RU2630668C1 - Substance of proteolithic ferment based on protosubtilin gzh immobilized on chitosan and composition for treatment of purulent-necrotic wounds - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: proteolytic enzyme substance based on Protosubtilin GZH and a composition for purulent-necrotic wounds treatment are proposed. The proteolytic enzyme substance immobilized on chitosan activated by glutaraldehyde has an enzymatic activity of 17-19 units of neutral protease activity/g of wet weight. The composition for treatment of purulent-necrotic wounds contains the proteolytic enzyme substance based on Protosubtilin GZH as the main component, T-2 emulsifier, Tween 20 and 0.05% chlorhexidine bigluconate solution as the target additives, vaseline oil as a base.

EFFECT: increased purity of the proteolytic enzyme and increased wound healing properties of the composition.

2 cl, 1 dwg, 4 tbl, 10 ex

Description

The invention relates to a proteolytic enzyme (protease) of Bacillus subtilis, immobilized on a carrier and composition for the treatment of purulent necrotic wounds based on it and can be used in medicine, biotechnology and biochemistry for protein hydrolysis.

Proteolytic enzymes accelerate the biosynthesis of collagen and other muscle proteins, normalize the level of indicators of redox processes, thereby significantly reducing wound healing time, have a powerful anti-inflammatory and anti-edematous effect (Diagnosis and treatment of wounds / Edited by Yu.G. Shaposhnikov. - M. : Medicine, 1984, p. 200, 218) [1].

Known methods of immobilization by adsorption of the enzyme on matrices with a highly developed surface (Immobilized enzymes. Under the editorship of IV Berezin. T. 1, M., Publishing House of Moscow State University, 1976, S. 79-140) [2]. The disadvantage of these methods is the low bond strength of the enzyme with the carrier, as a result of which the enzyme can diffuse from the carrier into the solution during operation.

A known method of producing a water-soluble immobilized enzyme preparation (USSR author's certificate No. 730694, IPC С08В 37/02, publ. 30.04.80) [3], which includes the stages:

- activation of the carrier - dextran with a pier. mass of 20 kDa, in the reaction of periodic oxidation (oxidation state of the carrier γ = 6-48%);

- preparation of the reaction mixture: oxidized dextran is dissolved in water to a concentration of 50 mg / ml and mixed with a solution of the terrilithin enzyme in 0.1 M borate buffer pH 8.5;

- enzyme immobilization: the reaction mixture is stirred for 2 hours at 21 ± 1 ° C, then a 0.1 M solution of borate buffer containing sodium borohydride is added and stirred for another 40 minutes;

- desalination of the immobilized enzyme preparation by dialysis in the cold (4 ± 2 ° C) against distilled water for 48 hours;

- separation of excess unbound enzyme by gel filtration;

- freeze drying.

Get the drug with a specific activity of 7.4 PE / mg.

The disadvantages of this method are: the complexity of the process (the long process of obtaining an activated carrier, the gel filtration process is difficult to scale, a significant investment of time, energy and raw materials), the enzyme during operation can diffuse with the carrier into the bloodstream during bleeding of the wound surface and cause allergic reactions . In addition, there is some inconvenience during use: the solution dries after 0.5-1 hours and the dressing must be moistened or the dressing changed.

A known method of obtaining a water-soluble immobilized proteolytic complex (USSR author's certificate No. 1442548, IPC С12N 11/00, publ. 07.12.88) [4], which consists in the following:

Protosubtilin G10X, which is a complex of bacterial neutral and alkaline proteases from you. subtilis, dissolved in 0.05 M borate buffer, pH 8.4 at 2-4 ° C for 30 minutes, and the insoluble precipitate was separated by centrifugation at 7000 g for 30 minutes. To the obtained supernatant add a 10% aqueous solution of polyethylene oxide per mol. weighing 600-2000 Yes, mainly 1500 Yes, in the ratio of the carrier - protosubtilin 1: (7.5-8.5). The mixture of the enzyme with the carrier is irradiated with bremsstrahlung γ-radiation at a dose of 2.3-2.7 mrad. In this case, a water-soluble immobilized enzyme preparation with a proteolytic activity of 80-120 PE / ml is obtained. The drug is a clear yellowish liquid, which is stored at 4-8 ° C.

A known method of obtaining a water-soluble immobilized proteolytic complex (RF patent No. 2137835, IPC C12N 11/00, publ. 09/20/1999) [5], including the preparation of a reaction mixture containing protosubtiline with polyethylene oxide mol. weighing 1500-4000 Yes, followed by irradiation of the resulting mixture with bremsstrahlung γ-radiation, characterized in that the ratio of the enzyme-carrier is set to 1: (2-6), before the irradiation, the ballast proteins are salt precipitated by irradiation by sequential addition to the reaction mixture first, sodium phosphate to a final concentration of 0.45%, and then calcium chloride to a final concentration of 0.63%. The target product is subjected to freeze drying in the presence of polyethylene oxide with a mol. weighing 4000 Yes in an amount of 50-300 mg / ml.

The disadvantages of the known methods [5, 6] are:

- radiation modification of bacterial proteases with polyethylene oxide leads to heterogeneity of the obtained product (the method of immobilization does not exclude the desorption of the complex of proteases from the matrix, since the enzyme in this case is not covalently linked to the carrier),

- the enzyme during operation can diffuse with the carrier into the bloodstream during bleeding of the wound surface and cause allergic reactions.

In addition, there is some inconvenience in use: the solution dries after 0.5-1 hours and the dressing must be moistened or changed.

Known drug "Dressing for the treatment of wounds" (RF patent No. 2219954, IPC A61L 15/08, publ. 12/27/203) [6], consisting of a perforated film of chitosan (1.0-6.0 h) in polyvinyl alcohol (60 , 0-95.0 h), containing biologically active additives, including proteases (0.5-30.0 h), glutaraldehyde (0.1-2.0 h) and organic acid (0.5- 4.0 h).

However, enzymes, glutaraldehyde and organic acid during use of the dressing can diffuse into the bloodstream when the wound surface is bleeding and cause allergic reactions (as noted in the description, “desorption of biologically active substances from the chitosan film” occurs). The activity of enzymes is weakly manifested in 60-95% alcohol.

A known method of obtaining funds for the treatment of purulent-necrotic wounds (RF patent No. 1814764, IPC A61K 47/48, publ. 03.20.1995) [7], which consists in processing a 2% chitosan gel with a 2% solution of glutaraldehyde in phosphate buffer at pH 7.5, taken in the ratio 1: 1, keeping at a temperature of 18-20 ° C for 30 minutes, washing and passing through it the hepatopancreas extract of king crab containing a complex of enzymes: TNK-aza, RNA-ase, alkaline phosphatase, phosphodiesterase type 1 and protease.

However, in the known method of obtaining funds, it is technologically difficult to pass liquids through gels and provide accurate concentrations of chitosan (to obtain funds, a 2% chitosan gel is used). If a 1% gel is used, the suspension of the carrier is obtained liquid and less suitable for the preparation of ointment forms. With a concentration of chitosan of 3-4%, the density of the carrier increases sharply. The activation time of the matrix for 30 min is clearly not enough [8], and a 2% chitosan gel has short shelf life, because prone to dry.

The closest analogue (prototype) of the claimed substance of the proteolytic enzyme Bacillus subtilis is a preparation of immobilized protease Bacillus subtilis on a carrier in the form of aminoethyl cellulose activated by glutaraldehyde, described in a method for producing an immobilized proteolytic complex, which involves activating aminoethyl cellulose with glutamate glutamate. The result is a complex of immobilized protease with a specific activity of 32-40 units. act / g dry weight (RF patent No. 686377, IPC A61K 37/48, publ. 15.12.1993) [8].

However, this substance has insufficient purity of the enzymatic complex before immobilization on a carrier (the Protosubtilin GX preparation used in the method contains the following excipients: sodium chloride, chalk, cornmeal, Bacillus subtilis waste products. Calcium chloride precipitation is used for purification (the supernatant is collected) and ethanol (use a precipitate.) However, not all impurities having amino groups are removed from the substance and can reduce the yield of immobilized protease. The drug "Profesim-10 ml" isp It is used in the form of a suspension, which requires the creation of a “wet chamber” by applying a gauze dressing. Practice has shown that this form of the drug is inconvenient in the treatment of burns and wounds, since when changing dressings, granulation of the wound is damaged, which does not contribute to its rapid healing.

The closest analogue (prototype) of the claimed composition for the treatment of purulent necrotic wounds is a preparation of the Bacillus subtilis protease (Profesim) chemically immobilized on aminoethyl cellulose in the composition based on polyethylene glycol (PEG 5000) and glycerin (RF patent No. 2150936, IPC A61K 9/06, publ. 06/20/2000) [9] (wt.%):

Profesim with an activity of 1.0-2.5 pe / g 20,0 Polyethylene glycol with mol. weighing 5000 25.0 Glycerol 25.0 Chlorhexidine bigluconate 0.2 Water Up to 100.0

However, the wound healing composition has an insufficient rate of wound healing, as well as a liquid consistency of ointment (insufficient viscosity). The last factor allows the drug to spread (spread) over a significant surface of the body, reducing the amount of active substance on the wound surface.

The technical result of the claimed invention is to increase the wound healing properties of the substance and composition of the drug based on it by increasing the degree of purification of proteolytic enzymes and using chitosan activated by glutaraldehyde as a carrier, as well as improving the rheological properties (viscosity) of the ointment form of the drug composition.

The specified technical result is achieved by the creation of a substance of a proteolytic enzyme based on Protosubtilin GZH, used as the main component of the composition for the treatment of purulent-necrotic wounds, with an enzymatic activity of 17-19 units. neutral protease / g wet weight and obtained by suspending the specified dry enzyme in phosphate buffer A in an amount of 0.1 mol / l at pH 7.6, purification by salt precipitation to a final concentration of 0.1 mol / l and removing the precipitate by centrifugation, gel filtering the enzyme on a column of acrylex P-15, using phosphate buffer A, activating chitosan with a 25% solution of glutaraldehyde for 5-6 hours, followed by immobilization on the activated chitosan of the purified proteolytic enzyme GZH at a temperature 6-8 ° C for 18 hours, sequentially washing the resulting substance with water, a solution of Tris-HCl at a concentration of 0.1 mol / l at pH 7.6, a solution of sodium phosphate at a concentration of 0.5 mol / l at pH 7, 4 and suspending the substance in a Tris-HCl solution at a concentration of 0.01 mol / L at a pH of 7.4 containing 0.9% NaCl.

The specified technical result is also achieved by the fact that the composition for the treatment of purulent-necrotic wounds, comprising a substance of the proteolytic enzyme (protease) of Bacillus subtilis immobilized on a carrier, target additives and a base, according to the invention, as a substance of the proteolytic enzyme (protease) of Bacillus subtilis, immobilized on the carrier it contains a substance according to claim 1 of the claims, as the target additives is an emulsifier T-2, tween 20 and a 0.05% solution of chlorhexidine bigluconate, and as a base - petroleum jelly th with the following quantitative content of components (wt.%):

a substance according to claim 1 of the claims 29.0-30.0 emulsifier T-2 12.0-13.0 twin 20 16.0-17.0 0.05% chlorhexidine bigluconate solution 19.0-20.0 liquid paraffin the rest is up to 100%

In accordance with GOST 20264.2-88, page 2 (http://www.internet-law.ru/gosts/gost/28652/), the ability of the enzyme to convert sodium caseinate into 1 min at a temperature of 30 ° C is taken as non-precipitated trichloroacetic acid state in an amount corresponding to 1 μmol tyrosine. Proteolytic activity is expressed by the number of units indicated in 1 g of the test drug.

The invention is illustrated by an electrophoregram of the complex of proteolytic enzymes shown in FIG. 1. Electrophoresis in 12% SDS page under denaturing conditions, Coomassie R-250 staining, where: K - a complex of proteolytic enzymes; M - molecular weight markers (20-150 kDa).

Example 1. Characterization of the substance of the proteolytic enzyme (protease) of Bacillus subtipis immobilized on chitosan activated by glutaraldehyde.

The substance of the proteolytic enzyme based on Protosubtilin GZH is used as the main component of the composition for the treatment of purulent-necrotic wounds, has an enzymatic activity of 17-19 units. neutral protease / g wet weight and obtained by suspending the specified dry enzyme in phosphate buffer A in an amount of 0.1 mol / l at pH 7.6, purification by salt precipitation to a final concentration of 0.1 mol / l and removing the precipitate by centrifugation, gel filtering the enzyme on a column of acrylex P-15, using phosphate buffer A, activating chitosan with a 25% solution of glutaraldehyde for 5-6 hours, followed by immobilization of the purified proteolytic enzyme Protosubtilin GZH on activated chitosan at a temperature of 6-8 ° C for 18 hours, sequentially washing the resulting substance with water, a Tris-HCl solution at a concentration of 0.1 mol / l at pH 7.6, a sodium phosphate solution at a concentration of 0.5 mol / l at pH 7.4 and suspension substances in a Tris-HCl solution at a concentration of 0.01 mol / L at a pH of 7.4 containing 0.9% NaCl.

Example 2. Composition for the treatment of purulent-necrotic wounds in the form of an ointment with a minimum content of components (wt.%):

the substance described in example 1 29.0 emulsifier T-2 12.0 twin 20 16,0  0.05% chlorhexidine bigluconate solution 19.0 liquid paraffin the rest is up to 100%

Example 3. The composition "PROTOSAN" in the form of an ointment for the treatment of purulent-necrotic wounds (wt.%):

the substance described in example 1 30,0 emulsifier T-2 13.0 twin 20 17.0 0.05% chlorhexidine bigluconate solution 20,0 liquid paraffin the rest is up to 100%

To obtain the substance according to example 1 and the composition according to examples 2 and 3, the following preparations were used: Protosubtilin GZH, TU 9291-029-13684916-2010 (Sibbiopharm, Russia), chitosan (Apifarm, Russia). (You can use chitosan from the company Bioprogress CJSC, Schelkovo, Russia). Neutral protease activity was determined by the Anson method in modification (Gracheva I.M., Grachev Yu.P., Mosichev M.S. et al. Laboratory workshop on the technology of enzyme preparations. - M .: Light and food industry (1982) . SS. 36-41) [10]. The degree of chitosan deacetylation was determined by potentiometric titration (Kuchina Yu.A., Dolgopyatova N.V., Novikov V.Yu., Sagaidachny V.A., Morozov N.N. Instrumental methods for determining the degree of chitin deacetylation // Vestnik MGTU, t. 15, No. 1, 107-113 (2012)) [11]. Used acrylic P-15, ("Reanal", Hungary); reagents (glutaraldehyde, benzoquinone, salts) of the chemically pure grade.

Example 4. Preparation of an enzyme preparation for immobilization.

To clean the enzymes, 5 g of Protosubtilin GZH powder were suspended in 15 ml of sodium phosphate buffer A (0.1 mol / L; pH 7.6). Insoluble components were removed by centrifugation (8000g, 20 min). Added 1 ml of a solution of calcium chloride to a final concentration of 0.1 mol / L, stirred for 20 minutes and the resulting precipitate was removed by centrifugation. To remove low molecular weight colored components and salts from the solution, gel filtration was performed on a column (55 ml) with P-15 acrylex using buffer A. As a result, 20-22 ml of a complex of proteolytic enzymes containing 125-135 mg of protein and neutral protease activity were obtained 480-520 units (24-26 units act./ml), (3.85 units act./mg protein versus 2 units act./mg in the prototype (RF Patent 686377 [8]).

In FIG. 1 shows the electrophoregram of a complex of proteolytic enzymes. Electrophoresis in 12% SDS page under denaturing conditions, Coomassie R-250 staining. Where:

K - a complex of proteolytic enzymes;

M - molecular weight markers (20-150 kDa).

Example 5. Activation of the media. For a comparative experiment, 3 g of chitosan powder was placed in water, then washed on glass filters with buffer B (0.2 mol / L; pH 7.8). Each sample was suspended in 20 ml of buffer B.

1 ml was added to the first portion, 4 ml of a 25% solution of glutaraldehyde (2.5 and 10 mM) was added to the second portion and stirred for 5-6 hours at room temperature. After that, samples of activated chitosan were washed with buffer B on glass filters.

Example 6. Immobilization of the enzyme complex. Each portion of the activated carrier was introduced into 10 ml of an enzyme solution (250 units of act. Neutral protease). Incubation was carried out for 18 hours with stirring at a temperature of 6-8 ° C. Then, the obtained immobilized preparations were washed successively with water, Tris-HC1 solution (0.1 mol / l, pH 7.6), sodium phosphate solution (0.5 mol / l; pH 7.4). And suspended in Tris-solution HCl (0.01 mol / L, pH 7.4) containing 0.9% NaCl.

Example 7. Analysis of the obtained samples by activity

The obtained samples of immobilized enzymes were evaluated by the mass and activity of a neutral protease.

The results are presented in table 1. From the data of table 1 it is seen that at a ratio of glutaraldehyde of 10 mM per 15 mM of deacetylated chitosan groups, the protein almost completely immobilized and the specific activity of the neutral protease was observed up to 17 units. act./g wet mass, which corresponds to 57 units. act. / g dry weight against 32-40 units. act. / g in the prototype (RF Patent 686377, class A61K 37/48 [8]). At the same time, the activity of the immobilized enzyme remained up to 70% (170 from the initial 250 units of act.). The preparation obtained by activation at a glutaraldehyde ratio of 10 mM per 15 mM of deacetylated chitosan groups was called “Protosan”.

After storage at plus 37 ° C for 7 days, the preparations retained their activity up to 90%.

Example 8. Obtaining immobilized proteases on activated chitosan in an amount of 30 g for the subsequent preparation of an ointment form

7.5 g of Protosubtilin GX powder was suspended in 22 ml of sodium phosphate buffer A (0.1 mol / L; pH 7.6). Insoluble components were removed by centrifugation (8000g, 20 min). Added 1.5 ml of a solution of calcium chloride to a final concentration of 0.1 mol / L, stirred for 20 minutes and the resulting precipitate was removed by centrifugation. To remove low molecular weight colored components and salts from the solution, gel filtration was performed on a column (75 ml) with P-15 acrylex using buffer A. The result was 30-32 ml of a complex of proteolytic enzymes containing 370-400 mg of protein and neutral protease activity 740-800 units Act.

9 g of chitosan powder was placed in water, then washed with buffer B on glass filters (0.2 mol / L; pH 7.8). The sample was suspended in 60 ml of buffer B.

12 ml of a 25% glutaraldehyde solution (10 mM) were added and stirred for 5-6 hours at room temperature. After that, a sample of activated chitosan was washed with buffer B on glass filters.

The activated carrier was added to 30 ml of the enzyme solution. Incubation was carried out for 18 hours with stirring at a temperature of 6-8 ° C. Then the obtained immobilized preparation was washed successively with water, Tris-HCl solution (0.1 mol / l, pH 7.6), sodium phosphate solution (0.5 mol / l; pH 7.4). and suspended in a Tris-HCl solution (0.01 mol / L, pH 7.4) containing 0.9% NaCl.

Received 30 g of a wet preparation with an activity of 17-19 units. act./g wet weight

Example 9. Obtaining the ointment form of the drug immobilized proteases "Protosan ointment"

To obtain the ointment form of the drug "Protozan-ointment" used the components in the ratios shown in table 2.

Vaseline oil, emulsifier T-2 and Tween-20 were mixed at plus 50-55 ° С until a homogeneous suspension was cooled to 35-40 ° С and a complex of immobilized proteases in chlorhexidine solution was added to it with vigorous stirring. Stirring was continued until the ointment was completely homogeneous, 20-30 minutes. Ointment was packaged in 10 g in plastic bottles or gripper - packages with zip-lock latches.

The developed ointment form had a denser consistency than that prepared on a solution of polyethylene glycol with glycerol (prototype RF patent No. 2150936, [9]) and did not spread over the surface. It was called Protozan Ointment.

Example 10. The study of the wound healing effect of the complexes was investigated on a thermal burn model. The experiment used rats of males of the Wistar strain weighing 250-300 g of 6 animals in the group. On a clipped back section of 3 × 3 cm ² under barbamyl anesthesia (50 mg / kg, ip), the animals were thermally burned. For this, a device with an established temperature scale and an electric soldering iron was used, at the end of which a metal plate with a size of 1.0 × 1.5 cm ² was fixed. The plate size was chosen so as to obtain a burn surface area comparable to that in the prototype (patent No. 2150936, [9]). The exposure time of a contact plate heated to 190-200 ° C was 10 seconds.

Experimental animals were divided into 4 groups. In rats of the first experimental group, the burn wound was treated with daily applications of the Protosan suspension, rats in the second with applications of the Protozan-Ointment ointment, rats in the third group with applications of a suspension of chitosan (without an immobilized protease complex), and rats in the fourth group with applications of the ointment base. Samples were applied at a rate of 1 g / 5 cm ^{2 of the} surface of the wound. The condition of the wound was recorded by measuring its area, applying a transparent stencil to the wound. The rate of wound healing (SZR) or Popova index, expressed as a percentage, was calculated by the formula:

SZR = (S-Sn) × 100 / S × t,

where S is the value of the area of the wound in the previous measurement, Sn is the value of the area of the wound at the moment, t is the number of days between the first and subsequent measurement (Rational Antimicrobial Pharmacotherapy: A Guide for Practitioners / Edited by V.P. Yakovlev, S.V. Yakovleva. - M., 2003) [12]. The general condition of the animals was evaluated based on behavioral responses, appetite, and body weight. The results are presented in table 3.

For comparison, the speed of wound healing (SZR) in the prototype (patent No. 2150936, [9]) was calculated and the results are presented in table 4.

From the presented results (tables 3 and 4) it can be seen that the rate of wound healing (SZR) in animals treated with the protease complex immobilized on protozan chitosan significantly differs from the results of the prototype group treated with the “profesim” suspension (first columns of the table). The rate of wound healing (SZR) in animals treated with the ointment form of "protosan ointment" also significantly differs from the results of the prototype group treated with 20% ointment "profesim" (second column of the table). This indicates a more pronounced reparative effect of the complex of proteases immobilized on chitosan. Complete healing of wounds in animals of the experimental group (“protosan ointment”) was completed on the 10th day of the experiment, while in animals of the experimental group of the prototype (20% ointment “profesim”) - on the 11th day. From table 4 it is seen that when using the matrix - chitosan for the treatment of wounds, the healing rate was significantly different from the results of the control group. Complete healing of wounds in animals of the control group was completed on the 17-18th day of the experiment with the formation of a rough scar, which absorbed more slowly (5-6 days) than in the experimental groups (1-3 days). Both complexes (“protozan” and “protozan-ointment”) did not cause changes in the motor reactions of rats, did not have a toxic effect on the animal organism during the observation period.

Claims (3)

1. The substance of the proteolytic enzyme based on Protosubtilin GLC, used as the main component of the composition for the treatment of purulent-necrotic wounds, having an enzymatic activity of 17-19 units of neutral protease activity / g wet weight and obtained by suspending said dry enzyme in phosphate buffer A in an amount 0.1 mol / l at pH 7.6, purification by salt precipitation to a final concentration of 0.1 mol / l and removing the precipitate by centrifugation, gel filtration of the enzyme on a P-15 acrylex column using phosph buffer A, activation of chitosan with a 25% solution of glutaraldehyde for 5-6 hours, followed by immobilization on the activated chitosan of the purified proteolytic enzyme Protosubtilin GZH at a temperature of 6-8 ° C for 18 hours, sequential washing of the obtained substance with water, Tris solution -HCl at a concentration of 0.1 mol / l at pH 7.6, a solution of sodium phosphate at a concentration of 0.5 mol / l at pH 7.4 and suspension of the substance in a solution of Tris-HCl at a concentration of 0.01 mol / l at pH 7.4 containing 0.9% NaCl. 2. A composition for treating purulent necrotic wounds, comprising a substance of the proteolytic enzyme Bacillus subtilis immobilized on a carrier, target additives and a base, characterized in that as a substance of the proteolytic enzyme (protease) Bacillus subtilis immobilized on a carrier, it contains a substance according to claim .1 of the claims, as target additives is an emulsifier T-2, tween 20 and a 0.05% solution of chlorhexidine bigluconate, and as a base - vaseline oil, with the following quantitative content of components, wt.%: a substance according to claim 1 of the claims 29.0-30.0 emulsifier T-2 12.0-13.0 twin 20 16.0-17.0 0.05% chlorhexidine bigluconate solution 19.0-20.0 liquid paraffin the rest is up to 100%

Images