CA1217134A

CA1217134A - Proteolytic cover of wounds

Info

Publication number: CA1217134A
Application number: CA000464332A
Authority: CA
Inventors: Jaroslava Turkova; Jiri Stamberg; Vladimir Pitrak; Lenka Jelinkova; Karel Sebesta; Mojmir Sebestik
Original assignee: Czech Academy of Sciences CAS
Current assignee: Czech Academy of Sciences CAS
Priority date: 1983-09-29
Filing date: 1984-09-28
Publication date: 1987-01-27
Also published as: JPS6094916A; GB8423913D0; FR2552667B1; CS249311B1; GB2147206B; DE3435718A1; FR2552667A1; GB2147206A; DD258121A3; SE8404769L; SE8404769D0; HUT35956A

Abstract

ABSTRACT OF THE DISCLOSURE:

The invention pertains to a proteolytic cover of wounds in the form of powder, which serves for covering and treatment of ulcerous anc necrotic wounds. It is based on the original combination of the proteolytic and absorption principles of cleaning of infected ulcerous wounds. According to the invention, the cover consists of porous spherical cellulose with particle size 0.05 to 0.5 mm, which contains an immobilized protease. The method of production warrants that the obtained products contain only the covalently bonded protease and no undesirable toxic compounds or soluble por-tions with the proteolytic activity, which could penetrate into blood circulation and develop an alergic reaction as antigens. The immobilized enzymes act in cleaning of the wound by dissolving undesirable proteins, in particular fibrin, necrotic tissues, pus and others. The porous structure of hydrophilic spherical particles of cellulose warrants a high absorption capacity, which is exhibited by sorption of an exudate into the material and suction of pus infected with bacteria from the wound into the inter-particular space of powder layer. The combination of proteolytic and absorption principles enables to achieve the fast cleaning of infected necrotic defects, formation of clean granulations, and fast healing of the wound. The invention may be utilized in pharmaceutical production.

Description

Proteolytic cover of wounds The invention pertains to a proteolytic cover of wounds in the form of a powder which serves for coating and treat-ment of ulcerous and necrotic wounds.
The powders consisting of various bases (starch, talc, etc.) and bactericides or bacteriostatics, in particular antibiotics, sulfonamides and antimycotics, are recommended for this purpose in the common pharmaceutical practice.
Recently, absorbing covers in the form of powders, which contain a hydrophilic polymer as a main component, proved suitable in the treatment of ulcerous wounds. Their effect consist in the removal of liquids from the surface of wound by sorption in a porous structure of the effective polymeric component and by suction of capillary forces into voids between particles of the powder layer. The exudate together with bacteria, substances causing inflammation, or also toxins are removed from the wound in this way. In particular, prepara-tions based on crosslinked dextrans (B.S. Jacobsson et al., Scand. J. Plast. Reconstr. Surg. 10, 65-72 (1976)) are used in medical practice. Promising results were attained also with polymer compositions based on cellulose, which contain a highly hydrophilic polymeric component, for example carboxymethyl-cellulose as an additive (H. Dautzenberg et al., Absorbing cover of wounds; Czechoslovak certificate of authorship No. 237,604).
Further development of powders with cleaning effect in 3~

treatment of ulcerous wounds has been directed to cellulose powders containing immobilized enzymes of protease type (Collection of papers: Immobilized proteolytic ferments in heating of ulcerous-necrotic processes (in Russian), AN SSSR, Sibirskoe Otdel., Institut tsitol gii i Genetiki, Novosibirsk, 1981). In this case, the enzymatic action is used to attain cleaning effects. The proteolytic covers of wounds cause a hydrolytic decomposition and dissolution of necrotic tissues and pus on the wound surface thus removing also the medium for growth of bacteria and interrupting suction of toxic products from the wound.
Practical utilization of the proteolytic principle requires a solution of numerous problems in the preparation of the efficient cover which would not develop harmful side effects.
This means, in human medicine, above all to prevent cellulose or derivatized cellulose from entering into the blood circula-tion, because the human organism does not have enzymatic systems for the removal of such compounds at its disposal. However, the preparations of proteolytic cover used so far employ only conventional cellulose types, i.e. ground ion-exchanging dusts with a fibrous structure containing fine fractions of dust which may penetrate into blood.
To attain the maximum efficiency of covers, the proteolytic principle should not be employed without using the previously known absorption principle at the same time. Recent solutions comprise, however, only the conventional type of cellulose with a high crystallinity and low porosity as the starting material, which provides covers with a relatively low absorp-tion capacity.

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In addition, such ~rocedures for the immobiliza-tion of proteases in cellulose have to be found for the utilization of proteolytic principle, which would exclude a release of the enzyme from matrix and penetration of its soluble form into blood circulation, where it would develop an alergic reaction as an antigen.
The above said shortcomings in the utilization of proteolytic principle are met by a new cover for wounds according to the invention.
The invention pertains to a proteolytic cover of wounds in the form of powder, which serves for covering and treatment of ulcerous and necrotic wounds. It is based on the original combination of the proteolytic and absorp-tion principles of cleaning of infected ulcerous wounds.
According to the invention, the cover consists of porous spherical cellulose with particle size 0.05 to 0.5 mm, which contains an immobilized protease. The method of production warrants that the obtained products contain only the cova-lently bonded protease and no undesirable toxic compounds or soluble portions with the proteolytic activity, which could penetrate into blood circulation and develop an alergic reaction as antigens. The immobilized enzymes act in clean-ing of the wound by dissolving undesirable proteins, in particular fibrin, necrotic tissues, pus and others. The porous structure of hydrophilic spherical particles of cellulose warrants a high absorption capacity, which is exhibited by sorption of an exudate into the material and suction of pus infected with bacteria from the wound into the interparticular space of powder layer. The combination of proteolytic and absorption principles enables to achieve the fast cleaning of infected necrotic defects, formation of clean granulations, and fast healing of the wound.
In particular the present invention relates to a composition of matter which can be used as a proteolytic -cover for wounds, which consists of spherical particles of diameter 0.05 to 0.5 mm, preferably o 1 to 0.3 mm, based on (a derivatized) cellulose provided with one or more immobilized enzymes of protease type e.g. selected from the group which comprises chymotrypsine, trypsine, and subtilisine. In another aspect the present invention pro-vides a method for producing a composition o~ matter, useful as a proteolytic cover for wounds, from bead cellulo-se, prepared by the procedure according to Czechoslovak Patent no. 172,640, where the bead cellulose swollen in water, which never has been dried, of particle size 0.07 to 0.7 mm, prefera~ly o.14 to 0.4 mm, is perfectly freed of toxic contaminants by washing, distillation with steam or both activated for bonding of an en~.yme, modified by immobilization of one or more proteases selected from the group compri.sing chymotrypsine, trypsine, and subtilisine, alternately washed with a buffer of pH 8.5 to 9.5 and a buffer of pH 4 to 5 until the washings have proteolytic activity zero, then washed with a buffer of pH 7.5 to 8.5, and dried in this medium to 0.1 to 15~ of the residual water content in th~ dry substance, preferably by lyophili-sation,a composition of matter of the above defined particle size thereafter being recovered by any suitable means.
The regular spherical shape of individual particles, which form the new cover for wounds, and the chosen particle size and the distribution of particle size (0.05-G.5 mm, preerably 0,1-0.3 mm), warrant an easy handling both in production and in application, a smooth flow of powder during its spreading on wounds, and, in particular, meet the requirement to prevent penetration of cellulose into blood circulation.
The production of new cover advantageously employs the regenerated spherical cellulose prepared according to Czechoslovak Patent no. 172,640. Its advantage is a high porosity which facilitates the activation for enzyme bind-ing and the immobiliæation of proteases. The porous hydro-philic character of the bead cellulose carrier is retained even after activation, immobilization and drying. Lyophili-sation proved a suitable drying method which removes waterin a considerate way with respect to the bonded enzyme and provides the dry derivatized cellulose with a sufficient absorption power.
The bead cellulose prepared by the procedure described in Czechoslovak Patent no. 172,640 is thoroughly freed of soluble portions, in particular of all impurities with toxic effects which cause its cohtamination during preparation (decomposition products of xanthogenate groups, residues of a disperse medium, e.g. of chlorobenzene). For this purpose, it is washed with water at 50-90C and/or with ethanol. An effective removal of chlorobenzene occurs by distillation with steam.
Several methods known from literature can be used to activate the cellulose for protease binding (Handb ok of , / _ . _ . . . .

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Enzyme Biotechnology, Ed. ~. Y~iseman; E. ~orwood Ltd~ Chich~-ster 1975), but such methods should be chosen which form a sufficiently stable bond between enzyme and cellulose and do not contaminate the product with toxic co~pounds becoming effective during application of the coYer, for example; by releasing or action in the direct contact. A suitable method is the periodate oxidation~ which is relatively simple and easy to carry out and provides products of a suffi~ient porosity which exhibit a high absorption effectO
Yarious proteases are suitable for immobilization, parti-cularly trypsine, chymotrypsine and subtilisine, but also thermolysine5 papaine, and others. Binding of the enzyme to cellulo~e activated by periodate oxidation, î.eO ~he cellulose containing reactiYe dialdehyde groups, is carried out in two steps: the compounds of cellulose with the enzyme of a Schiff--base type are formed first~ which are then stabilized by reduction of the ~nreacted aldehyde groups with sodium boro-hydride.
An important stage in the preparation of the new type o~
coYer is the perfect removal of solub~e portions containing proteases from the modified cellulo~e beads. I~ is attained by the repeated alter~ation o~ a wea~ly alkaline (pH 8~5-9.5) ~nd a weakly acidic buffer (pH 4-5) in a static or column arrangement. ~or this purpose7 thsy are suitable~ e.g~ the 0.1 M borate buffer ~ontaining 1 M NaCl tp~ 9~ and the 0.1 acetate buf~er containing 1 M NaCl 5pH 4.53~ or both these buffer~ with~ut NaCl, which are applied on the cellulose prsduct after immobilization until the lîquid phase ha~
.

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a proteolytic activity ~ero. ~he alternation of buf'~er~ of lower and higher pH and variable ionic strength ~acilitates the elution of ccvalently non~onded protein by the alternating suppression of electrostatic and hydrophobic interactions.
To obtain a perfectly stable preparation with the immobilized enzyme, the beads are even-tually washed with a borate buf~er, e.gO, of pH 8, and lyophilized in a suspDnsion with this bu~fer~ Under ~uch conditions~ the resulting product contains only the covalently bonded enzyme and no ~oluble portion ~f it, which could penetrat~ i~to blood during treatment and could become an antigen.
The proteolytic co~er with immobilized chymotrypsine was tested in clinical practiceO It exhibited the proteolytic acti~ity to necrotic tissue, pus and fibrin and, at the s~me time, did not harm a healthy tissue at all~ The cover ~YaS
noted for the hi~h absorption capacity in agreement with the hydrophilicity and porous structure of cellulo~e matrixO It sucked up the exudate ~rom a wound, which contained clea~ed tissue necroses and bacteria. Binding o~ the enzyme t~ carrier pre~ent~ its autolysiæ and los~ of its acti~ity during appli-caticn~ The extended proteolytic acti~ity of chymotryp~ine led t~ the release of necroses from wound in a large extent and to the stepwise cleani.ng of wo~nd~ The early in~ected secretion wa~ actively absorbed between the particles of powder thu~ preventing the circumference of wound from maceration and reducing the nutrient ~ur~ace ~or the ~rowth of bacterial in~ection. The original combination of the proteolytic and abssrption principle~ enabled to achieYe the ~a~t cleaning o~

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infected necrotic defects, formation of clean granulations and fast healing of the wound.
It has been proved that the new cover is suitable for all kinds of ulcerous and necrotic wounds, including the surgi-cal wounds healing per secundam, for abcesses, prolonged andnonhealing ulcers of various origin (varicose, X-ray and trophic ulcers~, defects after decubital necroses, infected and necro-tic defects after acral amputations, incisions and necrecto-mies at diabetic gangrenes and gangrenes of arteriosclerotic origin, carbuncles, ulcerous burns of the 2nd and 3rd degree, infected open fractures, treatment of amputation stumps, decom-posed and ulcerous tumours. No harmful side effects were observed in the application of the new cover.
The following examples of performance illustrate the method for production of covers according to the invention and their applications without limiting the scope of invention.
Example 1 Preparation of the cover with bonded chymotrypsine a) Introduction of aldehyde groups by oxidation with sodium periodate The bead cellulose swollen in water and never dried before with porosity P = 90~ (i.e. the overall content of pores in volume ~), which was prepared according to Czechoslovak Patent no. 172,640, was used as the starting material. Cellulose was perfectly freed of impurities by washing with hot water (90, 5h, twentyfold excess) and by distillation with steam (4h). The filtered cellulose (1000 g) was dispersed in 5 1 0.1 M sodium periodate and stirred at laboratory temperature for 45 min. After completion of the oxidation, the oxidized cellulose was immediately washed with about 20 1 distilled water (on Buechner funnel), transferred into a column and washed with distilled water until the electric conductivity of effluentbecame equal to the conductivity of distilled water (overnight).

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b) Binding of chymotrypsine to oxydized cellulose The washed oxidized cellulose (1000 g) was dispersed in 1 litre 0.1 M borate buffer (pH 9) containing 5 g chymotrypsine (the proteolytic activity of the solution was 5.35 JA280/min-ml as determined by means of a solution of denaturated haemo-globin, pH 8). The suspension was stirred at laboratory tempe-rature. The progress of binding was followed as the decreasing proteolytic activity of the binding solution. After an hour, the proteolytic activity decreased to o.l JA2~0/min.ml and bonding was stopped by suction off the binding solutionO
The rate of chymotrypsine binding increases with increasing pH, however, the solubilization of cellulose increases at the same time depending on time of oxidation~ The chosen procedure was worked out on the base oE numerous comparative experiments and compromises between the amount of bonded enzyme and a loss of cellulose caused by solubilization.
c) Reduction of cellulose with bonded chymotrypsine to stabilize Schiff bond between the carrier and enzyme and remove unreacted aldehyde ~roups The cellulose with bonded chymotrypsine was suspended in 1 litre 0.1 M borate bufEer (pH 9) with dissolved 500 mg NaBH4. The reduction was stopped after 20 min of stirring at laboratory temperature by suction oEf the reducing solution .

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and once more rep~ated in the sa~e way with the new ~olution of sodium borvhydride. The reduction vras carried out twice by addition of the l~aBH4 solution of lower concentration to prevent the loss of proteolytic activity of chymotrypsine by the contingent reduction o~ disulfide bridges.
d ) Washing and lyophilisation o~ cellulose with bonded chymotrypsine The cellulose with bonded chymotrypsine was washed, a~ter completion o~ reduction, alternately with 2 1 Ool M borate buffer containing 1 ~ NaCl (pH 9~, 2 1 0~1 ~ acetate buffer containing 1 ~ NaCl (pX 4.5~, and further with the same volumes of both buffers without NaC10 The cellulose ~Jith bonded chymotrypsine ~as then trans~erred into a column~ where it was washed with all above mentioned buffers repeatedly alY~ays until the proteolytic activity of e~luent was zero. The cellu-lose with bonded chymotrypsine was eventually washed with 0.25 M borate buffer o~ pH 8 and lyophilised also in the su~pension with this buffer. The obtained preparation contained 5 mg active chymotrypsine on 1 g dry cellulose~ The comparative amino acid analysis o~ the prepared sample and a ~ample addi-tionally washed with 6 M guanidine hydrochloride and distilled water proved that all chymotrypsine is covalently bonded to cellulose and therefore cannot penetrate into blood during application and become an antigen.
Example 2 Preparation of the cover with covalently bonded trypsine I~obilization of trypsine ~ras carried out in the same way ~2~3~

as in example 1. The obtained preparation contaIned 8.2 mg actiYe trypsine in 1 g lyophilised cellulose. Because of it~
narrower specificity, trypsine clea~es proteins at lower number of sites (it cleaves proteins only behind basic amino acids lysine and arginine).
Example 3 Preparation o~ the coYer with covalently bonded subtilisine The immobilization of bacterial p~oteinase was carried out in the same way as in example lo The obtained preparation contained 11~3 mg active enzyme in 1 g lyophilised preparation.
~xample 4 The proteolytic cover with chymotrypsine bonded according ~o example 1 was ap?lied to 7 patients ~or the period ~f 1 to weeks with a very good eflec~ at 6 patients. The effect is ch~racterized by retreat of purulent secre~ion, cleaning o~
the de~ect base and release of adhering necroses of which those of larger extent were surgically removed by necrectomy. The defect was rinsed with hydrogen peroxide solution and 2 per~
mille solution of Chloramine before further application of the preparationO Vi~id red granulation~ and epithelisation of the defects from circumference,:vllowed by their reduction in size, were achieved by this procedure. ~ local application o~
Panthenol spray int~ epithelising defect was mostly used after the treatment ~va3 ~inished~, One case showed ~ decrease of purulent secretion, o~ fifth of the def~ct was cleaned to vivid granulations ? but deeply reacting adhering necroses occurred in the r~maining parts of de~ect in the terrain of chronic ischaemia caused by the combination of diabetic microangiopathy and oblitering arte-riosclerosis of peripheral arteries, so that an insufficient blood supply of neighbouring tissues wa~ the limiting factor of healing o~ the defectO ~our weeks after the ~pplication of ~he preparation was stopped, the defect made progress in the sense of abcessing diabetic phlegmon~
Example~ of application of the preparation:
1) J~., man, age 72~ report no. 8357/83; a defect of diameter 3 cm after amputation of the 4th toe of right leg for the diabetic gangrene with wet necroses on the bottom and a puru lent secretion. After 2-week application of the preparation~
predominan~ly clean granulations occur with isolated adhering necrose~ in the medial edge of wound ~ without purulent secretion.

2) V.S~, man, age 60, report no. 6417/83; the state after a~putation of the right shin ~or diabetic gangrene, where the defect of diameter 5 cm and depth 20 5 cm arose af~er abce~sing phlegmon, which had necroses on the lower lateral part of amputation stump-and al30 medially scabby granulat;ons in ~he whole range of wound 10 cm long and 1 cm wide with purulent secretion. After 4-week application of the prepara-tion, almost complete healing was achie~ed, except a de~ect of diameter 2 cm and depth 1 cm in the lateral part of stump with clean granulations and progressi~e epithelisation of the G ircumference~

3) F.N~, man, age ~6, report no. 3376/83; a defect after ampu-tation of 1st to 3rd toes including the heads of corresponding metatarses for diabetic gangrene, ~ize 5-4 cm~ partly with scabby granulations and partly with adhering necroses;

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the depth of defect was in it~ lateral part up to 1 cm. After

4-week application o~ the preparation, clean granulations were obtained in the whole region of defect with the progressive epithelisation of circu~ference and reduction to diameter of 3 cm. The same patient, ~ N., man, age 66, after amputation of the distal half of 4th toe including the head of 4~h meta-tarse; a defect aro~e of diameter 3 cm with a scabby lower part; after a week application of the preparation, the defect cleanly granulates without secretion and with sound surroundings.
4) F.Yi~ man7 ag~ 77, report ~o. 2~4/84, with the defect after amputation of the 2nd, 3rd and 4th toe for diabetic gangrene of size 5x4 cm and depth up to 3 cm with an abundant purulent secretion and extensive adhering necroses in the whole ran~e o~ wound; after 4 week application of the preparation, clean granulations were formed in one ~ifth of the defect; the necro-tic process remained Ylithout limits in other parts due to poor blood supply in leg periphery at the diabetic microangiopathy and oblitering arteriosclerosis, but the purulent secretion in the defect ceased; the progress in defect occurred first after the application of preparation was stopped.

5) P~. M.K3~ man, age 56, report noO 9568/83, with the defect after incision of an abc~ssing phlegmon of the interphalangal joint of the 1st to~ 7 ~hich re~ched intraarticularly, of diameter 205 cm, with necroses and purulent secretion; after a week application of ~he preparation, the secretion receded~
the defect was cleaned to clean granulations and the surrounding of defect get calm~ ~

6) ~,.P~ 3 woman, age ~8~ report no. 1994~83, with a defect aftar amputation of the 1st and 2nd toes including the heads o~

corresponding metatarses for diabetic gangrene of size Sx3 cm, with a scabby lower part, adhering necroses and purulent secretion in the distal field of wound. After 4-week application of the preparation, clean granulations were obtained in the whole range of wound, the purulent secretion was suppressed and the defect was reduced to 3x2 cm by the advanced epithelisation.

7) M.T., woman, age 77, report no. 17741/83, with two defects in an amputation stump of shin after amputation for a diabetic gangrene followed by the abcessing phlegmon of stump, which caused liqhefaction of the wound in whole region and defects of the size lOx2-4 cm with a purulent secretion, scabby granulations and extensive necroses. After 4-week application of the preparation, the granulations became clean, necroses were released, the secretion suppressed and epithelisation advanced from the circumference of the defects so that these ; were medially reduced to diameter of 3 cm and laterally to diameter of 1.5 cm.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition of matter suitable for use as a proteolytic cover for wounds, wherein the said composi-tion consists of spherical particles of cellulose of diameter 0.05 to 0.5 mm, said cellulose being a derivatized cellulose provided with one or more immobilized enzymes of the protease type selected from the group comprising chymotrypsine, trypsine and subtilisine.

2. A composition of matter as defined in claim 1, wherein the spherical particles have a diameter of 0.1 to 0.3 mm.

3. A composition of matter as defined in claim 1, wherein said cellulose is regenerated cellulose.

4. A composition of matter as defined in claim 2, wherein said cellulose is a regenerated cellulose.

5. Method for producing a composition according to claim 3, wherein a regenerated bead cellulose swollen in water and not dried beforehand, with a particle size of 0.07 to 0.7 mm, is freed of toxic impurities by washing, distillation with steam or both, activated for binding of an enzyme thereto, modified by the immobilisation of one or more proteases selected from the group comprising chymo-trypsine, trypsine and subtilisine, alternately washed with a buffer of pH 8.5 to 9.5 and a buffer of pH 4 to 5 until the proteolytic activity of washing liquid is zero, then washed with a buffer of pH 7.5 to 8.5, and dried in this medium up to 0.1 to 15% of the residual water content in the dry substance.

6. A method as defined in claim 5, wherein the wet, swollen, regenerated bead cellulose has a particle size of 0.14 to 0.4 mm.

7. A method as defined in claim 5, wherein the product is dried by lyophilisation.

8. A composition of matter suitable for use as a proteolytic cover for wounds, wherein the said composi-tion consists of spherical particles of cellulose of diameter 0.05 to 0.5 mm, said cellulose being a derivatized cellulose provided with one or more immobilized enzymes of the protease type.

9. A composition of matter suitable for use as a proteolytic cover for wounds, wherein the said composi-tion consists of spherical particles of cellulose of diameter 0.05 to 0.5 mm, said cellulose being provided with one or more immobilized enzymes of the protease type.

10. Method for producing a composition according to claim 9, wherein a bead cellulose swollen in water and not dried beforehand, with a particle size of 0.07 to 0.7 mm, is freed of toxic impurities by washing, distillation with steam or both, activated for binding of an enzyme thereto, modified by the immobilisation of one or more proteases, alternately washed with a buffer of pH 8.5 to 9.5 and a buffer of pH 4 to 5 until the proteolytic activity of washing liquid is zero, then washed with a buffer of pH
7.5 to 8.5, and dried in this medium up to 0.1 to 15% of the residual water content in the dry substance.