RU2268751C2 - Medical bandage containing proteolytic enzyme complex including collagenolytic proteases from crab hepatopancreas - Google Patents

Abstract

FIELD: wound-healing facilities.

SUBSTANCE: invention provides wound coating from chemically modified cellulose, to which enzymes, including collagenolytic ones constituting enzyme complex of crab hepatopancreas, are added by way of chemical bonds formation. Method of making such bandage is also claimed.

EFFECT: enhanced therapeutical activity maintenance.

4 cl, 1 tbl, 3 ex

Description

The invention relates to medicine, specifically to medical dressings containing enzymes, in particular with a collagenolytic effect.

Enzyme-containing medical dressings are widely known. You can refer, for example, to the application of Great Britain No. 2240040, which refers to a medical dressing of partially oxidized cellulose-containing material in the form of gauze, to which enzymes with proteolytic, collagenolytic and / or bacteriolytic activity are attached due to the formation of chemical bonds.

In the patent of the Russian Federation No. 2127128 a method for producing a wound cover is described, which consists in mixing a collagen solution with a complex of proteolytic enzymes isolated from king crab hepatopancreas and having collagenolytic activity, followed by structuring, in some cases, in formaldehyde vapor. The disadvantages of the method include the fact that the mixing of the enzyme with its substrate (i.e., collagenolytic with collagen) forces very strict control of pH and temperature; in addition, formaldehyde, traces of which are inevitably present in the coating, has a carcinogenic effect on the mucous membranes (nose and, possibly, wounds).

The aim of the invention is the creation of a wound cover, devoid of the above disadvantages and retaining high therapeutic activity.

This goal was achieved due to the fact that in the wound cover, consisting of a base containing a complex of proteolytic enzymes from crab hepatopancreas with collagenolytic activity, chemically modified cellulose was chosen as the base.

Also claimed is a method of obtaining such a bandage, comprising the following steps:

- partial oxidation of cellulose in the form of medical gauze to an aldehyde content of 0.13 ÷ 0.25 mmol / g;

- preparation of a solution of hepatopancreas enzymes concentration of 0.4 ÷ 1.5 mg / ml;

- introducing the obtained enzyme solution in the bath module 5-10 into the interaction with partially oxidized cellulose for 1-3 hours at a temperature of 15 ÷ 25 ° C and a pH of 6.8 ÷ 8.5;

- separation of the material with a modified protein, washing with a buffer solution to remove physically adsorbed protein and drying to constant weight or, alternatively, the interaction of the enzyme solution obtained above with phosphoric cellulose ether under the above conditions.

Collagenase, i.e. enzymes capable of cleaving native collagen molecules under physiological conditions are well known and widely used. This is especially true for microbial collagenase from Clostridium his. ("Biochemistry", 1984, vol. 23, p. 3085-3091) and collagenases from mammalian tissues (Extracellular Matrix Biochemistry, N.Y., Elsevier Publishers, 1984). More recently, collagenolytic proteinases from crab Uca pugilator (Biochemistry, 1973, vol. 12, p. 1814-1822), Kamchatka crab Paralithodes camchatica (Biochem. Biophys. Res. Communs., 1990, vol. 166, No. 3, p. .1411-1420) and the crab-chipper Chionoecetes opilio ("Reports of the USSR Academy of Sciences", 1991, vol. 317, No. 2, pp. 488-484).

It is noted that collagenolytic enzymes of various origins significantly differ from each other in substrate specificity, and collagenolytic proteases belong to the trypsin superfamily.

To isolate collagenolytic proteases, hepatopancreas crab was extracted with aqueous buffer solutions containing NaCl and Ca ²⁺ -ion, followed by ultrafiltration and microfiltration and gel filtration on a column with Sephadex G-50 and obtaining a mixture of 9 proteins having a molecular weight of 23 to 36 kDa and showing collagenolytic activity. Enzymes from crab hepatopancreas are characterized by broad specificity: they are able to hydrolyze both native collagen and elastin, and standard protein substrates (gelatin, casein) in a wide temperature range (4-40 ° C) and pH (4.5-10.5) .

Due to the fact that the isolated proteins act as an “ensemble”, it becomes possible to use them together for the treatment of wounds with different and changing wound environments.

For immobilization, we used a solution of the proteolytic complex with a pH from 6.0 to 8.5, because the use of solutions having a pH below 6.0 and above 8.5 leads to complete and irreversible inactivation of the complex during immobilization, drying and storage of the immobilized preparation; an increase in immobilization time above 3 hours does not lead to immobilization of an additional amount of active protein, and when immobilized for less than 0.5 hours, the bulk of the active complex remains in solution, which leads to technological losses; when using a complex solution with a concentration of less than 0.4 mg / ml when immobilizing, the resulting material has an enzymatic activity less than the required therapeutic value, especially at the end of the shelf life of the drug, and with an increase in the complex concentration in the solution for immobilization of more than 1.5 mg / ml, most the drug remains in solution, which leads to a significant cost overrun of the enzyme preparation; the process at temperatures above 25 ° C leads to inactivation of the enzyme complex, and lowering the temperature below 15 ° C requires additional energy consumption.

The invention is illustrated in detail by the following examples of its practical implementation.

EXAMPLE 1. Prepare 1 l of a solution of a complex of hepatopancreas crab (manufactured by Bioprogress CJSC Biokombinat, Moscow Region), containing 1.0 g of a substance, which is filled with 100 g of gauze, partially oxidized with periodation to an aldehyde content of 0.18 mmol / g After 2 hours, the material is squeezed from the excess solution and dried to obtain the finished product having the following characteristics:

- proteolytic activity for casein 0.5 PE / g (Kunitz method);

- tryptic activity for hydrolysis of benzoylarginine ethyl ester of paranitroanilide 0.50 μmol / g;

- chymotryptic activity for the hydrolysis of acetyl tyrosine ethyl ester 0.08 μM / g;

- activity against collagen hydrolysis of 30 PIECES / g (according to the Mandeyl method).

EXAMPLE 2. In 1 liter of 1/15 M phosphate-potassium sodium buffer (pH 7.8), 1.0 g of the crab hepatopancreas complex is dissolved with 100 g of cellulose phosphate ester (2.2 mEq / g of phosphate groups) ); after 3 hours, the product is squeezed on a roll and dried. The resulting product has the following characteristics:

- proteolytic activity in casein 0.62 U / g;

- tryptic activity for hydrolysis of benzoylarginine ethyl paranitroanilide ethyl ester 0.65 mol / g;

- chymotryptic activity for the hydrolysis of acetyl tyrosine ethyl ester 0.10 μmol / g;

- activity against collagen hydrolysis of 35 PIECES / g (according to the Mandeyl method).

EXAMPLE 3. From the material obtained according to example 1, cut napkins measuring 7 × 10 cm, which were used in experimental conditions for the treatment of purulent wounds. The results of treatment (for 40 patients) are shown in the table, in which data on the treatment of wounds in the traditional way are given for comparison.

Table 1. The onset of healing, days. No. Studied samples Wound cleansing Wound healing one. Traditional treatments 13.2 ± 0.7 24.5 ± 0.7 2. Napkins according to example 3 2.7 ± 0.2 13.2 ± 0.1

Claims (4)

1. A wound cover, consisting of a base containing a complex of proteolytic enzymes from crab hepatopancreas, with collagenolytic activity, characterized in that chemically modified cellulose is selected as the base. 2. The wound coating according to claim 1, characterized in that the partially oxidized cellulose containing aldehyde groups is selected as the chemically modified cellulose. 3. The wound coating according to claim 1, characterized in that the chemically modified cellulose is selected chemically modified gauze containing residues of phosphoric acid. 4. A method of obtaining a wound cover according to any one of claims 1 to 3, comprising reacting chemically modified cellulose with collagenolytic enzymes in an aqueous buffer solution, characterized in that a complex of enzymes from crab hepatopancreas is used as the named enzymes, the reaction is carried out at pH 6.0-8.5, a temperature of 15-25 ° C for 1-3 hours at an initial concentration of the complex of 0.4-1.5 mg / ml and a bath module of 5-10.