CS250018B1 - A method of preparing a biologically active cover for the early - Google Patents

Abstract

The purpose of the solution is a method of preparing a biologically active wound dressing. The stated purpose is achieved by crosslinking powdered polysaccharide such as starch, cellulose, agar, lichenan with a particle size of 0.06 to 0.3 mm with 2-chloromethyloxirane to a hydrophilic gel with a swelling degree of 4 to 15 ml. g"1 is activated with 2-chloromethyloxirane and after activation, proteolytic enzymes such as chymotrypsin, trypsin, subtilisin and lysozyme are chemically bound to the resulting oxirane groups, then unbound enzymes are removed from the gel by washing and the washed gel is drained with an organic solvent and after its evaporation, the powdered product is sterilized by radioactive χ-radiation. The preparation can find application in surgical departments, in clinics specializing in civil and professional injuries and in veterinary medicine

Description

The present invention relates to a method for preparing a bi-active active cover in the form of a backfill.

For the purpose of treating surface wounds and in medical practice, powders prepared from scratch, mastenoa or other galenic bases and an active ingredient with a bactericidal action, such as antibiotics, suilfoamides and the like, are commonly used. The treatment of purulent and necrotic wounds has recently highlighted the sorption effect of some polymers, which, by virtue of their hydrophilic character, are able to take off liquids from the surface early by aspiration of exudate, bacteria and their metabolites into the capillaries of the internal gel structure.

Preparations based on cross-linked a-1,6-gil, cane, dextran [B. S. Jacobson: Scand. J. Plast. Reconstr. Surg. 10, 65-72 (1976)], or cellulose derivatized in a substitution reaction to ether [DTA 1,932,753],

Recently, the efficacy of the powders thus prepared has been further enhanced by the binding of enzymes from the group of hydrolases with the proolytic activity of the modified biopolymers by covalent bonding. [Immobilizovanyje proteolytičeskije ferments in the treatment of gnojmo-necrotic processes, proceedings of rabotts, AN USSR, Sibirsikoje otdelenije, Institute of Citology and Genetics !, Novosibirsk (1981).)

The presence of immobilized proteases in such treated preparations causes the decomposition of necrotic tissue in the vicinity of the early and pus on its surface, and the resulting degradation products soak up the hydrophilic gel into its internal structure, thereby simultaneously removing the bacterial growth environment and preventing toxins from penetrating the organism.

The preparation and properties of an early proteolytic cover is described on the basis of polyaldehyde-modified peiric cellulose modified in the oxidation step and subsequent coupling of the proteolytic enzyme with the primary amino group of the cellulose and finally stabilizing the Schiff bond between the enzyme and the hydrophilic carrier by reduction. 249 311.

Said process for the preparation of an early proteolytic cover is technically demanding, especially with regard to the chemicals used, low yields in the immobilization of enzyme preparations and the need to stabilize the resulting schiff bonds.

The disadvantage is the necessity to use sophisticated methods of drying the prepared preparation by freeze-drying, as well as the fact that the use of said preparation only removes bacteria in the early presence - by their sorption into the internal structure of the gel.

It is an object of the present invention to provide a powdered polysaccharide such as starch, cellulose, agar, lichenate having a particle size of 0.006-0.3 mm crosslinked with 2-chloromethyl oxirane to a hydrophilic gel having a swelling degree of 4-15 ml. g ^-1 is activated at a temperature of 10 to 25 degrees C for 2 to 4 h, in the presence of sodium hydroxide with 2-chloromethyloxirane, after which an activated gel containing 0.05 to 0.3 mmol is activated. The g- ¹ oxirane functional groups are washed into a neutral reaction and reacted with a solution of proteolytic enzymes such as chymotrypsin, trypsin, substillizine and lysozyme at a ratio of proteolytic enzyme to lysozyme activity of 2 to 4 to 1, at 20 to 40 ° C for 3 to 6 h, after which the polysaccharide gel with the bound enzymes is freed of free enzymes by washing, then the gel is drained by washing with an organic solvent, preferably ethanol and / or acetone, and the dry gel is finally sterilized by radioactive χ-radiation.

The biologically active early dressing prepared on the basis of cross-linked polysaccharides can be dried by evaporating excess water in a vacuum evaporator or by sucking off excess water on a frit and suspending polysaccharide gel particles in ethanol or acetone, and after removing excess organic solvent in vacuum to dry at a temperature of 40 to 60 ° C to a residual moisture of 30 to 50 mg of water per 1 g of gel.

The thus prepared and dried biologically active wound dressing ensures easy handling in production and use, uniform powder flow when applied to the wound, preventing the entry of gel and enzyme proteins into the bloodstream and the absence of small particles, so-called " fines gel biopolymer fraction.

Particles of 0.06 to 0.3 mm in size do not get caught in the surface structure early. Microbiota lysis Gram positive bacteria responsible for the suppurative character of the wounds are provided by immobilized lysozyme. The sorption capacity of the carrier gel is up to 15 ml. g ^-1 , which is substantially more than the sorption capacity of native biopolymers or pearl cellulose.

When applying a biologically active wound dressing prepared on the basis of mobilized chymotrypsin or trypsin and lysozyme on a gel of cross-linked cellulose, hydroxyethylcellulose, starch or agar, a marked proteolytic effect on necrotic tissue characterized by 300-600 mg of hydrolyzed protein (bovine or 1'-human albumin) serum, denatured collagen) 1 g dry gel weight per hour.

The gels used showed a high sorption ability when aspirating exudates from the early (up to 15 g exudate per 1 g dry weight of gel) and immobilized lysozyme caused a 95% decrease in the observed staphylococci and streptoicocci (characterized by 150 mg lysed Micrococcus luteuis and up to 100 mg aureus (dry weight per hour 1 g dry weight gel).

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Example 1

100 g of microcrystalline cellulose cross-linked with 10 wt. 2-Chloromethyloxirane with a swelling volume of 5 ml. g ^-1 and a particle size of 0.05 to 0.3 mm is made alkaline for 1 h at 20 ° C with 100 ml of an aqueous solution of sodium hydroxide at a concentration of 20% by weight. 25 ml of 2-chloro-methyloxane are then added to the reaction mixture and the mixture is left to react for 6 hours at 15 DEG C. with stirring.

Next, the reaction mixture is neutralized with an aqueous acetic acid solution to the phenolphthalein indicator, and the resulting salts are washed with distilled water. After washing the unreacted chemicals, a solution of chymotrypsin and lysozyme in phosphate buffer (10 mmol.l ^-1 , pH 7.6, 300 ml) with a chymotrypsin activity of 1,300 mg hydrolyzed bovine serum albumin per lh and lysozyme activity 650 was added to the eluted gel. mg of lysed Micrococcus luteus cells per h. The suspension thus prepared is shaken on a recirculating shaker (frequency 1 Hz, amplitude 20 cm) for 6 h at 20 ° C.

Unreacted chymotoypsine and lysozyme were eluted with 2,000 ml phosphate buffer pH 7.6 (10 mmol.l ^-1 ), then 2,000 ml phosphate citrate 50 mmol.l ^-1 ; pH 5.5j and finally distilled water (4000 ml). The dewatering of the gel was ensured by successive elution of the gel with ethanol-water mixture (1000 ml: first 30 parts by volume of ethanol and 70 parts by volume of water; then by 70 parts by volume of ethanol and 30 parts by volume of water; 4 volumes of water).

The dewatered gel was dried first in air and then in a rotary evaporator at 50 ° C. Sterilization of the finished iproteolytic cover for early was ensured by gamma-irradiation, with a dose of 25 kGy in polyethylene sealed bags. The proteolytic capsule thus prepared did not contain free oxirane groups or non-flammable chlorine form. The unreacted 2-chloromethyl-oxirane was decomposed to glycerol and eluted at the gel treatment. The unreacted methyloxirane substituent on the gel surface hydrolyzed to a 1,2-dihydroxypropyl substituent.

Example 2 such that the polysaccharide (used hydroxyefylcelulóza powder crosslinked with 2-c.hlórmetyioxiránom the swelling volume 15 ml. ^G-1 and activation of the 2-chiórmetyloxiránom is performed at preferably 25 ^{DEG C} for 3 hours.

Example 3

The procedure of Example 1, except that crosslinked agar with 2-chloromethyloxiranini having a degree of flaking of 0.03 cross-links per anhydroglucosyte unit and a swelling volume of 15 ml was used as the enzyme support. g ^-1 . A mixture of subtilisin and lysozyme was used for immobilization as described in Example 1. The dried preparation had an activity of 300 mg hydrolyzed bovine serum albumin per hour per g dry weight and 150 mg dry Micrococcus luteus cells lysed per hour g immobilized preparation at 37 C.

Example 4

The procedure is as described in Example 1 except that papermaking is used for immobilization and potato starch, beaded with 2-chloromethylloxirane to a swelling volume of 5 ml, is used as the enzyme carrier. g ^-1 . Enzyme binding was performed at 40 ° C for 3 h. Drainage was provided as described in Example 1 except that aqueous acetone solutions were used. The resulting enzyme preparation had an immobilized papain activity of 50 mg hydrolyzed bovine serum albumin per hour for 1 g and 12.5 milligrams of Micrococcus luteus lysed cells per hour for 1 g dry preparation at 37 ° C.

Example 5

The procedure is as in Example 1 except that crosslinked α1,6-glucan-pulluan having a cross-linking degree of 0.03 and a swelling volume of 15 ml is used as carrier. g ^-1 . The resulting activity of the immobilized enzyme preparations was 290 mg hydrolyzed bovine serum albumin per hour per g dry weight and 143 mg lysed Micrococcus luteus cells per hour per g dry immobilized preparation.

The invention has potential applications in surgical departments, clinics specialized in civil and professional injuries, but also in veterinary medicine.

The procedure of Example 1 with the difference

Claims (1)

A method for preparing biologically active ingredients, characterized in that the powdered polysaccharide, the alcohol starch, the cellulose, the agar, the leiche with a particle size of 0.06 to 0.3 mm are crosslinked: with 2-chloromethyloxirane to a hydrophilic goal with a swelling degree of 4 to 15 ml. g "1 is activated at a temperature of 10 to 25 ° C for 2 to 4 h in the presence of sodium hydroxide with 2-chloromethyl oxide, and then the activated gel containing 0.05 to 0.3 mmol of oxirane functional groups is washed to a neutral state and reacted with the flow of proteolytic enzymes such as chymo-trypsin, trypsin, subtilisin, and lysozyme in the proteolytic enzyme activity to lyosome 2 to 4 to 1 at 20 ° C to 40 ° C for 3 hours up to 6 h, the polysaccharide gel with enzyme bound free of enzymes for washing by washing, the gel is finally sterilized by radioactive γ-irradiation by dewatering with organic solvent, preferably ethanol and / or acetone, and dry gel. 7, Most Price 2,40 Kčs