(19)
(11)EP 1 459 738 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
09.02.2011 Bulletin 2011/06

(21)Application number: 02806416.0

(22)Date of filing:  24.12.2002
(51)Int. Cl.: 
A61K 9/19  (2006.01)
A61K 41/00  (2006.01)
A61P 7/02  (2006.01)
A61K 38/48  (2006.01)
C12N 11/08  (2006.01)
A61P 9/10  (2006.01)
(86)International application number:
PCT/RU2002/000552
(87)International publication number:
WO 2003/059326 (24.07.2003 Gazette  2003/30)

(54)

PHARMACEUTICAL COMPOSITION HAVING THROMBOLYTIC, ANTI-INFLAMMATORY AND CYTOPROTECTIVE PROPERTIES

PHARMAZEUTISCHE ZUSAMMENSETZUNG MIT THROMBOLYTISCHEN, ENTZÜNDUNGSHEMMENDEN UND ZYTOPROTEKTIVEN EIGENSCHAFTEN

COMPOSITION PHARMACEUTIQUE POSSEDANT DES PROPRIETES THROMBOLYTIQUES, ANTI-INFLAMMATOIRES ET CYTOPROTECTRICES


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SI SK TR

(30)Priority: 26.12.2001 RU 2001135876

(43)Date of publication of application:
22.09.2004 Bulletin 2004/39

(73)Proprietor: Scientific Future Management SFM
630056 Novosibirsk (RU)

(72)Inventors:
  • ARTAMONOV, Andrei Vladimirovich
    Novosibirsk, 630070 (RU)
  • VERESCHAGIN, Evgeny Ivanovich
    Novosibirsk, 630083 (RU)
  • GRISHIN, Oleg Vitalievich
    Novosibirsk, 630005 (RU)
  • TROITSKY, Alexandr Vasilievich
    Novosibirsk, 630074 (RU)

(74)Representative: Meissner, Peter E. et al
Meissner & Meissner Patentanwaltsbüro Hohenzollerndamm 89
14199 Berlin
14199 Berlin (DE)


(56)References cited: : 
WO-A-93/20838
RU-C1- 2 003 346
RU-C1- 2 146 139
RU-C2- 2 158 603
GB-A- 2 147 206
RU-C1- 2 137 835
RU-C1- 2 150 936
US-A- 5 133 968
  
  • VERESCHAGIN E I ET AL: "Radiation technology in the preparation of polyethylene oxide hydrophilic gels and immobilization of proteases for use in medical practice" ARCHIVES OF PHARMACAL RESEARCH (SEOUL), vol. 24, no. 3, June 2001 (2001-06), pages 229-233, XP009073483 ISSN: 0253-6269
  • GONCHAR A M ET AL: "Immobilization of bacterial proteases on water-solved polymer by means of electron beam" RADIATION PHYSICS AND CHEMISTRY, ELSEVIER SCIENCE PUBLISHERS BV., AMSTERDAM, NL, vol. 48, no. 6, December 1996 (1996-12), pages 795-797, XP004070757 ISSN: 0969-806X
  • PALUBINSKAS V I ET AL: "Trypsin-like enzyme from Streptomyces 771. Purification and properties of native and immobilized enzyme." APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY. JUN 1984, vol. 9, no. 3, June 1984 (1984-06), pages 231-241, XP009073571 ISSN: 0273-2289
  • "Thermostability of soluble and immobilized subtilisins after their modification by dextrans and dextrins" ENZYME ENGINEERING, PLENUM PRESS, NEW YORK. NY, US, no. 5, 1980, pages 423-426, XP002078116 ISSN: 0094-8500
 
Remarks:
The file contains technical information submitted after the application was filed and not included in this specification
 
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Field of the Art



[0001] The present invention relates to medicine, particularly to pharmacology and drugs based on enzyme preparations, and can be used in complex therapy for treating ischemic heart disease, ischemic cerebral strokes, rheumatoid processes and other diseases accompanied by ischemic, thrombotic, and non specific inflammation phenomena.

[0002] This invention encompasses the field of enzymatic hydrolysis of thrombogenic proteins, mainly fibrin, structureforming protein of all thrombi, irrespective of the location and size of peptides. More particularly, the invention relates to compounds containing proteolytic enzymes possessing fibrinolytic properties, immobilized on (attached to) hydrophilic water-soluble polymers. The invention is also associated with methods of using such compounds for fibrinolytic therapy in treating acute myoc-rdial infarctions, ischemic cerebral strokes, and also with therapeutic methods directed to the correction (improvement) of hemorheological characteristics in treating ischemic injuries of organs and tissues of different etiology, including mi-crothrombosis.

State of the Art



[0003] Known in the art are various medicinalpreparations used for treating ischemic heart disease andischemic cerebral strokes. As a rule, at the final stage of these diseases the main pathogenetic factor is intravascular thrombosis. In present-day medical practice thrombolytic medicinal preparations have become widely used as etiotropic therapy means for treating acute myocardial infarctions and ischemic cerebral strokes. There are known pharmaceutical preparations, such as streptase, streptokinase, tissue plasminogen activator (alteplase) and fibrinolysine (Methodological Recommendations for Carrying out Early Therapeutic Measures in Patients with Acute Myocardial Infarction, Communication of the American Cardiologic College and American Heart Association. V.N. Ganyukov (Ed.), Collection of Papers, Novosibirsk, 1998, p. 100 (Russian translation)). All these preparations, directly or as a result of activation of the anticoagulative system of blood, act on fibrin, leading to its destruction and, correspondingly, to lysis of the intra vascular thrombus. In spite of their high therapeutic effectiveness, these preparations have a pronounced side effect, namely, they can induce uncontrollable and dangerous hemorrhages, due to depletion of the coagulative system of blood (Saunders W.B. Indications for Fibrinolytic Therapy Trialists Collaborative Group.//Lancet Ltd., 1994, vol. 343, pp. 311-322). Besides; for such preparations as streptokinase and alteplase it is difficult to select an adequate therapeutic dosage, because of the existence of an individual antiserum activity to streptococcus in the human organism, that leads to the inactivation of these preparations.

[0004] Medicinal preparations are known, which are capable of diminishing the inflammatory reaction. The most widespread of these preparations are non-steroidal anti-inflammatory preparations, such as aspirin, indomethacin, sodium diclofenac, and others (Nasonov E.L., Tsvetkova V.S., and Tov N.L., Selective inhibitors of cyclooxygenase-2: new plospects of treating human diseases //Ter. Arkhiv, 1998, No. 5, pp. 8-14 (in Russian)). The known pharmaceutical preparations *are not free from essential disadvantages: they may cause stomach injuries with development of non-steroidal gastropathy, hemorrhages and ulcerations of the gastrointestinal tract mucosa.

[0005] The WO 93/20838 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.

[0006] The publication Archives of Pharmacal Research Vol. 24, No. 3, 229-233, 2001 deals with the development of a technology foor making a hydrophilic gel of polyethylene oxide reception in which radiating ability is employed to cause cross-linking of polymers in a water solution.

[0007] In Radiation Physics and Chemistry, Vol. 48, No. 6, pp. 795-797, 1996 the possibility of electron beam usage for proteases' immobilization on 1,4-polyalkylene oxide to obtain biologically active complex for multipurpose usage is disclosed.

[0008] The publication in Applied Biochemistry and Biotechnology, Vol. 9, No. 3, pp. 231-241, 1984 discloses the usage of ionizing radiation technology to attached enzymes to polymeric compound by creating reactive groups on the latter.

[0009] In Enzym Engineering, Plenum Press, New York, No. 5, pp. 423-426, 1980, the thermostability of soluble and immobilized subtilisins after their modification by dextrans and dextrins is disclosed.

[0010] Pharmaceutical preparations are known, which have cytoprotective properties, such as preductal (cardioprotective effect) and blockers of H2 receptors (gastrozepine, ranitidine). The known preparations have a weak cytoprotective effect and prove to display pharmacological action on prolonged administration (Brottier L., Barat J.L., Combie C. et al., Therapeutic value of a cardioprotective agent in patients with severe ischaemic cardiomyopathy //Eur. Heart J., 1990, vol. 11, pp. 207-212).

[0011] At present no data can be found in scientific and medical literature concerning the provision of pharmaceutical compositions producing multipurpose synergic effect on the main pathogenetic links of ischemia, inflammation and thrombosis in combination with cytoprotective properties.

[0012] Different methods for the immobilization of enzymes, including proteases, on a number of polymeric carriers were reported. For instance, in the work by R.I. Salganik et al. "Immobilized Proteolytic Enzymes in Treating Purulent Processes", Novosibirsk, pp. 3-8, 1981 (in Russian) a method is described for treating purulent wounds, abscesses and phlegmons with the help of proteolytic enzymes attached covalently to solid granules of cellulose. The use of liquid polymers with attached proteases is described by O.A. Peretyagin et al. in "Oftalmologiya", 1987, No. 3, pp. 145-148 and by Gonchar et al. in "Veterinariya", 1989, No. 4, pp. 52-55. In most cases a bifunctional chemical reagent was used as the binding agent, in which one chemical group was linked to the amino acid residue of the enzyme protein, while another reactive group was linked to the polymeric carrier. A number of chemical procedures for the covalent attachment of various proteins to solid carriers were investigated. However, proteases attached to solid granules or fibers cannot be used for treating injuries or diseases which lead to intravascular thrombosis and are accompanied by negative changes of hemorheology in the microcirculatory channel of tissues and organs.

[0013] Therefore, compounds are required, which effectively hydrolyze proteins, predominantly those responsible for thrombosis in blood vessels of different diameters, which constitute an etiological factor of the development of such diseases as acute myocardial infarction and ischemic cerebral stroke. At present such compounds containing nontcxic thrombolytics and substances with hemorheological corrective functions have not been synthesized for complex therapy of ischemic heart disease, hypertensive disease and rheumatoid diseases. Simple, inexpensive and convenient methods are also required for simultaneously attaching proteases to a complex of polymeric carriers and providing sterilization of the end product. Ideal methods are those which can provide compounds having different viscosity and aggregate state, suitable for different purposes and different ways of administration.

Essence of the Invention



[0014] The herein-presented investigation meets the above-said requirements and also offers relative advantages over other medicinal preparations and methods of preparing pharmaceutical compositions featuring a complex synergic therapeutic effect on various links of the pathogenesis of ischemic heart disease. The presented investigation, the claimed pharmaceutical composition, and methods of preparing thereof ensure the obtaining of high therapeutic effect in treating non-specific inflammatory processes owing to cytoprotective, thrombolytic and anti-ischemic effects.

[0015] The proposed invention is directed to the provision of compositions for selective hydrolysis of thrombogenic proteins, consisting of active proteases attached to a combination of hydrophilic water-soluble polymers. Said compositions predominantly consist of subtilysines immobilized on hydrophilic polymers.

[0016] This invention contributes to the development of methods of making such compositions by attaching proteases to water-soluble polymers to produce a mixture and simultaneously sterilize said mixture by irradiation, particularly with the help of a stream of accelerated electrons or gamma-radiation, as well as other kinds of ionizing radiation, including laser radiation sources.

[0017] The proposed invention is directed to novel pharmaceutical compositions as defined in the claims for the hydrolysis of thrombogenic proteins, consisting of active proteases attached to hydrophilic polymers. Such compositions are useful for the removal of thrombi comprising fibrin and other formations. The proposed invention is also directed to novel compounds for correcting pathological changes in tissues and organs, originated as a result of thrombosis and ischemia, as well as to the correction of non-specific inflammatory reactions, and as an auxiliary means for treating and preventing gastropathies developing as a result of administering non-steroidal anti-inflammatory preparations. Proteases immobilized on hydrophilic polymers according to the invention selectively degrade thrombogenic proteins, mainly fibrin, while live cells and functionally active proteins remain intact and undamaged. For effective hydrolysis of the proteins of thrombotic masses, it is preferable to have a polyfunctional mixture of proteases which are capable of recognizing and hydrolyzing various peptide linkages. Natural proteases include, for instance, subtilysine, trypsin, chemotrypsin, papain and other enzymes of animal and bacterial origin, having fibrinolytic and proteolytic activity.

[0018] Though proteases are obtainable from any sources, including animal and vegetable ones, bacterial proteases are especially preferable. Compared with other types of proteases, bacterial proteases are usually less costly and are available in unlimited amounts. Preferable bacterial proteases of the proposed invention can be reproduced by Bac subtilis and are known as subtilysines. A mixture of neutral and/or alkaline subtilysines is preferable, because the mixture can selectively degrade a variety of peptide linkages and preserve activity at pH from 6.0 to 10.0.

[0019] The proteases according to the proposed invention are attached to a combination of water-soluble polymers which act as carriers for proteolytic enzymes. The water-soluble polymers are polyethylene oxide (PEO), synonym: polyethylene glycol (PEG), having a molecular weight of 1500 kDa (PEO-1500), and dextran having a molecular weight of 30-40 kDa. Polyethylene oxides and dextrans having a smaller or greater molecular weight can also be used instead of or together with PEO-1500 and dextran having a molecular weight of 30-40 kDa. Other suitable water-soluble polymers and their combinations that also can be used include, e.g., polyvinyl alcohol, polyvinylpyrrolidone, and the like.

[0020] The compositions according to the proposed invention can be in any non-solid or solid form or in a combination of aggregate forms. It is preferable to use lyophilized powders (substances) in various combinations with water-soluble polymers as the compositions, depending on the field in which the compound is planned to be used.

[0021] For the provision of the overall therapeutic properties, antibodies, therapeutic disinfectants, glucocorticoids, tissue regeneration stimulants, and other therapeutic agents can be added to the given composition. To the benefit of the case in hand, such agents should not be mixed in a considerable amount with proteases, unless their effect on the specific and therapeutic activity of the claimed pharmaceutical composition has been circumstantiated (investigated).

[0022] The present invention is also directed to methods of preparing compositions as defined in the claims by attaching proteases to water-soluble polymers and simultaneously sterilizing the mixture by radiation. Preferable kinds of radiation are accelerated electrons or gamma-radiation.

[0023] Briefly, the technology is as follows: a solution of a mixture of proteases, preferably subtilysines, and water-soluble polymers, namely polyethylene oxide and dextran, is subjected to the action of electrons emitted by an electron accelerator in a dose which promotes attaching thereof to a polymeric carrier and simultaneously sterilizing the obtained compound. On the other hand, gamma-rays emitted by Co60 can be used instead of an electron bunch with the provision of attaching and sterilizing the components. A sufficient radiation dose can be selected by a person skilled in the art by calculating the conditions for the provision of the mixture sterility and inessential effect on the proteolytic activity of the enzymes.

[0024] Finally, the proposed invention is also directed to methods of cleaning the vascular bed from thrombi by controlling or using effective amounts of the claimed composition for the removal of this material. The compositions according to the present invention are effective when used as preparations for pharmaceutical purposes in different ways and fields, such as medicine, stomatology, veterinary medicine, and personal care.

[0025] In this connection, the compositions according to the present invention are particularly eligible for treating ischemic heart disease and its complications, as well as ischemic cerebral strokes, rheumatoid diseases and other pathologies, in whose pathogenesis there take place an inflammatory reaction, ischemia of tissues, disturbances of hemorheology and vascular microcirculation because of thrombosis. The claimed pharmaceutical composition is also suitable for enzymatic destruction of viscous biological fluids, such as secreted mucosae (e.g., bronchial secret), therefore it is effective in treating pulmonary diseases for the prevention and treatment of bronchial obstruction. The claimed pharmaceutical composition is also applicable for the enzymatic destruction of proteins and peptides of necrotic tissues which are always present in traumatic and infectious-inflammatory processes. Therefore this composition is also effective in treating pyoinflammatory diseases of different etiology and localization.

[0026] These compositions can be applied to the focus of injury by any method known in the medicine. Such methods include, for instance, parenteral administration, intracavitary and external use, aerosol spraying, syringing through a trocar, catheters, bronchoscope, or other suitable use. The method of administration will depend, at least partially, on the nature of the injured area and on the type and quantity of thrombogenic or necrotic tissues to be removed. The predominant method of administration is parenteral administration of the preparation (compositions).

Brief Description of the Drawings



[0027] Fig. 1 demonstrates the thrombolytic properties of the claimed composition, investigated on a model of thrombolysis in vitro.

[0028] The claimed composition in the standard therapeutic dose of 50 physiological units/ml (50 PhU/ml) reliably excels fibrinolysine (p < 0.02); trypsin (p < 0.01); and spontaneous thrombolysis in physiological solution (p < 0.01).

[0029] Fig. 2 demonstrates that the thrombolytic properties of the claimed composition are preserved as the thrombus "age" increases to 7 days. Fibrolysine does not act on the 7-days thrombus. During the first two hours the action of fibrolysine does not differ with certainty from the spontaneous background lysis in physiological solution. By the end of the 4th hour the claimed composition dissolves the thrombus completely, while fibrinolysine lyses only 20% of the mass of the "old" thrombus.

[0030] Fig. 3 demonstrates the anti-inflammatory properties of the claimed composition, investigated on a model simulating induction of an inflammation mediator - tumor necrosis factor (αTNF) in CBA-line mice with endotoxic shock. The activity of the αTNF was measured using L929 line cells and was expressed in activity units (AU):
  1. 1 - αTNF activity in intact animals;
  2. 2 - αTNF activity in animals after administration of endotoxin;
  3. 3 - αTNF activity in animals to which the claimed composition was administered one hour before the administration of endotoxin. As is seen, the claimed composition reduces 50-fold the αTNF activity.


[0031] Fig. 4 demonstrates the cytoprotective properties of the claimed composition, studied on a model of adrenaline myocarditis in rats. Data of histological morphometric investigations of the volume of necroses and dystrophic changes in the myocardium of the rats are presented. The volume of injuries was determined planimetrically and expressed in percent of the volume density which is equal to the volume of injuries/total value of tissue ratio multiplied by 100%.

[0032] K necr. - control animals to which after administering adrenaline 1 ml of isotonic NaCl solution was administered intraperitoneally twice a day.

[0033] TRB necr. - experimental animals to which after administering adrenaline 1 ml of the claimed composition was administered intraperitoneally twice a day.

[0034] The first two columns demonstrate positive effect of the claimed composition (approximately 1.5-fold reduction of the number of necroses in the cardiac muscle) by the end of the third day of treating the animals.

[0035] The next two columns demonstrate positive effect of the claimed composition (approximately 2.5-fold reduction of the number of dystrophic changes in the cardiac muscle) by the end of the seventh day of treating the animals.

[0036] The Examples which follow are intended to illustrate the formulation, the method of preparing and the pharmacological properties of the claimed composition, but not to limit the proposed invention.

Preferred Embodiments of the Invention


Example 1



[0037] The proposed pharmaceutical composition is prepared in the following manner:

a reaction mixture is prepared by dissolving protosubtilin, predominantly protosubtilin G3Kh and polyethylene oxide (PEO) having a molecular weight of 400-20000 Da (predominantly 1500 Da) in a solution of dextran having a molecular weight of 40-70 kDa (predominantly 40 kDa) in a 0.025 M sodium phosphate buffer solution with pH 7.5-8.2. The obtained mixture is purified by removing ballast proteins by salt precipitation and subsequent filtration. The resulting solution is subjected to irradiation with gamma-rays or a stream of accelerated electrons (with energy of 2.0 MeV) in a dose of 0.5-1.5 Mrad. The solution after irradiation is subjected to sterilizing filtration and packaged in 10 ml batches into 15 ml vials. Then the solution is lyophilized to a residual moisture content not exceeding 2%.



[0038] As a result, a composition is obtained, containing a protease complex from Bac. subtilis, immobilized on polyethylene oxide and dextran. The proteolytic activity of the obtained composition in one vial is from 500 to 1000.0 proteolytic units per gram (PU/g). The composition comprises a slightly yellowish porous homogeneous mass.

[0039] For therapeutic purposes a solution of the claimed composition is used (conditional appellation thereof being "Trombovazim"), which is prepared ex tempore by dissolving the contents of a vial in 10 ml of sterile isotonic solution of sodium chloride or in 10 ml of water for injections.

Example 2



[0040] The claimed composition can be prepared by another method, namely, by preparing separately an active component (component (1)) which contains a complex of proteases immobilized on polyethylene oxide and dextran, and a solvent which is a solution of polyethylene oxide (component (2)). The two-component formulation of the composition makes it possible to vary the activity of the claimed pharmaceutical composition by varying the active component/solvent ratio, so that individual schemes of treating can be selected. The two-component composition is prepared in the following manner:

Component (1) (active substance)



[0041] The reaction mixture is prepared by dissolving protosubtilin (predominantly protosubtilin G3Kh) and polyethylene oxide (PEO) having a molecular weight of 400-20000 Da (predominantly 1500 Da) in a solution of dextran having a molecular weight of 70-40 kDa (predominantly 40 kDa) in a 0.025 M sodium phosphate buffer solution with pH 7.5-8.2. The obtained mixture is purified by removing ballast proteins by salt precipitation and subsequent filtration. The resulting solution is subjected to irradiation with gamma-rays or a stream of accelerated electrons (with energy of 2.0 MeV) in a dose of 1.0 Mrad. The solution after irradiation is subjected to sterilizing filtration and packaged in 10 ml batches into 15 ml vials. Then the solution is lyophilized to a residual moisture content not exceeding 2%.

[0042] As a result, component (1) of the composition is obtained, which component contains a protease complex from Bac. subtilis, immobilized on polyethylene and dextran. The proteolytic activity of the obtained composition is from 500 to 1000.0 PU/g. Component (1) comprises a porous slightly yellowish homogeneous mass.

Component (2) (solvent)



[0043] A solution of polyethylene oxide (predominantly polyethylene oxide with a molecular weight of 1500 Da) is prepared in a 0.025 M sodium phosphate buffer solution with pH 7.5-8.2. The obtained solution is subjected to irradiation with gamma-rays or a stream of accelerated electrons (with energy of 2.0 MeV) in a dose of 1.0 Mrad. The solution after irradiation is subjected to sterilizing filtration and packaged in 10 ml batches into 15 ml vials.

[0044] As a result, a sterile solvent for component (1) of the claimed composition is obtained. The solvent comprises a slightly yellowish liquid.

[0045] For therapeutic purposes a solution of the claimed composition is used (conditional appellation thereof being "Trombovazim"), which is prepared ex tempore by dissolving the contents of a vial with component (1) containing immobilized proteases in 10 ml of component (2) (solvent).

[0046] Dissolution ex tempore of component (1) in the solvent (component (2)) gives the claimed pharmaceutical composition.

Example 3



[0047] 15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 10% solution of dextran having a molecular weight of 40 kDa in a 0.025 M sodium phosphate buffer with pH 7.5, 6.3 g of protosubtilin G3Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.0 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, a composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G3Kh 2.1
2. Dextran (molecular weight 40 kDa) 10.0
3. Polyethylene oxide PEO-1500 5.0
4. 0.025 M sodium phosphate buffer 82.9
The proteolytic activity of the composition is 850 PU/g.

Example 4



[0048] 15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 5% solution of dextran having a molecular weight of 40 kDa in a 0.025 M sodium phosphate buffer with pH 8.2, 6.0 g of protosubtilin G3Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.0 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, a composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G3Kh 2.0
2. Dextran (molecular weight 40 kDa) 5.0
3. Polyethylene oxide PEO-1500 5.0
4. 0.025 M sodium phosphate buffer 88.0


[0049] The proteolytic activity of the composition is 800 PU/g.

Example 5



[0050] 0.5 of polyethylene oxide PEO-1500 is dissolved in 300 ml of a 10% solution of dextran having a molecular weight of 40 kDa in a 0.025 M sodium phosphate buffer with pH 8.2, 5.7 g of protosubtilin G3Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.0 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, a composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G3Kh 1.9
2. Dextran (molecular weight 40 kDa) 10.0
3. Polyethylene oxide PEO-1500 0.5
4. 0.025 M sodium phosphate buffer 87.6


[0051] The proteolytic activity of the composition is 750 PU/g.

Example 6



[0052] 15 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 5% solution of dextran having a molecular weight of 40 kDa in a 0.025 M sodium phosphate buffer with pH 8.2, 7.5 g of protosubtilin G3Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.2 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, a composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G3Kh 2.5
2. Dextran (molecular weight 40 kDa) 5.0
3. Polyethylene oxide PEO-1500 5.0
4. 0.025 M sodium phosphate buffer 87.5


[0053] The proteolytic activity of the composition is 500 PU/g.

Example 7



[0054] 15 of polyethylene oxide PEO-4000 are dissolved in 300 ml of a 5% solution of dextran having a molecular weight of 70 kDa in a 0.025 M sodium phosphate buffer with pH 8.2, 7.5 g of protosubtilin G10Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a
dose of 0.8 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, a composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G10Kh 2.5
2. Dextran (molecular weight 70 kDa) 5.0
3. Polyethylene oxide PEO-4000 5.0
4. 0.025 M sodium phosphate buffer 87.5


[0055] The proteolytic activity of the composition is 1000 PU/g.

Example 8


Preparing the claimed composition consisting of an active component and a solvent (two-component formulation)


Component (1):



[0056] 0.5 of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 10% solution of dextran having a molecular weight of 40 kDa in a 0.025 M sodium phosphate buffer with pH 8.2, 5.7 g of protosubtilin G3Kh are added, the mixture is stirred at a temperature of 18-20°C for 30 minutes. Then ballast proteins are precipitated by salt precipitation techniques. For this to be done, added to the mixture in succession till complete dissolution are 1.3 g of sodium phosphate disubstituted to final concentration of 0.45% and 1.9 g of calcium chloride to final concentration of 0.63%. After the dissolution of calcium chloride an insoluble precipitate of calcium phosphate is formed in the reaction mixture, and this calcium phosphate adsorbs the ballast proteins. The mixture is maintained for 12 hours at a temperature of 4 to 8°C for complete precipitation of the ballast proteins. After that the reaction mixture is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.0 Mrad. After the irradiation the solution is subjected to sterilizing filtration, packaged in batches of 10 ml in 15 ml vials and lyophilized to a residual moisture content not exceeding 2%. As a result, component (1) of the composition is obtained, having the following formulation in weight percent:
1. Protosubtilin G3Kh 1.9
2. Dextran (molecular weight 40 kDa) 10.0
3. Polyethylene oxide PEO-1500 0.5
4. 0.025 M sodium phosphate buffer 87.6


[0057] The proteolytic activity of component (1) is 750 PU/g.

Component (2) (solvent):



[0058] 15.0 g of polyethylene oxide PEO-1500 are dissolved in 300 ml of a 0.025 M sodium phosphate buffer. Then the solution is filtered through paper filters ("white ribbon"). The volume of the filtrate is 300 ml. The obtained solution is subjected to gamma irradiation in a dose of 1.0 Mrad. After the irradiation the solution is subjected to sterilizing filtration and packaged in batches of 10 ml in 15 ml vials. As a result, component (2) of the composition is obtained, having the following formulation in weight percent:
1. Polyethylene oxide PEO-1500 5.0
2. 0.025 M sodium phosphate buffer 95.0


[0059] The dissolution of component (1) in the solvent (component (2)) and lyophilization give the claimed pharmaceutical composition.

Example 9


Investigation of the pharmacological properties of the claimed composition ("Trombovazim")



[0060] The pharmacological properties of the claimed composition (its conditional appellation being "Trombovazim") have been checked under laboratory conditions in vitro and in vivo.

[0061] The thrombolytic properties of the composition have been investigated on a model of thrombolysis in vitro (Figure 1). As can be seen from Figure 1, "Trombovazim" produces a sharply pronounced thrombolytic effect, reliably exceeding in the standard therapeutic concentration of 50 PhU/ml the effect of fibrinolysine (p < 0.02); trypsin (p < 0.01); and spontaneous thrombolysis in physiological solution (p < 0.01). It should be noted that as the "age" of the thrombus increases to 7 days, the thrombolytic properties of "Trombovazim" are preserved (Figure 2), while fibrinolysine practically does not act on the 7-days thrombus. During the first two hours the action of fibrolysine does not differ with certainty from the spontaneous background lysis in physiological solution. By the end of the 4th hour the claimed composition dissolves the thrombus completely, while fibrinolysine lyses only 20% of the mass of the "old" thrombus.

[0062] It is known that fibrinolysine is the most active fibrinolytic (2), while such preparations as streptokinase, urokinase, alteplase and tissue plasminogen activator are indirect fibrinolytics and their thrombolytic action is mediated by the activation of the fibrinolysis system and production of endogenous fibrinolysine, the latter leading to the lysis of the formed thrombus (1).

[0063] The specific thrombolytic activity of "Trombovazim" in vivo has been investigated on a model of carotid artery thrombosis induced in rats by the application of ferrous chloride.

[0064] The results of the effect of prophylactic administering "Trombovazim" (80 PU per animal) on the blood flow (ml/min) along ipsilateral carotid arteries after the application of ferrous chloride (0-15 min) in Wistar line rats are presented in Table 2.
Table 2
Group0 min15 min30 min45 min60 min75 min90 min
Control n = 9 3.8±0.3 2.3±0.4 0.9±0.5 0.4±0.2 0.5±0.4 0.2±0.1 0.2±0.1
Experiment n = 10 2.9±0.3 2.5±0.1 2.1±0.3 1.9±0.4 1.9±0.5 1.9±0.4 1.7±0.4


[0065] As is seen from the presented results, prophylactic administration of "Trombovazim" effectively limits the process of thrombosis in the carotid artery during the first 1.5 hours after the effect of ferrous chloride thereon.

[0066] The results of the effect of therapeutic administering "Trombovazim" (80 PU per animal) on the blood flow (ml/min) along ipsilateral carotid arteries after the application of ferrous chloride (0-15 min) in Wistar line rats are presented in Table 3.
Table 3
Group0 min15 min30 min45 min60 min75 min90 min
Control n = 8 3.3±0.4 2.6±0.6 2.8±0.9 2.1±0.7 2.3±0.7 1.2±0.4 0.9±0.4
Experiment n = 9 3.8±0.3 3.3±0.5 2.7±0.5 1.7±0.4 1.6±0.3 1.6±0.4 1.8±0.4


[0067] As is seen from the presented results, therapeutic administration of "Trombovazim" effectively inhibits the process of thrombosis in the carotid artery during the first 1.5 hours after the effect of ferrous chloride thereon. Besides, histological (morphometric) investigations have shown that upon therapeutic administration of "Trombovazim" after 24 hours the proportion of animals with complete occlusion of the carotid artery and with the presence of a thrombus in it reduces 3-fold, this being indicative of a pronounced thrombolytic activity of the preparation.

[0068] The anti-inflammatory properties of the claimed composition have been investigated on a model of inducing one of the main inflammation mediators - tumor necrosis factor (αTNF) in mice of the CBA line with endotoxic shock (Figure 3). The activity of the αTNF was measured biologically, using L929 line cells and was expressed in units of action (UA). Shown in Figure 3 are: the αTNF activity in intact animals (1) and in animals after endotoxin administration (2). The administration of "Trombovazim" one hour before the administration of endotoxin (3) reduces 50-fold the αTNF activity, this proving the anti-inflammatory properties of "Trombovazim".

[0069] The cytoprotective properties of "Trombovazim" have been studied on a model of indomethacin gastropathy (Table 1) and adrenaline myocarditis in rats (Figure 4).
Table 1
No. of animal in groupControl: 6.2 mg of indomethacin administered intragastrically 1 hour ed before experimental effect of intraperitoneal administration of 3 ml of physiological solutionExperiment: 6.2 mg of indomethacin administered intragastrically 1 hour before experimental effect of intraperitoneal administration of 3 ml of "Trombovazim"
Stomach, mm2 SHSbleeding, mm2 SB% of injuries, S%Sstomach, mm2 SHSbleed-ing, mm2 SB% of injuries, S%
1 910 17.3 1.9 830 29.2 3.5
2 685 26.9 3.9 778 13.6 1.8
3 650 24.0 3.7 845 12.2 1.4
4 784 18.4 2.3 745 8.54 1.1
5 726 34.3 4.7 823 7.6 1.1
6 927 22.7 2.5 769 0 0
7 699 46.6 6.7 778 0 0
8 853 17.0 2.0 575 10.2 1.8
9 892 34.1 3.8 653 16.2 2.4
10 - - - 568 0 0
X ± SD 792±106 26.8±9.9 3.5±1.5 715±151 9.8±9.0 1.3±1.2


[0070] From the presented Table it follows that the control and experimental groups of the rats are comparable in terms of the total stomach area (SH): there was no reliable difference between the characteristics. The area of bleedings (SB) in the groups reliably differed: in the group with preliminary administration of "Trombovazim" (experiment) SB is 3 times smaller than in control (p < 0.01). The same reliable relationship is observed when comparing the relative affected area (S%). Hence, "Trombovazim" displays a pronounced cytoprotective effect in the case of indomethacin injury of the stomach mucosa.

[0071] Figure 4 shows the data of histological morphometric investigation of the volume of necroses and dystrophic changes in the myocardium of rats with adrenaline myocarditis. The volume of injuries was determined planimetrically and expressed in percent of the volume density which is equal to the volume of injuries/total value of tissue ratio multiplied by 100%. In the control group (K), after administering adrenaline, 1.0 ml of isotonic NaCl solution was administered intraperitoneally twice a day; in the experimental group (TRB) 1 ml of "Trombovazim" was administered intraperitoneally twice a day. From the presented results it follows that after the development of adrenaline myocarditis the treatment with "Trombovazim" reliably reduces 1.5-fold the number of necroses in the cardiac muscle by the end of the 3rd day: (TBR necr.) compared with control K necr.). Dystrophic changes in the cardiac muscle in the experimental group (TBR dystr.) by the end of the 7th day are reliably 2.5 times smaller than in the control group (K dystr.).

[0072] The results presented in Table 1 and in Figure 4 prove that "Trombovazim" has pronounced cardioprotective and cytoprotective activity. The protective effect of "Trombovazim" reliably manifests itself in specific gastropathy caused by non-steroidal anti-inflammatory preparations, particularly by indomethacin, and the therapeutic effect of "Trombovazim" reliably manifests itself in acute adrenaline myocarditis, in whose pathogenesis the key role is played by acute ischemia, necrosis with the development of myocardial dystrophy.

[0073] The disintoxication, anti-ischemic and cytoprotective effects were investigated also on a model of the liver ischemia/reperfusion. For this purpose, in the experimental and control groups of Wistar line rats (10 rats in each group) the liver triad in the animals after laparotomy was clamped for 20 minutes, i.e., the regional lymph and blood circulation were completely blocked. Then the blood flow was restored, and the histological structure of the liver in the animals was investigated (the main quantitative criterion was the accumulation of leukocytes in regional lymphatic nodes as the characteristic of response to the cytotoxic injury of the liver tissue by the products of ischemia/reperfusion: peroxides and free radicals). The results of the investigation are presented in Figure 5.

[0074] As is seen from the presented results, "Trombovazim" produces a hepatoprotective effect on the model of liver ischemia/reperfusion, and this is an additional confirmation of the cytoprotective, disintoxication and anti-inflammatory properties of "Trombovazim".


Claims

1. A composition having thrombolytic, anti-inflammatory, disintoxication and cytoprotectiveactivity properties for treating diseases accompanied with ischemia, thrombosis, intoxication and / or inflammation, comprising active proteases immobilized on a mixture of water-soluble polyethylene oxide and dextran.
 
2. A composition according to claim 1, characterized in that immobilization of proteases on the mixture of water-soluble polymers is effected with the help of ionizing radiation, by acting therewith on an aqueous solution containing active proteases and a mixture of water-soluble polyethylene oxide and dextran.
 
3. A composition according to claim 1, characterized in that the ionizing radiation is a flow of accelerated electrons, gamma-radiation and UV-radiation.
 
4. A composition according to claim 1, characterized in that proteolytic enzymes obtained from microbiological, animal or vegetable starting material are used as the active protease.
 
5. A composition according to claim 4, characterized in that proteolytic enzymes produced by Bacillus subtilis are used as the active protease.
 
6. A composition according to claim 1, characterized in that it comprises a one-component medicinal form containing lyophilized active proteases immobilized on a mixture of polyethylene oxide and dextran, and also combinations with aspirin, indomethacin and diclofenac.
 
7. A composition according to claim 1, which comprises a two-component dosage form containing lyophilized active proteases immobilized on a mixture of polyethylene oxide with dextran and a solvent.
 
8. A composition according to claim 7, wherein a solution of polyethylene oxide activated by ionizing radiation is used as the solvent.
 
9. A composition according to claim 1, which is an effective therapeutic agent for treating: ischemic heart disease and acute myocardial infarctions, ischemic disturbances of cerebral circulation, arterial and venous thromboses, obstructive diseases of the lungs, gastrites and stomach ulcer, gynecological, stomatological and surgical diseases.
 
10. A composition according to claim 9, wherein the therapeutic effect is attained upon parenteral, oral, intracavitary and local administration.
 
11. A composition according to claim 1, producing a protective effect on the stomach mucosa upon combined administration with non-steroidal analgesics.
 
12. A method of preparing a composition of claim 1, comprising simultaneous attachment of proteases to the mixture of water-soluble polymers for the formation of a non-solid mixture, and radiation sterilization of the mixture.
 
13. A method according to claim 12, wherein the radiation is a flow of accelerated electrons, gamma-radiation or UV radiation.
 
14. A method according to claim 12, wherein said protease is subtilysine.
 


Ansprüche

1. Zusammensetzung mit thrombolytischen, entzündungshemmenden, entgiftenden und zellschutzaktiven Eigenschaften zur Behandlung von mit Ischämie, Thrombose, Vergiftungserscheinungen und/oder Entzündungen einhergehenden Krankheiten, bestehend aus auf einer Mischung von wasserlöslichem Polyethylenoxid und Dextran immobilisierten aktiven Proteasen.
 
2. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass die Immobilisierung der Proteasen auf der Mischung von wasserlöslichen Polymeren mit Hilfe ionisierender Strahlung bewirkt wird, indem diese auf eine wässrige Lösung, die aktive Proteasen und eine Mischung von wasserlöslichem Polyethylenoxid und Dextran enthält, angewendet wird.
 
3. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass die ionisierende Strahlung ein Strom von beschleunigten Elektronen, Gammastrahlung und UV-Strahlung ist.
 
4. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass als aktive Proteasen proteolytische Enzyme verwendet werden, die aus mikrobiologischem, tierischem oder pflanzlichem Ausgangsmaterial gewonnen wurden.
 
5. Zusammensetzung nach Anspruch 4, dadurch gekennzeichnet, dass als aktive Proteasen von Bacillus subtilis produzierte proteolytische Enzyme verwendet werden.
 
6. Zusammensetzung nach Anspruch 1, dadurch gekennzeichnet, dass sie aus einer Einkomponenten-Arzneiform besteht, die lyophilisierte, auf einer Mischung von Polyethylenoxid und Dextran immobilisierte aktive Proteasen und auch Kombinationen mit Aspirin, Indomethacin und Diclofenac enthält.
 
7. Zusammensetzung nach Anspruch 1, bestehend aus einer Zweikomponenten-Darreichungsform, die lyophilisierte, auf einer Mischung von Polyethylenoxid mit Dextran und einem Lösungsmittel immobilisierte aktive Proteasen enthält.
 
8. Zusammensetzung nach Anspruch 7, bei der als Lösungsmittel eine durch ionisierende Strahlung aktivierte Polyethylenoxidlösung verwendet wird.
 
9. Zusammensetzung nach Anspruch 1 als wirksames Therapeutikum zur Behandlung von ischämischen Herzkrankheiten und akuten Herzinfarkten, ischämischen Hirndurchblutungsstörungen, Arterien- und Venenthrombosen, obstruktiven Lungenerkrankungen, Gastritis und Magenkrebs, gynäkologischen, stomatologischen und chirurgischen Krankheiten.
 
10. Zusammensetzung nach Anspruch 9, bei der die therapeutische Wirkung bei parenteraler, oraler, intrakavitärer und lokaler Verabreichung erzielt wird.
 
11. Zusammensetzung nach Anspruch 1, die bei kombinierter Verabreichung mit nicht steroidalen Analgetika eine schützende Wirkung auf die Magenschleimhaut erzeugt.
 
12. Verfahren zur Herstellung einer Zusammensetzung nach Anspruch 1, umfassend die gleichzeitig stattfindenden Vorgänge Anlagerung von Proteasen an die Mischung wasserlöslicher Polymere zur Bildung eines nicht festen Gemisches und Sterilisation des Gemisches durch Strahlung.
 
13. Verfahren nach Anspruch 12, bei dem die Strahlung ein Strom von beschleunigten Elektronen, Gammastrahlung oder UV-Strahlung ist.
 
14. Verfahren nach Anspruch 12, bei dem als Protease Subtilysin verwendet wird.
 


Revendications

1. Composition aux propriétés thrombolytiques, anti-inflammatoires, désintoxicantes et cytoprotectrices pour le traitement de maladies accompagnées d'ischémie, de thrombose, d'intoxication et/ou d'inflammation, constituée de protéases actives immobilisées sur un mélange d'oxyde de polyéthylène et de dextrane hydrosolubles.
 
2. Composition selon revendication 1, caractérisée en ce que l'immobilisation des protéases sur le mélange de polymères hydrosolubles s'effectue à l'aide d'une radiation ionisante, en appliquant celle-ci sur une solution aqueuse qui contient des protéases actives et un mélange d'oxyde de polyéthylène et de dextrane hydrosolubles.
 
3. Composition selon revendication 1, caractérisée en ce que la radiation ionisante est un courant d'électrons accélérés, de radiation gamma et de radiation UV.
 
4. Composition selon revendication 1, caractérisée en ce que les enzymes protéolytiques obtenues à partir d'un matériau de départ microbiologique, animal ou végétal sont utilisées comme protéases actives.
 
5. Composition selon revendication 4, caractérisée en ce que les enzymes protéolytiques produites par Bacillus subtilis sont utilisées comme protéases actives.
 
6. Composition selon revendication 1, caractérisée en ce qu'elle est constituée d'une forme médicamenteuse monocomposant qui contient des protéases actives lyophilisées immobilisées sur un mélange d'oxyde de polyéthylène et de dextrane ainsi que des combinaisons avec l'aspirine, l'indométacine et le diclofénac.
 
7. Composition selon revendication 1, constituée d'une forme de dosage bicomposants, qui contient des protéases actives lyophilisées immobilisées sur un mélange d'oxyde de polyéthylène et de dextrane et d'un solvant.
 
8. Composition selon revendication 7, dans laquelle une solution d'oxyde de polyéthylène activée par radiation ionisante est utilisée comme solvant.
 
9. Composition selon revendication 1 comme agent thérapeutique efficace pour le traitement de maladies cardiaques ischémiques et d'infarctus aigus du myocarde, de troubles ischémiques de la circulation cérébrale, de thromboses artérielles et veineuses, de maladies pulmonaires obstructives, de gastrites et de cancers gastriques, de maladies gynécologiques, stomatologiques et chirurgicales.
 
10. Composition selon revendication 9, dans laquelle l'effet thérapeutique est obtenu par administration parentérale, orale, intracavitaire et locale.
 
11. Composition selon revendication 1 qui produit un effet protecteur sur la muqueuse gastrique par administration combinée avec des analgésiques non stéroïdaux.
 
12. Méthode de préparation d'une composition selon revendication 1, comprenant les processus simultanés de l'attachement de protéases au mélange de polymères hydrosolubles pour former un mélange non solide et de la stérilisation par radiation du mélange.
 
13. Méthode selon revendication 12 dans laquelle la radiation est un courant d'électrons accélérés, de radiation gamma ou de radiation UV.
 
14. Méthode selon revendication 12, dans laquelle la subtilysine est utilisée comme protéase.
 




Drawing















REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description