GB2431655A

GB2431655A - Antagonisation of anticoagulants

Info

Publication number: GB2431655A
Application number: GB0522644A
Authority: GB
Inventors: Anna Perchuc; Marc Heidl; Beatrice Buhler; Reto Schoni; Marianne Wilmer; Reto Stocklin; Laure Menin
Original assignee: Pentapharm AG
Current assignee: DSM Nutritional Products AG
Priority date: 2005-10-27
Filing date: 2005-10-27
Publication date: 2007-05-02
Also published as: GB0522644D0; WO2007066179A2; WO2007066179A3

Abstract

The present invention relates to new peptides of the general formula (I) R<1>-(B<1>)m-(B<2>)n-Tyr-(B<3a>)o(-B<3b>)p-Asnq-Asnr-Tyrs-Leut-B<4>-(Pro)u-Phe-Abu-B<5>-(B<6>)v-R<2>```(I) wherein R<1> represents hydrogen or (C1-C8)-acyl, R<2> represents OH, O-(C1-C8)-alkyl, NH2 or Ala-Asp-Pro-OH, B<1> to B<6> represent, independent of one another, a basic amino acid, such as arginine, homoarginine, lysine, ornithine, 2,4-diaminobutyric acid or 2,3-diaminopropionic acid m,n,o,p,q,r,s,t,u,v represent, independent of one another, zero or 1, and their salts as well as pharmaceutical compositions characterized by containing a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNNYLKPFCKK. The peptides and the pharmaceutical compositions can be used for the antagonisation of heparins, LMWH, pentasaccharide, danaparoid or other related anticoagulants. Reagent kits for in vitro analysis of blood or blood components are also described.

Description

New method for antagonisation of anticoagulants

Summary

Heparins and heparin related compounds are abundantly used in medicine in order to form an immediately acting inhibition of anticoagulation. This might be the case in order to treat a thromboembolic disorder, to prevent thromboembolism in a patient with elevated risk or in order to perform intervention during which blood is coming into contact with artificial surfaces (catheters, stents, cardiopulmonary bypass devices) which would induce clotting of blood and thus lead to embolism or clotting of the extracorporeal device.

In many situations it is desired to antagonize the inhibiting effect of heparins or heparin-related compounds. This may be in order to analyze in vitro blood clotting in a patient who is treated with heparins, e.g. for performing thromboelastography or thromboelastometry in patients during heart surgery. Another situation is the treatment of bleeding in a patient receiving heparins. In this situation one wants to end the heparin effect in order to restore blood coagulation capability to end the bleeding complication. A third option is the requirement to end the anticoagulation at the end of procedures that require anticoagulation of the patient, e.g. at the end of heart surgery. In this situation the contact of blood with the artificial surfaces of the cardiopulmonary bypass device ends, therefore the anticoagulation of the patient is not any more required. Therefore it is useful to reverse the anticoagulation which also reduces the risk of bleeding leading to transfusion requirements or even potentially to the need of surgical reexploration of patients.

The typical strategy for reversing the anticoagulant effect of heparin and related compounds is the use of protamine sulfate or hydrochloride. However protamin sulfate or hydrochloride has many shortcomings: as a relatively large animal-derived * * product it can induce severe allergic complications. In addition its potential to reverse :. smaller glycosaminoglycans such as low molecular weight heparin (LMWH) is limited. Is..

Another strategy is the use of polybrene and other polycationic compounds. However * polybrene due to its toxicity is not applicable in vivo and also for in vitro use it has :: several shortcomings: polybrene has a limited solubility and stability in vitro and thus cannot reverse large amounts of heparin (more than 2 U/mI).

Another strategy is the use of recombinant PF4, which has been proposed several : years ago. The production of this endogenous and quite large compound is expensive as it requires recombinant technology. It is not clear which effects PF4 has in vivo besides heparin neutralization and therefore the safety of the transfusion of large amounts might be dangerous.

Another strategy is the use of heparinase, an enzyme of bacterial origin which cleaves heparins to smaller chains. Heparinase again is a larger protein of bacterial origin which may induce an immune response and therefore either shorten half-life in repeated applications or induce allergic complications. In addition for the antagonisation of unfractionated heparin it must be noted that during the process of cleaving the large chains of unfractionated heparin (UFH) to smaller chains, heparinase produces transiently large LMWH, which may cause significant bleeding complications, as one molecule of unfractionated heparin (UFH) may be transiently converted in several molecules of LMWH. In this context it is important to note that heparinase is being used for the production of LMWHs from unfractionated heparin (UFH) in the pharmaceutical industry.

Another strategy which has been proposed for the antagonisation of heparins and related compounds is the use of low molecular weight fragments of protamine. The published data however has shown that low molecular weight protamin could be not totally devoid of any protamin related responses. Probably due to those problems it is still not present at the market as a heparin inactivating drug.

In conclusion an optimal strategy for the inhibition of heparins has not yet been found. During the analysis of the snake venom of Bothrops moo] eni we found that some phospholipases A2 bind and inactivate very effectively heparin. In addition we found that small synthetic peptides derived from the sequence of those phospholipases A2 also possess this property. It has been found that not only unfractionated heparin (UFH) but also the much smaller LMWH can be inhibited by the use of the synthetic peptides. Based on the identified heparin-binding sequence of PLA2 several groups of synthetic peptides could be derived and applied for heparin antagonisation.

Background

The present invention is concerned with the process of blood coagulation, more precisely with process of inhibition of blood coagulation and its antagonisation.

The serine proteinase inhibitor antithrombin III (ATIII) is the most important plasmatic inhibitor of activated coagulation factors. It is a key regulatory protein in the intrinsic pathway of blood coagulation. Its most important targets are free FXa, thrombin, FXIa, FXIIa and FIXa. ATIII attains its full biological activity upon binding poly-sulfated glycosaminoglycans, such as heparin [1]. Heparin catalyzes the ::::. interaction between ATIII and the coagulation factors, accelerating complex formation by 1000-fold.

S..... Heparin accelerated binding of ATIII to serine proteases of blood coagulation is mediated through a reversible binding interaction. Heparin is not consumed by this : interaction and, following formation of the ATIII-coagulation factor complex, heparin dissociates from the antithrombin molecule and is able to repeat the activation cycle, thus acting as a true catalyst [2].

The binding site for ATIII in heparin molecules is a sequence of several acidic sugar molecules. The shortest active chain is a pentasaccharide with a distinct pattern of carboxy and sulphate groups on the glycoside backbone. The presence of * the critical pentasaccharide unit is sufficient to mediate accelerated inactivation of factor Xa by ATIII. This domain alone is, however, not adequate to catalyze ATIII inactivation of factors IXa, XIa and thrombin.

Heparin, when administrated as a drug, enhances the inhibitory properties of ATIII, which can result in potent systemic anticoagulation. Heparin is used to treat patients with established thrombosis and to prevent the occurrence in individuals known to be at high risk. It is also used as an adjunct to fibrinolytic therapy.

However the unfractionated heparin (UFH), used as an anticoagulant drug, is characterized by highly variable pharmacokinetics. Bleeding risk as a consequence of anticoagulant overdosage necessitates monitoring of the patient's condition and an antidote reversing the strong anticoagulant effects is needed.

The use of other anticoagulants such as LMWHs does not require routine monitoring of the anticoagulant effect, as the pharmacokinetics are usually less variable. LMWH, containing only a part of the glycosaminoglycan heparin chain, is less active than unfractionated heparin (UFH) and it's use is often considered to be safer. But also with LMWH bleeding complications may occur under anticoagulant treatment, as suspected or demonstrated in cases of impaired clearance of the drug (e.g. due to renal dysfunction), unusual pharmacokinetics (e.g. children or strongly obese patients) or suspected or potential over-dosage.

Thus an antidote is needed to reverse the effects of heparin in the setting of cardiac catheterization, cardiac surgery, hemodialysis, or if serious bleeding occurs, during heparin anticoagulation [3]. Protamine sulfate or protamin hydrochloride have been used for many years to reverse the effects of heparin, but although being highly effective, due to their antigenicity they can cause hemodynamic changes and other serious side effects. Mixon and Dehmer reported that mild protamine reactions occur in up to 16% whereas the incidence of severe reactions varies from 0.2% to 0.3% in the overall population. Severe protamine reactions were reported in up to 27% of patients previously treated with neutral protamine Hagedorn insulin in one study, but a considerably lower incidence was found in other studies [3]. Apart of those adverse effects the complete antagonization of LMWH by protamine can not be achieved.

Thus, the ideal heparin antidote that most clinicians would prefer is a compound that provides all the advantages of protamine, yet lacks anaphylactic potential and preserves hemodynamic stability when being infused [4]. An efficient inhibition of LMWH would be also required.

State of the art Protamine (sulphate or hydrochloride) Protamine has been used for many years to reverse the anticoagulant effects of unfractionated heparin (UFH) and to alleviate heparin-induced bleeding risks.

::. The mechanism of heparin neutralization by protamine has been thoroughly investigated. It competes with antithrombin III (ATIII) for binding to heparin and due to ** its stronger affinity to heparin, protamine dissociates ATIII from the heparin-ATIII complex, reversing the anticoagulant function of heparin [5]. It has been used in the :": settings of cardiac catheterization, cardiac surgery, hemodialysis, or if serious bleeding occurs, during heparin anticoagulation [3].

Protamine consists of a family of heterogeneous and highly cationic proteins, obtained from fish sperm. It is a low molecular weight (4.5 kDa) protein rich in basic : amino acids such as arginine (nearly 67%). It interacts by high-affinity ionic binding (electrostatic interaction) with the poly-anionic unfractionated heparin (UFH) * 1 molecules, thereby counteracting their anticoagulant effect. Paradoxally it also prolongs activated partial thromboplastin time (aPTT), when given in excess. This effect may be due to direct interactions of protamine with clotting factors, particularly th rombi n.

Although protamine is highly effective, it is associated with rare but clinically significant adverse reactions, e.g. fatal cardiovascular responses [3], [6]. The combined use of heparin and protamine has been suggested as the major cause of morbidity and mortality by patients undergoing cardiopulmonary bypass [5]. Minor adverse reactions include: pruritus, flushing, urticaria, nausea, leukopenia and thrombocytopenia, while severe ones include: bronchospasm, elevated pulmonary arterial pressure, pulmonary edema, hypotension, cardiac arrest and circulatory collapse, which has a very high mortality rate [3].

Three types of reactions to protamine have been described [3]: I hypotension due to rapid administration, which can be, however, avoided by slow administration of protamine, 2. anaphylactic responses due to antibody production, which are rather unpredictable, not preventable, and always life-threatening, 3. catastrophic pulmonary vasoconstriction of unknown etiology.

The mechanisms of protamine-induced toxicity are complex and not yet completely understood. Available data indicate that severe adverse reactions could be mediated by the immune response against protamine as a non-human protein.

Like any foreign protein, protamine possesses immunogenic potential. The life threatening protamine induced toxicity by means of immunologic mediated pathway is attributed to the immunogenicity and antigenicity of protamine, which are the two principal events of protamine-induced immunotoxicity (protamin allergy) [5].

Low molecular weight protamine It is well recognized that small peptides with molecular weight in the range of 1.5 kDa or below are usually either weak or completely devoid of immunogenicity. It was concluded that small peptidic fragments derived from protamine by chemical or enzymatic digestion might be devoid of, or at least possess markedly reduced, immunogenicity and antigenitcity [5], [6]. On the other hand the effective binding of protamine to the pentasaccharide sequence in heparin, and displacement of the complexed ATIII by means of an ionic interaction, may not require the whole protamine molecule, but rather a small fragment encompassing an intact arginine rich sequence, for favourable electrostatic interaction [4], [5].

Thus, low molecular weight protamine (LMWP) fragments containing an intact :,:::. arginine sequence were prepared by enzymatic digestion of protamine by thermolysin (the protease that does not cleave the arginyl bonds). Their average . molecular weight ranged from 0.7 to 1.9 kDa. The ability of these fragments to neutralize the anticoagulant functions of unfractionated heparin (UFH) and low :: molecular weight heparin (LMWH) was studied [4],, [5], [6], [7].

A typical structural scaffold made by arginine clusters in the middle and non-arginine residues at the N-terminal of the peptide sequence was observed for all the .. : fragments, which were found to retain the complete heparin-neutralizing function of : protamine. It was found that retention of potency similar to that of protamine required * the presence of at least two arginine clusters (each containing 4 to 6 arginine residues) in the LMWP fragments; such as the sequence of VSRRRRRRGGRRRR (molecular weight of 1.88 kDa) . It was found to be essential to achieve a binding affinity strong enough to completely neutralize heparin. That finding was further validated by using a synthetic LMWP analogue, an octapeptide, CRRRRRRR (CR7), and it was found that its heparin-neutralizing ability was increased by changing from a monomeric to a dimeric structure of this analogue peptide.

Different experiments were performed to compare the ability of protamine, LMWP and its synthetic analogues to neutralize heparin[4], [5], [6], [7]. --4

The in-vitro efficacy was examined using an anti-Xa chromogenic assay. It was shown that LMWP was less potent than protamine, nevertheless it competed effectively with ATIII in binding to heparin.

Recombinant Platelet Factor 4 (rPF) [3] Platelet factor 4 (PF4) is a naturally occurring protein comprising 70 amino acids with a molecular weight of 7.8 kDa. It is synthesized in megakaryocytes and eventually stored for later release in the granules of platelets.

Under normal conditions PF4 released from circulating platelets immediately attaches to the heparan sulphate exposed on the endothelial cell surface. But PF4 plasma levels increase 10-to 30-fold after heparin administration, when PF4 bound to endothelial cell surface is freed.

Although the physiological role of platelet factor 4 (PF4) is not yet fully understood, it is confirmed that it binds to heparin and thereby effectively antagonizes heparin anticoagulation.

Complete neutralization of heparin was achieved with twice the concentration of rPF4 compared with protamine.

The major clinically significant adverse reactions caused by protamine after its administration to a heparinized patient were related to protamin-heparin complexes rather than to the protamine alone. Since the PF4 interaction with heparin is quite different, it does not cause complement consumption, haematological or hemodynamic abnormalities, nor would it likely cause an immunologic response.

However, it has been demonstrated that indeed antibodies, directed against the heparin-PF4 moiety, do form and are more important in the syndrome of heparin-induced thrombocytopenia.

Neither human study showed serious adverse effects related to rPF4; however several possibilities should be considered.

PF4 has been cloned by gene technology and is available as a recombinant full-length, human-sequence protein and the rPF used for the study was produced by : ***. recombinant DNA methods using Escherichia co/i as the source of the recombinant *0* protein. The amino acid structure of rPF is identical to that of naturally occurring PF4 ". from platelet a-granules, but proteins made in bacteria are not glycosylated and could have altered secondary structure leading to neoantigen formation.

rPF4, obtained by an expensive recombinant technology, has also been mentioned as a cofactor in the syndrome of heparin-induced thrombocytopenia.

Although rPF4 was initially being evaluated as a clinical alternative to protamine, it is not currently being developed for general clinical use. * S *

Heparinases [3] Heparinases are enzymes that selectively cleave, via an elimination mechanism, highly sulfated polysaccharide chains containing 1-4 linkages between hexosamines and 0-sulfated iduronic acid residues. They are heparin-degrading enzymes that cleave certain sequences of heparin/heparan sulfate specifically. They are extracted from the gram-negative soil bacterium, Flavobacterium heparinum and have also been purified and used for technical as well as therapeutic purposes.

Heparinase-l (Neutralase TM IBEX Technologies, Montreal, Quebec, Canada) -lyses heparin and its a-glycosidic linkages. Heparinase-Ill (IBEX Technologies, Inc., Montreal, Quebec, Canada) degrades heparan sulphate, a related compound. Animal studies demonstrated that heparinase-l normalized ACT levels increased by heparin application without significant hemodynamic effects.

Heparinases, however, obtained by a recombinant technology are expensive.

They can also degrade unfractionated heparin (UFH) causing the formation of LMWH and increasing the anticoagulant effects.

Synthetic Peptides WO 2005/014619 describes heparin binding peptides for administering to reduce plasma LMWH and heparin levels and to reduce the anticoagulant effects of heparin and LMWH. These peptides have a high molecular weight and are therefore not only more expensive but show also a tendency to unsufficient biological availability.

Summary of the invention

Surprisingly we found that some components of the venom of the Brasilian Lancehead snake Bothrops moojeni could inhibit the anticoagulant action of unfractionated heparin (UFH) in plasma. Those components were then purified and amino acid sequences of two new phospholipases A2 (PLA2s) have been determined.

Thus few peptides, representing the heparin binding site of the new PLA2s (characteristic 115-129 amino acid fragment of the C-terminal region), have been synthesized.

The whole protein, as well as the synthetic peptides, have been shown to bind in vitro heparin in plasma inhibiting its anticoagulant activity.

Description of the invention

A new Lys49 phospholipase A2 was found in a one of the HPLC fractions obtained during specific fractionation and purification of Bothrops moojeni crude : venom. The following sequences have been identified: 20 30 40 * I

SLVELGKMIL QETGKNPVTS -YGAYGCNCG VLGRGKPKDA

60 70 80

TDRCCYVHKC CYK---KLTD C NPKK DRYSYSWKDK

100 110 120

TIVC-GENNS CLKELCECDK AVAICLRENL DTYNKKYKNN

I

SI....

* 130 140

YLKPFCKK-A DPC

20 30 40

SLVELGKMIL QETGKNPLTS -YGAYGCNCG VGGRGKPKDA

60 70 80

TDRCCYVHKC CYK---KMTD C DPKK DRYSYSWKDK

100 110 120

TIVC-GENNS CLKELCECDK AVAICLRENL DTYNKKYKNN 140

YLKPFCKK-A DPC

The heparin binding structure is suggested to be as follows:

KKYKNNYLKPFCKK

Based on the above sequence and possible modifications (amino acids content and/or three dimensional structure) different peptides were synthesized (table 1).

Their ability to neutralize unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was assessed in vitro.

According to that aspect, the invention provides a phospholipase A2 and synthetic analogs corresponding to the general formula (I), R1(B1)m(B2)nTyr(B3a)o( B3b)pAsnqAsnrTyrsLeutB4(Pro)uPheAbuB5(B6)vR2 (I) wherein R1 represents hydrogen or (C1-C8)-acyl, R2 represents OH, O-(C1-C8)-alkyl, NH2 or Ala-Asp-Pro-OH, B1 to B6 represent, independent of one another, a basic amino acid, such as arginine, homoarginine, lysine, ornithine, 2,4-diaminobutyric acid or 2,3-diaminopropionic acid, m,n,o,p,q,r,s,t,u,v represent, independent of one another, zero or 1, as well as pharmaceutically acceptable salts of these compounds, which surprisingly interact in vitro with unfractionated heparin (UFH) as well as with low molecular weight heparin (LMWH) neutralizing their anticoagulant properties.

* The compounds of formula (I) together with acids can form mono-or polyvalent, :.. homogeneous or mixed salts, e.g. with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; or with appropriate organic aliphatic saturated or unsaturated carboxylic acids, e.g. aliphatic mono-or dicarboxylic acids, such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic :: acid, propionic acid, glycolic acid, succinic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, phthalic acid, citric acid, lactic acid or tartaric acid; or with aromatic carboxylic acids, such as benzoic acid or salicylic acid; or with aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid; or with heteroaromatic carboxylic acids, such as nicotinic acid; or with aliphatic or aromatic sulfonic acids, such as methanesulfonic acid or toluenesulfonic acid. Pharmaceutically tolerated * salts, in particular salts with acetic acid and/or lactic acid, are preferred.

The amino acids mentioned in formula (I) can have a L or D configuration, or represent a mixture of both configuration.

Preferred compounds of formula (I) are those wherein R1 represents hydrogen or acetyl, or R2 represents OH or Ala-Asp-Pro-OH, or B1 to B6 represent, independent of one another, arginine or lysine.

The following examples of the table I illustrate the invention without limiting its scope. 1 7

Sample Compound MW [glmol] 20-004E Ac-KKYKNNYLKPF-Abu-KK ADP-OH *6TFA 2793.66 20-005E AcKKYKNNYLKPFAbuKKOH*6TFA 2510.38 20-019 H-KKYKNNYLKPF-Abu-kKAbP-OH *7TFA 2865.65 20-020 H-KKYKNNYLKPF-Abu-KK-OH *7TFA 2582.36 20-021 Ac-KKYKNNYLKPF-Abu-KK-OH *6AcOH -2186.53 20-022 H-KKYKNNYLKPF-Abu-KK ADPOH*7AcOH _____ 2487.83 20-023 H-KKYKNNYLKPF-Abu-KK-OH *7AcOH --2204.54 20-024 H-KKYKNNYLKPF-Abu-KK-OH *7HCI -2039.41 20-025 H-KKYKNNYLKPF-Abu-KKADP-OH *7HCI 2322.7 20-062 H-KKYKNNYLKF-Abu-KK-OH *7ACOH 2107.45 20-063 H-KKYKNNYLKF-Abu-KK-OH *7HCI -1942.30 20-064 HYKNNYLKPFAbuKKOH*5AcOH __________ 1828.11 20-065 H-YKNNYLKPF-Abu-KK-OH *5HCI _____ 1710.15 20-066 H-KYKNNYLKPF-Abu-K-OH *5ACOH --______ 1828 11 20-067 H-KYKNNYLKPF-Abu-K-OH *5HCI __________ 1710.15 20-069 H-KKYKNNYLKPF-Abu-KKADP-OH *7AcOH 2487.85 20-070 H-KKYKNNYLKPF-Abu-KKADP-OH *7HCI --____ 2322.71 20-071 H-KKYKNNYLKPF-Abu-KK-OH *7ACOH -2204.57 20-072 H-KKYKNNYLKPF-Abu-KK-OH *7HCI --_______ 2039.42 20-073 H-RRYKNNYLKPF-Abu-KK-OH *7ACOH 2260.59 20-074 H-RRYKNNYLKPF-Abu-KK-OH *7HCI --2095 45 20-080 H-KRYKNNYLKPF-Abu-RK-OH *7AcOH -2260.59 20-081 H-KRYKNNYLKPF-Abu-RK-OH *7HCI 2095.45 20-082 H-KKYKKF-Abu-KK-OH *7ACOH --1602.90 20-083 H-KKYKKF-Abu-KK-OH *7HCI ___ 1437.76 20-134 -H-KKYKNNYKF-Abu-KK-OH *7AcOH -1994.29 20-1 35 H-KKYKNNYKF-Abu-KK-OH *7HCI 1829.14 *.S. _________________________________________________________________________ _ _______________ - 20-136 H-KKYKNNKF-Abu-KK-OH *7AcOH 1831.11 20-1 37 H-KKYKNNKF-Abu-KK-OH *7HCI -___ 166597 20-138 -H-KKYKNKF-Abu-KK-OH *7AcOH _________ 1717.01 20-139 H-KKYKNKF-Abu-KK-OH *7HCI _________ 1551.86 20-140 -H-KKYKKNYKKF-Abu-KK-OH *9AcQH 2310.61 20-141 -H-KKYKKNYKKF-Abu-KK-OH *9HCI 2044.31 ::: : Tab.1 Experiments performed The following abbreviations are used in the text and in Examples 1-4: Abu 2-amino-butyric acid Ac acetyl AcN acetonitrile AcOH: acetic acid Ala alanine Asn asparagine Asp aspartic acid Boc: tert.-butyloxycarbonyl DMF dimethylformamide DBU: 1,8-diazabicyclo[5]undec-7-ene(1,5-5) DIPEA: diisopropylethylamine Fmoc: fluorenylmethyloxycarbonyl Hyp: hydroxyproline Gly: glycine Leu leucine Lys lysine Phe phenylalanine Pro: proline RT: room temperature TBTU: O-(benzotriazol-1 -yI)-N, N, N', N'-tetramethyluronium-tetrafluoroborate tBu: tbutyl tert.-butyl TEA: trifluoroacetic acid Trt trityl Tyr tyrosine

Examples 1-4:

The following embodiments 1-4 describe the synthesis of the compounds of formula (I) of the present invention and of salts of such compounds. The eluates and products obtained according to the examples are analysed using HPLC-electrospray MS. The compounds can be manufactured according to known methods described hereinafter (general instructions from M. Bodanszky "The Practice of Peptide Synthesis", Springer, 2nd Edition, 1994). Accordingly, the suitable protected amino acid, e.g. Fmoc-Lys(Boc)-OH, is bound to a resin at the carboxy terminal end in a solid-phase synthesis. The side chain is protected with, e.g., Boc or t-butyl. If necessary, the protective groups are selectively split off in order to link up the further amino acid derivatives with the reagents commonly used in peptide synthesis until the desired chain is completely built up. Afterwards, the peptide or peptide analogue, respectively, is split off from the resin at the carboxy terminal end with simultaneous :. removal of all protecting groups. e.

Example 1: H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-OH *7TFA; H-KKYKNNYLKPF-Abu-KK-OH *7TFA 11.1: In a typical solid-phase synthesis protocol, the tetradecapeptide is obtained by repetitive coupling of 1.50 g (1.13 mmol, capacity: 0.75 mmol/g) of commercial available H-Lys(Boc)-2-chlorotrityl resin with 2.0 mmol of the amino acids Fmoc-Lys(Boc)-OH, Fmoc-Abu-OH, Fmoc-Phe-OH, Emoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH (2x), Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH (2x), 2.1 mmol TBTU, 4.2 mmol DIPEA and unblocking with 5% DBU in DMF (2 x 8 mm). The Peptide is cleaved from the resin with simultaneous removal of all protecting groups by 95% TEA. The solution is added dropwise to 250 ml of an ice-cooled 1:1 mixture of diethylether and petroleum ether followed by HPLC purification. Yield: 172mg (66.6 tmol, 5.9%).

Example 2: Ac-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-O H *6TFA; Ac-KKYKNNYLKPF-Abu-KK-OH *6TFA In a typical solid-phase synthesis protocol, the tetradecapeptide is obtained by repetitive coupling of 1.20 g (0.90 mmol, capacity: 0.75 mmol/g) of commercial available H-Lys(Boc)-2-chlorotrityl resin with 2.0 mmol of the amino acids Fmoc-Lys(Boc)-OH, Fmoc-Abu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH (2x), Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH (2x), 2.1 mmol TBTU, 4.2 mmol DIPEA and unbiocking with 5% DBU in DMF (2 x 8 mm). With the peptide chain fully assembled, the resin is treated with an acetylating mixture (8m1 DMF, imi Pyridine and I ml acetic anhydride) for 5 minutes. The Peptide is cleaved from the resin with simultaneous removal of all protecting groups by 95% TFA. The solution is added dropwise to 250 ml of an ice-cooled 1:1 mixture of diethylether and petroleum ether followed by HPLC purification. Yield: 116mg (46.1 j.imol, 5.1%).

Example 3: H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-OH *7 AcOH; H-KKYKNNYLKPF-Abu-KK-OH *7AcOH 23.1 mg (8.9 jtmol) of H-KKYKNNYLKPF-Abu-KK-OH *7TFA described in Example 1, is dissolved in 20m1 of an 1:1 mixture of AcN: water and treated with 1.Og of Biorad Ion exchange resin in acetate form for two hours. The resin is filtered off, the solution lyophylized. Yield 17.1 mg (7.8 jtmol, 87%).

Example 4: H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-O H *7 HCI; H-KKYKNNYLKPF-Abu-KK-OH *7HCI 23.7 mg (9.2 imol) of H-KKYKNNYLKPF-Abu-KK-OH *7TFA described in Example 1, is dissolved in 2Oml of an 1:1 mixture of AcN: water and treated with 1.Og of Amberlite IRA 400exchange resin in chloride form for two hours. The resin is filtered off, the solution lyophylized. Yield 14.8 mg (7.3.tmoI, 79%).

In a similar manner the compounds of the general formula (I) and those of the table 1 can be prepared.

In preliminary screening using FXa chromogenic substrate the following sequence was found to exhibit the best neutralizing properties against unfractionated heparin (UFH) and LMWH. Thus it was chosen for further experiments.

:.". Samples r Sequence MW [gimol] name 20-062 H-KKYKNNYLKF-Abu-KK-OH *7AcOH 2107.45 Example 5: Chromocienic test.

Materials: * a.

FXa Hyphen BioMed; France FXa chromogenic substrate Pefachrome FXa; Pentapharm Ltd.; Basel, Switzerland Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England (WHO Standard 97/578) LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England Antithrombin Ill Grifols Germany; Siemensstrasse, 18;D-63225 Langen/Hessen Protamine hydrochloride ICN Pharmaceuticals Germany GmbH; Frankfurt/Main, Germany Synthetic peptide Pentapharm Ltd.; Basel, Switzerland Purified PLA2 Athens Laboratories; Geneva, Switzerland I -. 10 The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 50mM Hepes buffer, pH 7.5. unfractionated heparin (UFH) and LMWH were used in a concentration of 0.5 U/mI, FXa -in a concentration of 2 jig/mI, FXa chrornogenic substrate -in a concentration of 4mM and antithrombin Ill (ATIII) -in a concentration of 3.75 U/mI. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/mI. The native, purified phospholipase A2 from B. moojeni venom (130 jig/mI), was also included in that test. 50mM Hepes buffer, pH 7.5 was used as a control.

In the following tables/descriptions peptides and purified phospholipase A2 are assigned as samples and protamine hydrochiorid as reference. Following measurements were performed: Sample! 3 mm FXa ____________ UFH reference Buffer incubation AT FXa Substrate Control without UFH/LMWH without -180 p1 3 mm 10 p1 20 p1 peptide/reference _______ __________ _______ _______ _____ Control with UFH/LMWH without 10 p1 160 p1 3 mm lOpl lOpl 20 p1 peptide/reference ______ __________ _______ __________ _______ _______ Sample with UFH/LMWH, with 10 p1 10 p1 150 p1 3 mm 10 p1 10 p1 20 p1 peptide/reference ______ __________ _______ __________ _______ _____ ____ Example 6: KC4 A micro ball coaqulometer -neutralization of unfractionated heparin * (UFH) and LMWH, comparison of the properties of the native PLA2, synthetic peptide * 20-062 and protamine hydrochloride. Is.. * S

After the preliminary tests, using a non-physiological system of FXa and its chromogenic substrate, measurements were performed in plasma, on the KC4 A *: : micro ball coagulometer.

Materials: Is . : PiCT activator Pentapharm Ltd.; Basel, Switzerland Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England (WHO Standard 97/578) LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England Protamine hydrochloride ICN Pharmaceuticals Germany GmbH; Frankfurt/Main, Germany CaCI2 HemosiL, Instrumentation Laboratory; Milano, Italy Synthetic peptide Pentapharm Ltd.; Basel, Switzerland Purified PLA2 Athens Laboratories; Geneva, Switzerland The experiments were performed as follows: Clotting was triggered using the PiCT test, which is described in detail in EP1240528). The test employs a mixture of FXa and RW-V (FV activator from Daboia russelii russelii venom) in combination with phospholipids. Plasma was spiked with 0.5U/mI unfractionated heparin (UFH) or LMWH.

Plasma was incubated 180s with all the needed reagents and then recalcified with CaCI2 used in a concentration of 25 mM. The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 50mM Hepes buffer, pH 7.5. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/mI and the native phospholipase A2 from B. moojeni venom -in a concentration of 130 tg/ml. 50mM Hepes buffer, pH 7.5 was used as a control.

tl Plasma with or without UFH/LMWH tl Sample or reference jil PiCT activator 3 mm. Incubation at 37 C tl CaCl2 to start coagulation The clotting times (CT) obtained are presented in the table 2.

CT in % (plasma CT in % (plasma CT in plasma with UFH) CT in plasma with LMWH) Samnle name with UFH compared to with LMWH compared to normal CT in normal CT in (sec) plasma without (sec) plasma without

____________ _____ _____ UFH ___________ LMWH

Control Buffer 125.10 406 133.90 461 Protamine 35.25 114 69.60 240 B. moojeni PLA2 71.25 231 139.05 479 Synthetic peptide (20-062)0.25 35.30 114 55.20 190 mM _____ ___________ ________ ___ ______ ___ Synthetic peptide 38 05 123 54 20 187 2O-062) 0.5 mM. -______________ ______________ Tab.2 * CT measured in plasma without unfractionated heparin (UFH) (30.85s) represents 100% of CT for UFH measurements.

** CT measured in plasma without LMW (29.05s) represents 100% of CT for LMWH : .. measurements. IS.. S...

... Example 7: KC4 A micro ball coaqulometer -neutralization of 1U LMWH, comparison of the properties of the synthetic peptide 20-062 and protamine h' hydrochloride.

Experiments were performed as in the example 2. LMWH was added to plasma to obtain an end concentration of 1 U/mI. The obtained results are shown in the figure 3. * I. * S * S. * I*.. -12

Example 8: Automated Coagulation Test System BCS (Behring Coagulation System).

The Automated Coagulation Test System BCS was used to evaluate the properties of the tested substances to neutralize the anticoagulant activity of unfractionated and low molecular weight heparin. PiCT assay was performed as described in example 2.

Materials: PiCT activator Pentapharm Ltd.; Basel, Switzerland Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England (WHO Standard 97/578) LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England Protamine hydrochloride ICN Pharmaceuticals Germany GmbH; Frankfurt/Main, Germany CaCI2 HemoslL, Instrumentation Laboratory; Milano, Italy Synthetic peptide Pentapharm Ltd.; Basel, Switzerland Purified PLA2 Athens Laboratories; Geneva, Switzerland Clotting was triggered using the PiCT test, which employs a combination of FXa and RVV-V (FV activator from Daboia russelii russelii venom) in combination with phospholipids. Plasma was spiked with 0.5U/ml unfractionated heparin. Plasma was incubated 180s with all the needed reagents and then recalcified with CaCl2 used in a concentration of 25 mM. The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 50mM Hepes buffer, pH 7.5. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/mI and the native phospholipase A2 from B. moojeni venom -in a concentration of 130 ig/ml. 50mM Hepes buffer, pH 7.5 was used as a control. The obtained results are shown in the figure 4.

Example 9: Rotem system (Thromboelastography measurements) with the native Mate na Is: DiaPlastin Pentapharm Ltd.; Basel, Switzerland in-TEM Pentapharm Ltd.; Basel, Switzerland star-TEM Pentapharm Ltd.; Basel, Switzerland ROTROL N Pentapharm Ltd.; Basel, Switzerland Synthetic peptide Pentapharm Ltd.; Basel, Switzerland : .. Purified PLA2 Athens Laboratories; Geneva, Switzerland Is.. Is..

Heparin not only affects the onset time of clotting, i.e. the begin of clot formation, but also the clot formation kinetics. Therefore we examined the effects of heparin inhibition by the inventive technique in addition with a method which quantifies continuously the clot formation kinetics.

So-called ROTEM analysis was applied, which is described in detail in US5777215.

In essence the system quantifies the clot formation. The clotting time represents the : time of the onset of clotting, the clot formation time, the time from onset of clotting till I..... S *

the formation of a clot of defined strength and the maximum clot firmness defines the maximum clot stability.

The experiments were performed as follows: tI star-TEM , 0.2 M buffered CaCI2 solution, pH 7.4 inTEM , ellagic acid solution an organic activator of the contact phase, resulting in activation of factors I XII, Xl, IX, VIII, V, X and thrombin or ex-TEM, a tissue factor solution derived from rabbit brain, activating hemostasis by the extrinsic pathway, i.e. by factors VII, X, V and thrombin p1 Purified PLA2 ROTROL N, lyophilized, standardized plasma tI produced from pooi donor plasma (+/-unfractionated heparin (UFH)) The activity of purified PLA2 was evaluated. The purified PLA2 was tested in a concentration of 10 mg/mI.

The results obtained are shown in figure 6.

In both measurements good heparin neutralizing properties of the purified PLA2 could be measured, even for high concentrations of unfractionated heparin (UFH) (5 and 10 U/mi), where no clotting could be observed in the performed tests.

Example 10: Accelerated stability testing of the native PLA An accelerated stability testing of the native, purified PLA2, dissolved in deionized water (10 mg/mI), was performed in Rotem system (Thromboelastography measurements). Experiments were performed as in the example 4.

tI star-TEM in-TEM pI or ex-TEM) p1 Purified PLA2 tI ROTROL N (+/-unfractionated heparin (UFH)) The PLA2 was stored at 37 C during a period of 2 weeks and at 2-8 C during a : period of 3.5 weeks. In both cases very good stability data was obtained. *.S.

" Example 11: Rotem system (Thromboelastography measurements) with synthetic peptide 20-062.

* Similar experiments were performed with the synthetic peptide 20-062, tested in a concentration of 5 mM, dissolved in deionized water.

The experiments were performed as follows: S.....

tl star-TEM in-TEM 20.il or ex-TEM or 20 il Purified PLA2 300.tI ROTROL N (+/-unfractionated heparin (UFH)) The results obtained are shown in figure 9.

Example 12: Cytotoxicity study [19] Cytotoxicity study was performed using normal human fibroblast cells (NHFO3) and the MTT-assay. MTT ((3-(4 5-d imethylth iazol-2-yl)-2, 5-d iphenyltetrazolium bromide) is a yellow, water-soluble tetrazolium dye that is reduced by living, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. The measurement of the MTT reduction is an indirect method of the viability assessment.

The amount of MTT-formazan product was determined spectrophotometrically once the MTT-formazans crystals have been dissolved in a suitable solvent.

The cytotoxicity was measured after a supplementation period of 72h. Sodium dodecyl sulfate (SDS) was used as a control. No cytotoxic effects were observed with the synthetic peptides up to the highest concentration tested 100 kiM, which corresponds to 200 -350.tg/ml, depending on the peptide.

Conclusions

In conclusion, the invention describes a new property of some phospholipases A2 purified from the venom of B. moojeni snake. Although it is well known that different PLA2s from snake venoms influence blood coagulation system, the use of those substances or their fragments to antagonize the anticoagulant effect of heparin in vivo or in vitro has never been proposed till now.

According to the literature, heparin is known to interact in a non-covalent, charge-dependent way with basic myotoxic phospholipases A2 (PLA25), leading to inhibition of their enzymatic and biological activities [20]. It was reported that heparin was used for neutralization of the myotoxic effects caused by some snake venom PLA2s [11], [12], [21], [22], [8], [23], [13], [24], [10], [15], [20], [25].

Lomonte et al. [8], [23] first described that heparin binds to a site comprising residues 115-129 of the Lys49 myotoxin II (MT-Il) from B. asper, resulting in the neutralization of its toxic actions. In the same paper he also reported that the synthetic peptide of 115-129 of this protein mimics the cytotoxic effects of the whole protein in vitro. Thus due to heparin binding properties of that region, the toxic activity : could be reduced or completely abolished. *1*. * .

Thus the advantages of the invention include: ** * S5 * a) Better inhibition of smaller glycosaminoglycans (LMWH) compared to the standard technique i.e. the use of protamine hydrochloride. To neutralize the action of 1 U/mI of LMWH preferably the concentration of 1.5-0.1 mM (3161.175-210.745 jtg/ml), most preferably of 0.3mM (632.235 p.g/mI), of the synthetic peptide 20-062 has been used.

S.....

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b) The use of synthetic peptides excludes substances of animal origin and therefore the risk of infection by known or unknown viruses, prions of animal origin.

c) The use of small synthetic peptides instead of large proteins of animal origin excludes the anaphylactic potential and the allergy response.

d) All the substances -the PLA2 as well as the synthetic peptides have very good solubility in water, in the opposite to polybren.

e) Very good stability could be proved for the PLA2, in the opposite to polybren.

f) The PLA2 as well as the synthetic peptides have been shown to reverse the action of even 5U of heparin in 1 ml plasma.

g) Synthetic production of the small peptides can be inexpensive in large scale.

h) No need for recombinant technology, used for production of PF4 and heparinases, which is much more costly and may sensitize patients against mouse or hamster proteins present due to the production procedures.

References 1. Tyler-Cross, R., et al., Structure-function relations of antithrombin Ill-heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide-pentasaccharide-docked complexes. Arch B iochem Biophys., 1996. 334(2): p. 206-213.

2. Freedman, J.E. and B. Adelman, Pharmacology of heparin and oral anticoagulants. Thrombosis and Hemorrhage, ed. J. Loscaizo and A.l.

Schafer. 1994, Boston: Blackwell Scientific Publications.

3. Mixon, TA. and G.J. Dehmer, Recombinant platelet factor 4 for heparin neutralization. Semin Thromb Hemost., 2004. 30(3): p. 369-377.

4. Chang, L.-C., et al., Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (II): in vitro evaluation of efficacy and toxicity. AAPS PharmSci., 2001. 3(3): p. E18.

5. Byun, Y., et al., Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine. ASAIO J., 2000. 46(4): p. 435-439.

6. Chang, L.-C., et al., Low Molecular Weight Protamine (LMWP) as Nontoxic Heparin/Low Molecular Weight Heparin Antidote (I): Preparation and Characterization. AAPS PharmSci., 2001. 3(3): p. E17.

7. Lee, L.M., et al., Low Molecular Weight Protamine as Nontoxic Heparin/Low Molecular Weight Heparin Antidote (III): Preliminary In Vivo Evaluation of Efficacy and Toxicity Using a Canine Model. AAPS PharmSci., 2001. 3(3): p. E19.

8. Lomonte, B., et al., Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification * of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J Biol Chem., 1994. 269(47): p. 29867-29873.

* 9. Lomonte, B., Y. Angulo, and L. Calderon, An overview of Iysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon, 2003. 42(8): p. 885-901.

* 10. Soares, A.M., et al., Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom. Arch Biochem Biophys., 2000. 378(2): p. 201-209. 0I*** * .

11. Melo, P.A. and G. Suarez-Kurtz, Release of sarcoplasmic enzymes from skeletal muscle by Bothrops jararacussu venom: antagonism by heparin and by the serum of South American marsupials. Toxicon, 1988. 26(1): p. 87-95.

12. Melo, P.A., et al., Antagonism of the myotoxic effects of Bothropsjararacussu venom and bothropstoxin by polyanions. Toxicon, 1993. 31(3): p. 285-291.

13. Melo, PA. and C.L. Ownby, Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins. Toxicon, 1999. 37(1): p. 199-215.

14. de Olive ira, M., et a I., Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by suramin. Toxicon, 2003. 42(4) : p. 373-379.

15. Oshima-Franco, Y., et al., Neutralization of the pharmacological effects of bothropstoxin-I from Bothropsjararacussu (jararacucu) venom by crotoxin antiserum and heparin. Toxicon, 2001. 39(10): p. 1477-1485.

16. Cardin, A.D. and H.J. Weintraub, Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis., 1989. 9(1): p. 21-32.

17. de Azevedo, W.F.J., et al., Crystal structure of piratoxin-l: a calcium-independent, myotoxic phospholipase A2-homologue from Bothrops pirajai venom. Toxicon, 1998. 36(10): p. 1395-1406.

18. Andersson, E., et al., Antimicrobial activities of heparin-binding peptides. Eur J Biochem., 2004. 271(6): p. 1219-1226.

19. Lindl, T., Zell-und Gewebekultur. 5th ed, ed. C. Fischer. 2002, Berlin: Spektrum Akademischer Verlag GmbH Heidelberg.

20. Ketelh ut, D. F., et a I., Isolation, characterization and biological activity of acidic phospholipase A2 iso forms from Bothrops jararacussu snake venom.

Biochimie, 2003. 85(10): p. 983-991.

21. Melo, P.A. and G. Suarez-Kurtz, Release of creatine kinase from skeletal muscles by Bothrops venoms: heparin potentiation of inhibition by antivenin.

Braz J Med Biol Res., 1988. 21(3): p. 545-548.

22. Melo, P.A., et a I., Antagonism of the myotoxic effects of Bothrops jararacussu venom and bothropstoxin by polyanions. Toxicon, 1993. 31(3): p. 285-291.

23. Lomonte, B., et al., Neutralization of the cytolytic and myotoxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family. Biochem Pharmacol., 1994. 47(9): p. 1509-15 18.

24. Ownby, CL., et al., Lysine 49 phospholipase A2 proteins. Toxicon, 1999.

37(3): p. 411-445.

25. Lomonte, B., Y. Angulo, and C. Santamaria, Comparative study of synthetic peptides corresponding to region 115-129 in Lys49 myotoxic phospholipase A2 from snake venoms. Toxicon, 2003. 42(3): p. 307-312. * S *IS. S'S. * * * I..

**5*SS * . SI. * . S *5 *

*4S*S5 * S F 17

Claims

What we claim is: 1. Compounds of the general formula (I), R1(

B1)m(B2)nTyr(B3a)o( B3b)pAsnqAsnrTyrsLeurB4( Pro)-Phe-Abu-B5-(B6)-R2 (I) wherein R1 represents hydrogen or (C1-C8)-acyl, R2 represents OH, O-(Ci-C8)-alkyl, NH2 or Ala-Asp-Pro-OH, B1 to B6 represent, independent of one another, a basic amino acid, such as arginine, homoarginine, lysine, ornithine, 2,4-diaminobutyric acid or 2,3-diaminopropionic acid, m,n,o,p,q,r,s,t,u,v represent, independent of one another, zero or 1.

2. Compounds according to claim 1 wherein R1 represents hydrogen or acetyl.

3. Compounds according to claim I wherein R2 represents OH or Ala-Asp-Pro-OH.

4. Compounds according to claim 1 wherein B1 to B6 represent, independent of one another, arginine or lysine.

5. Compounds according to one of the claims 1-4, thereby characterized that the compounds of formula (I) together with acids form mono-or polyvalent, homogeneous or mixed salts, preferably with inorganic acids, or with appropriate organic aliphatic saturated or unsaturated carboxylic acids, or with aromatic carboxylic acids, or with aromatic-aliphatic carboxylic acids, or with heteroaromatic carboxylic acids, or with aliphatic or aromatic sulfonic acids, preferably with acetic acid and/or lactic acid.

6. Compounds according to one of the claims 1-5, thereby characterized that they are present in pure isomeric forms and mixtures thereof.

7. Pharmaceutical composition for the antagonisation of heparins, LMWH, pentasaccharide, danaparoid or other related anticoagulants characterized by containing a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNNYLKPFCKK.

8. Pharmaceutical composition for the antagonisation of heparins, LMWH, pentasaccharide, danaparoid or other related anticoagulants characterized by containing a compound of the general formula (I) according to one of the claims I to 6.

9. Use of a compound according to one of the claims 1 to 6 for the antagonisation of *. anticoagulants. * *

10. Use of a compound according to one of the claims 1 to 6 for the antagonisation of heparins.

11. Use of a compound according to one of the claims 1 to 6 for the antagonisation of LMWH. * S. * * S ** *

**..** * * 12. Use of a compound according to one of the claims ito 6 for the antagonisation of pentasaccharide.

13. Use of a compound according to one of the claims 1 to 6 for the antagonisation of danaparoid.

14. Use of a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNN YLKPFCKK for the antagonisation of anticoagulants.

15. Use of a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNN YLKPFCKK for the antagonisation of heparins.

16. Use of a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNN YLKPFCKK for the antagonisation of of LMWH.

17. Use of a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNN YLKPFCKK for the antagonisation of pentasaccharide.

18. Use of a myotoxic phospholipase A2 (PLA2) or an other snake venom containing the common peptide sequence KKYKNN YLKPFCKK for the antagonisation of danapa roid.

19. Method according to one of the claims 14 to 18 where PLA2 has been derived from the crude venom of B. Moojeni or portion thereof 20. Method for antagonisation of heparins before the performance of in vitro tests including the step of adding one of the following compounds to the in vitro reagents: PLA2, an other snake venom containing the common peptide sequence KKYKNNYLKPFCKK or a compound according to one of the claims I to 6.

21. Reagent kits for in vitro analysis of blood or blood components containing one of the following compounds for the antagonisation of heparins and related anticoagulants: PLA2, an other snake venom containing the common peptide sequence KKYKNNYLKPFCKK or a compound according to one of the claims 1 to 6. * . * S... * S** * . * * S *55 S. *

*4**SI -19

Brief description of the figures

Fig.1 Neutralization of unfractionated heparin (UFH) anti-FXa activity assessed using FXa chromogenic substrate. Best neutralizing activity was measured for the crude PLA2 purified form B. moojeni venom (95%). Two concentrations of the synthetic peptide, corresponding to the heparin binding sequence of the PLA2, showed weaker unfractionated heparin (UFH) neutralization: 68% and 67%. In comparison, protamine hydrochloride, in the therapeutical concentration, neutralized 54% of anti-FXa activity of unfractionated heparin (UFH).

Fig.2 Neutralization of LMWH anti-FXa activity assessed using FXa chromogenic substrate. Best neutralizing activity was measured for the crude PLA2 purified form B. moojeni venom (63%). Two concentrations of the synthetic peptide, corresponding to the heparin binding sequence of the PLA2, showed weaker unfractionated heparin (UFH) neutralization: 43% and 48%. In comparison, protamine hydrochloride, in the therapeutical concentration, neutralized 45% of anti-FXa activity of unfractionated heparin (UFH).

Fig.3 The synthetic peptide 20-062 neutralized 1 U LMWH more efficiently than protamine hydrochloride. The normal CT in plasma without LMWH is expressed as 100%. The concentration of 1U LMWH/ 1 ml plasma prolongs CT up to 527%.

Best inhibition was obtained with the concentration of 0.3 mM of the 20-062 peptide (176%), while 3U and 5U of protamine hydrochloride were not as efficient (223% and 226% respectively).

Fig.4 All tested substances neutralized the anticoagulant activity of unfractionated heparin in plasma. The clotting times obtained were very similar for all three substances Fig.5 The anticoagulant activity of low molecular heparin was neutralized by protamine hydrochloride and slightly better by the synthetic peptide. Interestingly, the B. moojeni PLA2, showing good neutralizing properties against unfractionated heparin (UFH), did not neutralize LMWH. In the contrary to expected effects, it prolonged the clotting time. Thus, best LMWH neutralizing properties of synthetic peptide 20-062 could be established in plasma Fig.6 Using the in-TEM test and heparinized plasma, the effect of B. moojeni PLA2 on unfractionated heparin (UFH) was measured in the intrinsic pathway of blood coagulation. However clotting time prolongation could be observed in the measurements performed in plasma without addition of unfractionated heparin (UFH), very good unfractionated heparin (UFH) neutralization was obtained even for the concentration of 10 U unfractionated heparin (UFH) in I ml plasma.

** Fig.7 Using the ex-TEM test and the heparinized plasma, the unfractionated heparin (UFH) -influencing properties of B. moojeni PLA2 were measured in the extrinsic pathway of blood coagulation. A slight clotting time prolongation could be observed in the measurements performed in plasma without unfractionated heparin (UFH) addition. Also in the extrinsic pathway very good unfractionated heparin (UFH) neutralization was obtained even for the concentration of 10 U unfractionated heparin (UFH) in 1 ml plasma.

*.ss.. S *
2.0

Fig.8 The purified B. moojeni PLA2 shows very good stability during the period of 2 weeks at 37 C, as well as during the period of
3.5 weeks at 2-8 C. All performed measurements gave the same, high level of neutralization of unfractionated heparin (UFH) anticoagulant activity.

Fig.9 The synthetic peptide 20-062 neutralized anticoagulant activity of unfractionated heparin (UFH), measured in the in-TEM test.

Fig.1O Concerning the better unfractionated heparin (UFH) deactivation results obtained with B. moojeni PLA2 in the extrinsic system of blood coagulation, two concentrations of the synthetic peptide were checked in the ex-TEM test. Both showed good unfractionated heparin (UFH) neutralization properties, indicating however the concentration dependence of the anti-unfractionated heparin (UFH) activity. * .* ** * * **. * *.* * * * S..

S..... * . * S. * S *

** S...