Injection: 15 μ 1

⑤ Temperature: 25 ℃

⑥ Derecror: 280 nm

[Sterilization test]

The sterilizing power test was done about the product of Examples 1-5.

As a method, the cup method was adopted as the antimicrobial test method, and each sample was left in an incubator at 37 ° C. for 24 hours, and then titer was measured. Experimental conditions and results were as follows.

Experimental conditions and results

1) Used species: Micrococcus lysodeiitigus

Subculture

2) Medium: Use of agapeptone media with pH 6.5 ~ 6.6

3) Lowland measurement: Lowland was measured as Zone leader.

Experimental Example 4 [Method of H. P. L. C. Method by Acceleration Test]

The stability of this formulation was examined in a short period of time by accelerating and measuring the content of lysozyme chloride in the ointment of Example 6 by the H. P. L. C. method established according to Experimental Example 3.

3) Test Items and Test Methods

(1) Observation time

1) Acceleration test: 2 months, 4 months, 6 months at the start of the test

(2) Test method and test item

Claims (7)

A skin solvent containing 0.1 to 10% by weight of a water-in-oil emulsified lysozyme of PH 5.0 ± 2 containing a lysozyme or a salt thereof, an aliphatic gas, an emulsifier, an auxiliary emulsifier, a chelating agent, a humectant, and a matrix agent. Pharmaceutically stable water-in-oil, characterized in that lysozyme or its salts, aliphatic bases, emulsifiers, co-emulsifiers, chelating agents, humectants and matrix agents are added and the pH is maintained at pH 5.0 ± 2 and emulsified at 10-80 ° C. A method for producing an outer skin solvent containing 0.1 to 10 wt% of an emulsified (W / O) lysozyme. The method of claim 2, wherein the aliphatic base is selected from white petrolatum, liquid paraffin, cetyl alcohol, stearyl alcohol, stearic acid, scalene, emulsifiers are used natural lipids or synthetic emulsifiers, iso synthetic emulsifiers Characterized in that it is selected from glycerol mono C _12-18 fatty acid of fluorophyte adipate or HLB 8.0 to 13.5, or among ether or sucrose mono C _12-18 fatty acid ester or ether, PEG or PPG fatty acid ester or ether. . The co-emulsifier is an emulsifier having a nonionic HLB value of 10-13 or an alkyl cationic fatty acid, a polyamine alkylsulfonate, an oxazoline derivative, a betaine-based compound, an imidazole-based emulsifier or a tego-emulsifier. The method characterized by the choice of using. 3. The method according to claim 2, wherein the chelating agent is selected from alpha-keroglutamine, soy protein-based treatase, lysine or cysteine, and glutathione. The moisturizing agent of claim 2, wherein the moisturizing agent is hyaluronic acid or a salt thereof, kojic acid or a derivative thereof, chondroitin or a salt thereof, collagen, lecithin, 1,3-furophyllene glycol, polyglycerin, chitosan and chitingyu, sorbitol, PEG, horse Toss, squalene or squalane. The method of claim 2, wherein the matrix agent is selected from gelatin, polyoxymethylcellulose, sodium polyacrylate, carboxyvinyl polymer.