CS245160B1 - Proteolytic cover on wounds - Google Patents

Abstract

The present invention relates to a proteolytic capsule wounds on dextran base, in the form of backfill, serving to cover and treat purulent and necrotic wounds. According to the solution, the cover consists of reticulated dextran to a water content in the skimmed state of 2 to 10 g HoO / g dry matter, in spherical form of size individual particles of 0.05 to 0.5 mm, containing immobilized protease for example chymotrypsin, trypsin, papain or subtilisin. The dextran carrier contains only covalently bound protease, non-washable into the wound. Proteases immobilized by dissolving dextran undesirable protein substances, in particular fibrin, non-necrotic tissues, components pus, etc. New proteolytic cover except This also has a high absorption abilities, ie sucking exudate and pus infected with bacteria from wound to capillary spaces between particles and especially into the mass of highly porous particles. The proteolytic cover acts according to the invention rapid cleansing of necrotic defects, accelerates granule formation and healing early. The uses of the invention are in pharmaceutical form production.

Description

(54) Proteolytic wound cover

The invention relates to a dextran-based proteolytic wound cover in the form of a backfill for covering and treating purulent and necrotic wounds. According to the solution, the cover consists of a sieve dextran swelling to a water content in the centrifuged state of 2 to 10 g HoO / g dry matter, in a spherical form with a particle size of 0.05 to 0.5 mm, containing immobilized protease such as chymotrypsin, trypsin, papain or subtilisin. The dextran carrier contains only a covalently bonded protease and does not elute into the wound. Proteases immobilized with cross-linked dextran cleanse wounds by dissolving unwanted protein substances, especially fibrin, necrotoxic tissues, pus components, etc. In addition, the new proteolytic cover has a high absorption capacity, ie aspirates exudate and pus infected bacteria from the wound into the capillary spaces between particles into the very mass of highly porous particles. The proteolytic cover according to the invention causes a rapid cleaning of necrotic defects, accelerates the formation of granules and heals the wound.

Possible uses of the invention are in pharmaceutical production.

The invention relates to a proteolytic wound cover in the form of a free-flowing powder which is used to cover and treat purulent and necrotic wounds.

In the current pharmaceutical practice and for similar purposes, they recommend powders, often of the two-component type, for example starch or talc, and where the active ingredient is a bacteriostatic or bactericidal agent such as an antibiotic, antifungal or a suitable sulfonamide.

Further developments indicated from these conventional formulations to new type powders based on hydrophilic biopolymers. Their advantage is insolubility in water, preventing contamination of the wound with foreign substances. Biopolymer hydrophilic insoluble powders are well tolerated by the organism, or? they have no side effects. Their action consists in the interaction between the solid and liquid phases, ie, between the dust particles and the liquid component of the injured spinal cord. (The interaction is limited to the area of contact and is not transferred to other sites in the body. Depending on the type of interaction, two types of biopolymer wound dressings, adsorption and proteolytic, can be distinguished.

The first of them is phyllosic, the second has a chemical effect.

The adsorption cover sucks the aqueous component, especially the exudate, and with it the bacteria, substances causing inflammatory processes and toxins from the wound. The proteolytic cover with covalently bonded proteases acts by enzymatic hydrolysis of the protein components of festering wounds.

Its effect is manifested by hydrolytic decomposition and dissolution of necrotic tissues and pus, thus removing the environment supporting the growth of bacteria and limiting the suction of toxic products from the wound into a healthy organism.

An effective adsorbent cover is, for example, spherical cross-linked dextran, which is commercially available under the name Debrisan see Β. Ξ. Jacobason et al., Scand J. Plast. Recoňstr. Surg. 10, 65 (1976).

A proteolytic cover based on spherical cellulose and covalently bonded proteases, freeze dried and its use in medical practice is described by J. Turková et al. in MS.

AO 249311.

Both types of biopolymers have advantages over conventional backfill, but also limitations. Said dextran adsorption cover acts only physically, i.e. by sucking off water and aqueous solutions, eventually into the porous mass of the particles and into the interparticle, i.e. interstitial, proators of the backfill layer. It is, however, highly desirable to simultaneously aspirate the liquid components from the wound, i.e. to also exhibit an adsorption effect. sorption This, however, can only be achieved if special precautions are taken to prepare the cellulosic carrier and the cover itself, which are often complicated and expensive.

Cellulose has a natural tendency to crystallize on drying and to form low porous structures. If covers with high adsorption capability, high porosity are to be obtained, special drying procedures should be used at all stages of preparation.

The current drawbacks of the hydrophilic biopolymer insoluble shells are overcome by the proteolytic sheath of the invention

The subject of the invention is a proteolytic wound cover, which consists of spherical particles with a diameter of 0.05 to 0.5 mm, preferably 0.1 to 0.3 mm, and a base of derivatized cross-linked dextran with immobilized protease enzymes.

A distinctive feature of the novel proteolytic cover is the use of cross-linked dextran as a biopolymer base for immobilizing proteases. Unlike cellulose, cross-linked dextran shows high porosity without the need for special measures.

The porous structure of the dextran carrier can be controlled over a wide range according to the degree of crosslinking of the starting polymer. When drying the cross-linked swollen dextran in water, the porosity is not lost in the case of cellulose, and even in a conventional drying process, products can be recovered that swell in water to a high water content. The dextran-based proteolytic wound covers of the present invention have a high adsorption capability without the need for special measures.

Spherical cross-linked dextrans having a particle size of 0.05-0.5 mm are used to prepare a novel type proteolytic cover, which swells in water to a content of 2-10 g water / g dry matter. Water content means the swollen mass of the particles after centrifugation on a glass frit.

These requirements are met by the commercial product Debrisan and the corresponding chromatographic materials of the brand Sephadex resp. materials of a different origin.

Porous crosslinked dextrans have good chemical reactivity for enzyme binding and known or modified procedures can be used to immobilize proteases, see, for example, the Handbook of Enzyme Biotechnology. Alan Wieseman, Editors, E. Horwood,

London 1975. Procedures should be chosen which provide products with firmly bound enzymes not released during use. Lyophilization is preferably used to dry the product.

The method of use of the novel proteolytic cover in medical practice is similar to that already described in U.S. Pat. AO 24931 1.

In the following, the method of making the cover according to the invention is explained, but not limited, by way of example.

Example 1

Preparation of a cover with chymotrypsin bound

Cross-linked dextran manufactured by Sephadex G-100 and 2-amino-4,6-dichloro-s-triazine were used as carrier.

The cross-linked dextran was swollen in water and used in an amount corresponding to 7.5 g of dry matter. The activating agent was dissolved in 750 ml acetone at 50 ° C and an equal volume of water was added to the solution at the same temperature. The cross-linked dextran was stirred with 300 ml of the activation solution at 50 ° C for 5 minutes. A solution was prepared by mixing a solution of 15 g of sodium carbonate in 100 ml of water with 0.6 volume of 1 M HCl, and added 120 ml at 50 ° C to the activation mixture, which was stirred at 50 ° C for an additional 5 minutes. By adding concentrated hydrochloric acid, the pH of the active mixture was suddenly lowered below 7, the activated product was filtered off, washed with 50% aqueous acetone solution, water and stored in 0.1 M phosphate buffer at 2 ° C and pH 6.6.

Chymotripsin was bound to the activated carrier from a 1.5% solution in 0.2 M borate buffer, pH 8.75 at 24 ° C. After 12 hours, 195 mg enzyme / g carrier were immobilized.

Crosslinked chymotrypsin-bound dextran was washed alternately with 0.1 M borate buffer containing 1 M sodium chloride, pH 9, and 0.1 M acetate buffer containing 1 M sodium chloride, pH 4.5, to zero. proteolytic activity of the eluate. Finally, the sample was washed with 0.25 M borate buffer pH 8 and lyophilized.

Example 2

Preparation of trypsin-bound housing

Cross-linked dextran, the trade name Sephadex G 50, activated by listing of isothiocyanate groups, was used as the carrier.

g of cross-linked dextran was stirred with 4 g of 4-nitrophenylglycidyl ether in 20 ml of toluene and 80 ml of sodium hydroxide solution was added. The suspension was heated at 70 ° C for 18 hours. After washing, the support was swollen in 100 ml, 40 ml of 5M sodium hydroxide NaOH and 3 g of sodium dithionite were added and the reaction mixture was kept at 65 ° C for 1 hour. The product was washed, swelled in 3.5 M phosphate buffer pH 6.8, treated with a 10% solution of thiophosgene in carbon tetrachloride, washed and dried.

Trypsin was bound to an activated carrier from a 1.8% solution in 0.05 M borate buffer pH 8.6 in a nitrogen atmosphere at 5 ° C. After 15 hours, 200 g enzyme / g carrier was immobilized.

The product was washed, as in Example 1, by alternating buffers to zero proteolytic activity, eluate, saturated with the same borate buffer and lyophilized.

Example 3

Preparing the cover with bound papain

The carrier used was cross-linked dextran, manufactured by Debrisan, activated by imidocarbonate groups.

g of cross-linked dextran was left in contact with 40 ml of cyanogen bromide at 24 ° C and pH for 6 minutes. The product was then washed with 500 ml of cold 0.1 M sodium bicarbonate solution for 7 minutes, water and 0.1 M phosphate buffer pH 7.6.

Papain was bound to an activated support of 0.1 M phosphate buffer pH 7.6 at room temperature for 16 hours.

The product was finally treated in a similar manner to Example 1, i.e., washed thoroughly by alternating buffers so that the eluate did not exhibit proteolytic activity, and dried by lyophilization.

Claims (1)

SUMMARY OF THE INVENTION Proteolytic wound dressing characterized in that it consists of spherical particles with a diameter of 0.05 to 0.5 mm, based on a derivatized cross-linked dextran with immobilized protease enzymes. Severography, n. P., MOST Price 2,40 Kčs