EP1471935A2 - Criblage de proteases et nouvelle utilisation de proteases - Google Patents

Criblage de proteases et nouvelle utilisation de proteases

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Publication number
EP1471935A2
EP1471935A2 EP03708017A EP03708017A EP1471935A2 EP 1471935 A2 EP1471935 A2 EP 1471935A2 EP 03708017 A EP03708017 A EP 03708017A EP 03708017 A EP03708017 A EP 03708017A EP 1471935 A2 EP1471935 A2 EP 1471935A2
Authority
EP
European Patent Office
Prior art keywords
protease
proteases
plasminogen
acute
thrombosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03708017A
Other languages
German (de)
English (en)
Inventor
Rudy Susilo
Hans Christian Korting
Hans Günther GASSEN
Martin Hils
Ralf Pasternack
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
N Zyme Biotec GmbH
Original Assignee
N Zyme Biotec GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by N Zyme Biotec GmbH filed Critical N Zyme Biotec GmbH
Priority to EP03708017A priority Critical patent/EP1471935A2/fr
Publication of EP1471935A2 publication Critical patent/EP1471935A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)

Definitions

  • US Pat. No. 4,752,603 discloses a method for isolating a new plasminogen activator from the culture medium of human melanoma cells.
  • the isolated activator shows a thrombolytic effect and can be used for therapeutic treatment.
  • the isolation of this new activator is very complex and costly.
  • Another disadvantage is that the culture medium of human melanoma cells is enriched with calf serum, which in times of infectious diseases such as. B. BSE is undesirable.
  • active ingredients which can be used in the treatment and / or supportive therapy of wounds and which in particular promote the healing of wounds which are otherwise poorly or not at all healing. Furthermore, it would be desirable to provide such active ingredients that are relatively inexpensive and that no expensive processes are necessary for their production. It would be particularly desirable if active ingredients were available which are both fibrinolytic and collagenolytic and / or plasminogen-activating, preferably having all three of the properties mentioned above. Active ingredients that have all three of the properties mentioned would be particularly advantageously used in wound healing.
  • the present invention relates in particular to the use of protease from Streptomyces griseus, protease VIII, protease XVIII, ficin, proteinase K, protease XXIII, thrombin, metalloendopeptidase, endoproteinase, Asp-N, clostripain, protease XIX, chymopapain, papain, protease X, and / or trypsin in wound healing to achieve a fibrinolytic, collagenolytic and / or plasminogen-activating effect.
  • the protease from Streptomyces griseus is a protease mixture with the following pronase components: a) streptogrisin A (3.4.21.80, Streptomyces protease A, SGPA, pronase enzyme A), b) Streptogrisin B (3.4.21.80, Streptomyces protease B, SGPB, pronase enzyme B), c) Mycolysin (3.4.24.31, pronase component, Streptomyces griseus neutral proteinase).
  • the natural substrates of the protease from Streptomyces griseus are oligopeptides, which are hydrolyzed by reaction with water.
  • the substrate spectrum also includes casein and gelatin, which are also hydrolyzed by the protease from Streptomyces griseus.
  • this enzyme should not have amidase or esterase activity.
  • Protease VIII's natural substrates are proteins that are hydrolyzed by this enzyme.
  • the range of substrates extends to casein, hemoglobin, ovalbumin, gelatin, insulin and other proteins and peptides.
  • the proteases from S. griseus, the protease VIII, the proteinase K, the protease XXIII, Protease XIX and / or the trypsin have collagenolysis activity.
  • the protease from S. griseus, the protease XIII, the thrombin, the metalloendopeptidase, the endoproteinase Asp-N, the clostripain and / or the trypsin have a plasminogen-activating activity.
  • the protease from S. griseus and protease VIII can be used, which have both a collagenolytic, a fibrinolytic, and a plasminogen-activating activity.
  • proteases could have the activities mentioned.
  • trypsin surprisingly has both a plasminogen-activating and a collagenolytic activity.
  • proteinase K and protease XXIII have both fibrinolytic and collagenolytic activity.
  • protease from S. griseus and protease VIII which have both collageneolytic and fibrinolytic and plasminogen activating activity.
  • the activities mentioned above are connected in any way in such a way that it would have been expected that proteases which have one or the other of these activities could also have further activities.
  • the specific activities of the proteases according to the present invention for the plasminogen activation are preferably each greater than 1 U / milligram. Furthermore, the specific activities of the proteases according to the present invention for collagenolysis or fibrinolysis are greater than 0.1 U / milligram.
  • the proteases according to the invention can be used to degrade fibrin clots, activate matrix metalloproteases, break down an extracellular matrix, process glu-plasminogen to lys-plasminogen, activate and release growth factors and / or endogenous plasminogen to plasmin activate. All of the above activities, individually or in combination, are helpful or necessary in wound healing and thus also contribute to healing otherwise badly or slowly healing wounds.
  • the proteases according to the present invention can therefore be used to produce a pharmaceutical composition for the treatment and / or supportive therapy in wound healing.
  • the pharmaceutical composition preferably further comprises suitable carriers and / or excipients. Such suitable carriers and / or auxiliary substances are known in the field of wound healing.
  • proteases listed above have anti-thrombotic and anti-coagulant properties. These advantageous properties also enable the use of the proteases, in particular the protease from Streptomyces (S.) griseus, the protease VIII, the proteinase K and trypsin, as anti-thrombotic and anti-coagulant active ingredients for the prophylaxis and / or treatment of heart attack, thrombosis , Venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels, acute pulmonary embolism, acute and subacute arterial thrombosis, fresh or older clots in the case of venous thrombosis, deep venous thrombosis of the pelvis and extremities, early thrombosis in the area of the occlusive central vessels, amniotic occlusion, Burns and frostbite, disseminated intravascular coagulation in shock, acute arterial occlusion
  • the proteases are preferably used together with an anticoagulant.
  • proteases should be inexpensive to manufacture and easily available on the market; this criterion is met by commercially available proteases. - Only proteases that are active under physiological conditions should be included in the screening. That is, acidic proteases, such as pepsin, have not been used.
  • the selected proteases were tested for three activities that are particularly relevant for wound healing and whose combination can be expected to have a particularly good effect on wound healing, especially of slowly healing wounds:
  • Activation and release of growth factors e.g. B. the "basic fibroblast growth factor” bFGF, the “transforming growth factor” TGFß-1 or the “vascular endothelial growth factor” VEGF.
  • test methods based on microtiter plates should be developed, which should enable the selected proteases to be checked in a short time with little effort.
  • test methods should be developed that can be carried out both easily and on a large scale and also lead to clear results with high reproducibility.
  • screening methods have therefore been developed for screening for fibrinolysis or collagenolysis which do not have the above problems and which furthermore meet the various criteria mentioned above.
  • the present invention is also directed to a screening method for the identification of enzymes suitable for wound healing, the enzymes being examined for their ability to activate plasminogen, for fibrinolysis and / or for collagenolysis.
  • the screening method according to the present invention comprises at least the following steps:
  • the plasminogen activation is preferably measured photometrically by detecting a released dye by increasing the absorbance at 405 nm.
  • the dye released is the para-nitroaniline (pNA) cleaved from the peptide substrate by plasminogen formed from plasminogen.
  • the plasminogen-enzyme mixture is preferably in a 1: 1 (v / v) ratio. This mixture is further preferably incubated at 37 ° C. for 10 minutes.
  • the incubation of plasminogen, enzyme and peptide substrate is preferably carried out for 5 to 30 minutes, further preferably at about 37 ° C.
  • the investigation of the koUagenolysis activity comprises at least the following steps:
  • the photometric detection in the examination for a KoUagenolysemod is preferably carried out after incubation with the ninhydrin reagent for 10 to 25 minutes, preferably for 20 minutes, at about 60 ° C and cooling to room temperature by measuring the extinction difference at 550 nm.
  • the collagen and the enzyme to be examined are incubated for 30 to 90 minutes, preferably at about 37 ° C., before the reaction is preferably stopped by adding 10% trichloroacetic acid.
  • the insoluble components can be separated by centrifugation.
  • Another preferred embodiment of the screening method of the present invention comprises screening for a fibrinolytic activity of enzymes, this screening method being carried out according to the screening method described for collagenolysis, with collagen being replaced in each case by fibrin.
  • the enzymes to be examined for the screening method are preferably selected from the group of proteases.
  • Proteases which are active under physiological conditions are particularly preferably used for the screening.
  • Proteases which are commercially available are also preferably selected for the screening.
  • the present invention further relates to a protease obtainable by the screening method as described above.
  • the present invention also relates to the use of such a protease, obtained by the screening method described above, for the production of a pharmaceutical composition, for the treatment and / or supportive therapy in wound healing, in particular for providing a fibrinolytic, collagenolytic and / or plasminogen-activating activity.
  • the activity of the proteases with regard to collagenolysis or fibrinolysis is determined directly with the natural substrate collagen or fibrin.
  • the cleaved peptides are acid-soluble and can therefore be determined photometrically after sedimentation of the insoluble collagen.
  • the low sensitivity in the direct measurement of these peptides and amino acids was initially improved by staining with Folin-Ciocalteus phenol reagent.
  • the proteases to be tested partially contained substances which were also colored by the above phenol reagent. For this reason, the coloring was replaced according to the invention by the amino acid detection with ninhydrin.
  • proteases could be identified that have all three required activities (protease from S. griseus, protease VIII).
  • protease from Streptomyces griseus (a mixture of three proteases) and protease VIII (also referred to as subtilisin Carlsberg) are active with respect to all substrates tested.
  • the values of the specific activity of the protease from S. griseus can be classified as very high compared to all substrates. Trypsin also stands out because the enzyme has a particularly high plasminogen activation activity. Trypsin also has collagenolytic activity. In addition to high fibrinolysis activity, the herbal protease ficin has (albeit low) plasminogen activation (below 1 U / mg). Proteinase K is able to degrade both collagen and fibrin with medium or high activity.
  • thrombin could be used for the enzyme thrombin, which is active in the blood coagulation cascade Plasminogen activation activity can be measured. Furthermore, several enzymes with activities against the different substrates were discovered. The results are summarized in a table below.
  • Tab. 1 Compilation of the results of the protease screening with regard to the substrates plasminogen, collagen and fibrin.
  • the symbols used in the table mean:
  • Plasmin activity (after proteolytic activation of plasminogen):
  • protease screening show new ways of enzymatically assisted wound healing.
  • Proteases have been identified that are very potent candidates for wound healing, since they either have a co-agent, fibrinolytic or plasminogen-activating effect or even combine several of the three activities tested.
  • the protease mixture Protease from S. griseus is particularly interesting because it implements all three substrates with high activity.
  • the pharmaceutical compositions contain at least one protease selected from the group consisting of protease from Streptomyces (S.) griseus, protease VIII, protease XVIII, ficin, proteinase K, protease XXIII, thrombin, metalloendopeptidase, endoproteinase Asp-N, clostripain, protease XIX, Chymopapain, papain, protease X and trypsin, together with physiologically compatible carriers, auxiliaries and / or solvents, and optionally anti-coagulant active ingredients.
  • protease selected from the group consisting of protease from Streptomyces (S.) griseus, protease VIII, protease XVIII, ficin, proteinase K, protease XXIII, thrombin, metalloendopeptidase, endoproteinase Asp-N,
  • Proteases from Streptomyces (S.) griseus, protease VIII, proteinase K and / or trypsin are particularly preferred.
  • Heparin, heparin derivatives or acetylsalicylic acid are preferably added as additional anticoagulant active ingredients.
  • the proteases which can be used according to the invention are preferably used in external wound treatment in pharmaceutical compositions which are suitable for topical use.
  • the protease (s) are used in a concentration of 0.01-500 units per gram, preferably 0.1-500 U per gram of pharmaceutical composition, further preferably 0.5-250 U per gram of pharmaceutical composition and particularly preferably in a concentration from 1 - 150 U protease per gram of pharmaceutical composition used.
  • plasters or other dressing materials are used instead of semi-solid preparations in the form of, for example, ointments, creams, pastes, gels, etc., the concentration ranges given above per 2 cm 2 of plaster surface or surface of the dressing material apply.
  • the formulations suitable for topical use in particular ointments, creams, sprays, pastes, gels for wound treatment, especially poorly healing wounds, preferably contain 0.01-500, particularly preferably 0.1-500 U protease per gram of pharmaceutical composition.
  • the protease (s) are used in a concentration of 0.1 to 100,000 units per gram of formulation, preferably 100 to 80,000 units per gram of formulation and particularly preferably 1,000 to 50,000 units per gram of formulation.
  • the proteases are preferred in a concentration of 0.01 unit - 100 million 0.1 unit to 100 million units, further preferably 1 unit to 100 million units per 10 ml solution, further preferably 1 unit to 10 million units per 10 ml solution and particularly preferably 3 units to 5 million units per 10 ml solution and further particularly preferably 10 units - 1 million units of protease (s) used per 10 ml of solution.
  • the stated amounts of protease (s) are further preferably based on 1 ml of solution.
  • proteases are preferably used alone and not in combination with other proteases, but combinations of two or three proteases are definitely useful and usable for certain indications.
  • compositions according to the invention are prepared in a known manner with the customary solid or liquid carriers or diluents and / or the commonly used pharmaceutical adjuvants according to the desired type of application in a suitable dosage.
  • the preferred pharmaceutical formulations or preparations exist in a dosage form which is suitable for topical external application.
  • dosage forms are, for example, ointments, pastes, gels, films, dispersions, emulsions, suspensions or special formulations, such as, for example, nanodisperse systems in the form of liposomes, nanoemulsions or lipid nanoparticles, and also surfactant-free formulations, polymer-stabilized or solids-stabilized emulsions.
  • thrombosis When using the pharmaceutical compositions for the prophylaxis and / or treatment of heart attack, thrombosis, venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels, acute pulmonary embolism, acute and subacute arterial thrombosis, fresh or older clots in venous thrombosis of deep vein thrombosis Pelvic and extremities, early thrombosis in the area of the obliterated vessels, acute central vascular occlusion on the eye, disseminated intravascular Coagulation in shock, acute arterial occlusion of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, as well as for treatment after a heart attack, after a bypass operation, after angioplasty and after balloon dilatation, for thrombolytic therapy for acute heart attack, for recanalizing arteriovenous Preparations prepared for parenteral
  • compositions are, for example, protease-containing plasters or other dressing materials. These formulations are particularly suitable for topical use in wound treatment.
  • the screening methods which can preferably be used for the determination of the fibrinolysis activity, the collagenolysis activity and the plasminogen activation activity are presented below as examples.
  • the corresponding protease is pipetted into a fibrin suspension and incubated at 37 ° C.
  • the reaction is stopped by adding 10% trichloroacetic acid and the insoluble constituents are separated off by centrifugation.
  • the same volume of 3 M sodium acetate buffer pH 5.5 is added to 100 ⁇ l of the supernatant and incubated at 60 ° C. for 5 minutes.
  • the extinction difference used for the activity calculation is measured at 550 nm.
  • buffer A 100 mM sodium phosphate buffer pH 8, 0.36 mM CaCl 2 , 0.9% NaCl
  • the suspension thus obtained was washed with a Ultraturrax mixer treated three times for 30 s, level 5 (Ultra Turrax®, IKA T18 basic, dispersing tool S18-105, IKA Works, USA).
  • level 5 Ultra Turrax®, IKA T18 basic, dispersing tool S18-105, IKA Works, USA.
  • protease solution 50 ⁇ l of protease solution were added. This mixture was incubated at 37 ° C with shaking. After 0, 30, 60 and 90 min.
  • L-serine solutions in buffer A were prepared with the following concentrations: 0; 0.5; 1; 1.5; 2; 2.5; 3; 3.5; 4 mM. These solutions were stained with ninhydrin as described above and the absorbance at 550 nm was measured after transfer to microtiter plates.
  • 1 U fibrinolysis activity corresponds to the increase in extinction per minute, which is equivalent to the release or color intensity of 1 ⁇ mol L-serine.
  • proteases from S. griseus, Protease VIII, Protease XXIII, Protease XIX, Protease XVIII, Ficin, Metalloendopeptidase, Clostripain, Endoproteinase Glu-C, Protease XIII, Chymopapain, Chymotrypsin, Protease X, Bromelain, Kallikrein and Proteinase A were from Sigma , Deisenhofen, related; Trypsin, papain, endoproteinase Asp-N, thrombin, dispase I, endoproteinase Lys-C and elastase were from Röche, Mannheim, and proteinase K was supplied by QIAGEN, Hilden.
  • Protease VIII 0.097 U / mg
  • Papain 2.361 U / mg
  • Protease XXIII 0.105 U / mg
  • Protease X 0.032 U / mg
  • Protease XVIII 0.081 U / mg
  • Protease from S. griseus 4.048 U / mg
  • Ficin 1.149 U / mg
  • Chymopapain 0.166 U / mg
  • Proteinase K 1.344 U / mg.
  • No fibrinolysis activity could be determined for the following proteases: trypsin, chymotrypsin, bromelain, dispase I, protease XIX, protease XIII, thrombin, kallikrein, Elastase, Endoproteinase Glu-C, Proteinase A, Metalloendopeptidase, Endoproteinase Asp-N, Clostripain and Endoproteinase Lys-C.
  • proteases trypsin, chymotrypsin, bromelain, dispase I, protease XIX, protease XIII, thrombin, kallikrein, Elastase, Endoproteinase Glu-C, Proteinase A, Metalloendopeptidase, Endoproteinase Asp-N, Clostripain and Endoproteinase Lys-C.
  • the collagenolysis assay follows the same scheme as the assay for fibrinolysis activity.
  • No collagenolysis activity could be determined for the following proteases: chymotrypsin, bromelain, thrombin, papain, protease X, dispase I, protease XIII, protease XVIII, ficin, kallikrein, chymopapain, elastase, endoproteinase Glu-C, proteinase A, metalloendopeptidase, endoproteinase Asp-N, clostripain and endoproteinase Lys-C.
  • proteases chymotrypsin, bromelain, thrombin, papain, protease X, dispase I, protease XIII, protease XVIII, ficin, kallikrein, chymopapain, elastase, endoproteinase Glu-C, proteinase A, metalloendopeptidase, end
  • the inventive screening method for determining the plasminogen activation activity presented here is based on the use of synthetic peptide substrates. These are short amino acid chains that are linked to a chromophore. The chromophore is released by a protease-catalyzed reaction and can be determined photometrically. In addition to the high specificity of the substrates used, these processes also offer the advantage that the reaction can be followed directly.
  • pNA para-nitroaniline, dye
  • the assay developed with this substrate is carried out as follows: Plasmin is added to a Tosyl-Gly-Pro-Lys-pNA solution and incubated at 37 ° C. The course of the reaction can be monitored photometrically using the released dye at 405 nm.
  • proteases supplied in powdered form were dissolved in buffer, the proteases supplied in solution were used directly or, if necessary, diluted with buffer. 25 ⁇ l of the protease solutions were mixed with 25 ⁇ l plasminogen (obtained from Röche, Mannheim, concentration 20 mg / ml) and incubated at 37 ° C. for 10 minutes. The plasmin activity with respect to the substrate N-tosyl-Gly-Pro-Lys-pNA was then determined.
  • proteases from S. griseus, Protease VIII, Protease XXIII, Protease XIX, Protease XVIII, Ficin, Metalloendopeptidase, Clostripain, Endoproteinase Glu-C, Protease XIII, Chymopapain, Chymotrypsin, Protease X, Bromelain, Kallikrein and Proteinase A were from Sigma , Deisenhofen, related; Trypsin, papain, endoproteinase Asp-N, dispase I, endoproteinase Lys-C, thrombin and elastase were from Röche, Mannheim, and proteinase K was supplied by QIAGEN, Hilden.
  • proteases protease XXIII, protease XIX, chymopapain, endoproteinase Lys-C, chymotrypsin, papain, Dispase I, protease X, bromelain, kallikrein and proteinase K no plasminogen activation could be detected.
  • proteases identified in the individual screening processes are used for the pharmaceutical formulations:
  • Hydroxyethylcellulose 400 2.5 - 5.0 g of purified water to 100.0 g
  • the swelling time is 1 to 3 hours.
  • Polyhexanide can optionally be used as an antimicrobial active ingredient in a concentration up to
  • Hydroxyethylcellulose 400 e.g. Tylose ® H 300 or Natrosol 250 ® HX PHARM
  • Macrogol 400 30.0 - 32.5 g
  • 12.5 g Macrogol 4000 and 30.0 g Macrogol 400 (7.5 g Macrogol 4000 and 32.5 g Macrogol 400 for soft ointments) are heated in an ointment dish in a water bath until the macrogol has melted. After cooling, the corresponding amount of protease (s) dissolved in 7.5 g of purified water is added and then homogenized.
  • proteases are preferably used individually in the pharmaceutical formulations mentioned herein. However, combinations of two or three proteases can also be used, the amount then being understood in units per protease used or as the total activity of all proteases.
  • Hydroxyethyl cellulose 10,000 3.5 g optional preservation (sorbic acid / potassium sorbate 0.1-0.4%, PHB ester 0.1%). purified water ad 100.0
  • hydroxyethyl cellulose or instead hypromellose or methyl cellulose can alternatively be used in an amount of 0.5-15.0 g. gel
  • Hydroxyethyl cellulose 10,000 32.5 g optional preservation (sorbic acid / potassium sorbate 0.1-0.4% » PHB ester 0.1%)
  • Hydrophilic ointment (macrogol ointment)
  • Vaseline white to 100 g
  • Vaseline white to 100 g
  • Carbomer e.g. Carbopol 974p 15 g
  • Vaseline white 25 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%), PHB ester 0.1%)
  • one capsule with 0.25g powder / granules contains:
  • 100g pellets contain:

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne une nouvelle utilisation de certaines protéases dans le cadre du traitement pour une guérison de plaies, notamment leur utilisation lors de la fibrinolyse, de la collagénolyse et de l'activation du plasminogène. La présente invention concerne également des procédés de criblage pour préparer des protéases adaptées aux utilisations susmentionnées. En outre, cette invention concerne l'utilisation des protéases ainsi obtenues pour produire une composition pharmaceutique utilisée pour la guérison de plaies, pour la prévention et/ou le traitement d'un infarctus, d'une thrombose, d'une thrombose veineuse, d'une resténose, d'une hypoxie, d'une ischémie, d'une nécrose de coagulation, d'une inflammation des vaisseaux sanguins, d'une embolie pulmonaire aiguë, d'une thrombose artérielle aiguë et subaiguë, d'un caillot frais ou plus ancien en cas de thrombose veineuse, d'une thrombose veineuse profonde du bassin et des extrémités, d'une thrombose précoce dans la zone de vaisseaux désoblitérés, d'une oblitération de vaisseau central aiguë de l'oeil, de brûlures et de gelures, d'une coagulation intravasculaire disséminée en cas de choc, d'une oblitération artérielle aiguë des extrémités, d'une artériopathie occlusive chronique, d'une thrombose de shunt artério-veineux et pour le traitement après un infarctus, après une opération de pontage, après une dilatation par ballonnet, pour une thérapie thrombolytique en cas d'infarctus aigu, pour une recanalisation de shunt artério-veineux et pour une reperfusion d'artère coronarienne oblitérée en cas d'infarctus aigu et après une angioplastie avec ou sans insertion d'endoprothèse.
EP03708017A 2002-02-06 2003-02-06 Criblage de proteases et nouvelle utilisation de proteases Withdrawn EP1471935A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03708017A EP1471935A2 (fr) 2002-02-06 2003-02-06 Criblage de proteases et nouvelle utilisation de proteases

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP02002706 2002-02-06
EP02002706 2002-02-06
US35781002P 2002-02-21 2002-02-21
US357810P 2002-02-21
EP03708017A EP1471935A2 (fr) 2002-02-06 2003-02-06 Criblage de proteases et nouvelle utilisation de proteases
PCT/DE2003/000342 WO2003066088A2 (fr) 2002-02-06 2003-02-06 Criblage de proteases et nouvelle utilisation de proteases

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FR2966734B1 (fr) 2010-10-29 2014-07-18 Max Rombi Composition comprenant au moins une enzyme proteolytique pour son utilisation pour empecher la synthese des triglycerides

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