WO2012119744A1

WO2012119744A1 - Novel fibrinolysis inhibitors and medical application thereof

Info

Publication number: WO2012119744A1
Application number: PCT/EP2012/000966
Authority: WO
Inventors: Birgit FELSER; Dieter U. PREISS
Original assignee: Solution Shop Ag
Priority date: 2011-03-08
Filing date: 2012-03-03
Publication date: 2012-09-13
Also published as: DE102011013326A1

Abstract

The invention relates to fibrinolysis inhibitors and to the medical application thereof in antifibrinolytic therapy and prophylaxis. Known fibrinolysis inhibitors are used in surgical procedures that cause greater intraoperative or perioperative blood loss, or in procedures with extracorporeal circulation in order to reduce the extent of blood loss. The disadvantage is the occurrence of undesired side effects. In order to prevent such side effects, proteins, which contain amino acid sequences selected from the group consisting of SEQ ID NO: 1 (dBPS1 = duck Basic Protein Small 1), SEQ ID NO: 2 (dBPS2), SEQ ID NO: 3 (meleagrin), and SEQ ID NO: 4 (similar to meleagrin), are used in antifibrinolytic therapy and prophylaxis.

Description

New fibrinolysis inhibitors and their medical use.

The present invention relates generally to the field of hemostasis and, more particularly, to novel fibrinolysis inhibitors (antifibrinolytics) which may be used for therapeutic or prophylactic antifibrinolytic treatment. The invention relates to the therapeutic, even prophylactic, use of said fibrinolysis inhibitors and medical treatment methods in which said inhibitors are administered for therapeutic or prophylactic purposes to a human or animal organism. The invention further includes the use of the novel fibrinolysis inhibitors in the field of laboratory medicine.

Fibrin is formed in the post-vascular injury process of hemostasis during the so-called fibrin formation phase. The subsequent polymerization forms a wound-closing clot (thrombus). Fibrin is produced from soluble fibrinogen under the action of the enzyme thrombin, after it has been activated.

During the wound healing phase, the fibrinolytic system causes fibrin to be dissolved again as soon as it is no longer needed for wound healing. In fibrinolysis, the insoluble fibrin is converted into soluble fibrin cleavage products by enzymatically controlled proteolytic reactions. The central step here is the activation of plasminogen to the proteolytic enzyme plasmin, which causes the cleavage of fibrin. Surgery involving major intraoperative or perioperative blood loss or extracorporeal circulation (via the heart-lung machine) requires the use of antifibrinolytic agents to reduce the level of blood loss. As a result, a lower number of blood donations (donor blood) is needed than would be the case without administration of antifibrinolytic agents.

The above-mentioned surgical interventions in which antifibrinolytic agents are used to reduce blood loss include, in particular, cardiac surgery such as, for example, surgery. B. Bypass operations, Herklappen surgery or heart transplants, or surgery on the liver.

Furthermore, antifibrinolytic agents are used in local or generalized fibrinolysis, for example, local bleeding in gynecology and obstetrics or in dentistry.

The protease inhibitor aprotinin (Trasylol ^®, Bayer AG) was used as antifibrinolytic z. In open heart surgery or liver surgery to minimize blood loss and thus the need for blood transfusions. The antifibrinolytic action of aprotinin is due to the inhibition of the activation of plasminogen to the active plasmin.

Due to indications of unwanted side effects (especially renal complications, cerebrovascular events) or increased mortality, aprotinin (Trasylol ^® ) was withdrawn from the market worldwide. The application of aprotinin is only permitted under strict conditions in exceptional cases.

Some of the unwanted side effects, such as As the risk of renal failure, are probably due to the fact that aprotinin has a broad substrate specificity and therefore not only antifibrinolytic but also antithrombol tisch and anticoagulatory acts (by inhibiting streptokinase, t-PA and urokinase).

Antifibrinolytic agents that can be used instead of aprotinin include the synthetic ω-aminocarboxylic acids tranexamic acid (TA), ε-aminocaproic acid (EACA) and p-amino-methylbenzoic acid (PAMBA). These inhibitors are advantageous over aprotinin because they specifically inhibit fibrinolysis without affecting other proteolytic processes. However, after administration of TA, visual disturbances may occur (e.g., loss of vision, disturbance of color vision).

Whether the antifibrinolytic agents of the class of synthetic ω-aminocarboxylic acids mentioned above achieve the same efficacy in reducing blood loss in surgical procedures, especially in surgeries with extracorporeal circulation, as aprotinin is to be doubted. As mentioned above, the use of aprotinin under strict conditions is still permissible in exceptional cases. The drug is still available in certain countries, including the United States, for certain groups of patients with a justified medical need, indicating that highly potent fibrinolysis inhibitors are urgently needed. For example, in the search for new, better tolerated fibrinolysis inhibitors variants or analogues of aprotinin were considered (eg WO 2006/17355 A2 WO 2008/110301). By DIETRICH et al. A low molecular weight serine protease inhibitor (700 Da) with antifibrinolytic and anticoagulant properties has been described (DIETRICH W, NICKLISCH M, KOSTER A, VAN DE HOCHT A: CU-2010-A novel antifibrinolytic protease inhibitor displays, ie anticoagulant properties.) Anesth 2009; 108 (SCA Suppl.), 1-104, SCA86).

Furthermore, the serine protease inhibitor textileinin-1 isolated from snake venom has been proposed as a substitute for aprotinin (FLIGHT SM, JOHNSON LA, DU QS, WARNER RL, TRABI M, GAFFNEY PJ, LAVIN MF, DE JERSEY J, MASCI PP: Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss, British J. of Hematology 145, 207-211).

The aforementioned inhibitors have so far found no entry into clinical application.

The present invention therefore an object of the invention to provide novel drugs with antifibrinolytic activity for therapeutic use, especially in surgical procedures, wherein the above-mentioned disadvantages of the known antifibrinolytic agents are avoided or reduced. A further object of the present invention is to provide methods of therapeutic treatment by means of which the active substances mentioned are administered to humans or animals for the purpose of antifibrinolytic treatment, in particular for the reduction of blood loss during surgical interventions. The above object is achieved by providing a protein or a combination of at least two proteins, as defined below, for use in antifibrinolytic treatment or prophylactic antifibrinolytic treatment.

A protein of the invention has an amino acid sequence selected from the group consisting of

SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (according to appended sequence listing) and amino acid sequences having a sequence identity of at least 70% to one of the aforementioned SEQ ID NOS: 1 to 4 , consists.

Said combination comprises at least two proteins which each have one of the abovementioned amino acid sequences.

It has surprisingly been found that the abovementioned proteins, individually or in combination, have an antifibrinolytic action (that is to say as pyrolysis inhibitors), in which case no anticoagulant effect is produced or at most can be observed to a very limited extent. Because of these properties, the above-mentioned proteins having the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, individually or in combination, are advantageously for use in the antifibrin reaction. lytic therapy or antifibrinolytic prophylaxis, in particular for the reduction of blood loss in surgical interventions, suitable. Due to the lack of anticoagulant activity, the risk of side effects is reduced.

The proteins used in the present invention (including their variants) have a hemostatic effect. kung. They are advantageous because - unlike aprotinin - have no effects on the coagulation system and are free from serious side effects, in particular have no kidney-damaging effect. Furthermore, no immunological side effects are known.

Without wishing to be bound by any means, it is presently believed that the antifibrinolytic activity of the abovementioned proteins used according to the invention is due to a specific or selective inhibition of plasmin formation. This is also in accordance with the observation that the proteins according to the invention do not exert an anticoagulant effect.

The above-mentioned proteins having the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 are known in the art as noted in the Sequence Listing.

The isolation, sequencing and biochemical characterization of the proteins having the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2 has been described by NA NU OOL et al. (NAKNUKOOL, S., HAYAKAWA, S., SUN, Y., and OGAWA, "Structural and physicochemical characteristics of novel basic protein isolated from duck egg white"; Biosci Biotechnol., Biochem., 72 (8 ) 2008, p. 2082-2091).

NAKNUKOOL et al. The proteins "Duck Basic Protein Small 1" (dBPS1) and "Duck Basic Protein Small 2" (dBPS1) were isolated from the protein of duck eggs. The amino acid sequences of dBPS1 or dBPS2 correspond to the above-indicated sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The abovementioned sequences are among the accession numbers P85123 or P85124 can be called up in the Swiss-Prot database (Version No. P85123.1 or P85124.1). SEQ ID NO: 2 is identical to the sequence of Cygnin (P02785, Swiss-Prot).

The amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are as follows:

SEQ ID NO: 1 (= dBPSl)

QKKGFCAGYC SYSCAKTDEW TFHQTCGKMY CCIPPPKKG (39 aa) SEQ ID NO: 2 (= dBPS2)

QVRKYCPKVG YCSSKCSKAD VWSLSSDCKF YCCLPPGWK (39 aa)

The biological function of the proteins dBPSl and dBPS2 was previously unknown. The above-mentioned publication of

NAKNU OOL et al. contains no indication of possible functions of dBPSl and dBPS2.

The amino acid sequences SEQ ID NO: 3 and SEQ ID NO: 4 correspond to the proteins MeLeggrin and "Similar to meleagrin" known in the prior art (Accession No. P21376 (Swiss-Prot), XP 429908 (NCBI)). These proteins have sequence similarity to dBPS1 and dBPS2 (NAKNUKOOL et al., Supra, FIG. 4). Also with respect to these proteins, a biological function is not yet known.

SEQ ID NO: 3 (meleagrin)

QVLKYCPKIG YCSSKCSKAE VWAYSPDCKV HCCVPANQKW (40 aa)

SEQ ID NO: 4 ("similar to meleagrin")

VLKYCPKIGY CSNTCSKTQI WATSHGCKMY CCLPASWK K (40 aa) Note: The sequence shown above is a partial sequence of the sequence "similar to meleagrin" (accession no.

XP 429908; Total length 63 aa), with amino acids 1-40 corresponding to amino acids 24-63 of the total sequence from "similar to meleagrin".

The said proteins having the sequences SEQ ID NO: 1 to SEQ ID NO: 4 have a high sequence similarity to one another. Furthermore, they are characterized by the presence of 6 cysteine residues, which allow the formation of three intramolecular disulfide bridges, resulting in a very compact and stable protein folding (see NAKNUKOOL et al., Supra, Fig. 4).

According to the present invention, either a single protein having one of the sequences SEQ ID NO: 1 to SEQ ID NO: 4 is used for said therapeutic purpose, or a combination of at least two proteins, each of which is one of Sequences SEQ ID NO: 1 to SEQ ID NO: 4 has been used.

The present invention is not limited to those proteins whose sequence is identical to one of the sequences SEQ ID NO: 1 to SEQ ID NO: 4. It is known to those skilled in the art that some or more amino acids in a polypeptide sequence can be replaced (or added or deleted) with other amino acids or chemically modified without affecting the biological function of the protein. Therefore, the present invention also encompasses functional variants (such as, for example, derivatives, allelic variants, fragments, homologs, analogs, fusion proteins) which have an SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 have similar amino acid sequence and the have mentioned antifibrinolytic action. It is to be expected that such variants (homologs) can be isolated, for example, from the egg whites of eggs of other bird species.

Preferred variants are those which differ from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by conservative amino acid substitutions. Conservative substitution is understood to mean the replacement of one amino acid with another amino acid having similar physicochemical properties. Such conservative substitutions are known to those skilled in the art. Preferably, the number of substituted, inserted or deleted amino acids is less than 15, in particular less than 10, particularly preferably less than 5.

Furthermore, it is plausible to those skilled in the art that also sequences which have less than the full length of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, may have the mentioned antifibrinolytic activity , Furthermore, the present invention also encompasses proteins which contain a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, this sequence being N-terminal and / or C terminal is extended by further amino acid residues.

In particular, the present invention extends to functional variants (such as homologues or allelic variants) having amino acid sequences which are similar to any of SEQ ID NO: 1 to SEQ ID NO: 4, wherein the sequence identity is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%. The determination of the sequence identity or similarity (in "% identity") of two proteins can be determined by known algorithmic methods such as. Using programs such as "FASTA" ¹ , ¹¹ BESTFIT ^1,2 or "BLAST / BLASTP ³ ". Programs of the BLAST program family are accessible, for example, via the homepage of the NCBI

(www.ncbi.nlm.nih.gov). If the sequence to be compared is considerably longer than SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, then the identity (in%) will not be on the entire length but on the respective subarea. Otherwise, the programs mentioned are executed with the parameters specified by the basic settings of the respective program. A similarity of x% identity exists if at least one of the mentioned programs yields this value.

^! pearson WR, Method in Enzymology, 183, 63-99 (1990);

2 Smith and Waterman, J. Mol. Biol. 147, 195-197 (1981);

3Altool S F et al. , J. Mol. Biol. 215, 301-410 (1990), Nucleic Acids Res. 25, 389-3402 (1997).

In general, the above-mentioned proteins used for the therapeutic purposes of the present invention are low molecular weight (especially less than 10 kDa) basic proteins having at least two disulfide bridges and having an antifibrinolytic activity.

The molecular mass of the proteins used according to the present invention is preferably in the range from 4 to 10 kDa, in particular in the range from 4 to 6 kDa. The determination of the molecular weight can be known to the person skilled in the art Way z. B. by SDS-PA6E (in the presence of a reducing agent) can be determined. In case of doubt, the value determined by mass spectrometry applies.

The isoelectric point (pI) is above 7.0, in particular above 8.0, preferably above 9.0.

The primary sequences of the proteins used according to the invention preferably contain at least 4, in particular 6, cysteine residues, which permit the formation of two, preferably of three, cysteine bridges.

Further suitable for the present invention are those functional variants of the proteins defined above, which are modified by glycosylation, acylation, pegylation, etc.

Methods for determining the antifibrinolytic activity by determining the inhibition of piasmin formation (see example) are known to the person skilled in the art. Since activation of plasminogen to the proteolytic (fibrin-splitting) enzyme plasmin is the central step in fibrinolysis, piasmin formation may serve as a measure of fibrinolysis.

In this case, it is generally possible to proceed as described in the following exemplary embodiment: Plasmin formation is investigated in the presence of t-PA (tissue-specific plasmid gene activator). After addition of a suitable substrate, the enzymatic activity of the plasmid is determined in the experimental batch and compared with a corresponding control batch (without addition of an inhibitor). As "antifibrinolytic effect" is associated with The present invention means any reduction in plasmin activity relative to a corresponding control experiment without the addition of the protein to be tested (inhibitor).

The measurement of plasmin activity may, for example, by means of thromboelastography or thrombelastometry (Rotationsthromboelastometrie, RED ^®). In these standard methods known in the art, the coagulation properties of the blood are examined by viscoelastic measurements. The clot firmness (MCF) and the subsequent dissolution (lysis) of the clot can be determined. The lysis index (LI) indicates the decrease within 60 minutes after clot formation; it is thus a measure of the fibrinolytic effect (fibrinolysis).

In particular, an antifibrinolytic effect in the context of the present invention is present when the time required for the dissolution of a clot (under otherwise identical conditions) is at least 1.1 times, preferably 1.5 times the time required without addition of inhibitor, or if the mass of a clot remaining after a defined period of lysis (eg 60 min) corresponds to at least 1.1 times the mass, preferably 1.5 times the mass, of a clot which, without added inhibitor, is present during said lysis Period under otherwise identical conditions.

Proteins which can be used according to the invention and which have the sequences SEQ ID NO: 1 (dBPS1), SEQ ID NO: 2 (dBPS2), SEQ ID NO: 3 (meleagrin) or SEQ ID NO: 4 (similar to meleagrin), or corresponding variants a seguenzid At least 70% may be obtained, for example, as follows:

- By extraction or isolation of a naturally occurring biological material, in particular from the protein of bird eggs (preferably duck eggs). This can be done, for example, according to the method described by NAKNUKOOL (NAKNUKOOL et al., Supra, p. 2083, "Protein Purification"). According to this method, the protein diluted with water (from duck eggs) is adjusted to pH 6.0 by adding HCl, and the supernatant remaining after centrifugation is separated on an SP Sepharose column. Fractions with molecular masses less than 9.4 kDa are collected, dialysed (3500 MWCO) and then purified on Sephacryl S-300. The purity of the purified protein can be controlled by SDS-PAGE.

by means of genetic engineering methods (by operative linking of a nucleotide sequence coding for SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 with an expression control sequence of a suitable expression vector, transformation or transfection of suitable host cells with the recombinant vector, expression of the protein in the host cells, recovery of the expressed protein from the host cells or the culture medium).

- by chemical synthesis.

According to the present invention, the above-mentioned proteins can be used both individually and in combination. According to a preferred embodiment, a combination of proteins comprising a first protein comprising the amino acid sequence SEQ ID NO: 1 or an amino acid sequence having a sequence identity of at least 70% to SEQ ID NO: 1 and a second protein comprising the Amino acid sequence SEQ ID NO: 2 or an amino acid sequence having a sequence identity of at least 70% to SEQ ID NO: 2 comprises. Preferably, the molecular weight of the first protein and the second protein are each in the range of 4 to 10 kDa, in particular 4 to 6 kDa.

With regard to the relative proportions of the two proteins in the combination, it is preferred that the mass fraction of the second protein corresponds to at least 1.5 times, preferably at least twice, more preferably at least three times the mass of the first protein.

According to a particularly preferred embodiment, the combination of proteins according to the invention contains

dBPS1 (SEQ ID NO: 1) as said first protein and

dBPS2 (SEQ ID NO: 2) as said second protein.

The two proteins dBPS1 and DBPS2 can be present in equal or approximately equal proportions in the combination (that is, in the ratio 1: 1). According to a further preferred embodiment, the mass fraction of the second protein (dBPS2) is at least 1.5 times, preferably at least twice, more preferably at least three times the mass of the first protein (dBPS1).

According to a further preferred embodiment, the abovementioned combinations contain at least dBPS1 and dBPS2, these two proteins being isolated from the egg white of duck eggs.

The present invention further includes the possibility of using, instead of isolated or purified proteins, extracts or mixtures containing said proteins, alone or in combination.

Furthermore, the present invention also encompasses proteinaceous, antifibrinolytically active compositions (eg mixtures, extracts, partially purified fractions) which contain constituents derived from the egg whites of avian eggs, preferably from duck eggs, these constituents being at least one basic protein having a molecular mass of at most 15 kDa, in particular not more than 10 kDa,

for use in a method for antifibrinolytic treatment or for antifibrinolytic prophylaxis, preferably for the treatment or prophylaxis of blood loss occurring during or as a result of surgery, particularly preferably in open heart surgery and / or when using heart-lung machines.

The said proteinaceous compositions which can be prepared from the egg whites of bird eggs have an antifibrinolytic action which can be determined by means of a known method as described above. The preparation or isolation, for example, according to the

NAKNUKOOL et al., Supra, p. 2083, "Protein Purification".

Preferably, the basic protein contained in the proteinaceous compositions, or at least one the basic proteins, at least 4, in particular 6 cysteine residues on.

Furthermore, the isoelectric point of the protein (s) contained in the proteinaceous compositions is in each case greater than 7.0, preferably greater than 8.0, particularly preferably greater than 9.0.

The molecular weight of the basic protein (s) contained in the proteinaceous compositions is preferably in the range of 4 to 10 kDa, especially 4 to 6 kDa.

In a particularly preferred embodiment, the proteinaceous composition described above contains two of said basic proteins in combination, the molecular weight being 4 to 6 kDa each.

According to another preferred embodiment, the basic protein or proteins has an amino acid sequence as defined above with reference to SEQ ID NO: 1 to SEQ ID NO: 4.

The proteinaceous compositions described above, which are obtainable from the egg white of bird eggs, can be formulated as pharmaceutical formulations (medicaments) in a manner known to those skilled in the art.

The application of the above-defined protein (s), or said protein-containing composition, in a method for antifibrinolytic treatment or for antifibrinolytic prophylaxis is generally carried out in such a way that a protein of the invention or a Combination of proteins (as described above), or said protein-containing composition, by means of a suitable pharmaceutical dosage form, ie as a pharmaceutical preparation (= drug, medicament), is administered to a human or animal organism to be treated.

Suitable formulations, pharmaceutically acceptable excipients, carriers, vehicles, etc., are well known to those skilled in the art of pharmaceutical formulations. In particular, administration may be by oral, intravenous, intramuscular, intraperitoneal, subcutaneous or topical route of administration.

Particularly preferred are formulations for parenteral (eg, intravenous, intramuscular, intraperitoneal) administration, such as, for example, As injection and infusion solutions, suspensions, emulsions, lyophilisates, powder). As a vehicle for liquid formulations may, for. As water, isotonic solutions, saline solutions, dextrose solutions, etc. can be used. If necessary, stabilizers, preservatives, buffers, etc. may be added.

Accordingly, the present invention provides pharmaceutical compositions (ie, medicaments) containing a protein of the invention or a combination of proteins as defined above. Such a pharmaceutical preparation contains a therapeutically effective amount of a protein or a combination of proteins as described above together with a carrier or vehicle. Carriers or vehicles suitable for pharmaceutical preparations are known to the person skilled in the art. The active ingredient content of a medicament according to the invention is preferably in the range of 1-95% by weight, in particular 5-70% by weight. Depending on the particular dosage form, the intended dosage and the specific treatment purpose, the respective suitable active ingredient content may also be outside the stated percentage ranges. The term "active ingredient" refers to the above-defined proteins of the invention and combinations of proteins.

The above-defined proteins, as well as the preparations containing such proteins (medicaments) are provided according to the present invention for use in a method for antifibrinolytic treatment or for antifibrinolytic prophylaxis.

Particularly preferred is the use for the antifibrinolytic treatment or prophylaxis of blood loss occurring during or as a result of surgery, in particular in open-heart surgery and / or in the use of cardiopulmonary bypass machines (eg bypass surgery, heart valve surgery or heart transplantation) surgical interventions on the liver). In these cases, by administering the proteins according to the invention (as defined above) a reduction of the intraoperative blood losses can be effected, so that a smaller number of blood preserves is needed or can be completely dispensed with.

The amount of the dose to be administered depends, inter alia, on the coagulation status of the patient to be treated, the type and duration of the operation, the course of the operation, etc., and must be assessed individually in each individual case according to professional judgment. auxiliaries. If necessary, two or more doses may be administered in chronological order.

As the "therapeutically effective amount" of the protein (s) to be administered is meant an amount that results in a beneficial effect (for example, on intraoperative blood loss). This amount can vary considerably depending on a variety of factors, such as gender, age, body weight of the patient, and so on. The selection and, if necessary, adaptation of the appropriate dose is one of the abilities of the experts. As an orientation value for an "effective dose" may be assumed to range from 0.0001 to 100 mg / kg or 0.01 to 10 mg / kg (body weight).

In addition to the mentioned use for antifibrinolytic treatment or prophylaxis in operations, in particular in open-heart surgery and / or in the use of cardiopulmonary machines, the antifibrinolytic proteins or proteinaceous compositions according to the invention can be used in various other indications, such , As in local or generalized fibrinolysis, especially in local or generalized hyperfibrinolysis, for example, in severe trauma with major blood loss, in local bleeding in gynecology and obstetrics or in dentistry, as well as in traumatic hemorrhagic shock states.

The above-described anti-fibrinolytic proteins and proteinaceous compositions according to the present invention may further be used for the prophylaxis or therapy of disseminated intravascular coagulopathy (DIC; also called consumption coagulopathy). The present invention further relates to methods of inhibiting fibrinolysis comprising at least one step of administering to a subject or animal in need of antifibrinolytic treatment a therapeutically effective amount of a protein or a combination of proteins or a proteinaceous composition , each as defined above, is administered.

The present invention further relates to methods for the treatment or prophylaxis of a disease or condition associated with bleeding or blood loss and which require hemostatic treatment. Such a method according to the invention comprises a step of administering to a person or animal in need of hemostatic treatment a therapeutically effective amount of a protein or a combination of proteins, or a proteinaceous composition, each as described above.

In a preferred embodiment, said condition is a blood loss that occurs during or as a result of surgery, particularly in cardiac surgery and / or in the use of heart-lung machines.

According to another embodiment, the present invention relates to the use of a protein or a combination of proteins, or a proteinaceous composition, each as defined above, or a pharmaceutical preparation containing such a protein or combination, to inhibit or downregulate piasm formation without affecting the coagulation system, especially without interfering with thrombin generation.

This use is of importance for the therapeutic treatment of various diseases or disease states associated with increased or undesired fibrinolysis and which require inhibition or down-regulation of piasm formation without affecting the coagulation system.

The present invention relates primarily to the therapeutic or prophylactic treatment of humans, d. H. in the field of human medicine. Moreover, the present invention may also be advantageously used in veterinary medicine, for example for the treatment of livestock or pets, for the treatment of the same (or corresponding) diseases, or

Disease conditions as described above.

According to a further embodiment, the present invention relates to the use of a protein or a combination of proteins, or a proteinaceous composition, each as defined above, for laboratory medical purposes, in particular for the inhibition of fibrinolysis in laboratory medical test methods.

Furthermore, the present invention relates to a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (dBPSI), SEQ ID NO: 2 (dBPS2), SEQ ID NO: 3 (Meleagrin ) And SEQ ID NO: 4 (similar to meleagrin) and amino acid sequences which have a sequence identity of at least 70% to one of the aforementioned SEQ ID NOS: 1 to 4, or a grain at least two proteins, each having one of the abovementioned amino acid sequences, for use as a medicament. In particular, a protein or a combination according to one or more of the further or preferred embodiments described above is also suitable as said protein or as said combination of proteins.

The present invention further relates to a proteinaceous composition which contains constituents derived from the egg whites of avian eggs, preferably from duck eggs, which constituents comprise at least one basic protein having a molecular mass of at most 15 kDa, in particular not more than 10 kDa Use as a drug.

In particular, a protein-containing composition according to one or more of the further or preferred embodiments described above is also suitable as said protein-containing composition.

Formulations suitable for the preparation of the above medicaments according to the invention, pharmaceutically acceptable excipients, carriers, vehicles, etc., are well known to those skilled in the art of pharmaceutical formulations. Preferably, the medicaments of the invention are formulated as medicaments for oral, intravenous, intramuscular, intraperitoneal, subcutaneous or topical route of administration. Particularly preferred are formulations for parenteral (eg, intravenous, intramuscular, intraperitoneal) administration, such as, for example, As injection and infusion solutions, suspensions, emulsions, lyophilisates, powder). As a vehicle for liquid formulations may, for. Water, isotonic solutions, Saline solutions, dextrose solutions, etc. are used. If necessary, stabilizers, preservatives, buffers, etc. may be added.

Examples

The invention will be explained in more detail with reference to the examples described below:

In the experiments described below, a solution was used which contained the proteins "dBPS1" and ⁿ dBPS2 "in the ratio (w / w) of approximately 1: 3. This solution is referred to below as" dBPS ".

1) Inhibition of piasmin formation

Normal plasma (NP) obtained from healthy volunteers was treated with different concentrations (up to x g (ml)) of the protein combination "dBPS" according to the invention.

To study the fibrinolytic system, piasmin formation was measured, which serves as a suitable measure of fibrinolysis. Plasmin causes the cleavage of fibrin, as mentioned above.

Piasmin formation was assayed in the presence of t-PA (tissue-specific plasminogen activator, 200 U / ml). The enzyme t-PA causes the conversion of plasminogen into plasmin, which catalyzes the fibrinolysis.

The enzymatic activity of the plasmid was determined after addition of a suitable substrate.

FIG. 1 shows that the formation of plasmin by concentration of the proteins according to the invention (dBPS) is concentration-dependent. was inhibited compared to the control experiment "without dBPS".

2) No impairment of thrombin formation

To study the blood coagulation system, thrombin formation was measured which serves as a convenient measure of coagulation. Thrombin causes the formation of fibrin from soluble fibrinogen, as mentioned above.

Thrombin formation was carried out in such a way that calcium was added in excess to the previously citrated NP, in the presence of factor III (= tissue factor, concentration: 5 μm), coagulation-promoting phospholipids (4 μm) and a thrombin-suitable substrate.

Note: Factor III is a coagulation factor that initiates the formation of thrombin from prothrombin.

FIG. 2 shows that the formation of thrombin was not impaired by the addition of the proteins according to the invention (dBPS), and that these proteins therefore have no influence on the coagulation system, in particular bring about no anticoagulatory effect.

In further experiments, it was confirmed that trasylol (apatinin) acts as an anticoagulant in the test system described above under (2), as already known. This undesirable effect is presumably due to the fact that aprotinin has an inhibitory effect on a wide variety of proteases and therefore also inhibits various proteases involved in blood coagulation. sequence Listing

Sequence listing

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<222> (16) ...

<300>

<301> Naknukool, p. , Hayakawa, S., Sun, Y. and Ogawa, M.

<302> Structural and physicochemical characteristics of novel basic proteins isolated from duck egg white

<303> Biosci. Biotechnol. Biochem.

<304> 72

<305> 8

<306> 2082-2091

<307> 2008

<308> P85124 Swiss -Prot / üniProtKB

<309> 2007-04-17

<400> 2

Gin Val Arg Lys Tyr Cys Pro Lys Val Gly Tyr Cys Ser Ser Lys 1 5 10 15

Cys Ser Lys Ala Asp Val Trp Ser Leu Ser Ser Asp Cys Lys Phe

20 25 30

Tyr Cys Cys Leu Pro Pro Gly Trp Lys

35

<210> 3

<211> 40

<212> PRT

<213> Meleagris gallopavo

<220> <221> DISULFID

<222> (6) ... (

<220>

<221> DISULFID

<222> (12) ... (28)

<220>

<221> DISULFID

<222> (16) ... (32)

<300>

<301> Odani, S., Koide, T., Οηο, Τ., Takahashi, Y. and

Suzuki, J ..

<302> Covalent structure of a low molecular mass

protein, meleagrin, present in turkey (Meleagris gallopavo) ovomucoid preparation

<303> J. Bioc em.

<304> 105

<305> 4

<306> 660-663

<307> 1989

<308> P21376 Swiss-Prot / UniProtKB

<309> 1991-05-01

<400> 3

Gin Val Leu Lys Tyr Cys Pro Lys Ile Gly Tyr Cys Ser Ser Lys 1 5 10 15

Cys Ser Lys Ala Glu Val Trp Ala Tyr Ser Pro Asp Cys Lys Val

20 25 30

His Cys Cys Val Pro Ala Asn Gin Lys Trp 35 40

<210> 4

<211> 40

<212> PRT

<Gallus gallus

<300>

<308> XP 429908 GenBank / NCBI

<309> 2006-11-16

<400> 4

Val Leu Lys Tyr Cys Pro Lys Ile Gly Tyr Cys Ser Asn Thr Cys

5 10 15

Ser Lys Thr Gin Ile Trp Ala Thr Ser His Gly Cys Lys Met Tyr

20 25 30

Cys Cys Leu Pro Ala Ser Trp Lys Trp Lys

Claims

claims

A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1

(dBPS1), SEQ ID NO: 2 (dBPS2), SEQ ID NO: 3 (meleagrin), SEQ ID NO: 4 (similar to meleagrin) and amino acid sequences having a sequence identity of at least 70% to one of the aforementioned SEQ ID NOS: 1 to 4, or a combination of at least two proteins each having one of the abovementioned amino acid sequences,

for use in a method for antifibrinolytic treatment or antifibrinolytic prophylaxis.

2. protein or combination of proteins according to claim 1, characterized in that the sequence identity is at least 80%, preferably at least 90%, more preferably at least 95%.

3. protein or combination of proteins according to claim 1 or 2, characterized in that said amino acid sequence has at least 4, in particular 6 cysteine residues.

Protein or combination of proteins according to claim 3, characterized in that the protein (s) has / has two or three disulfide bridges.

5. protein or combination of proteins according to any one of the preceding claims, characterized in that the molecular weight is in each case in the range of 4 to 10 kDa, in particular 4 to 6 kDa.

6. protein or combination of proteins according to any one of the preceding claims, characterized in that the isoelectric point is greater than 7.0, preferably greater than 8.0, more preferably greater than 9.0.

7. A combination of proteins according to any one of the preceding claims, characterized in that it comprises a first protein having the amino acid sequence SEQ ID NO: 1 or an amino acid sequence having a sequence identity of at least 70% to SEQ ID NO: 1, and a second A protein comprising the amino acid sequence SEQ ID NO: 2 or an amino acid sequence having a sequence identity of at least 70% to SEQ ID NO: 2.

8. A combination of proteins according to claim 7, characterized in that the molecular weight of the first protein and the second protein is in each case in the range of 4 to 10 kDa, in particular 4 to 6 kDa.

A combination of proteins according to claim 7, characterized in that it contains as said first protein dBPS1 (SEQ ID NO: 1) and as said second protein dBPS2 (SEQ ID NO: 2).

10. A combination of proteins according to any one of claims 7 to 9, characterized in that the mass fraction of the second protein at least 1.5 times, preferably at least twice, more preferably at least three times the mass of the first protein corresponds.

11. Protein or combination of proteins according to any one of the preceding claims for antifibrinolytic treatment treatment, in particular for antifibrinolytic prophylaxis, in operations.

12. protein or combination of proteins according to any one of the preceding claims for antifibrinolytic treatment or for antifibrinolytic prophylaxis, in particular for the treatment or prophylaxis of a blood loss occurring during or as a result of surgery, preferably in open heart surgery and / or in the use of cardiopulmonary Machines, as well as for antifibrinolytic treatment in severe traumas with greater blood loss, in local bleeding in gynecology and obstetrics or in dentistry or in traumatic hemorrhagic shock states, as well as for the prophylaxis or treatment of disseminated intravascular coagulopathy.

Protein or combination of proteins according to any one of the preceding claims, characterized in that the protein or the combination of proteins is contained in a pharmaceutical composition.

14. Protein or combination of proteins according to any one of the preceding claims, characterized in that the / the protein (s) was prepared by genetic engineering methods from a host cell transformed or transfected with a recombinant expression vector (s).

15. protein or combination of proteins according to any one of the preceding claims, characterized in that the Protei (s) of animal material, in particular from the protein of bird eggs, isolated (n).

16. A pharmaceutical preparation which contains a protein or a combination of proteins according to any one of the preceding claims, for antifibrinolytic treatment or for anti-fibrinolytic prophylaxis, in particular for the treatment or prophylaxis of blood loss occurring during or as a result of surgery, preferably in open heart surgery and / or in the use of cardiopulmonary machines, as well as for antifibrinolytic treatment in severe traumas with greater blood loss, local bleeding in gynecology and obstetrics or in dentistry or in traumatic hemorrhagic states of shock, as well as for the prophylaxis or therapy of a disseminated intravascular coagulopathy.

17. Use of a protein or a combination of proteins according to any one of the preceding claims, or a pharmaceutical preparation containing such a protein or combination, for antifibrinolytic treatment or for anti-fibrinolytic prophylaxis, in particular for the treatment or prophylaxis of during or as a result an operation occurring blood loss, preferably in open heart surgery

and / or in the use of heart-lung machines, and for antifibrinolytic treatment in severe trauma with greater blood loss, in local bleeding in gynecology and obstetrics or in dental wedge or traumatic hemorrhagic shock states, as well as for the prophylaxis or therapy of a disseminated intravascular Coagulopathy.

18. Use of a protein or a combination of proteins according to any one of the preceding claims, or a pharmaceutical preparation containing such a protein in or containing such combination for inhibiting or downregulating piasm formation without affecting the coagulation system, in particular without interfering with thrombin formation.

19. A protein-containing composition which contains constituents derived from the egg whites of avian eggs, preferably from duck eggs, which constituents comprise at least one basic protein having a molecular mass of at most 15 kDa, in particular not more than 10 kDa, for use in a method for antifibrinolytic Treatment or for antifibrinolytic prophylaxis, in particular for the treatment or prophylaxis of occurring during or as a result of surgery blood loss, preferably in open heart operations and / or in the use of cardiopulmonary machines, and for antifibrinolytic treatment in severe trauma with major blood loss, in local bleeding in gynecology and obstetrics or in dentistry or in traumatic hemorrhagic shock states, as well as for the prophylaxis or therapy of disseminated intravascular coagulopathy.

20. The composition according to claim 19, characterized in that the basic protein or at least one of the basic proteins has at least 4, in particular 6 cysteine residues.

21. The composition according to claim 19 or 20, characterized in that the isoelectric point of the protein (s) is greater than 7.0, preferably greater than 8.0, more preferably greater than 9.0.

22. The composition according to any one of claims 19 to 21, characterized in that the molecular weight of the basic protein or the basic proteins in each case in the range of 4 to 10 kDa, in particular 4 to 6 kDa, is located.

23. A composition according to any one of claims 19 to 22, characterized in that it contains two of said basic proteins, wherein the molecular weight is 4 to 6 kDa each.

Composition according to any one of Claims 19 to 23, characterized in that the basic protein (s) has an amino acid sequence as defined in Claims 1 to 3.

25. A method of inhibiting fibrinolysis comprising the step of administering to a subject or an animal in need of an antifibrinolytic treatment a therapeutically effective amount of a protein or a combination of proteins according to any one of claims 1 to 15 or a composition according to any one of claims 19 to 24.

26. A method of treating or preventing a disease or condition associated with bleeding or loss of blood and which requires hemostatic treatment, the method comprising a step of administering to a person or an animal a hemostatic condition Treatment is required, a therapeutically effective amount of a protein or a combination of proteins according to any one of claims 1 to 15 or a composition according to any one of claims 19 to 24 is administered.

27. The method according to claim 26, characterized in that said disease is a generalized or local hyperfibrinolysis.

28. Method according to claim 26, characterized in that said condition is a blood loss which occurs during or as a result of a surgical procedure, in particular during cardiac surgery and / or when using heart-lung machines.

29. Use of a protein or a combination of proteins according to any one of claims 1 to 15 or a proteinaceous composition according to any one of claims 19 to 24, for laboratory medical purposes, in particular for inhibiting fibrinolysis in labormedizinischen test methods.

30. A protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1

(dBPSI), SEQ ID NO: 2 (dBPS2), SEQ ID NO: 3 (meleagrin), SEQ ID NO: 4 (similar to meleagrin) and amino acid sequences having a sequence identity of at least 70% to one of the aforementioned SEQ ID NOS: 1 to 4, or a combination of at least two proteins, each having one of the aforementioned amino acid sequences, for use as a drug.

31. Protein or combination of proteins for use as a medicament according to claim 30, characterized in that the protein or the combination of proteins in a ner embodiment according to one or more of claims 2 to 15 is present.

32. A protein-containing composition comprising constituents derived from the egg whites of avian eggs, preferably from duck eggs, said constituents comprising at least one basic protein having a molecular mass of at most 15 kDa, in particular of at most 10 kA,

for use as a medicine.

33. Composition for use as a medicament according to claim 32, characterized in that the composition is present in one embodiment according to one or more of claims 20 to 24.