JPS6094916A - Protein decomposing coating and manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention This invention relates to a powdered proteolytic dressing for the covering and treatment of ulcerative and necrotic wounds.

(Prior Art) For this purpose, in common medical practice, various bases (starch, talc, etc.) and bactericides or anti-A111 are used.
Powders consisting of fungicides, especially antibiotics, sulfonamides and antifungals, are recommended.

Recently, powdered absorbent coatings containing hydrophilic polymers as the main component have proven suitable for the treatment of ulcerative wounds. The effect of this consists in effective absorption of the polymer component in the porous structure and removal of fluid from the wound surface by suction by capillary forces into the spaces between the particles of the powder layer.

In this way, exudate is removed from the wound along with debris, inflammatory substances, or even blue-purple. On the ship, cross-linked dextran-based K-type W products [B, S.

Jacobson et al., 5cond, J. Plas.
t, Reconstr.

Surg, 10 65-72 (1976)':] is used in medical practice. Cellulose-based polymer compositions containing new, highly aqueous polymer components, such as carboxymethyl cellulose, as additives also give good results (H, Dautzenberg et al.) Abs
ovbing cover of wounds ++,
Czechoslovakia patent &'l PV 4129-82
).

Other developments in powders with a cleansing effect in the treatment of ulcerative wounds have focused attention on cellulose powders containing immobilized enzymes of the protease type [immobilized proteolytic enzymes (0 shea) in the fever of the ulcer-necrotic process. (word),
AN 5SSR, 5ibirskoeOtde1. ,1
nstitut tsitologii i Gene
Tiki.

Novosibirsk, 1981). In this case, enzymatic action is used to obtain the cleaning effect. The proteolytic coating of the wound hydrolytically destroys and dissolves necrotic tissue and pus on the wound surface, thus removing the medium for bacterial growth and removing the necrotic tissue and pus from the wound surface. Interferes with absorption from wounds.

Practical application of the proteolytic principle requires solving many problems in the preparation of effective coatings that do not cause harmful side effects. In human therapy, this means preventing cellulose or cellulose derivatives from entering the blood circulation. This is because humans do not have enzyme systems to remove such compounds.

However, in the production of proteolytic coatings that have been used in the past, only conventional types of cellulose, such as ground ion-exchange dust with a fibrous structure containing a fine fraction of dust that can enter the blood, are used. is used.

(Problem Solved by the Invention) In order to obtain the maximum effectiveness of the coating, the proteolytic principle should not be used without simultaneous use of the already known absorption principle. However, current solutions use conventional cellulose with high crystallinity and low porosity as the starting material, resulting in coatings with relatively low absorption capacity.

Furthermore, in order to apply the principle of proteolysis, a method of immobilization of proteases in cellulose was found, in which the enzyme is released from the matrix and its lysate enters the blood circulation. If the enzyme lysate enters the bloodstream, it will act as an antigen and cause an allergic reaction.

The above-mentioned drawbacks when using the proteolytic principle are eliminated in the novel wound dressing of this invention.

The subject of the invention is a cellulose derivative with a diameter of 0.05 to 0.5τ with an immobilized enzyme of the protease type selected from the group consisting of chymotrypsin, trypsin and subtilisin. ! fit, preferably 0.1-0.
This is a proteolytic wound dressing consisting of spherical particles of 3 rrrm. Another subject of the invention is a method for producing proteolytic dressings of wound reservoirs from beaded cellulose produced by the method of Czechoslovakia Patent No. 172,640, which method comprises: The swollen and undried beaded cellulose with particle size 0.07-0.7 mm, preferably 0.14-Q, 4 mm is completely free of blue contaminants by washing or distilling with steam. , active for enzyme binding 4<1. modified by immobilization of a protease selected from the group consisting of chymotrypsin, trypsin and O.subtilisin, and proteolyzed the wash solution using a buffer solution at pH 8.5-9.5 and a buffer solution at pH 4-5. Washing alternately until the active +'j2 is O, then with a buffer of pH 7.5 to 85, and in this medium the residual water content in the dry matter is o,
i to 1.5%, preferably by freeze-drying.

The regular spherical shape of the individual particles constituting the new covering of the wound reservoir, as well as the selected particle size and particle size distribution (0.05 to 0.5 +o+, preferably 0.1 to 0 .3 ttrm) meets the requirements of easy handling in production and application, ensuring smooth flow of the powder while dispensing it to the wound and preventing cellulose from entering the sap circulation.

In the production of the new coating, regenerated spherical cellulose prepared according to the Czechoslovakia Patent No. 172,640 is used. This advantage is due to the high porosity, which promotes the activation of enzyme binding reservoirs and the mobilization of proteases. The porous hydrophilic nature of the beaded cellulose support is retained even after activation, immobilization and drying. Freeze-drying is a suitable drying method to remove water as appropriate in view of the bound enzymes and results in dry cellulose g-isomers that do not have sufficient absorption capacity.

The beaded cellulose produced by the method described in Czechoslovakia Patent No. 172,640 does not contain any soluble moieties, which can lead to its contamination during the preparation process (decomposition formation of xanthogenate groups). does not contain any impurities that have a toxic effect (e.g. residues of chlorobenzene). For this purpose, 50℃~90℃
Wash with water and/or ethanol at ℃. Effective removal of chlorobenzene occurs by distillation using steam.

In order to activate cellulose for the binding of proteases there are several known methods known from the literature (Handb
Oook of Enzyme Biotechnolo
gy.

Edited by A. Wisemon; E. Hor, woo
Chichester, 1975) can be used to form a sufficiently stable bond between the enzyme and the cellulose and the product is released during application of the coating or to the action in direct contact. Methods must be chosen to avoid contamination with well-functioning barrel compounds. A suitable method is periodate oxidation, which is relatively simple and easy to carry out and yields products with efficient porosity exhibiting high absorption efficiency.

For immobilization, proteases such as trypsin, chymotrypsin and subtilisin are suitable, but
Additionally, thermolysin, papain, and other proteases are suitable. Attachment of the enzyme to cellulose activated by periodate oxidation, ie cellulose containing reactive dialdehyde Me, takes place in two steps. That is, a Schiff base-type bond between the cellulose and the enzyme is performed, and then the unreacted aldehyde groups are reduced by sodium borohydrate to stabilize it.

The heavy-duty step in producing a new type of coating is
The goal is to completely remove protease-containing soluble moieties from immobilized cellulose beads.

It can be used statically or in a column at a weak base (pH 85
~9.5), and weak acidity (1) (4-5)'f: achieved by repeating alternately. For the purpose of
For example, 0, 1 M borate buffer (pH 9) containing IM NaCl, and 0, 1 M borate buffer (pH 9) containing IM NaCl.
This is preferably done by applying 1 M acetate buffer (pH 4,5) or both buffers without NaCl onto the cellulose product after immobilization until the protease activity of the liquid phase reaches zero. . Alternating buffers of low and high pH and varying ionic strength promotes elution of non-covalently bound proteins through alternating inhibition of electrostatic and hydrophobic interactions. In order to obtain a completely stable preparation with immobilized enzyme, the beads are finally washed with, for example, a pH 8 borate buffer, suspended in this buffer and lyophilized. Under such conditions, the resulting product contains only covalently bound enzymes and no malleable moieties that would enter the blood and become antigens during treatment.

A proteolytic coating with immobilized chymotrypsin was tested clinically. It showed protease activity against necrotic tissue, pus and fibrin, and at the same time did not damage any healthy tissue. This coating had high absorption capacity due to the hydrophilic and porous structure of the cellulose matrix. .

This sucked up exudate from the wound, including dislodged tissue necrosis and bacteria. Binding of the enzyme to the carrier prevents its autolysis and loss of its activity during application. Chymotrypsin's prolonged protease activity leads to significant prevention of necrosis from the wound and gradual cleansing of the wound.

Initially, contaminated secretions are actively absorbed between the particles of the powder, thereby avoiding the chance of wound maceration and reducing the nutrient surface for the growth of infectious bacteria.

The first combination of proteolytic progenitors and absorption principles achieves removal of infected necrotic debris, formation of clean granulation, and rapid healing of wounds.

The new covering can be used to treat long-term dissecting ulcers of various causes (varicose veins, Lack,
Incision 15i4 and debridement in diabetic and atherosclerotic gangrene, Kerbunkel, second and third degree follow-on burns, infected open fractures, treatment of stumps, disintegrated lung cancer and decomposed Il: Suitable for all types of ulcerative and necrotic wounds, including ulcers. No harmful side effects were observed in the application of this new coating.

The following example illustrates the method of manufacturing the object of the invention and its use, but is not intended to limit the scope of the invention.

Example 1. Preparation of coatings with bound chymotrypsin (,)
Introduction of aldehyde groups by oxidation using sodium periodate A water-swollen, undried, porosity P = 90% (pores) prepared according to Czechoslovakia patent gH7z, 64o (volume of total content) Beaded cellulose was used as the starting material. Wash cellulose with hot water9
Impurities were completely removed by distillation with steam (4 hours) and distillation with steam (4 hours). Filtered cellulose (100OF) was dispersed in 51 ml of 0.1M passed sodium iodate and stirred for 45 minutes at laboratory temperature. After the oxidation is complete, the oxidized cellulose is immediately washed with about 20 t of distilled water (on a Puchner funnel), transferred to the column, and washed with distilled water until the conductivity of the eluate is that of distilled water (-night). did.

(+,) Binding of chymotrypsin to oxidized cellulose Washed and oxidized cellulose (100o, 9) at 511
1 t of 0, 1 M borate buffer containing chymotrypsin (
The protease activity of this solution was 5.35 j A280/min·d as measured by a denatured hemoglobin solution pH 8). The suspension was stirred at laboratory temperature. The progress of binding was followed by the decrease in protease activity of the binding solution. After 1 hour, the protease activity decreased to O-1j A280 /min·ml and the reaction was stopped by removing the binding solution with suction. The binding rate of chymotrypsin increases with increasing pH;
At the same time, the solubilization of cellulose depends on the oxidation time f;!
Add 1. The method chosen was based on a number of comparative experiments and a compromise between the appearance of bound proteins and the loss of cellulose caused by solubilization.

(,) Reduction of cellulose with bound chymotrypsin to stabilize thick bonds between support and enzyme and remove unreacted aldehyde groups.
0.1M boron fgH% 4il with 1i7 of NaBH4
Solution (pH 9) 1 was suspended therein. After stirring for 20 minutes at laboratory temperature, the reduction was stopped by suctioning off the reducing solution and the same procedure was repeated with a fresh solution of sodium borohydrate. Reduction with low concentration of Na
Two runs were performed by adding BH4 solution to avoid loss of protease activity of chymotrypsin due to concomitant reduction of disulfide bridges.

(d) Washing and lyophilization of the cellulose with bound chymotrypsin After completing the reduction, the cellulose with bound chymotrypsin was washed with 2 t of 1 M borate buffer (PH9) containing 1 M NaC, 2 t of 1 M borate buffer (PH9), 0 containing 1 M NaCl. , 1
Washed with M#=acid buffer (pH 4,5) 2z and the same volume of both buffers without NaCl. The cellulose with bound chymotrypsin was transferred to the column where it was washed repeatedly with all the buffers mentioned above until in all cases the protease activity of the eluate was 0.

The cellulose with bound chymotrypsin is finally pH
8 of 0.25M borate buffer and lyophilized in suspension in this buffer. The resulting preparation contained 5 parts of active chymotrypsin on 19 parts of dry cellulose. Comparative amino acid analysis of the prepared samples and those further washed with 6M guanidine hydrochloride and distilled water revealed that all chymotrypsin was covalently bound to cellulose and therefore entered the blood during application and became an antigen. It has been proven that this is not the case.

Example 2. Preparation of coatings covalently bound to trypsin The j7i determination of trypsin was carried out as in Example 1.

The obtained preparation was placed in lyophilized cellulose of 8.
2 m! ? of active trypsin. Because of its narrower specificity, trypsin cleaves proteins at fewer sites (trypsin cleaves proteins only after the basic amino acids lysine and arginine.

Example 3. Preparation of coatings with covalently bound zuptinisin Immobilization of bacterial proteinases was carried out as in Example 1.

The obtained sample contained 11.3 m9 in the freeze-dried sample of IIi.
It contained active enzymes.

Example 4 A proteolytic coating with chymotrypsin bound according to Example 1 was applied to 7 patients for 1 to 4 weeks, with very good effects obtained in 6 patients. This effect is characterized by reprocessing of purulent secretions, removal of the defect, and release of bar necrosis, which is largely surgically removed by debridement. Defect U: The application of this preparation was to wash with a 2) ml solution of hydrogen peroxide solution and chloramine/living red granulation and epithelial formation from around 4° and the subsequent defect. A size reduction was achieved with this method. used topical application of nonsenol spray to the epithelial defect, most often after treatment had ended.

One case shows a decrease in acid secretion, with a deficiency of 11
5, viable granulation or deep adherent necrosis occurs in the residual part of the defect in the area of chronic ischemia caused by a combination of diabetic granulation/abnormality and obstructive arteriosclerosis of peripheral arteries; Inadequate blood supply of the tissue was the limiting factor for defective oil painting. Four weeks after stopping the application of the preparation, the defect developed in the form of an abscess diabetic syncytitis.

Example of standard application (1) J.T., male, 72 years old, report t+' No. 8357
/83; Diameter 3Qn defect after amputation of the fourth toe of the right foot in a diabetic gangrene tumor with fundal necrosis and purulent secretion. After 2 weeks of application of the preparation, very clean granulation occurs without purulent secretions and with isolated adhesion areas in the mid-edge of the wound.

(2) V.S., male, 60 years old, report number Jj56417
/83; Condition after amputation of right shin due to diabetic gangrene, abscess conjunctivitis, diameter 5c111. A defect with a depth of 2.5 on occurred. This one is 10 cm long and 1 cm wide with necrosis on the lower side of the cut stump and further purulent secretions.
Unsightly granulation was removed over the entire area of the wound of rr1. After 4 weeks of application of the standard, almost complete exhaustion was achieved. However, there was a defect with a diameter of 2Qn and a depth of 1m on the side of the stump with clean granulation and gradual epithelial formation around it.

(3) F, N, male, 66o, report number 3
376/83; loss after amputation of 1st to 3rd toes including the corresponding metatarsal head due to diabetic scarring, size 5-4 cr
n.

Partially dirty granulation and partially adherent necrosis; the depth of the defect is 1crr1° on its side.
After weeks of application, gradual epithelialization of the periphery and diameter 3c
Clean granulation occurred in the entire defect area with reduction to m. Same patient, male.

66o, after removal of half of the fourth toe, including the head of the fourth metatarsal,
A defect with a diameter of 3CjH occurred at the dirty bottom.

After applying the preparation for one week, the defect granulated well without secretion and with a healthy periphery.

(4) F, V, I male, 77o, report number 254/84
; Diabetic scar nodame No. 1. Post-amputation defects of the second and third toes, up to 5 x 4 cm in size and 3 crn in depth, with profuse RQ secretion and extensive adherent necrosis throughout the wound area. Standard 'f: After 4 weeks of application, defective IA
Beautiful granulation was formed. Due to the lack of blood supply in the foot capillaries in diabetic tubular abnormalities and arteriosclerosis obliterans, purulent secretion in the defect ceased, although an unrestricted necrotic process remained in other areas. Purulent discharge in the defect occurred shortly after application of the preparation was stopped.

(5) PhMrlM, male, 56 o, report number 9568/83. Defect after incision of 11 cases of interphalangeal interphalangeal fasciitis of the first toe, reaching intraarticular, diameter 2.5
ctn.

After one week of application of the preparation, with necrosis and purulent secretion, the secretion decreased, the defect became a clean granulation, and a skin formed around the defect.

(6) M.P., female, 78 years old, report number 1994/
83; 1st, including the corresponding metatarsal head due to diabetic scarring
and post-amputation loss of second toe, size 5×3 on,
Dirty lower area, adherent necrosis, and purulent secretion of the distal portion of the ff1J wound. After 4 weeks of application of the preparation, clean granulation occurred throughout the wound area, oxidation+Q secretion was inhibited, and the defect was reduced to 3×2 cm by epithelialization.

(Power M, T,, female, 77 o, report number 17741/
83; 2 defects in the cut end of the shin after amputation for diabetic gangrene and subsequent abscess conjunctivitis of the stump, wound liquefaction in the entire area, defect size 10 × 2-4 Q
n, with purulent secretion, messy granulation, and large necrosis.

After applying the preparation for 4 weeks, the granulation became clear, necrosis and death were loose, secretion was suppressed, and the epithelium developed from around the defect, resulting in an average reduction of the apical diameter to 3 cm, and the lateral It was reduced to a diameter of 1.5 cm.

Patent originator Czeskoslovenska Arik Demie Beto Patent application agent Patent attorney Aomizu Ro Patent attorney Kazuyuki Nishidate Patent attorney Fukumoto Private patent attorney Akira Yamaguchi Patent attorney Masaya Nishiyama Continuation of page 1 o Inventor Lenka jelinkova che oa intermediary karel siebesta che0 inventor moimir sieves chetake

Claims (1)

Claims: 1. Diameter 005-0.5 based on cellulose derivatives with immobilized enzymes of the protease type selected from the group consisting of chymotrypsin, trypsin and subtilisin.
A proteolytic wound dressing consisting of 111111 spherical particles. 2. Diameter 0.05-0 based on cellulose derivative with immobilized enzyme of protease type selected from the group consisting of chymotrypsin, trypsin and subtilisin
．． A method for producing a proteolytic wound dressing consisting of 5 runs of spherical particles, the method comprising washing and soaking regenerated beaded cellulose with a particle size of 0.07 to 0.7w+n that has been swollen in water and not dried. or completely removed by distillation with steam, activated for enzyme binding and modified by immobilization of a protease selected from the group consisting of chymotrypsin, trypsin and subtilisin, pH 8.5. The proteolytic activity of the washing solution was 0 when using a buffer solution of ~9.5 and a buffer solution of pH 4-5.
It is characterized by washing alternately with a buffer solution of pH 7.5 to 85, and drying in this medium until the residual moisture content in the dried product is 0.1 to 15 cm. How to do it.