CS264607B3 - A method for producing a proteolytic wound cover - Google Patents

Abstract

The solution concerns a new method of producing a proteolytic wound dressing in the form of a filling, used for covering, treating and especially cleaning purulent and necrotic wounds, which significantly improves the original method of production according to Czechoslovak Administrative Procedure Act No. 249 311. Instead of drying the immobilized enzyme by lyophilization, drying in suspension, or at elevated temperatures, is introduced. In contrast to the original procedure, simpler equipment can be used. The process is less energy-intensive, shortens production times and is generally cheaper.

Description

The invention relates to a method of producing a proteolytic wound dressing in the form of a dressing, which serves to cover, treat and especially clean long-term suppurating and necrotic wounds.

Recently, dressings based on hydrophilic polymers, such as dextran or cellulose in finely grained spherical form, have proven effective in treating necrotic purulent wounds. Their effect was initially only absorption, i.e. it consisted in sucking the liquid component from the suppurating wound into the inert porous mass of the hydrophilic polymer and into the capillary interparticle spaces of the dressing layer.

The effectiveness of the dressings was significantly increased when their absorption effect was supplemented by a proteolytic effect. Czechoslovak Patent No. AO 249 311, for example, describes a proteolytic dressing prepared from pearl regenerated cellulose (prepared according to Czechoslovak Patent No. AO 172 640), in which chymotrypsin was immobilized by covalent bonding. The obtained product was tested in clinical conditions, where it showed not only proteolytic activity on necrotic tissue, pus and fibrin, but in accordance with the hydrophilicity and porous structure of the cellulose matrix it also excelled in high absorption capacity, i.e. it sucked out exudate containing split tissue necrosis and bacteria from the wound. By combining both principles, proteolytic and absorption, rapid cleaning of infected necrotic defects, formation of clean granulations and rapid wound healing were achieved.

Compared to older absorbent covers, the proteolytic cover is significantly more effective, but its preparation is more complex and expensive. Complications arise from the presence of an active enzyme in the structure of the new preparation. Although the catalytic efficiency of an enzyme is generally derived from the structure of the active center, i.e. from a certain mutual position of several neighboring amino acid residues in the enzyme molecule, it is known that it depends entirely on the conformation of the polypeptide chain in the entire macromolecule. In order for an enzyme to be catalytically active, its native structure in an aqueous environment must be preserved, i.e. not only its primary structure (amino acid sequence), but also its secondary, tertiary and possibly quaternary (determining the native conformation). When manipulating the enzyme, one must be concerned primarily about thermal denaturation, which can lead to irreversible changes in the tertiary and quaternary structure, or even to the complete destruction of the native conformation and the formation of disordered statistical tangles of polypeptide chains.

For these reasons, all operations with active enzymes are carried out at relatively low temperatures and when separating the enzyme in a solid state, the most gentle known method is preferably used, i.e. freeze sublimation (lyophilization). Also when preparing the proteolytic wound dressing, it was necessary to respect the peculiarities of enzymatic preparations, avoid working at high temperatures and recommend lyophilization for the final drying of the product. Although this is a safe method and has been proven by numerous experiences, it complicates the production apparatus, prolongs it and generally increases its cost.

Systematic attention was paid to eliminating these shortcomings and after performing the measurements described in the following, it was surprisingly found that the shortcomings of the previous process can be avoided if the new method of producing the proteolytic coating according to the present invention is used.

The subject of the invention is a method of producing a proteolytic wound dressing according to Czechoslovak Patent Application No. AO 249 311, whereby the pearl regenerated cellulose swollen in water, never dried, with a particle size of 0.07 to 0.4 mm, is completely freed from toxic impurities by washing, or possibly by steam distillation, is activated for enzyme binding, is modified by immobilization of proteases from the chymotrypsin, trypsin, subtilisin groups, is alternately washed with a buffer of pH 8.5 to 9.6 and a buffer of pH 4 to 5 until the proteolytic activity of the washing liquid is zero, characterized in that the polymer product washed with a buffer of pH 7.5 to 8.5 is dried in this environment to 1 to 15% of the residual water content in suspension in an air stream at 20 to 90 °C for 10 to 150 minutes.

It has been shown that suspension drying is more advantageous than freeze-drying due to its easier feasibility. It does not require vacuum (vacuum sources, perfectly sealed apparatus), nor deep cooling of the dried product. The consequence of these differences is a lower energy consumption of the process and simpler, i.e. less expensive apparatus.

The removal of water from the pearl polymer product in a fluidized bed during suspension drying is more efficient than from a stationary freeze-dried bed. The higher temperature also has a positive effect, so the suspension drying time is significantly shorter than in the case of freeze-drying. While in the latter case it ranged between 48 and 70 hours, the product was dried to the desired degree at room temperature in 2 hours and at temperatures from 50 °C in just 20 minutes.

The possibility of drying at relatively high temperatures while perfectly preserving the activity of the immobilized enzyme is surprising. Although the literature reports cases of increasing the stability of enzyme activity by immobilizing them on a polymer carrier, the possibility of short-term exposure to temperatures of 70 to 90 °C observed by us can be considered extraordinary. The observed phenomenon can be explained by the very good stabilization of the protease in the porous cellulose carrier, probably due to its biopolymer structure and perfect covalent immobilization, which is given special attention in Czechoslovak AO 249 311.

A significant technical effect of the new production method is a significant improvement in the preparation of proteolytic wound dressings. The new method provides a preparation for the treatment of necrotic suppurating wounds with the same effectiveness as the original procedure according to Czechoslovak Patent Application No. AO 249 311. However, unlike the original method, it requires simpler, less expensive equipment, is less energy-intensive and shortens production times, so it is cheaper.

The new production method is further explained in detail below, but not limited to, exemplary embodiments.

In the experiments carried out, attention was paid primarily to comparing lyophilization with suspension drying.

Freeze-drying was carried out on three different apparatuses. The first one (A) was of laboratory type, approx. 100 g was used for drying, the sample was frozen at -40 °C and during drying it was heated to room temperature with self-heating. The second apparatus (B) was of commercial type (type designation KS-30, cooling unit HZ 12/50). The operating mode consisted of subcooling to -40 °C (2 hours) and drying at 25 °C (48 hours). The third apparatus was also of commercial type (C) LZ 45 Prigera). The dried product was first frozen at -40 °C and heated to 25 °C during the actual operation (70 hours in total).

The suspension drying was carried out on Uniglatt type devices (D, E, F), usually used in pharmaceutical production. The apparatus (D) was a pilot plant and was filled with 100 to 1000 g of dried product. The apparatuses (E, F) were of the production type and were filled with 2 to 5 kg of dried polymer product.

The experimental conditions used in the individual examples and the measurement results obtained are given in Tables 1 and 2.

Table 1

Comparison of freeze-drying and suspension drying

Example No. batch number Type ^and drying Equipment^ Moisture product, % Swelling ^c g H^O/g dry. Enzyme activity 1 28 L A 7.3 2.0 1.6 2 29 L B 11.4 2.1 1.8 3 X L C 12.2 1.4 1.3 4 29 In D 8.9 2.4 1.8 5 X In D 13.5 1.5' 1.8 6 X In D 14.5 1.4 1.5 7 29 In E - 1.7 1.8

Continuation table

Example Batch Type ^and Apparatus ¹³ Humidity Swelling ⁰ Enzyme. number number drying prod., % g H ₂ O/g dry. activity

In F

In F

5.7 1.8 1 .-2

9.9 2.1 1.6 ^a drying by lyophilization (L) and in suspension (V) ^see description of the apparatus used in the text ^c water content of the product after contact with excess water and centrifugation ^given as the increase in absorbance at 280 nm per 1 min. per 1 g of product on hemoglobin as substrate (ML Ansen, Gen, Physiol. 22, 79, 1939).

All experiments in suspension were carried out at room temperature, only example 6 at 40° C. The lyophilization time was an average of 3 days, suspension drying 120 minutes.

Table 2

Drying in suspension at different temperatures

Example no. Temperature °C Swelling g H ₂ O/g dry matter Enzymatic activity 10 25 1.6 . 1.8 11 35 1.6 1.8 12 40. 1.6 1.8 13 50 1.6 1.7 14 60 1.8' 1.7 15 70 1.7 1.7

Explanations see Table 1. - Experimental conditions: batch 29, apparatus D, inlet air temperature measured. Drying time for temperatures of 25 to 40 °C was 120 minutes, for temperatures of 50 °C and above 20 minutes.

Claims (1)

OBJECT OF THE INVENTION A method for producing a proteolytic wound cover according to U.S. Pat. AO 249 311, wherein the pearled regenerated cellulose swollen in water, never dried, with a particle size of 0.07 to 0.4 mm, is completely free of toxic impurities by washing or distillation with water vapor, activated for enzyme binding, modified by immobilization of proteases from the group of chymotrypsin, trypsin, subtilisin, alternately washing with a buffer of pH 8.5 to 9.5 and a buffer of pH 4-5 to zero proteolytic activity of the washing liquid, characterized in that the polymer product washed with a buffer of pH 7.5 to 8 1.5 in this medium is dried to 1 to 15% residual water content, suspended in an air stream at 20 to 90 ° C for 10 to 150 minutes.