GB2240040A - Immobilised enzyme-containing wound dressings - Google Patents

Abstract

Dressings for wounds comprise enzymes covalently bonded to a carrier which is suitable for topical application to the wound, eg. textiles, fibres and powders of natural or synthetic polymers.

Description

h :;->:2'400.4C A - 1 1 Enzyme - Based Physioloqical Dressinqs The present

invention relates to a material having biological activity, a method for preparation thereof, and also to a dressing based thereon.

katerial having biological activity is widely usable for treating trailmatal frostbites, tro-ohic ulcers, pyo-necrotic -wounds, diseases which are accompanied by formation of necrotic sections and accumulation of thick, viscous exudate, and can be applied in surgery, dentistry, urology, proctology, gastroenterology. In addition to that, the material having biological activity is applicable in veterinary, biotechnolo&7,for instance, as a sorbent for recovering trypsin- d-l-antittrypsin inhibitors.

It is lacqvn in medical practice to use native proteolytic, enzyme preparations for treating pyo-inf lammatory diseases. However, said enzymes are expensive and scarce. Regardless of a certaLl effectiveness, said enzymes are not resistant aGainst 4-1-hibitors -7hich are contained in -.7ound dischar.el against p.111 a--.d temmerature changes. Moreover, said enzymes possess antige- 0 nic (allergic) and pyrogenic pro-oerties.

Y - Knowra in the art 'safilm having biological activity and 2.

co_-,ainL-_,t- a carrier such as cellulose triacetate and secondary acetate and a -proteolytic enzy--ae, f or instance, tr,-)Psin, ochyaotrypsin. The ratio of said components is as follo-,-,7s:

cellulose triacetate (secondary acetate" - 80-995 by weighz enzyme balance (SU Inventor's Certificate 1711o. 70013,58, November;30, 1979).

Said film is prepared by emulsifying an aqueous solution of proteolytic enzyme in a cellulose ester solution in a waterimmiscible organic solvent, followed by forming film. In the obtained -Film, the proteolytic enzyme is in the form of finest inclusions.

Said film is capable of producing a long necrolytic effect due togradually releasing small doses of enzyme from the surface of the film.

-However, bein; released from the film, the enzyme has the praperties of a native material with all its disadvantages, that is, non-stability against inhibitors, against p1H and temperature changes, antigenicity and pyrogenicity. Along with that, the need for an enzyme is high - up to 20116 by weight. ApPlication of said film is restricted by highly possible enzyme inactivation in organic solvents which are used for foming films or monofilaments. The very film can be applied only as a draining material, it cannot be used as a dressing material.

r n in the art is also a granulated material having a Maovir, 0 biological activity and containing a carrier such as dextran, chitin, chitosan and the like comi-pounds, and an enzyme such as chy- motrypsin, trypsin, immobilized thereon, at the following weigInt ratios:

carrier 80.0-80.5.Vt by weight 3.

enzyme balance (laid-open French Patent No. 21--56222, June 14, 1985).

Said material has a prolonged action, and the enzyme has no possibility to penetrate into a blood channel and manifest antigenicity.

Said material is also disadvantageous in that it has an increased enzyme content - up to 2W% by, weight, and is expensive. Moreover, said material needs special installations and apparatus for its storing in specially prepared buffer solutions at lowered temperatures (at 2 0 c).

Known in the art is a method for preparing a material having a biological activity, which c=prises the following steDs:

preparing an aqueous solution of a proteolytic enzyme, Draferably trypsin; preparing a solution of cellulose ester, in the present case, cellulose triacetate, in a water-immiscible organic solvent; emulsifying the first solution in the second one; forming the material into medicinal formulations such as plates, film, etc., using a dry-forming method; and holding the obtained medicinal formulations in a vacuum drier at a temerature of 250C (SU Inventor's Certificate No. 700138, November 30, 1979).

Prepared by said method have been the materials having a biological activity, at the following weight ratios, in per cent by weight:

cellulose triacetate or secondary acetate - 8C-99 trypsin - balance (1-20).

4.

111he art-lmown metbod bas the follawins essential disadvan- taSes:

considerable need fortheenzyme (up to 20/), partial loss of the enzy=e activity during emulsifidation in an organic solventg the absence of a chemical bond between an enzyme and a carrier owing to the fact that the enzyme is included physically into the structure of the material; release of theenzyme into wound exudate in the form of a native preparation, which may result in a negative side-effect, for instance, allergic reactions; use of special equipment such as a vacuum drier.

The material in the form of plates prepared by the known method cannot be used as a dressing material since it does not possess the required sanitary-and-hygienic properties such as Q hya.

groscopicity, air permeability, elasticitY$ etc., and can only be used as a draining material.

Known in the art is also a method for preparing a material having a biological activity, which comprises the following steDs:

preparing material (carrier) particles of enass linked Dextran, Chitin, chitosan, by swelling them in water, preparing an activating solution of 2-ain:Lno-4,6-dichloros-triazine in acetone followed by dilution with water at 500C.

activating the material With an activating solution at 500C and agitating it; adding to the activating solution containing said =aterial, an aqueous solution of sodium carbonate and hydrochloric acid and stirr:L.ig at 50OG; 4 5.

1 filterin- the resulting 0 washing. the remaining material with an aqueous solution of -o give an activated material which can be acetone and water t stored in a form in a phosphate buffer at a temperature of 2 0 0; preparing an enzyme solution, for instance, chymatrypsin, in a buffer; immobilizing the enzyme on the activated material such as linked dextran, chitin, chitosan; washing the material containing immobilized chymotrypsin in turn with borate and acetate buffer solutions containing sodium chloride of different concentrations; washing the material with a borate buffer without sodium chloride; and lyophilizing the material with the i=obilized enzyme (laid-open French Patent No. 2556222, June 14, 1985; laid-open West-German Patent No. 3444746, June 20, 1985; Czechoslovakian Inventor's Certificate No. 249311, 1988).

Said methods were used to obtain the materials having a biological activity, the weight ratio of the components being as follows, in per cent by weight:

enzyme - 19.5-20 carrier - balance.

The above method has the following essential disad vantages:

considerable need for the enzyme (up to 201/o) multi-stage preparation technology and, as a result, low efficiency and high price, ^ a rare and exotic raw =aterial, for instance, chitin use oL from crab shells or fro= mycelium of Penicillium chz7sogenum 6.

fu-lI9US; and need for special eauipment for storin- the activated naterial and for carrying out the lyophilization step.

The material obtained by the art-knowin zethod and havin a biolo-ical activity cannot be used in a dressing, it can only be used as a powder or a suspension since in -.12e ready form said material is a powder cmtain:Lng the immobilized enzyme having a particle size of 0.01-0.5 m.

Known in the art is a dressing having a biological activity and consisting, successively, of a layer,.aii absorbing layer and a wound-ne7ighbouring layer (British Patent No. 2092006, February 11, 1986). Said dressing comprises a thin layer of the enzyme applied to the inner side of the wound-neighbouring layer. The protec tin.g layer can be made in the f o= of a woven or non-woven =aterial.

The absorbing layer can be zade from cotton or viscose (c. otton wool).

The wound-nRighbouring layer is made in the f orm of woven or non-woven textile materials or perforated polyester, polyamide, polyurethane, polyetbylene, polypropylene and cellulose triacetate film, and contains metallic copper or copper comDounds.

Contact of the wound discharge with the prior art dressing reveals all the negative properties of the enzyme as a native one, in particular, the non-resistance against inhibitors which are contained in the wound discharge, a,-ainst pH and temperat-ure changes, antigenicity and pyrogen, icity, and high cost due to the increased need for the enzyme.

1 7.

"lie ob4ec-'Lj of the present invention is -,,o provide a materia ti having a bioloSical activity, which enslires the -prolonGed action of medicines contained therein, capable of being stored for a long time when packed under usual conditionsretaining all. the sanitary-and-hygienic properties characteristic of a-dressing material, providing a medical effect within a shorter period at a lower content of a biologically active compound, and capable of being effectively used as & dressing.

Another object of the invention is to provide a method of pre paring said biologically active material, which enables to obtain the material to be used as a dressing, is technologi- cally simple, not expensive due to using available materials and easily carried out industrially.

A further object of the invention is to provide a:

dressing which ensures a high medical effect at a considerably smaller need for the enzyme, is resistant against the inhibitors which are contained in wound discharge, against pH and tempe rature changes, arfd, has no antigenic and pyrogenic properties.

Said object has been-achieved by providing a new material having a biological activity, containing a carrier and an enzyme immobilized thereon, which,according to the invention, comprises as said carrier, a textile material, said material containing an enzyme immobilized thereon which is connected to the textile carrier by means of a covalent bond, at the following ratios of components, per cent by weight:

enzyme - 0.02-0-50 carrier -99-98-99-50- 8.

instance, woven or non-woven or knitted cloth with an enzyme immobilized thereon.

The textile material is used for the reason that it has a aeak medical ef f ect since said material drains the sound, absorbs microbes and toxins, protects the wound surface from contamination,pre,vents from the excessive loss of moisture.

Said textile material can be made from natural, for instan e, cotton, linen, silk, wool fibers, or from synthetic fibers, for instance, polyacrylonitrile, polycaproamide, polyurethane f ibers, or from r-rian Ma-ae f ibers, f or instance, triacetate, cellulose secondary acetate, viscose fibers.

According to the invention, the enzyme immobilized on the textile material is bound by means of a covalent bond to said textile material, at the following ratio of said components of the claimed material, in pecent by weight., enzyme - 0.02-0-50 textile material - 99-98-99-50.

Said ratios are determined by the fact that the use of the material having a biological activity and containing less than 0.02% by weight of enzymet reduces sharply the medical effect (more than 1.5 as much), and the use of the material containing more than 0.501"o by weight of enzyme does not considerably reduce the period of treatment (the average treatment period is 14+2 da:7, however, the cost of the material and treatment increases sharply.

In medical practice, it is advisable that the period of a single application of the claimed material to the wound be 4896 hours.

The medical efficacy of the material while storing it in i 2 i i 1 9.

a packed state under usual conditions (at room temperature of about 2000) is retained for a period of not less than 5 years.

The use of the material according to the invention enables to reduce the period of healing wounds by an average factor of 1.5 at doses which are 10-30 times less as compared with th e terms of healing wounds by means of art-known materials having biological activityg or native enzymes. The claimedmaterial is not toxic and causes no allergic reactions.

According to the invention, it is advisable that the textile ca.rrier be made of. natural fibers, which provides f or the better conditions of applying the material to the wound surface, close to comfortable conditions.

In order to enlarge the range of raw materials, it is advisable that, according to the invention, the textile carrie= be made. Of synthetic or man made fibers.

According to the invention, it is also advisable that the material contain, as an enzyme, proteolytic (hygrolytin, protosubtilin, terrilytinitrypsin) or collagenolytic (collagenase), or bacteriolytic (lysozyme) enz7mes which enable to cleave denaturated proteins having ali-fe rent btrudtures and to Dromote the cleaning of the wound. The material obtained according to the invention, depending on the form of the carrier,-can, for example, be in the form of napkins, bpqadageS, dressings.

It is also advisable according to the invention that the material having a biological activity be in the form of lint having a fiber length of 1.015. mmt which allows to use said =aterial La stomatology and for treating deep wounds, includ n- I iz j)unctured -,)u---,len-V aounds of complex configu--ation.

"'Ine material obtained accordinc- to the invention can also L. 0 10.

be in the fo= of now-der havins a particle size of 0.001 Q tin- extensive to 1.00 mm, which allows to use it for -u::ea-, wound surfaces of complex configuration.

The material is suitable for treating pyo-necrotic wounds, acute and chronic forms of periodontitis, wounds having complex configurations and cavities, hollow organs of a live organism having narrow excretory channels, for instance, for treating inflammatory processes in the urinary bladder cavity.

The approbation of the claimed material under the conditions of tests and clinical observations has demonstrated a high biological and therapeutical activity. The special studies have shown that the highest therapeutic activity of the claimed material wbich is in contact with the wound surface, lasts for 4 days from. the monent of application.

The test animals showed no signs of allergic reactions after applying the material having biological activity and prepared according to the invention. No such changes of heinostasis functions as "the level of fibrinogen degradatim products and fibrinolytic activity were observed.

The study of wound inoculation before and after apply- ing the claimed material, has shown that the range of the sown microorganisms and the virulence thereof reduced due to a change for a less pathogenic flora, as well as the resistance to antibiotics. Application of the claimed material reduces the level of bacterial d- isseminatio-n of wound surfaces, the period of preparing patients for plastic o- perations and the period of stay of patients at stationary hospitals by 10 days on the average.

During the whole period of treatment, the urinalyses and bood counts of the patients were controlled, and their temperature 1 was taken. None of the cases showed any signs of general or local complications due to the application of the claimed material.

The claimed material can be used, for instance, as a dressing in the form of a bandage comprising at least three successive layers such as a protecting layer, absorbing layer and a wound.neighbouring layer.

The dressing has several embodiments, for instance, it may comprise one or two layers of the claimed textile material having a biological activity, provided with the immobilized enzyme which is bound by means of a covalent bond to said material.

In all the preferred embodiments, the protecting layer of the dressing has been prepared according to the inven- tion frm a textile, woven or non-wover, or knitted cloth.

In all the preferred embodiments of the dressing, the wo-und-neighbouring lzyer is made, according to the present invention, of a material having a biological activity and comprising a textile carrier made from natural or synthetic, or man made fibers, and an enzyme immobilized on said carrier, for instanceg collagenolytic enzyme such as collagenase, a proteolytic enzyme such as hygrolyting protosubtilin, terrilytin, trypsin, or a bacteriolytic enzyme such as lysozyme. Said enzyme is thereby bound by means of a covalent bondto said carrier, and the weight ratio of the carrier to the enzyme is 99-98-99-50:0.02-0-507-o by weight. The textile material of which the wound-neighbouring layer is made is a knitted or woven or non- woven cloth.

There is an embodiment of the dressing in which 12.

the latter com-orises one layer of the material having a biologi cal activity, which is a wound-neighbouring layer. and the absorbing layer is made of a textile woven or non-woven or knitted cloth without any biologically active compound.

In Olie embodiment when the dressing Bas two layers of the material which has a biological activity, the absorbing layer is made al the material comprising a bacteriolytic enzyme, for instance, lysozyme, which is woven or nonwoven or knitted textile cloth. The weight ratio of the enzyme to the textile material is 0.02-0-50:99-98-99-95% by weight. Besides, the absorbing layer can be made in the form of lint (cotton wool) with the immobilized bacteriolytic enzyme, having fibers 1.0-15 mm long, or from the powder of the same materialL having a particle size of 0.001-1 mm.

A dressing is provided with the means which enable to fix it on the patient's skin, for instance, an adhesive tape, a Velcro tape, etc.

A dressing may be prepared using conventional technologies of textile and clothing industries.

Application of the dressing according to the invention during the period of several hours after the trailma (injury) enables to prevent from infectirgand to reduce the treatment period by 2- 4 days.

The above-described material having a biological activity is prepared, according to the invention, by activating a chemi cally inert carrierg in the present case, a textile carrier in the form of a cloth, followed by immobilizing on to the activated carrier a biologically active compound, in the present case, 0 13.

1 of an enzyme such as protosubtilin7 hygrolytin, terriliting trypsin, lysozyme, collagenase Activation comDrises the introduction of reactive functional groups into the textile carrier. The activation process depends on the chemical structure (naturall artifiefal, synthetic fibers) and the textile structure of the carrier (the method of weaving, thickness and number of thread folds, density of the te-xtile material, etc.). The activation proce.s is preferably continued until the amount of reactive functional groups reaches 0.0625,,,.125 mg-equiv. per gram of a c&rrier.

A cellulose containing textile carrier may be activated by oxidation thereof with an alkali periodate, for instance, sodium periodate. to.,;ive cellulose dialdehyde. The activated textile carrier preferaby contains o_o625-3-125mg-equiv. of reactive functional groups per gram of a carrier.

Activation of a textile carrier from synthetic fibers (a carrier containing no cellulose), for instance, from Dolycaproamide fibers, rmy be carried out by hydrolyzing a carrier with an acid, followed by washing the resulting carrier and reacting the hydrolyzed carrier with a solution of a compound containi reactive functional groups until the amount of the latter in the carrier reaches 0.0625-0-3125 mg/equiv. per gram of the carrier. It is advisable that strong inorganic acids be used thereby, for instanqe hydrochloric or sulphuric acid. It is advisable that the treatment of the hydrolyzed carrier from synthetic fibers be carried out by a solution of gluta=ic aldehyde, followed by Cate solution, with washing it in water with a dimethyl sulL subsequent washing of the carrier in ethyl alcohol wid in a 14.

er.

nhos-ohate buff -hP- immobilization of the biologically active com-poiind i.,7k t ile carrier is preferably )resent case, an enzyme, on the activated text effected from buffer solutions having a pH ol. 6.4-7.5 at -room temperature for 8-16 hours. Due to reacting the reactive groups of the textile carrier with free functional groups of biologically active compounds, in the present case, enzymes, a covalent chemical bond betweeithe textile carrier and the enzyme is formed thereby, which results in a textile material with chemically (by means of covalent bonds) immobilized biologically activ compound having a prolonged effect. The immobilization process is continued until the textile material having a biological activity and containing, in the present case, preferably 0.02-0.5%, of the enzyme of the weight of the material, is obtained.

The obtained cloth is squeezed out, the physically adsorbecl enzyrne is washed off, then the cloth is dried, placed into a tight container, for instance, sealed into polyethylene bags, and sterilized by gammaradiation using the methods known per se.

Before sealing into polyethylene bags, the',material, if necessary, can be shaped into napkins, lint, powder, etc., using the methods known per se.

The claimed process is distinguished over the art-known ones by its simplicity and low cost, which also concerns the devices required for its embodiment.

The al&imed method enables to obtain the material which combines the properties-of a t-herapeutic preparation and a dressing, and whichretains all the" sanitary-and-hygienic and physica.-chemical properties of the starting dressin; -material.

1 15.

The thus obtained material having a biological activity retains a high therapeutic efficacy while being stored under usual conditions in packages for aperiod of at least years.

Further details of the claimed method with respect to different types of textile carriers are given in the examples.

The material prepared by the described method was tested under clinical conditions being used for treating 3200 patients having pyo-necrotic diseases, frostbites, trophic ulcers, etc.

The claimed material was applied locally following the procedure as described below: after a preliminary surgical treatment such as dissection of the pyo-necrotic area, Cpening of pockets, dissection of intersections and forming a single cavity, the proposed material having a biological activity, for instance, in the form of napkins, cotton wool (lint), and impregnated with a physiologic salt solution, was incorporated: Into the wound.

It has been found that when the claimed material is applied, the period of healing is reduced to around 15+2.1 days, whereas while curing by a conventional method using a hypertonic salt solution containing antiSeptic, a control group of patients (3110 patients), this period was 26+2.2 days, that is, application of the claimed material enables to accelerate the course of treatment 1.5 times as much on the average.

None of the cases of clinical application of the claimed material showed any, toxic or allergic reactions.

The claimed material is essentially atrauTnatic due to the presence on the surface thereof of an enzyme attached by means of a covalent bond. The force of breaking off the material from 1 1 16.

the wound surface is on the averase half that of the non modified commonly applied dressing material.

The results of the clinical application of the claimed material have shown the following:

high effectiveness thereof for treating pyo-necrQtic processes: the curing Deriod was reduced 1.3-1.8 times as much; high atraumaticity: the force of breaking the material off the wound surface is half as much; reduction of the need for enzymes: 10-30 times less as that-of native enzymes for treating the same diseases; not less than 23 times as that of the art-known analogs (according to the analogs the amount of the enzyme is 0.5-20%, whereas in the claimed therapeutic material, the amount of the enzyme is-0.02-0.5,76).

The check-up of the stability of the claimed material after its sterilization by means.of Samna-radiation has shown that i said material retains its therapeutic effect while being stored in packages under normal conditions (2000) for a period of cLt least 5 years, all the physico mechanical and sanitary/ hygienic properties being reserved.

The claimed material has no side-effects and contraindications f or applicatim.

The clinical effectiveness of applying the claimed material having a biological activity, for instanceg containing immobilized bygrolyting in comparison with the conventional medicines for treating pyo-necrotic wounds in soft tissues is illustrated by the f ollowing results.

1 Table

Period of.treatment (days) Characteristic of the process ivith a material with a with a bypertonic of treatment having a biolo- native salt solutions Sical activity enzyme anti-Tpt:lc (with the immo bilized enzyme) Complete purif ication of wounds and emergende of islands of granulations Complete healing of the wound from the beginning of treatment 4.5 0.2 7.8+0.5 9.5 0.6 15.0+2.1 20.0+1.5 26.0+2.2 The clinical treatment has shown that the application of textile materials containing immobilized enzymes for treating the -catients having purulent surgical diseases of tissues enables to reduce the total period of curing 1-3-1-8 times as much.

Enzymotherapy considerab3y reduces the period of beginning of purification of wounds and emergence of granulations: 1.7-2.1 times as much.

To better canprehend the invention, provided below are;s,pecifir, examples illustrating the preparation method and the material having a bioloGical activity, as well as the dressing. Example -1 3.3 L of waterwere poured into a reactor, mixed with a sti&er, and 9.4 S of iodic acid wereal-ded thereto.

The sare amount of water was poured into another reactor and 1. S S of sodium hydroxide were added theret- o using a stirren 0 18.

Both solutions were agiltated unitil the crystals oJE' the acid and sodium hydroxcide were dissolved within 5-13 minutes.

Then the solutions were poured into a reactor, agitated for 3-6 minutes to give a sodium periodate solution having a pH of.5.

1) 1 Kg of textile woVen cloth (Inedical cotton gauze) were placed into the resulting solution and held (activated) at room tempera ture for 14 hours in darkness. After activating,. the cloth (dialdehyde cellulose) was squeezed outg washed with 4x10 1 of water and squeezed out again.

As a result of activation, the cotton cloth acquires reac tive functional groups, in the present case, aldehyde Sroups, containing 0.0625 mg/eq. per gram of the textile carrier. To prepare'the buffer solution, in the present case, a phosphate buffer solution, two reactors were also used. 3. 3 L af water were poured into one of them, and 5 g of potassium dibydropboapbate were added thereto under stirring. 37 G of sodium hydrophosphate were added under stirring to the same amount of water in the other reactor. Stirring was continued until the crystals were completely dissolved for 10-20 minutes.

2) Both solutions were then poured into one reactor to give a phosphate buffer solution having a pH of 7.5.

3) 0.4 G of enzyme, in the present case, hygrolytin, were added to the prepared phosphate buffer solution under stirring until it was completely dissolved for 10-20 minutes. 1 Kg of the activated cloth (dialdehyde cellulose) were placed into the resulting solution and'held at room temperature for 12 hou.-Asy during which period the enzyme immobilization process takes place due to appearing covalent chemical bonds. The cloth was washed till no protein was observed in the washings. '2hen i 1 19.

the cloth was squeezed out and dried in air for 24 hours to give the material, in the present case, cotton cloth having a biological activity and prolonged therapeutic action. The amount of the immobilized enzyme, in the present case, bygrolv-tin, determined by the enzyme loss from the solution, is 0.2 mg per gram of the textile cotton cloth, that Is, 0. 02975 of the weight of the cloth.

The dried material is formulated Into medicinal formulations for instance, napkins having the required size, sealed into dmWU polyetbylene bags andsterilized by 1r-radiation at a dose of 25 kGy using the methods known per se.

Exam-Dle 2 A solution of sodium periodate was prepared following the procedure of Example 1, and 80 g of medical cotton gauze were placed into it. The process was further carried out following the procedure of Example 1 to obtain a cloth containin 0.78 =g/ eq. of aldehyde groups per gram of the textile cloth.

A ph0SDhate buffer solution was prepared. following the procedure of Example 1, and 0.24 g of enzyme, in the present case, bygrolyting were added thereto. The process was further carried out following the procedure of Example 1.

The amount of Immobilized enzyme, i.e., hygrolytin, is 1.25 mg of the enzyme per gram of the textile carrier (0.125% Example 3 A solution of sodium periodate was prepared following the -procedure of Example 1, and 20 g of medical cotton gauze were placed into it. The process was further carried out following the procedure of Example 1 to obtain a cloth containing 3.125 mg--eq. of aldehyde groups per gram of the cloth.

A phosphate buffer solution was prepared following the 20.

-procedure of Example 1, and 0.192 - of e-nzyme such as bygrolytin 0 c were added thereto. The process was further carried out f ollowirs the procedure of Example 1.

The amount of the immobilized enzyme, i.e. hygrolytin, was mg per gram of the cloth Example 4 The process was carried out following the procedure of Example 1, except that used as an enzyme was terrlytin. The amount of the immobilized enzyme, in the present case, terrilytin was 0.0275 of the weight of the cotton gauze. Example 5 The process was carried out following the procedure of Ekample 1, except that used as an enzyme was collagenase. The amount of the immobilized enzyme, in the present case, collagenase, was O.OZIS of the weight of the cotton gauze. Exam-ple 6 The process was carried out following the procedure of Example 2, except that used as an enzyme was terriltin. The amount of the immobilized enzyme, i.e. terrilytin, was 0.120% of the weight of the cotton gauze. Example 7 The process was carried out following the procedure of Example 2, except that used as an enzyme was collagenase. The E; amount of immobilized collagenase was 0. 140% of tbe'weight of the textile gauze. Mixample 8 The process was carried out following the procedure of Example 3, except that used as an enzyme was terrilytin. The amount of immobilized terrilYtin was 0.5117,6 of the weight of 21.

the gauze. Exam-ole 9 The process was carried out following the procedure of Example 3, except that used as an enzyme was collagenase. The amount of immobilized collagenase was 0._57-o of tlie.weight of the gauze. ExamDle 10 The process was carried out following the procedure of Example 1, except that used as a textile material was gauze from cotton-and-viscose fiber. ExamDle 11 The process was carried out following the procedure of Exam-D16 2, except that used as a textile material was gauze from cotton-and-viscose fiber. Example 12 The process was carried out following the -procedure of Example 3, except that used as a textile carrier was gauze from cotton-and-viscose fiber. Mycample 13 The process was carried out following the procedure of Mcample 1, except that to obtain a buffer solution, 20.7 g of potassium dihydrophosphate and 11.8 g of sodium hydrophosphate were added to give a phosphate buffer solution having a PH of 6.5. Example 14 The process was qarried out following the procedure of Ebcample 1, except that in order to obtain a buffer solution, 11.9 g oA. potassium dihydrophosphate and 2,3.6 S of sodium hydrophosphat-e were added to give a phosphate buffer solution 22.

havinj a -DH of r,7. ExamiDle 13 'be procedure of The process was carried out followinG t Example 19 e.,.,ceDt that used as an enzyme was trypsin. Exam-ple 16 The process was carried out following the procedure of Example 1, except that a phosphate- citrate buffer solution having gL pH of 7.5 was used. The solution was prepared following the procedure as described above.

1.8 L of water were poured into the first reactor, and 19.06 g of citric acid monobydrate were added thereto under stirring. 8.25 L of water were poured into the second reactor, and 329.3 g of sodium hydrophosphate were added thereto under st irri n g. Stirring was continued until a complete dissolution was achieved, then both solutions were put together.

The process was further carried out follcwing the procedure of:,,xa=Dle 1. Example 17 The process was carried out following the procedure of Example 13, except that a phosphate- citrate, buffer solution was used. In order to obtain said solution, 71.7 9 of citric acid monohydrate were added to 1.8 1 of the water poured into one reactor under stirring, and 224.5 g of sodium hydrophospbate were added to 8.25 1 of the water poured into the other reactor, also under stirring. After the salts were dissolved, the solutions were put together to give a buffer solution having a pH of 6.5. The process was further carried out following the procedure of Example 13.

9 1 23.

Example 18.

The process was carried out following the procedure of Example 14, except that in order to obtain a phosPhatecitrate buffer solution, 38.1 G of citric acid monobydrate were added to 1.8 1 of the water contained in one reactor under stirring, and 290.5 g of sodium hydrophosphate were added to 8.25 1 of the water contained in.the other.reactor. After the salts were dissolved, the solutions were put together to give a buffer solution having a pH of 7. The process was further ca-rried out following the procedure of Example 14. Exam-Dle 19 L of 3N hydrochloric acid were poured into a reactor, and 1 kg of knitted fabric from polycaproamide fibers were placed into it. The temperature was adjusted to 600C, and the fabric was held at this temperatdre for 4 hours. Upon comple tion of hydrolysis,e) hydrochloric acid was cooled and dis-car ded. The knitted fabric was washed with water until no hydro chloric acid was observed in the washing.... Then 40 1 of %, R( glutaric aldehyde were poured into the reactor, and 1 kg of the knitted fabric subjected to hydrolysis were placed into it.

The temperature of the glutaric aldehycte, solution was adjusted-to 5011C and maintained for 4 hours. Then the glutaric aldehyde solution vas cooled and poured out.,. and after that the fabric was washed with water until there was no smell of Slutaric aldehyde. The process of fabric activation was completed.

As a result of activation, the knitted fabric acquires.reactive functional groups, in the present case, aldehyde groups, containing 0. 0625 mg-eq. per gram of textile fabric.

24.

Preparation of the phosphate buffer solution was carried Out following the procedure of Example 1. 0.66 G of enzyme, 0 - in the present case, terrilytin, were added to the obtained phosphate buffer solution having a pH of 7.5 and dissolved completely W4610-20 Tniniltes.

1 KS of activated knitted fabric from polycaproamide fibers were placed into the thus prepared terrjytin solution in the phosphate buffer solution having a volume of 6.6 1 and the enzyme concentration of 0.01%, and held at room temperature for 9 hours to effect the process of immobilization of the enzyme due to the emergence of covalent chemical bonds. Then the fabric was washed with a physiologic salt solution and water,tJ.11 no enzyme (protein), the amount whichof is controlled by the methods known per se, was detectedin the washings.

Then the f abric was squeezed out and dried in air f or 24 hours till the residual hiuddity thereof was not higher than 10/;j to give a material, i4 the present case, a knitted polycaproamide fabric having a biological activity and prolonged therapeutic action.

The obtained material contains 0.02% of immobilized terrilytin (0.2 mg of enzyme per gram of the carrier).

The dried material is formulated into medicinal fortaulationm for instance, napkins having the required size, sealed into double polyethylene bags and sterilized by I-radiation at a dose of 25 KG y using the methods known per se.

Example 20 1

An activated knitted polycaprOamide fabric was prepared following the procedure of Example 19, except that lOT) Glutaric j 25.

aldehyde was used. The amount of aldehyde groups in the fabric after activation was 0.156 mg-eq. per gram of the fabric.

1 G of the activated fabric were placed into a reactor containing 6.6 1 of a 0. 031,5 terrilytin solution in a phosphate buffer solution having a pH of 7.5 and held at room temperature for 8 hours. Further, the fabric was treated following the procedure of Example 19. The obtained material having a biological activity, contained 0. 6 ing of terrilytin per gram of the fabric, that is,0.06l-o of the weight of the fabric. The process was continued following the procedure of Example 19. Exam-o-l.e" 21 An activated knitted polycaproamide fabric was prepared following the procedure of Example 19, except that 15% glutaric aldehyde was used. The amount of aldehyde groups in the fabric after activation was 0.26 =g-eq. per gram of the fabric.

1 G of the activated fabric were placed into 6.6 g of.a.0.1% terrilytin solution in a phosphate buffer solution having a pH of 7.5 and held at room temperature for 10 hours. The fabric was further processed following the procedure of Example 19.

The obtained =aterial contained 2 =g of terrilyn pen gram of the carrier, that is, 0.21,75 of the enzyme based on the weight of the fabric. Example 22 The process was.conducted following the procedure of Example 19, except that used as an enzyme was tryosin. The obtained material contained 0.02% of immobilized trypsin on the textile carrier, In the present case, on the knitted fabric from pol-ycazroa=ide fibers.

26.

--'-,a--ole 23 The process was conducted following the procedure of Example 20, except thait: used as an enzyme was trypsin, and 1 kg of the activated knitted polycaproamide fabric ivere placed into 6.6 1 of a 0. 03,55 tryp sin solution in a phosphate buffer solution and held at room temperature for 9 hours. The process was continued following the procedure of Example 20.

The obtained material contained 0.06% of trypsin based on the weight of the polycaproamide knitted fabric. Example 2 The process was conducted following the procedure of Example 21, except that Immobilization was performed in a 0.15-,-, trypsin solution in a phosphate buffer solution.

The obtained matepial contained 0.3575 of trypsin based on the weight of the fabric. ExamDle 25 A polycaproamide knitted fabric was held in a reactor with dimethylt sulfate at a bath modulus of 10 for 10 hours at room temperature. The excess dimethylesulfate was poured out. The fabric was washed once with an ethyl alcohol at a bath modulus of 20 at a temperature of 400, then washed three times with a phosphate buffer having a pH of 7.5 at a temperature of 40C. The processed fabric contained reactive Imidoethe groups.

Immobilization of enzyme, in the present case, trypsin, was performed at a bath modulus of 10 from the enzyme solution in a phosphate buffer solution having a concentration of 0.041,15 for 16 hours at room temperature.

27.

T Men the fabric was washed with a physiologic salt solution and with water till no enzyme, the amount of whiew was controlled using the methods kno-9gn per se, was-observed in the washing water.

Further, the fabric was squeezed out and dried In air for 24 hours to give a material, in the present case, -.% knitted polycaproamide fabric, having a biological activity and prolonged therapeutic action.

The obtained material contained 0.2% of immobilized trypsin. that is, 2 mg per gram of the carrier. Exam-Ple 2 The procedure of Example 25 was repeated, except that immobilization of an enzyme, in the present caseltrypsin, was performed in a 0.00% trypsin, solution In a phosphate buffer solution at a bath modulus of 10 for 12 hours to give a material containing 0.04% of immobilized trypsin (0.4 mg per gram of the carrier). Exam-ole 27 The procedure of Example 25 was repeated except that immobilization, of trypsin, was performed in a 0.024% trypsin solution in a phosphate buffer solution at a bath modulus of 10 for 10 hours to give a material containing 1.2 mg of trypsin per gram of the carrier (0.12% of trypsin per gram of the carrier). Example 28 The procedure of Example 19 was repeated, except that used as an enzyme was lysozyme to give a material containing 0.02% by weight of immobilized lysozyme. Exammle The procedure of Example 20 was repeatedg except that used 1 28.

as an enzyme was lysozyme. The matexial contained 0.2%, by weight of immobilized lysozyme. Example 30 The procedure of Example 21 was repeated, except that used as an enzyme was lysozyme. The material contained O.";r,, by weight of immobilized lysozyme. Example 31 The procedure of Example 19 was repeated, except that used as an enzyme was protosubtilin to give a material containing 0.020% by weight of immobilized protosubtilin. Example 2 The procedure of Example 20 was repeated,except that used as an enzyme was protosubtilin. The mate3:ial contained 0.06% by weight of immobilized protosubtilin. Example 33 The procedure of Example 21 was repeated, except that used as an enz77ne was protosubtilin. The obtained material contained O.a,-'j by weight of immobilized protosubtilin. Example 34 The sterile material having a biological activity, obtained f ollowing the procedure of Example 1, is a woven, medical, cottongauze and hygrol tin immobilized thereon and bound thereto by means of covalent bonds. The material contains 0.2 mg of hygro1 tin per gram of woven cotton gauze.

The material is made in the form of medical napkins and is usat le for treating abscesses, phlegmons, postoperative complications of a surgical suture.

While using said material having a biological activi'vy, the prolonged therapeutic action of an enzyme is ensured for 48-96 i 1 i i i i i i 1 i S9 g 1 1 29.

hours,whereas the life period of a native enzyme which is used for treating the same disease, is not more than 1 hour. After a therapeutic application of said material having a biological activity, the complete healing of a wound is observed after 17 days. In some cases, during the first hours after applying a bandage, the patients had a light feeling of burning, pricking of the wound surface under the bandages. No allergic reactions of the patients' organisms were observed.

It has been concluded that there is a covalent type of bond between the carrier and the enzyme of the claimed material having a biological activityq proceeding from the fact that after treating the material with the solutions of salts, for instance, sodium chloride, or buffer solutions, for instance, a phosphate buffer solution, the enzymatic activity thereof is retained. During the process of such treatment, physicEilly-_Wff-_Sorbed proteins (enzymes) are washed out of the carrier.

After five years of storing under usual conditions said material retains its therapeutic properties. Example 35 The material having a biological activity, obtained according to Example 1, had a composition analogous to that of Example 1, except that said material contained 1.25 mg of hygrolytin per gram of woven cotton gauze. The therapeutic application of said woven gauze with immobilized hygt-0- 13ft-in which was bound thereto by means of a covalent bondq produced a prolonged therapeutic action which lasted 48-96 hours against an hour- long period of action of the native enzyme.

After applying said material having a biological activity, a complete healing of the wound was observed after 15 days.

30.

The other results viere analogous to those of Example 34. Exam-ple 36 The material having a biological activity, obtained according to Example 1, bad a composition analogous to that of Example 1, except that said material contained =g of bygrolytin per gram of woven cotton gauze. After applying said material having a biological activity, a complete healing of the wound was observed after 14 days. The other results were analogous to those of Example 34. Exam-P1 e 37 The m-aterial having a biological activity, obtained according to Example 1, had a composition analogous to that of Example 1, except that used as a textile carrier was Imitted medical cotton gauze. The results of medical application of said material are the same as those of Example 34. Example 38 The material having a biological activityg obtained according to Example 1, bad a compositim analogous to that of Example 1, except that used as a textile carrier was a non-woven fabric frca cotton fibers. The results of medical application of said material are the same as those of Example 34. ExaMD1e 39 Thematerial having a biological activity, obtained 0 according to Example 1, bad a composition analogous to that of Exanple 1, excent that used as a textile carrier was a abric. The results of medical application of said linen L material are the same as those of L-cample 34.

31.

3:cariole 40 The material havinG a biological activity, obtained accordinZ to Exanple 1, had a composition analogous to that W of 'Example 1, except that used as a textile carrier was a woven, natural silk fabric. The results of medical application of said material are the same as those of Example 34. Exam-ole 41 The material having a biological activity, obtained according to Example 19, bad a composition analogous to that of Example 19, except that used As a textile carrier was a knitted polycaproamide fabric.

The results of medical application of said material were the same as those of Example 34. Exam.ole 42 The material having a biological activity, obtained according to Example 19, bad a composition analogous to that of Example 19, except that used as a textile carrier was a knitted polyurethane fabric.

The results of medical application of said material were the same as those of Example 34. Exam-ple 43 The material having a biological activity, obtained according to Example 1, bad a composition analogous to that of Exa=ple 1, except that used as a textile carrier was a knitted viscose fabric.

The results of medical application of said material were the same as those of Example 34. Exa.mDle 44 The material having a biological activity, obtained 32.

according to Example 1, had a composition analogous to that of Example 1, except that used as a textile carrier was a secondary acetate cellulose knitted fabric.

The results of medical application of said material were the same as those of Example 34. Example 43 The material having a biological activity, obtal=ed according to Example 21, had a composition analogous to that of Example 21, except that it contained lysozyme. Said material contained 2 mg of lysozyme per gram of the textile carrier. The application of said material containing immobilized lysozyme which was bound to the carrier by mean of a covalent bond, produced a prolonged therapeutic action which lasted 48-96 hours against an hour-long period of therapeutic action of the native enzyme.

After a medical-application of said material, a complete healing of the wound was observed after 15 days.

Treatment of purulent wounds in a hydration phase with a textile material containing immobilized lysozyme, reduces the period of elimination of the wound infection, of cup ping the alternative purulent inflammation, promotes the purification of wounds from pyo-necrotic tissues, stimulates the synthesis of glycogen and nucleic cells (DNA and RNA) in the wound cells, activates the -proliferation of connective tissue cells (fibro blasts), accelerates collagenosis, the contraction of edges, the contraction and e-oithe--lization of+jewound.

Exam-pie 4 aving a biological activity, obtained OTbe material b ' according to Example 1, had a composition analogous to that of Example 5. Said material contained a collagenolytic enzy=e such 33.

4 as collagenase in an amount of 0.2 mg of collagenase per gram of the textile carrier.

After a medical application of said material, a complete healing of the wound was observed after 16 days. The other, results of the medical application of said malierialWe.re analogous to those of Examples 34 and 35. Example 47 The material having a biological activity, obtained according to Example 15, had a composition analogous to that of Example 1.

Said material was in the form of lint having fibers 1.0 mm long; it is usable for treating purulent, deep, puncture wounds,, inflammatory processes in the rectum.

The medical effect, that is, a complete healing of wounds was observed after 17 days. Exam-Ple 48 The material having a biological activity, obtained according to Example 15, had a composition analogous to that of Example 1.

Said material was in the form of lint having fibers 8.0 mm long; it was used to accelerate the treatment of dental infection foci (chronic granulated periodontitis).

The material in the form of lint produces an antiexudative effect, it actively promotes the outflow of exudate through a root channel (even curved, narrowq but obligatorily permeable canals). Example__49 The material having a biological activity, obtained according to.'j:, Xample 15, had a composition analogous to that of A 1 34.

ExamDle 1.

Said material was in the form of lint; it was used for treating the inflammatory process in the rectum.

Themedical effect was observed after 16.5 days. Exam-ple 50 The material having a biological activity, obtained according to Example 15, had a composition analogous to that of Example 1.

Said material was in the form of a powder having a particle size of 0.001 m=; it was used for treating postoperative c qmplications of surgical sutures.

After a medical application of said powder having a biological activity, including an enzyme avtivity, a complete healing of the suture was observed after 17 days. Exammle 51 The material having a biological activity, obtained according to Example 15, had a composition analogous to that of Example 1.

Said material was in the f om of a powder hav:L-1c, a CD particle size of 0.5 mm; it was used for treating pyo-necrotic processes of complex localization and_cavities difficult of access.

The medical effect was observed after 17 days. Example 52 The material having a biolorical activity, obtained according to Example 15, had a composition analogous to that of 22caiaole 1.

Said =aterial was in the form of a powder having a particle size of LO =; it was used for treating pble-97-ons f i 35.

The medical effect was observed after IS-5 da,7s. Examnle 53 Dressing in the form of a bandage consisting of three la7ers and a fixing means such as an adhesive tape.

The bandage contained successively a protectinglayer, an absorbing layer and a wound-neighbouring layer, the latter being made of the material having a biological activity according to Example 21.

The dressing was applied at the stage of pre-medical aid for treating the trauma of soft tissues of gluteal region, having a size 6x4 cm and up to 4 cm deep. When the patient was taken to the clinic 12 hours later, no edema and perifocal inflammation was observed; the inner surface of the wound was clean and the tissues were viable. The complete healing of the wound was observed after the surgical treatment in 14 days. Example 5 Dressing in the form of a bandage consisting of three layers and a fixing means such as an adhesive tape.

The bandage contained successively a protecting layer, an absorbing layer and a wound-neighbouring layer, the latter being made of the material having a biological activity according to Example 21, and the absorbing layer being made of the material having a biological activity according to Example 30- The dressing was applied at the stage of pre-medical aid for treating the trauma of soft leg tissues having a size 4x3 cm and-up to 2 cm. deep. When the patient was taken to the clinic 8 hours later, no edema and perifocal inflammation was observed; the inner surface of the wound was clean and the 36.

tissues were viable. After the surgical treatment, the complete bealing of the wound was observed in 12 days.

Examole 52 Dressing in the form of a bandage conssting of three layers and a fixing means such as an adhesive tape.

The bandage contained successively a protecting layer, an absorbing layer and a wound-neighbouring IaFjer, the ldtter bein- made of the material having a biological activity according to Example 21, and the absorbing layer being made of the material having a biological ativity according to Example 30, said material being lint having fibers 10 cm long.

The dressing was applied at the stage of pre-medical aid for treating the trauma of soft tissues of gluteal region, having a size of 5x2 cm and up to 3 cm deep. When the patient was taken to the clinic 10 hours laterg no edema and perifocal inflammation was observed. After the surgical treatment, the complete healing of the wound was observed in 12,5 days.

37

Claims (23)

1. A substance for dressings comprising an enzyme covalently linked to a physiologically acceptable, insoluble carrier therefor.

2. A substance according to claim 1, wherein the enzyme is present in a ratio of between about 0.02 and about 0.5% W/W.

3. A substance according to claim 1 or 2. wherein the carrier is a textile.

4. A biologically active substance comprising a carrier and an enzyme immobilised thereon, wherein the carrier is a textile material and the enzyme is covalently bound thereto. the ratio of components being substantially (weight percent) 0.02-0.50 enzyme: 99.98-99.50 carrier.

5. A substance according to any preceding Claim. wherein the textile is made from natural, synthetic or man-made fibre.

6. A substance according to any preceding Claim. wherein the textile is in the form of a woven, non-woven or knitted fabric.

7. A substance according to any preceding claim, comprising proteolytic, collagenolytic and/or bacteriolytic enzymes.

8. A substance according to any preceding claim, wherein the substance is in the form of lint or powder.

38

9. A substance according to any preceding claim. wherein the substance is lint having fibres of 1.0-15 mm length.

10. A substance according to any of claims 1 to 8,. wherein the substance is a powder of particle size of between 0.001 and 1.0 mm.

11. A dressing comprising a substance according to any preceding claim.

12. A dressing in the form of a bandage comprising successively arranged protecting and absorbing layers and a layer bearing against the wound. wherein the dressing comprises at least one layer made of an active substance according to any of claims 1 to 10.

13. A dressing according to Claim 12, wherein the layer bearing against the wound is made of the active substance, and the absorbing layer is made of a textile containing no biologically active compound.

14. A dressing according to Claim 12 or 13, wherein the absorbing layer also comprises active substance.

15. A method for preparing a substance according to any of claims 1 to 10. comprising activating the carrier, immobilising the enzyme thereon using a buffer solution, followed by washing, drying and forming. the carrier being activated by forming therein reactive functional groups to about 0. 0625-3.125 m 2 eq. per gram of carrier. the enzyme from a buffer solution having a pH of 6.5-7.5 being immobilised on the activated carrier at room temperature for about 816 hours to form a covalent chemical bond between the carrier and the enzyme, after which the carrier is squeezed out, washed, preferably with with water. until the washings contain 1 1 1 k 39 substantially no biological activity, dried at ambient temperature. and optionally formed. placed into a tight container and sterilised.

16. A method according to Claim 15, wherein sterilisation is effected by gamma irradiation at a dose of 25 kGY.

17. A method according to claim 15 or 16 ' wherein the carrier comprises cellulose. and activation is performed by oxidation with an alkali periodate to give dialdehyde-cellulose containing reactive functional aldehyde groups in an amount of about 0.0625-3.125 mg eq. per gram of the carrier.

18. A method according to Claim 15 or 16, wherein the carrier contains no cellulose and is activated by acid hydrolysis, followed by washing the carrier with water and reacting the hydrolysed carrier with a solution containing reactive functional groups to form in the carrier reactive functional groups in an amount of about 0.0625-0.3125 mg-eq. per gram of the carrier.

19. A method according to any of Claims 15 to 18, wherein the hydrolised carrier is further treated with a solution of glutaric aldehyde. followed by washing the carrier in water.

20. A method according to any of claims 15 to 19, wherein the hydrolyzed carrier is further treated with a dimethyl sulphate solution, followed by washing the carrier in ethyl alcohol and in a phosphate buffer.

21. A substance or material comprising a carrier and at least one enzyme covalently bound thereto, substantially as described herein before. with particular reference to the accompanying Examples.

22. A dressing comprising a substance or material according to Claim 21.

23. A method for preparing a susbstance or material according to claim 21. substantially as described hereinbefore, with particular reference to the accompanying Examples.

Published 1991 atIbe Patent Office. State House. 66/71 High Holborn. LondunWCIR477. Further copies maybe obtained from Sales Branch. Unit 6. Nine Mile Point. Cwmfelinfach. Cross Keys. Newport. NPI 7HZ. Printed by Multiplex techniques lid, St Mary Cray. Kent.