WO1993020838A1

WO1993020838A1 - Method and compositions for treatment of pyonecrotic processes

Info

Publication number: WO1993020838A1
Application number: PCT/US1993/003542
Authority: WO
Inventors: Rudolf I. Salganik; Alexander M. Gonchar; Alexander V. Troitsky
Original assignee: Rufeld, Inc.
Priority date: 1992-04-20
Filing date: 1993-04-15
Publication date: 1993-10-28
Also published as: AU4104093A; MX9302286A

Abstract

The present invention is directed to compounds useful for hydrolyzing proteins in pyonecrotic matter comprising a protease attached to a hydrophilic gel or to the gel in combination with water-soluble polymer. The compound preferably comprises subtilisins covalently attached to polyethylene glycol (PEG): gel combined with PEG in liquid form. The invention is further directed to methods of preparing a gel by electron beam radiation, and sterilizing the mixture with radiation, preferably by electron beam or gamma-ray radiation. The present invention also relates to methods of hydrolyzing proteins by administering an effective amount of the proteolytic compounds of the present invention.

Description

METHOD AND CO1MPOSITIONS FOR

TREATMENT OF PYONECROTIC PROCESSES

BACKGROUND OF THE INVENTION

This invention relates generally to the field of enzymatic hydrolysis of proteins in necrotic tissues. More specifically, the invention relates to compounds containing proteolytic enzymes attached to hydrophilic gels or to the gels in combination with water-soluble polymers. The invention also relates to methods of preparing and using such compounds to hydrolyze proteins of necrotic tissues and purulent matter from wounds, burns, abscesse and other purulent cavities .

The effective treatment of suppurative processes has been a goal of medical researchers for many years. The terms "suppurative" and "pyonecrotic" are used interchangeably herein t mean the softening and liquefaction of inflamed tissue, resulting in necrotic tissue and the production of pus. Such pyonecrotic matter may result from injuries or diseases such as, for example, purulent pleuritis, peritonitis, mastitis, endometritis, osteomyelitis, sinusitis, abscesses, otitis, dental caries, pulpitis, periodontosis, paradentosis and other purulent and/or pyonecrotic processes . The convention methods of treating pyonecrotic processes usually involve the removal of necrotic tissues and purulent matter from body cavities and surfaces by surgical methods, or in the case of dentistry by drilling teeth, followed by the application of various antibiotics and disinfectants. Surgical debridement of suppurative wounds, however, generally involves the traumatic removal of healthy tissues in addition to the pyonecrotic tissue.

A number of investigators have worked on the development and application of enzymatic preparations for removing necrotic tissues and purulent material from wound surfaces, abscesses and body cavities. For example, French Pat. No. 2556222 discloses a method for removing necrotic tissues from wounds by applying a pulverized preparation containing solid particles of polymers with immobilized proteases. Compositions containing proteases have also been tested for preventing dental caries. For example, Fe. Rep. of Germany Patent Nos. 2265537 and 1944308 provide methods of preventing dental plaques by applying preparations containing a number of enzymes, including proteases.

Native proteases, however, are active in solution only for a relatively short time, -.approximately 40 to 90 minutes, due to the denaturation and autolysis of the enzymatic proteins. Attachment of proteases to solid polymers is known to increase the stability of proteolytic enzymes and to maintain their enzymatic activity for an expended period of time. Various methods for immobilizing enzymes, including proteases, on a number of polymeric carriers have been reported. For example, proteolytic enzymes attached covalently to solid granules of cellulose for treating suppurative wounds, abscesses and phlegmons are described in R. I. Salganik et al. "Immobilized proteolytic enzymes in treatment of pyonecrotic processes", Novosibirsk, pp. 3-8 (1981) . An application of liquid polymers with attached proteases is described in O. A. Peretyagin et aϊ. Oftalmologich. Zh., 1987, 3, pp. 145-148 and Gonchar et al., Veterinary, 1989 No. 4, pp. 52-55. In most cases, bifunctional chemical reagents were used as linking agents in which one chemical group is attached to an amino acid residue of the enzymatic protein while the other reactive group is linked to the polymeric carrier. A number of chemical procedures for the covalent attachment of various enzymes to solid carriers have bee developed. Proteases attached to solid granules or fibers, however, are not suitable for treating injuries and diseases resulting in pyonecrotic tissue in small cavities and canals, for example, those formed in bones affected by osteomyelitis or in carious teeth. The solid carriers are not capable of completely filling cavities or recesses nor do they effectively penetrate certain canals, such as small dental root canals. The proteases attached only to the liquid polymers do not stay for a sufficient time on would surface or in the body cavity being absorbed, sucke in and soaked up by the bandages .

In addition, sterilization of enzymatic preparations for medical and veterinary applications is usually accomplished by filtering enzymatic solutions through bacteria-retentive membranes . Consequently, the attachment of protease to polymeric carriers and sterilization are usually performed as separate steps.

Accordingly, compounds that effectively hydrolyze proteins while leaving functionally active proteins intact are needed. A simple, inexpensive and convenient method of simultaneously attaching proteases to polymeric carriers while providing sterilization is also needed. Ideally, the method can produce compounds of varying viscosity that are useful for a variety of purposes. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention is directed to compounds for selectively hydrolyzing proteins of pyonecrotic matter comprising an active protease attached to hydrophilic gels or to gels in combination with water—soluble polymers. The compound preferably comprises subtilisins covalently attached to hydrophilic gel, or to the gel in combination with liquid polymer. (NOTE: Herein and henceforth, it is implied by this statement that the proteases are attached to both the gel and water soluble polymer.) The invention is further directed to methods of preparing such compounds by simultaneously attaching a protease to hydrophilic gel or to the gel in combination with water-soluble polymer to form a mixture and sterilizing the mixture with radiation, particularly by electron beam or gamma-ray radiation. The present invention also relates to methods of hydrolyzing proteins in pyonecrotic matter by administering an effective amount of the proteolytic compounds of the present invention. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel compounds for hydrolyzing proteins in pyonecrotic matter comprising an active protease attached to hydrophilic gel or to the gel in combinatio with water-soluble polymer. Such compounds are useful for removi pyonecrotic material, which includes necrotic tissues and purule substances such as pus from suppurative wounds, cavities and oth difficult to treat areas. By removing pyonecrotic matter from affected areas, the compounds of the present invention eliminate the medium in which pathogenic microorganisms reproduce, thus preventing the production of toxic products by such microorganisms.

The immobilized proteases of the present invention selectively degrade proteins in pyonecrotic matter, while leavin live cells and functionally active proteins intact . An "active protease" means a protease having such activity. Such proteases recognize and cleave peptide bonds formed between amino and carboxyl groups of a number of amino acids. For the effective hydrolysis of proteins in pyonecrotic matter, a mixture of proteases capable of recognizing and hydrolyzing different pepti bonds is preferred. Those skilled in the art will be able to select suitable proteases having such properties. Suitable proteases include, for example, a subtilisin, trypsin, chymotrypsin, papain, streptokinase and the like.

Although proteases from any origin can be used, including animal and plant proteases, bacterial proteases are particularly useful. Relative to other types of proteases, bacterial proteases are usually less expensive and are generally available in ^•unlimited quantities. The preferred bacterial proteases of the present invention can be produced by Bacillus subtilis and are known as subtilisins. A mixture of neutral and alkaline subtilisins is preferred since a mixture will selectively cleave a number of different peptide bonds and is active over a pH range of about 6.0 to 10.0.

The proteases of the present invention are attached to a hydrophilic gel or to the gel in combination with water-soluble polymer, which act as carrier for the proteolytic enzymes. The gel is prepared more preferably from polyethylene glycol (PEG) , and the water-soluble polymer is more preferably a PEG having a molecular weight of 1500 kDa (PEG-1500) . Polyethylene glycols of lower or higher molecular weight can also be used in place of or in addition to PEG-1500. Other suitable water-soluble polymers that also can be used include, for example, polyvinyl alcohol, dextran, polyglycan and the like.

The compounds of the present invention may be in any non- solid form or combination of forms. Preferably, the compounds are hydrophilic gels or gels in different combinations with water- soluble polymers depending on the intended use of a particular compound. Those skilled in the art will know the useful forms that can be used for an intended use.

For example, a combination where the PEG-gel attached proteases are in small amounts may be desirable for use as a spray. The spray can be used as part of the treatment regimen fo bronchiectasis, purulent bronchitis or bronchopneumonia. This form may be desirable when attempting to penetrate small cavities, canals and other difficult to reach areas.

For other injuries and diseases, a gel may be more suitable. For example, to remove pyonecrotic matter for the treatment of dental caries, pulpitis, periodontitis, paradentosis, small dental carious cavities, tiny root canals, dentinal tubules and gingivitis, a gel can conform to the shape of the cavity, crevice or pocket, thus forming a tight seal that can be easily removed several hours later. The gel can also serve to protect the affected area from adverse conditions and pathogens in the environment.

A combination of gels and liquids may be desired for other injuries and diseases, such as deep surface wounds for example. In such cases, the gel can act on the affected surface area, while the liquid penetrates into the subsurface regions. The optimal ratio of gel to liquid will depend on the intended use and can be determined by those skilled in the art. The same or different proteases can be attached to the gel and liquid forms, again depending on the intended use.

The compounds of the present invention have several desirable properties that make them particularly appropriate for the treatment of various injuries and diseases. For example, the compounds selectively degrade proteins of necrotic tissue, while leaving live cells and functionally active proteins intact. In % addition, the compounds are stable at temperatures up to about 0°C.^" The proteolytic activity of the preferred PEG compounds is stable for at least 100 hours in a temperature range of about 30- 0°C. The proteolytic activity of the preferred PEG compounds is stable for at least 100 hours in a temperature range of about 30- 40°C, particularly at 37°C. Furthermore, the use of the-preferred

PEG and PEG-gel immobilized bacterial proteases in amounts exceeding normal therapeutic doses^"are non-toxic. The compounds are not cytopathogenic, mutagenic or teratogenic. There is also a reduced risk of allergic reaction if the compounds are administered directly into body cavities. Finally, the compounds do not change blood coagulability or provoke alterations in the number of blood cells or serum protein fraction ratios.

To provide additional therapeutic properties, antibodies, therapeutic disinfectants, glucocorticoids, stimulants of tissue regeneration and other therapeutic agents can be added to the compound. To be useful, such agents should not significantly interfere with or impair the proteolytic activity of the compound nor should the compound significantly affect the therapeutic activity of ^uc agents.

The prsrent invention is further directed to methods of preparing the compounds by attaching a protease to hydrophilic gels with or without water-soluble polymers to form non-solid mixtures and simultaneously sterilizing the mixtures with radiation. The preferred forms of radiation are electron beam radiation and gamma-ray radiation. Briefly, a mixture of proteases, preferably

and a gel, preferably PEG-gel, in combination with water-soluble polymer, preferably PEG, are irradiated with electrons emitted by an electron accelerator at a dose that facilitates the attachment to the polymeric carriers an simultaneously sterilizes the resulting compounds. Alternatively gamma-rays emitted by cobalt-60 can be used in place of electron beams to promote the attachment of the components and for sterilization. Sufficient radiation doses can be selected by those skilled in the art to sterilize the mixture while not substantially affecting the proteolytic activity of the enzymes.

Gels of various viscosity can be prepared by varying the amount of polymerization of the water-soluble polymer. A desired amount viscosity or density can be obtained by controlling the amount of excess water evaporated during the process or by any methods known in the art.

Finally, the present invention is further directed to method of debriding pyonecrotic matter by administering or applying an effective amount of the compound to remove such matter. The compounds of the present invention are effective when used as preparations for pharmaceutical, hygienic and cosmetic purposes i several diverse fields such as medicine, dentistry, veterinary medicine, and personal care. In this regard, the compounds of th present invention are particularly useful for the treatment of suppurative wounds caused by injuries or diseases. Diseases that may result in such wounds to the body include, for example, abscesses, pleuritis, peritonitis, endometritis, sinusitis, otitis, mastitis, phlegmons, bursitis, trophic ulcers, tympanitis, nasopharyngitis and salpingitis. The compounds are also useful in dentistry for the treatment of dental caries, pulpitis, periodontitis, paradentosis, an the debridement of the small dental carious cavities, tiny root canals, and dentinal tubules.

The compounds are additionally useful for opthalmological purposes, such as the selective removal of the non-transparent layer of dead cornea tissues and restoration of the cornea transparency, as well as the treatment of suppurative wounds affecting the eye and nasolacrimal canals. The compounds can also be used to enzymatically clean contact lenses of various types.

Several other utilities are contemplated. For example, the compounds, particularly the PEG gel alone or with appropriate additives, can be formulated into preparations for use as toothpaste, body oil for use with ultrasound diagnostic devices, and components of cosmetics, lotions, creams, emulsions, hair dressing, chewing gum, mouthwash and the like. The compounds of the present invention, particularly the PEG compounds, also have utility as agar substitutes to grow microorganisms and certain phytophagous insect larvae to produce biological insecticides. Such agar substitutes can also be used to grow human, animal and plant cells as well as bacteria and plant microbes. For industrial utility, the compounds can be used as lubricants, cleaners and carriers of various other compounds.

The compounds can be administered by any method known in the art-. Such methods include, for example, topical application, aerosol spray, by administering with a syringe, trocar, catheter, bronchoscope or other suitable applicators. The method of administration will depend, at least in part, on the nature of th affected area and the type and amount of necrotic tissue and purulent material to be removed. For example, in treating absces of inner organs, purulent pleuritis, peritonitis, sinusitis, otitis, mastitis, endometritis, in addition to others, the compounds can be administered into the affected areas by syringes trocars, catheters, tampons or turundae. In other cases, the compound can be applied to the surface of suppurative wounds, coated on or impregnated in suitable dressings such as gauze or other materials if required, or sprayed, for example, into the respiratory tract.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I The PEG gel was prepared from the commercial preparation of PEG with a molecular weight of 1,500 kDa (PEG-1500) . 100 g of PEG-1500 was first melted in a container immersed in a water bath at 40°C and thereafter diluted with 2 liters of distilled water. 2% formic acid was added to the PEG solution to give a final concentration of 0.1-0.001 M formic acid. To form a gel, the PEG solution was irradiated by accelerated electrons emitted by a linear electron accelerator (ILU-6, Institute of Nuclear Physics, Novosibirsk, U.S.S.R-) . Any linear electron accelerator known to those skilled in the art can be used as long as sufficient electron energy is emitted. The electron energy was 2 MeV and total dose was 10.0 Mrad. The Pd (power) was 40-500 krad/sec. The irradiation dose for producing the PEG gel depends on the concentration of the PEG in solution. Table 1 below provides a representative number of PEG concentrations and corresponding doses. The irradiation dose can be reduced by adding 0.1 M formic acid.

The thickness of the layer of the solution of PEG was not more than 5 mm. The PEG gel formed under irradiation was homogenized and then boiled to evaporate the-excess water to decrease the initial volume by 30-50%. The amount of excess water evaporated depends on the desired gel viscosity.

Proteases produced by Bacillus subtilis, referred to as subtilisins (Protosubtilisin GlOx, Berdsk Chemical Factory, Berdsk, U.S.S.R.) were used for attachment to the PEG gel. Protosubtilisin GlOx preparations contain neutral and alkaline subtilisins that are active over a wide range of pH (7.7 - 10.0) and are capable of cleaving a multitude of peptide bonds.

About 5 g of Protosubtilisin GlOx were dissolved in 100 ml 10- PEG-1500 in 0.1 M acetate-phosphate buffer (pH 8.2) and stirred for 30 minutes at 2-4°C, followed by centrifugation at 7,000 x g for 15 minutes. Proteolytic activity of the supernatant was not lower than 2,000 PU/ml as measured by conventional metho known to those skilled in the art. The supernatant was filtered through a 0.45 micron bacteria-retentive membrane to free the mixture of subtilisin-producing bacterial cells. Ten (10) ml of the filtered supernatant containing proteolytic enzymes .were mixe with 100 g of the PEG gel and 20 ml of sodium phosphate buffer (p 8.3). The mixture was irradiated, while stirring, with electrons emitted by a linear electron accelerator as indicated above at an electron energy of 2 MeV (Pd of 40-500 krad/sec) for a total dose of 1.0 Mrad to covalently attach the proteases to the dissolved PEG and PEG gel and simultaneously to sterilize the mixture. Alternatively, gamma-rays emitted by cobalt-60 with a total dose of 1.0 Mrad can be used for attaching the proteases to the polymeric substrate. The abbreviation used for the compound containing proteases attached to either PEG or PEG gels prepared by this method is referred to herein as "PGP." \ -

EXAMPLE II

^" The stability of PGP was studied. 100 ml of PGP prepared according to Example I were incubated for 100 hours at 37°C. The stability of PGP was tested every 12 hours. A 10 ml sample of PGP was centrifuged at 7,000 x g for 15 minutes. The resulting supernatant was separated from the gel. The proteolytic activities of the PEG gel and the supernatant were measured. It was found that the proteolytic activities of both parts decreased by about 15~ during the first 12 hours and then remained stabilized for the remaining 98 hours.

EXAMPLE ITT

The cell lines used to evaluate cytotoxicity of PGP were Hela cells derived from human cervix carcinoma (ATCC No. CCL 2) and primary fibroblasts from human foreskin. The cells were grown to confluence in 60 x 15 mm dishes. The cell onolayers were washed free of growth medium (199 Medium, Flow Laboratories) supplemented with fetal calf serum and 5 ml of the serum—free growth medium containing PGP at concentrations of 200, 100, 50 and 25 PU/ml were added. The cultures were incubated for 24 hours at 37°C. The cells were then examined microscopically for cytotoxic effects (CTE) . Controls were done in the same manner except that they were not incubated with PGP. -5

TABLE 2

Cell Type PGP concentration. PUtml medium

o = cells exhibiting normal morphology ± = less than 10% of cells showed CTE

The data presented in Table 2 demonstrate that PGP in a dos of up to 200 PU/ml has relatively little cytotoxic effect, while doses of lesser amounts have no observable cytotoxic effect.

EXAMPLE IV To study the in vivo effect of PGP on mucosa tissues, 5 ml o the PGP preparation were injected under general anesthesia into the peritoneal cavity of 10 Wistar male rats (280-300 g body weight) . In 24 hours, the peritoneal mucosa studied by macroscopic observation appeared as clean, smooth and glittering as the controls, with no adhesions observed. Similar results were observed in microscopic studies. In these studies, no toxic effects on the mucosa cells or on their proliferation activity were observed after administration of PGP .

EXAMPLE V The effectiveness of PGP for treating pyonecrotic wounds in rats was also studied. Wounds of 3.0-3.5 cm² in size in the sacrolumbar area were made under general anesthesia in Wistar mal \ - rats (180-200 g body weight) . The wounds were infected by common vivarium microflora such as proteus vulgaris and staphylococcus aureus and in three days clear-cut manifestations of suppurative processes were observed. 20 rats of the experimental group were treated by PGP. During the first 7 days after the appearance of suppurative conditions,, dressings with PGP were changed every day (1 ml or 50 Pϋ of PGP was applied per 10 cm2 of wound surface) in 20 experimental rats. After the first 7 days, the dressings were changed every two days according to the same procedure. Rats in the control groups (18-20 animals in each group) were treated in the same manner with nitrofurazone solution (1:5000) diluted in distilled water, 0.5% solution of trypsin in saline or no treatment at all. Body weight changes, motor activity, the wound debridement and healing time periods and the content of RNA and hydroxyproline amounts were measured.

When the wounds were treated with PGP, debridement (of necrotic tissues and pus) occurred in an average of 5 days, with trypsin treatment it took place in 10 days, with nitrofurazone treatment - in 15 days, and without treatment, in 19 days. Healing periods of the various treatments corresponded with the debridement period. Increase of RNA and hydroxyproline amounts in wound tissues was the highest under PGP treatment. The increase of UNA indicates an increased amount of healing of connective tissues and intensity of protein synthesis associated with such healing. EXAMPLE VI

^" The effectiveness of PGP for treating purulent peritonitis was studied. Purulent peritonitis was induced in male Wistar rat (280-330 g body weight) by implanting an amputated vermiform process, also known as an appendix, into the peritoneal cavity o each rat. Purulent peritonitis was observed in all rats in three days. In 14 days, all non-treated animals were dead as a result purulent peritonitis. In the peritoneal cavities of these control rats, 3-5 ml of turbulent exudate, fibrinous depositions on the peritoneal mucosa were observed as well as gas build-up i the intestinal loops. The amputated vermiform process was remove from the experimental animals three days after the implantation. In the experimental group receiving only carbenicillin treatment, which included evacuation of the exudate from the peritoneal cavity and injection with carbenicillin (100 mg in 1 ml of distilled water) , 30% of the animals died within a few days and 14 days 85% of the animals were dead. When 5 ml of the PGP preparation were administered for two hours through a catheter into the peritoneal cavity two times with an interval of 10 hours between each administration in addition to the carbenicillin, onl 25% of the animals were dead within 14 days. The first PGP treatment was given 5 days after the removal of the appendix when manifestations of peritonitis were clearly observed. Table 3 summarizes the results of this study. Groups of rats with purulent peritonitis

Non-treated

Treated by carbenicillin

Treated by PGP and

carbenicillin

EXAMPLE VII

The effectiveness of PGP for hydrolyzing proteins of necroti tissue was studied in patients having various pyonecrotic diseases. In some patients, the PGP compound was applied topicall and covered the surface of purulent wounds, such as trophic ulcers, phlegmons, panaritiums and covered with dressings. In other patients with paraproctitis, mastitis, or endometritis, the PGP was administered through catheters, trocars, or syringes into the purulent cavities of abscesses. Data demonstrating the i efficiency of PGP for hydrolysis of proteins are presented in Table 4. A 0.5% solution of non-immobilized trypsin diluted in physiological saline solution was used as a control.

The debridement of purulent wounds and cavities with the PGP preparation substantially accelerated their healing when compared to the control.

EXAMPLE VII The action of the PGP was studied j-n vitro on 24 extracted human teeth affected by caries. The dental carious cavities of 12 teeth were washed with physiological saline solution followed by the administration of 0.1 ml of PGP into each cavity. The cavitie were sealed with temporary fillings and the teeth were incubated at 37°C for 24 hours. The fillings were then removed and the cavities were washed by water jet. To study the effectiveness of the treatment, inner walls and bottoms of the carious cavities were stained for 1 minute with 0.1-0.2 ml of 1% aqueous methylene blue solution. This dye is known to intensively tincture necrotic tissues and weakly tincture healthy tissues. The microsections of the dyed teeth were prepared for detailed analysis. PGP completel to removed the intensely tinctured dentine from the carious cavities leaving intact the weakly tinctured healthy dental tissues.

EXAMPLE IX

The effectiveness of PGP for treatment of caries in humans was studied in clinical studies involving 245 male and female outpatients, with ages ranging from 20 to 45 years. The diagnosis was established on the basis of anamnesis, clinical, instrumental and electroodontological data. The patients had medium or deep dental caries and in all cases the central part of the tooth was affected.

About 0.1 ml PGP was introduced into each dental carious cavity and sealed with a temporary filling. The temporary filling was prepared by mixing water dentine (25% zinc sulfate, 10% kaolin, 65% zinc oxide) with the PGP in a ratio of 1:2. After 24 hours, the temporary filling was removed and any remains of disintegrated necrotic tissue were removed from the carious cavity mechanically or by water jet.

The clinical effect of PGP was regarded as positive if the walls and bottom of the carious cavity were bright yellow with solid boundaries when probed after the removal of any disintegrated filling. The absence of significant staining of solid dental tissues with 1% aqueous solution of methylene blue indicated that the PGP treatment was effective in cleaning the cavities. -2- \

A single introduction of the PGP into the dental carious cavity produced positive results in 86% of the patients. In 14S the cases, the favorable effect was obtained after the second application of the PGP. From this number of patients, in spite o a substantial improvement, 5 patients (2%) were given an additional treatment.

EXAMPLE X The compatibility of PGP with antibiotics was tested. Non- diluted PGP prepared according to Example I and PGP diluted in th nutrient medium (Difco) in concentrations of 1:10, 1:100 and 1:1000 were mixed with 0.1 ml of 10 S reptococcus aureus or Proteus mirabilia cultures in each tube. The tubes were incubated for 24 hours at 37°C. The microorganisms were then transferred to a nutrient agar and the number of colonies counted. Similar experimental studies with various antibiotics were conducted. One cf the following antibiotics was added to the nutrient medium in separate studies: gentamicin (10 mg/ml), kanamycin (30 mg/ml) , benzyl penicillin (10 UA/ml) . The studies demonstrate that PGP does not possess antibacterial activity, PGP does not interfere with the antibacterial activity of the antibiotics tested, and th antibiotics tested do not inhibit the proteolytic activity of PGP

EXAMPLE XI For application of PEG-immobilized proteases in an aerosol form to spray, for example, the respiratory tract during the ^•2.1. treatment of bronchiectasis, purulent bronchitis, or bronchopneumonia, the PEG gel was omitted. To prepare the liquid form of PEG-immobilized proteases (PIP), 5 g of Protosubtilisin GlOx was dissolved in 100 ml of 10% PEG-1500 in 0.1 M acetate- phosphate buffer (pH 8.2), stirred for 30 minutes at 2-4° C and centrifuged at 7,000 x g for 15 minutes. Proteolytic activity of the supernatant was not less than 1000 PU/ml. The mixture was irradiated by electron beam (total^' dose 1.0 Mrad) according to the procedure of Example I for attaching the proteases to the dissolved PEG-1500 and for sterilization. The final activity of the PIP was 250-500 PU/ml. The proteolytic activity of the concentrated PIP can be preserved for two years at 2-4°C. To apply it as a spray, PIP was diluted ten-fold with saline solution to give a final specific activity of 25-50 PU/ml. The diluted PIP was prepared just prior to its use since in this concentration the proteolytic activity is less stable.

EXAMPLE XII

The PEG gel prepared according to Example I was used instead of agar to grow bacteria cells. For use as an agar substitute, the components of the nutrient medium (peptone, protein hydrolysates, saccharose) were added to the PEG-1500 and thoroughly mixed. The Petri dishes (d = 10 cm) were filled with the mixture to provide a layer thickness of not more than 5 mm and sealed in polyethylene envelopes. The mixture was then irradiated by accelerated electrons emitted by a linear electron accelerator as described in Example I for PEG polymerization and sterilization. The electron energy was 2 MeV for a total dose of 3 Mrad. The growth efficienc of Staphylococcus aureus, Proteus vulgaris and Escherichia coli o PEG-gel was comparable to those on the agar medium.

EXAMPLE XIII

The PEG gel prepared as described in Example I was used as a agar substitute to grow plants. The Bs Gamborg medium which is known in the art was added to the PEG-gel. Apexes of 7-day alfalf seedlings were placed on the medium (pH 5.6-5.8). In 20 days the growth of plants on PEG gel-Bs Gamborg medium (approximately 1.5 cm) was comparable to those on the agar-Bs Gamborg medium by comparing the number of leaves on each plant.

EXAMPLE XIV The PEG gel was used as a lubricant on the skin of a patient for diagnostics and therapeutic ultrasound and electrocardiography. The PEG gel prepared as described in Example I, with 5% glycerol, PEG alone or with polyvinyl alcohol or other hydrophilic polymer, and a dye were added. The studies of the PEG gel in comparison with the industrially produced Sonogel, Echo-ge N 100 and SG Scanning gel have demonstrated that the PEG gel is comparable to these commercially available lubricants. The PEG ge of the present invention was found to have good lubricating properties and optimal viscosity. It did not irritate the -2.-Ϊ patient's skin and was not allergenic. In addition, it was found to be safe for the sensing elements of the equipment.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

2.SQ &LMS.WHAT IS CLAIMED IS:

1. A compound for selectively hydrolyzing proteins in pyonecrotic matter, comprising an active protease attached to a gel or a gel mixed with a water soluble-polymer.

2. The compound of claim 1, wherein the attachment of the protease to a gel or to a gel mixed with water-soluble polymer is achieved by radiation.

3. The compound of claim 2, wherein said radiation is electron beam radiation or gamma-ray radiation.

4. The compound of claim 1, wherein said gel is made by irradiation of PEG by an electron beam from an electron accelerator.

5. The compound of claim 1, wherein said protease is stabl up to a temperature of 70°C.

6. The compound of claim 1, wherein said protease is stabl for at least 100 hours at a temperature range of about 30°C to about 40°C.

7. The compound of claim 1, wherein said protease is a subtiiisin, trypsin, chymotrypsin, papain or streptokinase.

8. The compound of claim 7, wherein said protease is a subtiiisin.

9. The compound of claim 1, wherein said gel is polyethylene glycol, dextran or poϊygl can gel.

10. The compound of claim 9, wherein said gel is polyethylene glycol gel.

11. The compound of claim 1, wherein said water-soluble polymer is polyethylene glycol, polyvinyl alcohol, dextran or polyglycan.

12. The compound of claim 11, wherein said water-soluble polymer is polyethylene glycol.

13. The compound of claim 1, wherein said compound further comprises an effective amount of a therapeutic agents.

14. A method of preparing the compound of claim 1, comprising simultaneously attaching a protease to a gel, or gel mixed with water-soluble polymer to form a non-solid mixture and sterilizing the mixture with radiation.

15. The method of claim 14, wherein the radiation is electron beam radiation of gamma-ray radiation.

16. The method of claim 14, wherein said protease is a subtiiisin and said gel is polyethylene glycol gel while said water-soluble polymer is polyethylene glycol.

17. A method for hydrolyzing proteins, comprising administering an effective amount of a compound comprising a protease attached to a gel, or gel mixed with a water-soluble polymer to hydrolyze said proteins.

18. The method of claim 17, wherein the compound is administered by topical application, aerosol, syringe, trocar, catheter, bronchoscopy or turundae.

19. The method claim 17, wherein said protease is a subtiltisin, trypsin, chymotrypsin, papain or streptokiriase.

20. The method of claim 19, wherein said protease is a subtiiisin.

21. The method of claim 17, wherein said gel is polyethylene glycol, dextran or polyglycan gels.

22. The method of claim 17, wherein said gel is a polyethylene glycol gel.

23. The method of claim 17, wherein said water soluble polymer is polyethylene glycol, polyvinyl alcohol, dextran or polyglycan.

24. The method of claim 23, wherein said polymer is polyethylene glycol.