CS264607B3 - Production method of proteolytic wound cover - Google Patents

Abstract

The solution concerns a new production method a proteolytic wound cover in the mold backfill to cover, treat and especially purulent and necrotic cleaning wounds that substantially improve the original method of production according to MS. AO No. 249,311. Instead of drying the immobilized enzyme by lyophilization introducing a fluid drying, optionally at elevated temperature. Unlike the original a simpler apparatus can be used. The process is less energy intensive reduces production times and is generally cheaper.

Description

BACKGROUND OF THE INVENTION The present invention relates to a method for producing a proteolytic wound dressing in the form of a backfill which serves to cover, treat and, in particular, clean long-lasting purulent and necrotect wounds.

Recently, hydrophilic polymer based powders have been proven useful in the treatment of necrotic purulent wounds. dextran or cellulose in finely granular spherical form. Their effect initially was only absorbent, i.e. to suck the liquid component from the festering wound into the inert porous mass of the hydrophilic polymer and into the capillary interparticle spaces of the backfill layer.

The efficacy of the covers was substantially increased when their absorption effect was supplemented by a proteolytic effect. Cs. For example, AO 249 311 discloses a proteolytic cover prepared from pearl regenerated cellulose (prepared according to US AO 172 640) in which chymotrypsin has been immobilized by a covalent bond. The product obtained was tested under clinical conditions, where it exhibited not only proteolytic activity on necrotic tissue, pus and fibrin, but also, in accordance with the hydrophilicity and porous structure of the cellulose matrix, excelled in its high absorption capacity, ie. Combination of both proteolytic and absorption principles resulted in rapid cleansing of infected necrotic defects, creation of clean granulations and rapid wound healing.

Compared to older absorbent shells, although proteolytic shells are significantly more efficient, their preparation is more complex and costly. Complications are derived from the presence of an active enzyme in the structure of the novel preparation. Although the catalytic activity of an enzyme is generally derived from the structure of the active center, ie from a certain relative position of several adjacent amino acid residues in the enzyme molecule, it is known that it is entirely dependent on the conformation of the polypeptide chain throughout the macromolecule. In order for the enzyme to be catalytically active, its native structure must be maintained in an aqueous medium, ie not only its primary structure (amino acid sequence), but also secondary, tertiary and possibly quaternary (determining native conformation). When handling the enzyme, it is necessary to worry in particular 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 strands of polypeptide chains.

For this reason, all operations with active enzymes are carried out at relatively low temperatures, and the separation of the enzyme in the solid state preferably uses the most gentle known method, i.e. freeze-drying. Also, when preparing the proteolytic wound dressing, it was necessary to respect the peculiarities of the enzymatic preparations, avoid working at high temperatures and recommend lyophilization for the final drying of the product. Although it is a safe and proven way of experience, it complicates, prolongs and increases the overall cost of production.

Removal of these drawbacks has been the subject of continuous attention, and after performing the measurements described below, it has surprisingly been found that the drawbacks of the prior art can be avoided by using the novel proteolytic cover method of the present invention.

The subject of the invention is a process for the production of a proteolytic wound cover according to U.S. Pat. AO 249 311, where 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 with the groups chymotrypsin, trypsin, subtilisin, alternately washing with a buffer of pH 8.5 to 9.6 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 In this environment, 5 is dried to 1 to 15% of the residual water content in the air stream at 20 to 90 ° C for 10 to 150 minutes.

Floating drying has been shown to be preferable to freeze-drying for ease of feasibility. It does not require vacuum (vacuum sources, perfectly tight apparatus) or deep cooling of the dried product. The result of these differences is less energy consumption of the process and simpler, ie less expensive apparatus.

The removal of water from the beaded polymer product in the fluidized bed during fluidized bed drying is more efficient than from the immobilized lyophilized layer. Higher temperatures are also beneficial, so that the drying time in the fluidized bed is considerably shorter than in the case of lyophilization. While in the latter case it was between 48 and 70 hours, the product was dried to the desired degree at room temperature in 2 hours and at 50 ° C in as little as 20 minutes.

Surprisingly, the possibility of drying at relatively high temperatures while perfectly preserving the activity of the immobilized enzyme. Although there are reports of increased stability of enzyme activity by immobilization on a polymeric carrier in the literature, the possibility of short-term exposure to temperatures of 70-90 ° C observed by us can be considered extraordinary. This phenomenon can be explained by the very good stabilization of the protease in the porous cellulose carrier, probably due to its biopolymer structure and to the perfect covalent immobilization found in the Czech Republic. AO 249 311 pays special attention.

An important technical effect of the new production method is a substantial improvement in the preparation of proteolytic wound dressings. The new method provides a preparation for the treatment of necrotic suppurative wounds with the same efficacy as the original procedure of US. AO 249 311. However, unlike the original method, it requires a simpler, less expensive apparatus, is less energy intensive and shortens production times, making it cheaper.

In the following, the new production method is explained in more detail, but not limited, by way of example.

In the experiments carried out, attention was paid mainly to the comparison of freeze-drying with fluidized-bed drying.

Lyophilization drying was performed on three different apparatuses. The first one (A) was of the laboratory type, ca 100 g was dried for drying, the sample was frozen to -40 ° C and warmed up to room temperature during the drying process. The second apparatus (B) was of the operating 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). Also the third apparatus was of the operating type (C) LZ 45 Priger). The dried product was first frozen at -40 ° C and heated to 25 ° C for 70 hours.

Fluid drying was performed on Uniglatt (D, E, F) type apparatuses typically used in pharmaceutical manufacturing. The apparatus (D) was pilot operated and filled with 100-1000 g of dried product. The apparatuses (E, F) were of the process type and filled with 2-5 kg of dried polymer product.

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

Table 1

Comparison of freeze drying and drifting

Example Ref. batch no. Type ^and drying Aparatura ^ Humidity prod.,% Swelling ^c g H 2 O / g dry. Enzymat. ‘Activity 1 28 L AND 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 ^a Apparatus ¹³ Humidity Swelling ⁰ Enzymat. no. no. 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 ^and freeze-drying (L) and fluid (V) - see description of apparatus used ^c the water content of the product after contact with excess water and centrifugation given as absorbance increment at 280 nm per 1 min. per 1 g of product per hemoglobin substrate (ML Ansen, Gen, Physiol. 22, 79, 1939).

All floatation experiments were carried out at room temperature, only Example 6 at 40 ° C. The lyophilization time was 3 days on average, and the drying time was 120 minutes.

Table 2

Floating drying at different temperatures

Example no. Temperature ° C Swellability g H ₂ O / g dry. Enzymat. 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 Dec 70 1.7 1.7

For explanations see Tab. 1. - Experimental conditions: batch 29, apparatus D, temperature of the inlet air measured. The drying time for temperatures of 25 to 40 ° C was 120 minutes, for temperatures from 50 ° C to 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.