WO2015018343A1

WO2015018343A1 - Medicine composition for wound therapy

Info

Publication number: WO2015018343A1
Application number: PCT/CN2014/083809
Authority: WO
Inventors: 杜涛; 科恩⋅艾文⋅凯尔曼
Original assignee: 南京禾沃医药有限公司
Priority date: 2013-08-06
Filing date: 2014-08-06
Publication date: 2015-02-12
Also published as: CN103417581A; CN103417581B

Abstract

The present invention provides a medicine composition for wound therapy, comprising phosphorylated starch as an active ingredient, hyaluronate and honey. The phosphorylated starch is prepared from starch, a phosphorylation agent, and at least one conjugate agent through reaction.

Description

Pharmaceutical composition for wound treatment

Technical field

The present invention relates to pharmaceutical compositions for wound healing and uses thereof, and more particularly to pharmaceutical compositions useful for treating medical conditions such as acute and chronic wounds, burns and surgical wounds associated with infections. Background technique

Chronic non-healing wounds: Clinically, wounds are classified as acute or chronic depending on the time of healing. A normal response to tissue damage is a timely and orderly repair process that results in sustained recovery of anatomy and functional integrity. In contrast, in chronic ulcers, the healing process is prolonged and incomplete, and proceeds in an unorganized manner, resulting in poor anatomical and functional outcomes. Most chronic ulcers are associated with a few well-defined clinical conditions, especially chronic venous hypertension, diabetes and pressure ulcers. These diseases generally account for about 70% of all chronic ulcers. The incidence of chronic ulcers continues to increase due to factors such as prolonged life and a logarithmic increase in the incidence of type 2 diabetes worldwide. In addition to these non-healing skin wounds, there are many other diseases characterized by excessive protease activity and long-term disease, such as stomatological diseases such as gingivitis.

Proteolytic Environment for Chronic Trauma: Normal healing involves a complex cascade of interaction events involving many cell types, soluble factors, and matrix components. Healing can be arbitrarily divided into stages of temporally overlapping agglutination, inflammatoryization, fibrous tissue formation, and eventual remodeling. Cytokines (growth factors) are generally believed to be essential regulators of the repair process. Therefore, proteases destroy cytokines can delay healing. Usually, in the inflammatory phase, polymorphonuclear leukocytes (PMNs) are the first white blood cells to appear. They produce various proteases such as collagenase (MMP-8) and elastase to help remove damaged matrix and promote healing. In acute and chronic wounds, various cytokines are important in angiogenesis, contraction, collagen synthesis/degradation, and subsequent wound closure. Under normal conditions, epithelial (keratinocyte) cells allow wound closure for final closure, thereby further assisting in open wound closure. The healing of chronic wounds is very different from normal healing. Due to the high levels of persistence of PMNs, the same proteases that are part of the normal healing process become excessive. These proteases then destroy the matrix, growth factor and growth factor receptor sites. Acne is a typical example of chronic wounding.

Bacterial contamination delays wound healing by several different mechanisms, such as the continued production of inflammatory mediators, which leads to prolonged inflammatory responses leading to host damage and delayed healing. In addition, bacteria and host are fine The cells compete for the nutrients and oxygen necessary for the wound healing process. Wound infection can also cause tissue hypoxia, impede granulation tissue growth, reduce the number of fibroblasts and collagen production, and destroy re-epithelialization. Therefore, solving the problem of infected wound cleansing is the main goal of wound care and plays an important role in wound treatment.

Chronic Trauma Treatment: Over the past 20 years, many manufacturers have listed "Advanced Wound Care Products" for various chronic wounds. Includes hydrocolloids, hydrogels and calcium alginate. However, there is little evidence that these products can alter the rate of healing or the cell biology of the wound.

To date, there has been a lack of effective pharmaceutical or pharmaceutical compositions for treating wounds, particularly wound healing. Summary of the invention

The present invention addresses the above problems and shortcomings associated with wound treatment. The Applicant has found through long-term repeated studies that the combination of the phosphorylated starch, hyaluronate and honey of the present invention as an active ingredient unexpectedly produces a synergistic effect for treating wounds with unexpected technical effects, thereby The present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for wound treatment comprising the following as an active ingredient:

(a) a phosphorylated starch prepared by reacting a starch with a phosphorylating agent and at least one conjugate;

(b) hyaluronate;

(c) Honey.

The pharmaceutical composition of the present invention has a masking activity of 55% or more, preferably 60% or more and 70% or more for elastase, and has a masking activity of 75% or more for collagenase.

In the present invention, the hyaluronate is selected from the group consisting of sodium, potassium and zinc salts of hyaluronic acid, gp, sodium hyaluronate, potassium hyaluronate and zinc hyaluronate. Preferred hyaluronate salts have a molecular weight of less than 750,000 Daltons, such as from 150,000 to 225,000 Daltons. A small molecular weight hyaluronate is chosen to ensure that there is no condensation on or in the skin. Hyaluronate can come from manufacturers such as Skymart, China Shandong Freda Biochemical Co., Ltd., etc.

In the present invention, the honey is selected from the group consisting of manuka honey, jellybush honey, or honey from rewarewa, Kunzea ericoides and Nothofagus solandri. Preferably, the selected honey has a water content of 17% or less; preferably, the selected honey has an Aw of 0.94 or less, preferably 0.86 or less. When honey powder is desired, the powdering method of honey is a method of powdering honey by a method well known in the art. Once powdered, the powder of the resulting honey can be stored for use Mix or blend directly.

In the present invention, the conjugated agent is selected from the group consisting of urea, thiourea, biuret, cyanuric acid, urea formaldehyde, melamine; the phosphorylating agent is any compound that can phosphorylate starch, including disodium hydrogen phosphate. , sodium dihydrogen phosphate, sodium metaphosphate, diammonium hydrogen phosphate, sodium orthophosphate, sodium tripolyphosphate, sodium hexametaphosphate, tetrasodium pyrophosphate; the starch may be corn starch, potato starch, pea starch, tapioca starch , rice starch, wheat starch, concanava starch, breadfruit starch, sorghum starch, sago starch, etc.; corn starch is preferred.

In the present invention, preferably, the phosphorylated starch may have a structure of the following formula (I):

[-St-(OP0 ₃ "H) _k (OH) ₁ (0-Z) _m (0-W) _p ]

Formula (I)

among them:

"St" means a subunit of starch, such as C ₆ H ₄ 0 _2;

k+1+m+p-3;

k is equal to or less than 0.5;

1, m and p are numbers from 0 to 3, including 0 and 3 and can be fractions;

"Z" is selected from the group consisting of C(0)NH _{2 ;} C(S)NH ₂ ; C(0)NH ₃ .A or C(S)NH ₃ .A, wherein "A" is selected from the group consisting of phosphoric acid and sulfuric acid , anions of nitric acid, hydrochloric acid and perchloric acid; C(0)-St; and C(S)-St;

W is a condensation product -CH ₂ R, wherein R is selected from the group consisting of: -NH-C(0)-NH ₂ ; -NH-C(0)-NH-CH ₂ -OSt; -NH-C(S)-NH ₂ -NH-C(S)-NH-CH ₂ -OSt; melamine group; melamine group in which NH ₂ group is bonded to CH ₂ -OSt; any combination thereof and its chain extended form, and wherein W may be substituted Or unsubstituted; for example, a starch derivative, wherein w is a thiourea formaldehyde condensation product;

And wherein the negative charge on P0 ₃ - is neutralized by a cation such as Na+, K+, Η+,

Li+.

In the present invention, the mass ratio of phosphorylated starch, hyaluronate and honey is 5-10: 3-6: 2-4, preferably 7-8: 4-5: 2.5-3.5, more preferably 8: 5: 3.

The pharmaceutical compositions of the present invention are prepared in any form acceptable for use in wound treatment using conventional pharmaceutically acceptable excipients, such as in the form of powders, ointments, salves, gels, preferably ointments and salves.

The amount of the pharmaceutical composition of the present invention the skilled person according to conventional techniques may be determined, preferably over a wound with such an amount of the active ingredient in an amount of at least about 6-12mg / cm ^3, such as 12mg / cm ³ or more , 15 mg/cm ³ , 20 mg/cm ³ . Usage can be 1-2 times a day.

It is still another object of the present invention to provide the use of the pharmaceutical composition for wound treatment for the preparation of a medicament for wound treatment, in particular for the preparation of a medicament for wound healing. The wound is selected from Acute wounds, chronic wounds, burns, and surgical wounds, wherein preferably the chronic wound is selected from the group consisting of ulcers, acne, pressure ulcers, such as diabetic ulcers.

The phosphorylated starch of the present invention can be prepared by the reaction of a starch with a phosphorylating agent and at least one conjugate.

As used herein, "phosphorylating agent" refers to any compound that phosphorylates starch or cotton. Examples of phosphorylating agents which can be used in the present invention are, for example, a phosphorylating agent solution containing ammonium dihydrogen phosphate; sodium dihydrogen phosphate (MSP); sodium metaphosphate (SMP) (Na6(P0 ₃ ) _{6 ;} monoammonium phosphate (MAP) (NH ₄ H ₂ P0 ₄ ) (also known as ammonium dihydrogen phosphate); diammonium phosphate (DAP) (NH ₄ ) ₂ HP0 ₄ (also known as ammonium phosphate); other ammonium phosphates; Phosphorylating agents (eg, MAP, etc.); binary phosphorylating agents (eg, DAP, etc.); sodium orthophosphate, potassium, and lithium, and any combination thereof (see, eg, US Pat. No. 2,993,041); tripolyphosphate (see, For example, U.S. Patent 2,884,413); alkali metal phosphates or combinations of these salts (see, e.g., U.S. Patent 2,884,412); sodium metaphosphate, sodium tripolyphosphate, sodium hexametaphosphate, tetrasodium pyrophosphate (see, e.g., U.S. Patent 2,884,418). Other phosphorylating agents; etc. The phosphorylating agents mentioned are non-limiting examples. A combination of phosphorylating agents such as phosphoric acid and any phosphate, pyrophosphate, orthophosphate and/or medium may be used. The combination of the agents is such that the pH ranges between 1.5 and 10.0 (preferably 3.0 to 9.0). No lower pH, because phosphoric acid at low pH causes carcination and carbonization of cotton and starch at a curing temperature of 130 to 160 ° C. The phosphorylating agent can be in any ratio with a conjugate (eg urea, etc.) Mixed use.

Examples of conjugates which can be used in the present invention are, for example, urea, thiourea, biuret, cyanuric acid, urea formaldehyde, melamine and other compounds containing active nitrogen. It is particularly preferred to use urea for the derivatization of starch.

The ratio of conjugate to phosphorylating agent may range from about 10:1 to 1:2, with a preferred ratio of about 3:1. The ratio of starch to solution may be from about 0.1:10 to 1:1, a preferred example being about 2:5. The ratio of starch to phosphorylation agent, the ratio of starch to conjugation rate depends on the composition of the solution. Preferably, the ratio of the conjugated agent to the phosphorylating agent is determined first, and then other reagents are determined. For example, in a preferred embodiment, the ratio of starch to conjugate is 1 : 10 - 1 : 1, preferably 1 : 3.

For the mixing of the conjugate, phosphorylating agent and starch, the temperature is in the range of about 10 to 30 ° C (preferably 20-25 ° C), for example mixing at room temperature, preferably for a short period of time, for example about 1 to 10 minutes. Most preferably, it is about 2 to 3 minutes. For the spreading and drying of the material after mixing, and the subsequent curing, the conditions depend on the amount of material. For an amount of material of about 2 to 5 grams, the spreading and drying after mixing can be carried out at a temperature of about 70 to 95 ° C (preferably 80 to 90 ° C) for about 10 to 30 minutes (preferably about 10 to 15 minutes). Subsequent curing is preferably carried out at a temperature of from about 130 to 170 ° C (preferably from about 150 to 160 ° C) for about 3 to 15 minutes (preferably about 6 to 10 minutes). For example, in the present invention, a solution prepared by dissolving solid urea and ammonium dihydrogen phosphate (for example, monoammonium phosphate, MAP) and deionized water (for example, deionized water containing Triton X-100 Sigma Ultra) may be used. As another example, a phosphorylating agent, a conjugate, and a starch may be mixed into a solution or slurry, which may be cast onto a surface (preferably uniformly spread) and then dried (eg, at about 85-95 ° C) Drying at a temperature of about 30 minutes), then curing (for example, curing at about 160 ° C for about 6 minutes, preferably in a pressure oven), cooling, and peeling off the formed film from the surface, which can be used or A treatment (for example, processing into a fine powder). An example of treating a film into a powder is, for example, placing the film in a liquid (such as deionized water), allowing it to swell, then sedimenting, washing (for example with deionized water), filtering and drying (for example, at room temperature) Dry), in which a fine powder is produced.

Hyaluronic acid, also known as hyaluronic acid, hyaluronic acid and all its salts are internationally referred to as hyaluronan (hereinafter referred to as HA). HA is a macromolecular chain polysaccharide composed of a repeating unit of disaccharide units composed of D-glucose acetate and N-acetylglucose. At present, commercial HA is mainly derived from automatic material extraction and bacterial fermentation preparation. There is no species difference. The HA prepared from different sources is the same substance and has no immunogenicity. The molecular weight of HA is 5 to 8 million Daltons, which is a macromolecular single-chain glycan. The carboxyl group on the glucuronic acid group at a spatial distance and the plurality of hydroxyl groups in the disaccharide molecule impart a unique property to HA. A macromolecular network structure is formed in an aqueous solution, and a large amount of water is bonded to hydrogen molecules to form a solution, and the solution has excellent viscoelasticity and moisture retention. The larger the molecular weight of HA, the greater the viscosity of the solution, and the stronger its viscoelasticity and moisture retention. HA solution as a viscoelastic protective agent and lubricant for ophthalmology and orthopedics, as an anti-adhesion agent for abdominal abdominal pain surgery and as a medium for ophthalmic preparations. Many products have been marketed at home and abroad and have been used clinically for many years, but It has few products for wound care. According to the latest research data, in wound care, in addition to the physical effects of lubrication, moisturizing, and other covering dressings, HA also inhibits wound contracture during wound healing; regulates cell function and angiogenesis; improves local microcirculation; Mechanisms such as growth factor interactions play a role in promoting wound repair and reducing scars.

The use of honey in the treatment of wounds has long been known. Recent international medical literature has documented that honey can be effectively used as a dressing for wounds, burns, and skin ulcers. The observations recorded include inflammation, swelling, and rapid decline in pain and necrosis. Tissues are separated from carrion without debridement, and tissue growth is stimulated to repair the wound. As a result, healing occurs faster with minimal scar formation, usually without any skin grafting. These effects of honey are mainly due to the antimicrobial properties of honey, so that it does not cause tissue damage and actually helps to promote the healing process.

However, honey is relatively fluid, especially at body temperature, it is difficult to concentrate it in the desired area, and many wounds will ooze moisture, which will lead to a further dilution of honey, exacerbating retention problems, making honey The antibacterial activity is substantially reduced significantly, and the problem is difficult to solve. And the present application relates it to the phosphorus The combination of acidified starch and hyaluronic acid salt not only produces synergistic effects, but also clearly solves this problem. The pharmaceutical composition of the present invention can be prepared by any conventional method into any dosage form suitable for wound use, such as mixing the phosphorylated starch, hyaluronate, honey into a powder according to the ratio, and adding pharmaceutically acceptable The diluent or carrier may be prepared for use in a wound ointment, cream or ointment according to conventional formulation techniques.

In reviewing performance-improving therapeutic wounds, such as wound healing compositions, the Applicant has unexpectedly discovered that the phosphorylated starch, hyaluronate, and honey described herein can exhibit synergistic effects in a certain proportion of combinations. Significantly superior protease masking efficiency compared to phosphorylated starch alone, hyaluronate alone, or honey alone, or a combination of hyaluronate and honey (known in the art, high levels within the wound site) The presence of active elastase and collagenase has an important relationship with the onset of ulcers and subsequent healing failure, and is capable of simultaneously exerting antimicrobial activity against a broad spectrum of pathogenic microorganisms while achieving pain relief, cooling and wound healing.

As used herein, the terms "synergistic", "synergistic effect" and any grammatical conversion form thereof describe a synergistic effect occurring in a combination of two or more biologically active compounds, wherein the combination exhibited when the two compounds are used simultaneously The effect exceeds the cumulative effect of each compound when used alone. Thus, it is generally determined that there is "coordination" when the value indicating the effect of the combination of the two active agents is greater than the sum of the equivalent values obtained when each of these agents is acted upon alone.

The term "wound" as used herein shall be understood in its broadest sense to describe a damaged or damaged skin or mucosal area, whether or not it contains inactivated or eschar tissue, and includes any type of wound, including However, it is not limited to acute wounds, chronic wounds, surgical wounds, burns, and the like. The term "skin damage" as used herein describes damaged skin and is used interchangeably throughout the application with the term "wound."

The term "acute wound" describes a wound that is caused by traumatic wear, tearing or surface damage and that ultimately heals automatically through the normal stages of wound healing (eg, hemostasis, inflammation, proliferation, and remodeling) without complications. However, acute wounds can cause complications if they come into contact with pathogenic microorganisms that cause local infection.

The term "chronic wound" describes a wound that does not form in a clot that normally occurs in a patient that is affected in some ways and that is difficult to heal. Examples of chronic wounds are chronic skin ulcers such as diabetic ulcers, acne ulcers (pressure ulcers) and venous ulcers.

The phrase "acne ulcer", also known as "pressure sore", describes various factors such as pressure, friction, moisture, shear, temperature, age, self-control, and medicament for any part of the body, especially bone and cartilage areas such as Skin damage caused by the upper part of the bones, elbows, knees, ankles, etc. Although it is easy to prevent and completely curable at an early stage, hemorrhoid ulcers are usually fatal and reported in developed countries. One of the main iatrogenic causes of death. Acne ulcers can be caused by insufficient blood supply and insufficient reperfusion caused by blood re-entering the tissue. The most common microorganisms isolated from pressure sores are Proteus mirabilis, Group D Streptococcus, Escherichia coli, Staphylococcus, Pseudomonas, and Corynebacterium microorganisms.

The composition for wound treatment of the present application not only has excellent protease masking effect, but also has the advantages of quick wound healing, light scar, less toxic side effects, and comfortable moist feeling. detailed description

The invention will now be further described in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. All parts are parts by weight and all percentages are by weight.

Preparation example 1

Preparation of phosphorylated starch

3.00 grams of solid urea (99.5 % from Across, No. 140750010) and 1.00 grams of ammonium dihydrogen phosphate (monoammonium phosphate, MAP), (Across, reagent grade, No. 423385000), containing 0.1% by weight of Triton X- 100 Sigma Ultra (Sigma T9284) was dissolved in 6 ml of deionized water to prepare a solution of a phosphorylating agent and a conjugate. The solution was mixed at 35-40 ° C for 10 minutes, then 1.3 g of corn starch was added to the solution and mixed for a further 15 minutes at 35-40 ° C with good agitation. The highly viscous solution/slurry was then cast onto a Reynolds Wrap Release non-stick aluminum foil. The material was spread evenly with a blade and then dried in an oven at 85-95 for 30 minutes. The sample was then placed in a pressure oven for 6 minutes at 160 °C. The pressure oven ensures that heat can be transferred from the oven to the sample immediately. After taking out and cooling, the sample was peeled off from the peeled aluminum sheet. The resulting product is in the form of a translucent film (which can be used as such). In this example, the inventors placed the product in 400 ml of deionized water and allowed it to swell into a slurry to settle it; then washed with two additional portions of 400 ml of deionized water, filtered and dried to a fine temperature at room temperature. Powder. (Optionally, the same results were obtained using a Teflon-coated aluminum film instead of Reynolds Wrap). Preparation Example 2

Preparation of phosphorylated starch

Diammonium hydrogen phosphate (DAP) was used as a phosphorylating agent, together with urea. 3.00 grams of solid urea (99.5 % from Across, number 140750010) and 1.00 grams of DAP (Across, reagent grade, number 201825000) were used with 6 ml of deionized water containing 0.1 wt% Triton X-100 Sigma Ultra (Sigma T9284) Dissolved, a solution of a phosphorylating agent and a conjugate was prepared. The solution was mixed at 35-40 ° C for 10 minutes, then 1.3 grams of corn starch (Argol 0% food grade) was added to the solution, at 35-40 ° C with good agitation. Mix for another 15 minutes. The highly viscous solution/slurry was then cast onto Reynolds Wrap Release non-stick aluminum foil. The material was spread evenly with a blade and then dried in an oven at 85-95 for 30 minutes. The sample was then cured at 160 ° C for 6 minutes in a pressure oven. After taking out and cooling, the sample was peeled off from the peeled aluminum sheet. The resulting product is in the form of a translucent film (which can be used as such). The product was placed in 400 ml of deionized water, allowed to swell into a slurry, and allowed to settle; then washed with two additional portions of 400 ml of deionized water, filtered, and dried to a fine powder at room temperature (using Teflon coating) The coated aluminum film replaces the Reynolds Wra with the same result).

The product of Preparation 2 of the present invention can absorb more than 40 times its weight of fluid. Preparation Example 3

Preparation of phosphorylated starch

The phosphorylating agent sodium metaphosphate (SMP), (sodium hexametaphosphate, Across, No. 61217-5000) and the conjugated urea were used. A phosphorylating agent and a conjugate were prepared by dissolving 3.00 g of solid urea (99.5 % from Across, No. 140750010) and 1.00 g of SMP with 6 ml of deionized water containing 0.1% by weight of Triton X-100 Sigma Ultra (Sigma T9284). The solution. The solution was mixed at 35-40 ° C for 10 minutes, then 1.3 g of corn starch (Argo 100% food grade) was added to the solution and mixed for a further 15 minutes at 35-40 ° C with good agitation. The highly viscous solution/slurry was then cast onto a Reynolds Wrap Release non-stick aluminum foil. The material was spread evenly with a blade and then dried in an oven at 85-95 for 30 minutes. The sample was then reacted at 160 ° C for 6 minutes in a pressure oven. After taking out and cooling, the sample was peeled off from the peeled aluminum sheet. The resulting product is in the form of a translucent film (which can be used as such). The inventors placed the product in 400 ml of deionized water, swelled it into a slurry, and allowed it to settle; then washed with two additional portions of 400 ml of deionized water, filtered, and dried to a fine powder at room temperature (using special The fluorocarbon coated aluminum film replaced the ReynoldsWrap with the same results).

The product of Preparation Example 3 of the present invention can absorb more than 35 times its weight of fluid. Example 1

The phosphorylated starch powder prepared in Preparation Example 1, sodium hyaluronate (225,000 Daltons) and Manuka honey were mixed at a mass ratio of 7:4:2.5 to obtain a pharmaceutical composition powder of the present invention. Example 2

The phosphorylated starch prepared in Preparation Example 2, potassium hyaluronate (175,000 Daltons) and jellybush honey were mixed in a mass ratio of 5:3:2 to obtain a mixture, which was then prepared into an ointment. System The preparation method is as follows: Under aseptic operation, take the above mixture 0.025g, freeze-dry, grind to a particle size of less than 150 microns for use, then take white petrolatum 180g, liquid paraffin 20g, heat and dissolve the two, filter at 121 °C Sterilize for 20 minutes, cool to 25 °C, then gradually add white petrolatum and liquid paraffin cooled to 25 °C to the finely ground phosphorylated starch, sodium hyaluronate and jellybush honey under aseptic operation. The mixture is thoroughly mixed to obtain an ointment. Example 3

The phosphorylated starch powder prepared in Preparation Example 3, sodium hyaluronate and Manuka honey were mixed in a mass ratio of 8:5:3 to obtain a mixture, and the mixture was placed in a sesame oil which was 3 times the weight of the mixture. After 1 hour, an ointment was obtained. Comparative example 1

The phosphorylated starch powder prepared in Preparation Example 1 was mixed with sodium hyaluronate (225,000 Daltons) and Manuka honey in a mass ratio of 15:3:1 to obtain a pharmaceutical composition powder. Comparative example 2

Sodium hyaluronate and Manuka honey were mixed in a mass ratio of 5:3 to obtain a mixture of Comparative Example 2. The efficacy of the above examples was studied according to the following technique.

A, protease masking efficiency

a. Elastase The concentration of elastase in the initial solution and the solution after incubation is determined by: reacting elastase with N-methoxysuccinyl-ala-ala-val p-nitroanilide substrate, using A 96-well spectrometer (Bio-TEK, Microplate Reader; Model Power Wave HT) was operated at 550 nm. A known amount of elastase is used to generate a standard curve.

b. Collagenase The activity of the collagenase was monitored using an Molecular Probe assay kit (EnzCheck E-12055). This analysis enables accurate analysis of metalloproteinases and their inhibitors in a fast and high-throughput format. The enzyme kit comprises a gelatin fluorescent conjugate. The substrate is efficiently digested by gelatinase and collagenase to produce a highly fluorescent peptide. The increase in fluorescence is proportional to the proteolytic activity and can be monitored using a fluorescent microplate reader. The analysis was run on a Bio-Teck Multi-Detection Microplate Reader, Model FLx800, excited at 485 nm and detected at 530 nm.

c. Synthetic wound fluid Synthetic wound fluid is obtained by elastase from human leukocytes (Sigma E-8140) dissolved in HEPES buffer (pH 7.5, 0.1 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid [HEPES], 0.5 M NaCl, Triton X-100) Prepared. The solution was diluted to the elastase concentration of 25 mu/ml with the same buffer. This solution was used to evaluate the masking of elastase in the experiments reported herein.

d. Wound fluid from chronic wounds The wound fluid is obtained by extracting a sponge used on the wound. The fluid is squeezed from the sponge, placed in a test tube, and stored in a refrigerator. The elastase content of the wound fluid was determined by calibration with a standard curve of elastase (Sigma E-8140). Once the elastase content of the wound fluid was determined, it was diluted to a concentration of active elastase of 25 mU/ml. The dilution was carried out using HEPES buffer (pH 7.5, 0.1 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid [HEPES], 0.5 M NaCl, Triton X-100).

Analytical Procedures The cotton samples were then cut into 50 mg pieces, each placed in a 3 ml syringe. Keep the syringe upright and add 66 μl of diluted wound fluid to each sample. The samples were then incubated for 2 hours. Remove the liquid from the sample and place the ΙΟΟμ Ι liquid from each sample in the well of the 96-well plate. A 100 μM substrate (methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, 0.36 mg/ml) was then placed in each well containing the sample. Dynamic readings were taken at 405 nm in a spectrophotometer. The starch samples were analyzed by the same method except that after incubation, the starch slurry was filtered through a 5 micron Millex-SV needle filter and the eluate was analyzed by reaction with a substrate as described.

Protease masking efficiency The ability of each sample product to remove protease (elastase or collagenase) from the wound environment was determined by absorption techniques. The product sample is incubated with a volume of fluid comprising a protease that mimics the concentration of the wound fluid. After the incubation, the fluid is removed by mechanical means (extrusion or centrifugation) and the concentration of unbound elastase is determined. Activity is reported as % of elastase removed from the fluid. Acne wounds are simulated by incorporating neutrophil elastase or collagenase into the plasma. Typically 30-100 mg of the sample (calculated as the total amount of active ingredient in the case of the formulation) is incubated with 0.5 to 1.0 ml of simulated wound fluid for an incubation time of 1-3 hours. Ability to remove elastase

The elastase masking ability of each product is listed in Table 1 below and compared to corn starch, hyaluronate, honey, preparations (phosphorylated starch alone), comparative examples, etc.:

Sample 1 removed elastase%

Corn starch |8.4

Sodium hyaluronate .5

Potassium hyaluronate; Γ7.0 % of elastase removed

Manuka honey;;6.0

Jellybush honey 5.5

Preparation 1 | 55.9

Preparation Example 2;;64.5

Preparation Example 3;: 63.0

"Comparative Preparation 1;: 10.1

"Comparative Preparation Example 2.4

Comparative Preparation 3 1-5.3

Example 1 3⁄494

Example 2 ::95

;Example 3 96

Comparative example 1 ;70

Comparative example 2 il5

Note: The comparative preparations were prepared in the same manner as in the preparation examples except that the conjugate was not used.

As can be seen from Table 1 above, the samples of the preparation examples were significantly higher than the elastase masking effect of natural corn starch and much higher than the conventional phosphorylated starch (Comparative Preparation Examples 1, 2, 3). Comparative Example 3 produced negative data indicating that the sample not only failed to mask elastase, but it may also enhance the activity of elastase due to the altered pH, making the sample unfavorable for wound healing. However, the combination of phosphorylated starch, hyaluronate and honey of the examples of the present application produced a significant synergistic effect, with separate preparation examples 1-3 (phosphorylated starch), sodium hyaluronate alone, and individual transparency. Significant difference in the effect of potassium phytate, manuka honey alone, jellybush honey, sodium hyaluronate and manuka honey (Comparative Example 2); even if the drugs are the same but the ratio is different Although Comparative Example 1 also obtained a certain therapeutic effect, there was also a significant difference compared with the Examples 1-3 group.

B, the healing effect of the wound

Animal model

A total of 144 rats [72 males and 72 females] were selected to test the work of the pharmaceutical composition of the present application.

As group I of the test group and group II as a control group.

Table 2

Group 1 (test) group Π (control) N 72 72

Gender (M/F) 36/36 36/36

Age (week) 6-7 weeks 6-7 weeks Burns in animal models:

Test and control animals were used to generate burns. Special brass rods were used to create burns in rats. Brass rods were maintained for 15 ± 1 min in a 75 ± 2 ° C hot water bath. One day before the start of the experiment, the animals were shaved with an area of 2 X 2 X 2 cm. Remove the brass rod from the water bath and place it on the rat skin for a short period of 15-20 seconds, then immediately remove it. The rod diameter is selected in such a way that a wound of about 1 cm should be produced when the stick touches the skin of the animal. For each animal, a separate sterile bar was used to prevent any infection that may be present in the animal or any infection from one animal to another _0. After 30 minutes of burn on the rat, the above preparation was applied. Examples 1-3, pharmaceutical compositions of Examples 1-3, sodium hyaluronate alone, potassium hyaluronate alone, Manuka honey alone, jellybush honey alone, composition of Comparative Example 1, Comparative Example 2 composition. The study preparation and control were evenly applied to the burned area with a sterile cotton swab twice a day (each drug was used in an amount of 15 mg/cm ³ based on the total active ingredient) until the wound healed, or until the end of six weeks. . Animals are subjected to any possible basic burn management prior to application.

The primary purpose of the study was the duration of healing, and the duration of healing in Group I cases was significantly reduced by more than 50% compared to group sputum. A secondary end point of epithelialization was observed in the new formulation group compared to the control group. The difference in efficacy between Examples 1-3 was small, but with Preparation Examples 1-3, sodium hyaluronate alone, potassium hyaluronate alone, Manuka honey alone, jellybush honey alone, Comparative Example 1 -2 has a significant difference. No significant scar hyperplasia or any pigmentation change was observed in the pharmaceutical composition groups of Examples 1-3. The pharmaceutical compositions of Examples 1-3 were similar in duration of burn healing to animals, but the percentage of healing time was over 50% compared to control animals.

table 3

Group 1 (test) group π (control)

N 72 72

Gender (M/F) 36/36 36/36

Age (week) 6-7 weeks 6-7 weeks

Preparation 1 12 days (6 nos) 16 days (6 nos)

Preparation 2 22.5 days (6 nos) 15 days (6 nos)

Preparation 3 3 days (6 nos) 18 days (6 nos)

Sodium hyaluronate 15 days (6 nos) 17 days (6 nos) Potassium hyaluronate 14 days (6 nos) 16 days (6 nos)

Manuka Honey 13.5 days (6 nos) 16 days (6 nos)

Jellybush honey 14 days (6 nos) 17 days (6 nos)

Example 1 6 days (6 nos) 15 days (6 nos)

Example 2 6 days (6 nos) 16 days (6 nos)

Example 3 5 days (6 nos) 17 days (6 nos)

Comparative 1 10 days (6 nos) 16 days (6 nos)

Comparative 2 2 days (6 nos) 17 days (6 nos)

Note: 1. Healing is the criterion for clinically close to complete wound closure.

Based on the above healing time, it was concluded that the healing composition of the phosphorylated starch, hyaluronate and honey of the present invention in a certain ratio is significantly better than the single phosphorylated starch alone, sodium hyaluronate alone, The combination of potassium hyaluronate alone, manuka honey alone, jellybush honey alone, hyaluronate and honey indicates that the composition of the invention has a synergistic effect on burn wounds, resulting in faster burn wound closure , a better prognosis in patients with reduced hospitalization and burns, and even if the drugs were the same, the proportions of Comparative Example 1 obtained better therapeutic effects, but there was also significantness compared with the groups of Examples 1-3. difference.

Moreover, it has been confirmed by experiments that the pharmaceutical composition of the present invention is stable, has a long shelf life, and is produced for use in selected from acute wounds, chronic wounds, burns and surgical wounds, as well as ulcers, acne, pressure sores, such as diabetic ulcers. Unexpected synergy effects.

While the invention has been described in terms of its preferred embodiments, the embodiments of the invention

Claims

A pharmaceutical composition for wound treatment comprising as an active ingredient the following:

(a) phosphorylated starch,

(b) hyaluronate;

(c) honey;

Wherein the phosphorylated starch is prepared by reacting a starch with a phosphorylating agent and at least one conjugate.

The pharmaceutical composition according to claim 1, which has a masking activity of 70% or more for elastase.

3. The pharmaceutical composition according to claim 1, wherein said hyaluronate is selected from the group consisting of sodium salts, potassium salts and zinc

; n brn bu.

4. A pharmaceutical composition according to claim 1 wherein the honey is selected from the group consisting of manuka honey, jellybush honey, or honey from rewarewa, cancaca and beech.

The pharmaceutical composition according to claim 1, wherein the conjugated agent is at least one selected from the group consisting of urea, thiourea, biuret, cyanuric acid, urea formaldehyde, and melamine; and the phosphorylating agent is a starch phosphate Any compound, including disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium metaphosphate, diammonium hydrogen phosphate, sodium orthophosphate, sodium tripolyphosphate, sodium hexametaphosphate, tetrasodium pyrophosphate.

The pharmaceutical composition according to any one of claims 1 to 5, wherein the phosphorylated starch has a structure of the following formula (I):

[-St-(OP0 ₃ "H) _k (OH) ₁ (0-Z) _m (0-W) _p ]

Formula (I)

among them:

"St" means a subunit of starch;

k+1+m+p-3;

k is equal to or less than 0.5;

1, m and p are numbers from 0 to 3, including 0 and 3 and can be fractions;

W is a condensation product CH ₂ R, wherein R is selected from the group consisting of: -NH-C(0)-NH ₂ ; -NH-C(0)-NH-CH ₂ -OSt; -NH-C(S)-NH ₂ ; -NH-C(S)-NH-CH ₂ -OSt; melamine group; wherein NH ₂ group and CH ₂ -OSt Linked melamine group; any combination thereof and any chain extended form thereof, and wherein w may be substituted or unsubstituted;

And wherein the negative charge on P0 ₃ - is neutralized by the cation.

The pharmaceutical composition according to any one of claims 1 to 6, wherein the mass ratio of phosphorylated starch, hyaluronate and honey is 5-10: 3-6: 2-4, preferably 7-8 : 4-5: 2.5-3.5, more preferably 8: 5: 3.

The pharmaceutical composition according to any one of claims 1 to 7, wherein the composition is in the form of a powder, an ointment, an ointment or a gel, preferably an ointment and an ointment.

9. Use of a pharmaceutical composition according to any one of claims 1-8 in the manufacture of a medicament for the treatment of wounds, such as for wound healing.

10. Use according to claim 9, the wound being selected from the group consisting of an acute wound, a chronic wound, a burn and a surgical wound, wherein preferably the chronic wound is selected from the group consisting of an ulcer, an acne, a pressure sore, such as a diabetic wave.