GB2242681A - Hirudin fragments - Google Patents

Abstract

Hirudin fragments consisting of amino acids 1-52, 1-53 or 1-56 of hirudin possess antithrombotic properties. The fragments can be produced by recombinant DNA methods or enzymatic cleavage of parent hirudin, and may contain additional substitutions, additions or deletions.

Description

Enzvme inhibitors The present invention relates to novel anticoagulant polypeptides, to methods for the preparation thereof, to pharmaceutical compositions containing such polypeptides and to their use for the prevention or treatment of coagulation disorders.

Hirudin, the anticoagulant principle that occurs naturally in the leech Hirudo medicinalis, is a polypeptide of 65 amino acids with three disulphide bridges. Long known since the first investigations on leech saliva in 1884 and the pioneering work of Markwardt [Naturwissenschaften 42, 537 (1955); Methods Enzymol. 19, 924 (1970)], it was not until recently that the hirudin structure was elucidated by Dodt et. al. [FEB S Lett. 165 180 (1984)]. Hirudin exists in several naturally occuring variants designated as hirudin variant 1 (HV1), hirudin variant 2 (HV2), hirudin PA and other analogs.

Hirudin is the strongest thrombin inhibitor known, while other enzymes of the coagulation cascade are not affected. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, hirudin exerts its action directly on thrombin and, unlike heparin, does not act through antithrombin III. Furthermore, hirudin has a low toxicity and shows almost complete clearance via the kidneys in a biologically active form.

cDNAs and synthetic genes coding for hirudin or hirudin variants have been cloned and expressed in microbial hosts such as Escherichia coli and Saccharomvces cerevisiae (cf.

European Patent Applications No. 158564 and 168342). Although the expression products lack the sulphate monoester group at Tyr63 - and were therefore designated "desulphatohirudins" - they turned out to exhibit essentially the same biological activity as natural hirudin in which the tyrosine residue is present as sulphate monoester.

Although the high therapeutic potential of hirudins is generally acknowledged hirudins possess some properties bringing forth potential problems which remain to be solved. For example, hirudin has some susceptibility to C-terminal degradation by proteinases making the isolation and purification of microbially (via recombinant DNA technology) produced hirudin rather difficult. Furthermore, it is known that hirudin contains two functional domains which interact with independent sites of thrombin: the N-terminal core domain of hirudin binds to the active site region of thrombin thus inhibiting its catalytic activity while the C-terminal domain of hirudin binds to a non-catalytic site of thrombin which is required for the enzyme's fibrinogen recognition and hormonal (non-enzymatic) activities thus possibly inducing undesired side effects.Considering these shortcomings and the utmost usefulness of antithrombotic agents for the prophylaxis or therapy of thromboses and similar conditions there is a strong need for polypeptides which are comparable to hirudin with respect to antithrombotic activity and, simultaneously, eliminate one or more of the shortcomings of hirudin. It is an object of the present invention to provide such antithrombotically active polypeptides.

Surprisingly, it was found that certain fragments of hirudin display favourable properties which render them useful as anticoagulant agents.

Accordingly, the invention provides novel polypeptides consisting of amino acids 1-52, 1-53 or 1-56 of hirudin and mutants thereof.

The term "hirudin" is understood to comprise hirudin variants HV1, HV2 and HV3 (or PA) as described in European Patent Application No. 225633. Mutants of the hirudin fragments according to the invention are those deletion, insertion and substitution mutants in which the antithrombotic activity is retained.

Such fragments of hirudin or mutants thereof are especially those of the formula X1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn X2 Cys Ile Leu Gly Ser X3 Gly Glu X4 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro X5 Pro Gln Ser His X6 (I) (fragments of hirudin HV1 or mutants thereof), in which X1 represents the dipeptide radical Val-Val-, Ile-Thr- or Leu-Thr-, X2 represents Lys or Gln, X3 represents Asp or Asn, X4 represents Lys or Gln, X5 represents Lys or Arg, and X6 represents Asn, the dipeptide radical -Asn-Asp or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe, or Y1 Y2 Y3 Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Y4 Asp Val Cys Gly Gln Gly Asn Lys Cys Be Leu Y5 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Y6 (11) (fragments of hirudin HV1 mutants), in which Y1 represents Gly, Leu, Ile or Val, Y2 represents Thr, Arg, Lys or Val, Y3 represents Phe, Trp or Tyr, Y4 represents Ser or Asp, Y5 represents the hexapeptidyl radical -Gly-Ser-Asp-Gly-Glu-Lys- or the dipeptidyl radical -Asp-Gly-, and Y6 represents Asn, the dipeptide radical -A sn-Asp or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe, or Z1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gy Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Z2 Pro Glu Ser His Z3 (Ill) (fragments of hirudin HV2 and mutants thereof), in which Z1 represents the dipeptide radical Ile-Thr- or Val-Val-, Z2 represents Asn or Lys, and Z3 represents Asn, the dipeptide radical -Asn-Asn or the pentapeptide radical -Asn-Asn-Gly-Asp-Phe, or Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ee Leu Gly Ser Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Z4 (IV) (fragments of hirudin HV3), in which Z4 represents Asn, or the dipeptide radical -Asn-Gln.

Preferred compounds of the formula I are those in which X1 represents the dipeptide radical Val-Val- or Leu-Thr, X2, X4 and X5 each represent Lys, X3 represents Asp, and X6 represents Asn or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe.

Preferred compounds of the formula II are those in which Y1 and Y2 each represent Val, Y3 represents Tyr or Phe, Y4 iS Ser or Asp, Y5 represents the hexapeptidyl radical -Gly-Ser-Asp-Gly-Glu-Lys-, and Y6 represents Asn or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe.

In the preferred compound of the formula III Z1 represents the dipeptide radical Ile-Thr-, Z2 represents Lys and Z3 represents Asn.

In preferred compounds of the formula IV Z4 represents Asn.

In particular, the invention relates to compounds of the formula I in which X1 represents the dipeptide radical Val-Val- or Leu-Thr, X2, X4 and X5 each represent Lys, X3 represents Asp and X6 represents represents Asn or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe.

The compounds of the invention can be in the free form but also in the form of their salts.

As they contain free amino groups in several amino acid residues, the compounds of the invention can be in the form of acid addition salts. Suitable acid addition salts are in particular pharmaceutically acceptable salts with conventional pharmaceutically acceptable acids. Representative inorganic acids are hydrohalic acids (such as hydrochloric acid), and also sulfuric acid, phosphoric acid and pyrophosphoric acid.

Representative organic acids are in particular arenesulfonic acids (such as benzenesulfonic or p-toluenesulfonic acid), or lower alkanesulfonic acids (such as methanesulfonic acid), as well as carboxylic acids such as acetic acid, lactic acid, palmitic acid, stearic acid, malic acid, tartaric acid, ascorbic acid and citric acid. As, however, the compounds according to the invention also contain free carboxyl groups in several amino acid residues they can also be in the form of salts with inorganic or organic bases, e.g. sodium, potassium, calcium or magnesium salts, or also ammonium salts derived from ammonia or a pharmaceutically acceptable organic nitrogen-containing base. However, as they contain at the same time free carboxyl groups and free amino groups, they can also be in the form of inner salts. Pharmaceutically acceptable salts are preferred.

A first method for the preparation of a polypeptide according to the invention comprises culturing a microbial host strain which has been transformed with a hybrid vector comprising a DNA sequence coding for said polypeptide, which DNA sequence is controlled by a promoter, and isolating said polypeptide, and, if desired, converting an obtained polypeptide having free carboxy and/or amino groups into a salt or converting a salt obtained into the free compound.

A second method for the preparation of a polypeptide according to the invention and, additionally, of a polypeptide consisting of amino acids 1-49 of hirudin, comprises digesting hirudin or a mutant thereof with an endopeptidase selected from the group consisting of endopeptidase Asp-N, pepsin, chymotrypsin, elastase and thermolysin, amino acid 53 of the hirudin being aspartic acid in the case of Asp-N digestion, and isolating said polypeptide, and, if desired, converting an obtained polypeptide having free carboxy and/or amino groups into a salt or converting a salt obtained into the free compound.

1. Recombinant DNA technology Suitable microbial hosts include prokaryotic and eukaryotic hosts, for example bacteria such as strains of Bacillus subtilis or Escherichia coli, or yeast such as strains of Saccharomvces cerevisiae.

The transformed microbial host strains are cultured in a liquid medium containing assimilatable sources of carbon, nitrogen and inorganic salts, applying methods known in the art.

Various carbon sources are usable. Examples of preferred carbon sources are assimilatable carbohydrates, such as glucose, maltose, mannitol, fructose or lactose, or an acetate such as sodium acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as casamino acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore yeast extract, malt extract, corn steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or nitrate which can be used either alone or in suitable mixtures. Inorganic salts which may be used include, for example, sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally, the nutrient medium may also contain growth promoting substances.Substances which promote growth include, for example, trace elements, such as iron, zinc, manganese and the like, or individual amino acids.

Microbial host cells transformed with hybrid vectors tend to lose the latter. Such cells have to be grown under selective conditions, i.e. conditions which require the expression of a hybrid vector-encoded gene for growth. Most selective markers currently in use and present in the hybrid vectors according to the invention (infra) are genes coding for enzymes of amino acid or purine biosynthesis. This makes it necessary to use synthetic minimal media deficient in the corresponding amino acid or purine base. However, genes conferring resistance to an appropriate biocide may be used as well [e.g. a gene conferring resistance to the amino-glycoside G418]. Host cells transformed with vectors containing antibiotic resistance genes are grown in complex media containing the corresponding antibiotic whereby faster growth rates and higher cell densities are reached.

Host cells containing hybrid vectors with a constitutive promoter express the gene coding for the hirudin fragment controlled by said promoter without induction. However, if the gene is under the control of a regulated promoter the composition of the growth medium has to be adapted in order to obtain maximum levels of mRNA transcripts, i.e. when using the PH05 promoter in yeast the growth medium must contain a low concentration of inorganic phosphate for derepression of this promoter.

The cultivation is carried out by employing conventional techniques. The culturing conditions, such as temperature, pH of the medium and fermentation time are selected in such a way that maximal levels of the hirudin fragment are produced. A chosen yeast or E.

coli strain is preferably grown under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 250 to 350C, preferably at about 280C, at a pH value of from 4 to 7, for example at approximately pH 5, and for at least 12 hours to 3 days, preferably for such a period that satisfactory yields of the desired polypeptides are obtained.

If a signal sequence is included in the expression vector (see below), most of the produced hirudin fragment is secreted into the culture medium or the periplasmic space whereas only a minor part remains associated with the cell interior. The precise ratio (secreted compounds/cell associated compounds) depends on the fermentation conditions.

The hirudin fragments according to the invention can be isolated by conventional means.

For example, the first step consists usually in separating the cells from the culture fluid by mearis of centrifugation. The resulting supernatant can be enriched for secreted hirudin fragment by treatment with polyethyleneimine so as to remove most of the non-proteinaceous material, and precipitation of the proteins by saturating the solution with ammonium sulphate. Host proteins, if present, can also be precipitated by means of acidification with acetic acid (for example 0.1 %, pH 4-5). A further enrichment of the desired polipeptide can be achieved by extracting the acetic acid supernatant with n-butanol. Other purification steps include, for example, desalination, chromatographic processes, such as ion exchange chromatography, gel filtration chromatography, partition chromatography, HPLC, reversed phase HPLC and the like.The separation of the constituents of the protein mixture is also effected by dialysis, according to charge by means of gel electrophoresis or carrier-free electrophoresis, according to molecular size by means of a suitable Sephadex column, by affinity chromatography, for example with antibodies, especially monoclonal antibodies, or with thrombin coupled to a suitable carrier for affinity chromatography, or by other processes, especially those known from the literature.

If it is desired to isolate additional polypeptide which has accumulated in the periplasmic space, some supplementary purification steps are required: The desired polypeptide is recovered by enzymatic removal of the cell wall (infra) or by treatment with chemical agents, e.g. thiol reagents or EDTA, or by subjecting the cell wall to osmotic shocks which give rise to cell wall damages permitting the product to be released.

The transformed microbial host cells according to the invention can be prepared by recombinant DNA techniques comprising the steps of - preparing a hybrid vector comprising a DNA sequence coding for the hirudin fragment, which is controlled by a promoter, - transforming a microbial host strain with said hybrid vector, - and selecting transformed microbial host cells from untransformed host cells.

a. Hvbrid vectors The hybrid vectors according to the invention comprise a DNA sequence coding for the hirudin fragment according to the invention, which is controlled by a promoter.

The selection of a suitable vector is determined by the microbial host cell provided for the transformation. Suitable hosts are those specified above, especially strains of Saccharomyces cerevisiae, and bacterial strains, especially strains of Escherichia coli and Bacillus subtilis.

Examples of vectors that are suitable for the expression of the gene coding for the polypeptide according to the invention in an E. coli strain are bacteriophages, for example derivatives of the bacteriophage X, or plasmids, such as the plasmid colEl and its derivatives, for example pMB9, pSF2124, pBR317 or pBR322. Suitable vectors contain a complete replicon and a marker gene, which renders possible the selection and identification of the microorganisms transformed by the expression plasmids by means of a phenotype feature. Suitable marker genes impart to the microorganism, for example, resistance to heavy metals, antibiotics such as ampicillin or tetracyclin, and the like.

Several promoters can be used for regulating the expression cassette in E. coli. Especially promoters of strongly expressed genes are used. Suitable promoters are the lac, tac, trp and lpp promoters, furthermore the phage kN or the phage XpL promoter, and others. In the present invention, the preferred promoter for use in E. coli is the lpp and the lac promoter.

Vectors suitable for replication and expression in S. cerevisiae contain a yeast-replication origin and a selective genetic marker for yeast. Hybrid vectors that contain a yeast replication origin, for example the chromosomal autonomously replicating segment (ars), are retained extrachromosomally within the yeast cell after transformation and are replicated autonomously during mitosis. Also, hybrid vectors that contain sequences homologous to the yeast 2pL plasmid DNA can be used. Suitable marker genes for yeast are especially those that impart antibiotic resistance to the host or, in the case of auxotrophic yeast mutants, genes that complement the host lesions.Corresponding genes impart, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, HIS3 or the TRPl gene.

Preferably, yeast hybrid vectors furthermore contain a replication origin and a marker gene for a bacterial host, especially E. coli, so that the construction and the cloning of the hybrid vectors and their precursors can be carried out in E. coli. Promoters suitable for expression in yeast are, for example, those of the ADHI, ADHII, or PH05 gene, and also promoters involved in glycolysis, for example the.PGK or GAP promoter.

The promoter is operably linked to the gene coding for the hirudin fragment according to the irivention or, in particular, is operably linked to a signal sequence which in turn is linked in the proper reading frame to the gene coding for the hirudin fragment. The signal sequence is derived from a gene of the microbial host coding for a polypeptide which is ordinarily secreted. When E. coli is used as the host microorganism the ompA, lpp or ss-lactamase signal sequence may be chosen. Preferred signal sequences for use in yeast are, for example, the signal and prepro sequences of the yeast invertase, a-factor and repressible acid phosphatase (PH05) genes. Those combinations are favoured which allow a precise cleavage between the signal sequence and the hirudin fragment amino acid sequence.Additional sequences, such as pro- or spacer-sequences which may or may not carry specific processing signals can also be included in the constructions to facilitate accurate processing of precursor molecules.

Additionally, the hybrid vectors according to the invention include, downstream of the coding region of the hirudin fragment, a DNA sequence containing transcription termination signals. This is preferably the 3' flanking sequence of a gene derived from the selected microbial host which contains proper signals for transcription termination.

Suitable 3' flanking sequences are, for example, those of the gene naturally linked to the promoter used.

The hybrid vectors according to the invention can be prepared by methods known in the art, for example by linking the expression cassette consisting of a promoter operably linked to the DNA sequence encoding the hirudin fragment, a DNA sequence containing transcription termination signals and optionally, a signal sequence, or the constituents of the expression cassette to the vector DNA fragment(s) containing selective genetic markers and origins of replication for the selected microbial host in the predetermined order.

The DNA coding for the hirudin fragment according to the invention can be manufactured by methods known in the art. The methods for the manufacture of the DNA include conventional polynucleotide synthesis, the deletion of that part of the hirudin or hirudin mutant gene which codes for the C-terminal amino acids 53-65, 54-65 or 57-65 (or 53-66 or 54-66 in the case of hirudin HV3) by means of site-directed mutagenesis, or, for the synthesis of a DNA coding for a mutant hirudin fragment, excising a portion of the DNA comprising the codons for the undesired amino acid residues from the parental gene and replacing it with a DNA segment wherein said codons have been substituted with deoxyribonucleotide triplets coding for the desired amino acid residues.

b. Transformed microbial hosts The invention concerns furthermore a microbial host strain which has been transformed with a hybrid vector comprising a DNA sequence coding for a hirudin fragment according to the invention, which is controlled by a promoter, and to the method for the production thereof, comprising transforming said microbial host strain with said hybrid vector.

The microbial host strains are those specified above. The transformation with the hybrid vectors according to the invention is carried out, for example, in the manner described in the literature for S. cerevisiae [A. Hinnen et al., Proc. Natl. Acad. Sci. USA 75 1929 (1978)], B. subtilis [Anagnostopoulos et al., J. Bacteriol. 81, 741(1961)] and E. coli [M.

Mandel et al., J. Mol. Biol. 53, 159 (1970)]. The isolation of the transformed host cells is effected advantageously from a selective nutrient medium to which there has been added, for example, the biocide against which the marker gene contained in the expression plasmid imparts resistance. If, for example, the hybrid vectors contain the ampR gene, ampicillin is accordingly added to the nutrient medium. Cells that do not contain the hybrid vector are destroyed in such a medium.

2. Enzvmatic cleavage Endoproteinase Asp-N is known to cleave peptide bonds at the N-terminal end of the aspartic acid and cysteic acid residues. It has now surprisingly been found that endoproteinase Asp-N cleaves specifically and quantitatively the Asn52-Asp53 of hirudin HV1 although there are three additional X,-Asp bonds in the hirudin HVl molecule (Thr4-Asp5, Ser32-Asp33, Gly-Asp55). Even under the most vigorous digestion conditions no breakdown of these additional three X,-Asp bonds could be detected.The same result is obtained when different hirudin species containing an Asp53 residue, for example Asp53 mutants of desulphatohirudin HV2 or HV3, irrespective of the number and position of additional Asp residues, are digested with endoproteinase Asp-N: significant amounts of products resulting from the cleavage of a Xaa-Asp bond other than the Asn5Asp53 are never observed.

Pepsin preferentially hydrolyses peptide bonds at the NH2- or COOH-terminal ends of Phe, Tyr, Leu, Glu, Cys. However, pepsin hydrolysis of peptide bonds have also been reported to occur at the carboxyl side of all L-amino acids except for Pro. With hirudin, cleavage surprisingly occurs selectively at Phe56-Glu57 and Glu62-Tyr63, generating three hirudin fragments HV11-56, HVl57-62 and HV163-65. A minor cleavage at Asp53-Gly54 does also occur producing hirudin HVl 1-53 No cleavage within the core domain of hirudin is observed.

Chymotrypsin, elastase and thermolysin generally exhibit broad specificity toward protein substrates. Surprisingly, against hirudin, their cleavage sites all exclusively target at the C-terminal domain. Chymotrypsin and thermolysin selectively attack Gln49-Ser50 and Tyr63-Leu64. Elastase cuts Gln49-Ser50, His51-Asn52, Glu57-Glu58, Glu61-Glu62 and Tyr63-Leu64. All three proteinases produce the N-terminal core fragment of hirudin containing residues 1-49. No cleavage within the core domain of hirudin is observed.

The digestion of the substrate (hirudin) with AspN, elastase, thermolysin or chymotrypsin is carried out at room temperature or slightly elevated temperature, for example at a temperature range of from about 1 80C to about 350C, preferably at room temperature, in an aqueous buffer solution at pH 7.0-8.5, for example in an ammonium bicarbonate buffer pH 8.0, at a substrate/enzyme ratio of about 20 to 3000 (by weight). Depending on the specific conditions applied, especially the substrate/enzyme ratio, almost quantitative digestion is achieved within 4 to 24 hours. Satisfactory results are obtained at a substrate/enzyme ratio of 500-2500 in the case of Asp-N, 100-1000 in the case of elastase, 20-100 in the case of thermolysin, and 20-100 in the case of chymotrypsin.

In an analogous manner, the digestion of the substrate (hirudin) with the enzyme pepsin is carried out at room temperature or slightly elevated temperature, for example at a temperature range of from about 1 80C to about 35"C, preferably at room temperature, in a 0.005 to 0.02 N HCI solution, for example in a 0.01 N HC1, at a substrate/enzyme ratio of about 1000 to 3000 (by weight). Depending on the specific conditions applied, especially the substrate/enzyme ratio, almost quantitative digestion is achieved within 0.5 to 3 hours.

The digestion can be performed in homogeneous solution using a commercially available solution of the enzyme. Alternatively, it is also possible to perform the digestion in heterogeneous phase using an enzyme preparation immobilized on a solid support, such as the enzyme immobilized by reaction with tresyl (trifluoroethyl) sulfonyl or CNBr activated Sepharose 4B, CNBr or epoxy activated agarose, nitrophenyl chloroformate, trityl, N-hydroxysuccinimid or carbonyl-diimidazole agarose or carboxymethylcellulose hydrazide. The enzyme is bound to the matrix by using conventional techniques. This procedure is especially suitable for the large scale production of the hirudin fragments according to the invention.

The isolation and purification of desired polypeptides is accomplished by chromatographic means such as those mentioned above, especially HPLC, reversed phase HPLC, gel filtration and the like.

The enzymatically produced C-terminal fragment of hirudins starting with Asp53, Asp54 and Asp57 (for example desulphatohirudin HVl53-65) is a further object of the present invention. It can be separated from the large N-terminal fragment by the chromatographic methods mentioned above.

The hirudin starting polypeptides or mutants thereof in which, if required, amino acid 53 is aspartic acid are known or can be prepared in a manner known per se, for example by synthesizing the DNA coding for said polypeptide or mutant thereof using conventional polynucleotide synthetic methods, or introducing the desired mutation into an available DNA coding for a parent hirudin polypeptide or mutant thereof by means of site-directed mutagenesis, introducing the produced DNA in a suitable hybrid vector, for example one of the above-mentioned hybrid vectors, transforming a suitable host organism with the said hybrid vector, culturing the transformed host organism and isolating the desired hirudin polypeptide or mutant thereof.

Depending on the method employed, the compounds of the invention are obtained in the free form or in the form of acid addition salts, inner salts or salts with bases. The free compound can be obtained in known manner from the acid addition salts or salts with bases. In turn, acid addition salts and salts with bases can be obtained from the free compounds by reaction with acids or bases, e.g. with those acids or bases which form the above-mentioned salts, and by evaporation or lyophilisation. The inner salts can be obtained by adjusting the pH to a suitable neutral point.

Pharmaceutical compositions The novel hirudin fragments according to the invention have valuable pharmacological properties and can, like hirudins extracted from leeches, be used prophylactically or, especially, therapeutically.

The polypeptides according to the invention including the enzymatically produced C-terminal fragments of hirudins starting, for example, with Asp53 are similar to natural hirudin as regards their biological activity. They are completely specific to thrombin and exhibit no interactions with other proteinases of the blood coagulation system. The toxicity is extremely low. Similarly, no hypersensitivity reactions or allergic reactions are observed. Moreover, the polypeptides of the invention have an in vivo duration of action comparable with or better than that of native desulphatohirudin.

The novel polypeptides including the C-terminal fragments of hirudins according to the invention can therefore be used analogously to natural hirudins for the therapy and prophylaxis of thromboses and thromboembolisms, including the prophylaxis of post-operative thromboses, for acute shock therapy (for example for septic or polytraumatic shock), for the therapy of consumption coagulopathies, in haemodialyses, haemoseparations and in extracorporeal blood circulation.

The invention relates also to pharmaceutical compositions that contain at least one of the compounds according to the invention or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier and/or adjuncts.

These compositions can be used especially in the above-mentioned indications, when they are administered, for example, parenterally, such as intravenously, intracutaneously, subcutaneously or intramuscularly, orally or topically.

The invention relates also to the use of the novel compounds according to the invention and of pharmaceutical compositions containing them for the prophylactic and therapeutic treatment of the human or animal body, especially for the above-mentioned clinical syndromes, especially for inhibiting the coagulation of blood inside and outside the human or animal body.

The dosage depends especially on the specific form of administration and on the purpose of the therapy or prophylaxis. The size of the individual doses and the administration regime can best be determined by way of an individual judgement of the particular case of illness; the methods of determining relevant blood factors required for this purpose are familiar to the person skilled in the art. Normally, in the case of an injection the therapeutically effective amount of the compounds according to the invention is in a dosage range of from approximately 0.005 to approximately 0.1 mg/kg body weight. A range of from approximately 0.01 to approximately 0.05 mg/kg body weight is preferred.

The administration is effected by intravenous, intramuscular or subcutaneous injection.

Accordingly, pharmaceutical compositions for parenteral administration in single dose form contain per dose, depending on the mode of administration, from approximately 0.4 to approximately 7.5 mg of the compound according to the invention. In addition to the active ingredient these pharmaceutical compositions usually also contain a buffer, for example a phosphate buffer, which is intended to keep the pH value between approximately 3.5 and 7, and also sodium chloride, mannitol or sorbitol for adjusting the isotonicity. They may be in freeze-dried or dissolved form, it being possible for solutions advantageously to contain an antibacterially active preservative, for example from 0.2 to 0.3 % 4-hydroxy-benzoic acid methyl ester or ethyl ester.

A composition for topical application can be in the form of an aqueous solution, lotion or gel, an oily solution or suspension or a fat-containing or, especially, emulsified ointment.

A composition in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution of from pH 4 to pH 6.5 and, if desired, adding a further active ingredient, for example an anti-inflammatory agent, and/or a polymeric binder, for example polyvinyl-pyrrolidone, and/or a preservative.The concentration of the active ingredient is from approximately 0.1 to approximately 1.5 mg, preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a geL An oily form of administration for topical application is obtained, for example, by suspending the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an oil, optionally with the addition of swelling agents, such as aluminium stearate, and/or surfactants (ten sides) having HLB value ("hydrophilic-lipophilic balance") of below 10, such as fatty acid monoesters of polyhydric alcohols, for example glycerine monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate.A fat-containing ointment is obtained, for example, by suspending an active ingredient according to the invention, or a salt thereof, in a spreadable fatty base, optionally with the addition of a tenside having an HLB value of below 10. An emulsified ointment is obtained by triturating an aqueous solution of the active ingredient according to the invention, or a salt thereof, in a soft, spreadable base with the addition of a tenside having an HLB value of below 10. All these forms for topical application can also contain preservatives. The concentration of active ingredient is from approximately 0.1 to approximately 1.5 mg"preferably from 0.25 to 1.0 mg, in approximately 10 g of base.

Due to the extreme stability of the hirudin fragments according to the invention against enzymatic digestion by endopeptidases, especially pepsin, they can also be adminstered orally. For oral administration there are used tablets or gelatine capsules that contain the active ingredient together with conventional diluents, for example dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine, and lubricants, for example silica, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.

Tablets may also contain binders, for example magnesium aluminium silicate, starches, such as maize, wheat, rice or arrowroot starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, for example starches, agar, alginic acid or salts thereof, such as sodium alginate, and/or effervescent mixtures or adsorbents, colouring substances, flavourings or sweeteners. The composition for oral administration contain approximately from 1 % to 100 %, in the case of lyophilisates, up to 100 % of the active ingredient. The therapeutically effective amount of the compounds according to the invention in the composition for oral use is in a dosage range of from approximately 0.01 to approximately 0.2 mg/kg body weight.

The pharmaceutical compositions in question which, if desired, may contain other pharmacologically valuable substances, are manufactured in a manner known per se, for example by means of conventional mixing, dissolving or lyophilising processes.

In addition to the compositions described above that are intended for direct medicinal use in the body of a human or an animal, the present invention relates also to pharmaceutical compositions for medicinal use outside the living body of humans or animals. Such compositions are used especially as anticoagulant additives to blood that is being subjected to circulation or treatment outside the body (for example extracorporeal circulation or dialysis in artificial kidneys), preservation or modification (for example haemoseparation). Such compositions, such as stock solutions or alternatively compositions in single dose form, are similar in composition to the injection compositions described above; however, the amount or concentration of active ingredient is advantageously based on the volume of blood to be treated or, more precisely, on its thrombin content.In this connection it must be borne in mind that the active ingredients according to the invention (in free form) completely deactivate approximately 5 times the amount by weight of thrombin, are physiologically harmless even in relatively large amounts, and are eliminated from the circulating blood rapidly even in high concentrations so that there is no risk of overdose, even, for example, during transfusions. Depending on the specific purpose, the suitable dose is from approximately 0.01 to approximately 1.0 mg of the active ingredient/litre of blood, although the upper limit may still be exceeded without risk.

The following examples illustrate the invention but should not be construed as a limitation thereof.

Example 1: Hirudin HV11-52 Recombinant desulphatohirudin HV1 (cf. European Patent Application No. 225633) at a concentration of 5 mg/ml in 50 mM ammonium bicarbonate solution (pH 8.0) is digested at room temperature with endoproteinase Asp-N (Boehringer, Mannheim, FRG) at a substrate/enzyme ratio (by weight) of 2500. For time-course analysis, small aliquots of the digest are removed and mixed with equal volumes of 1 % trifluoroacetic acid in order to stop the reaction. The samples are then directly injected for reversed phase HPLC analysis [conditions: Vydac C-18, 5 clam; 230C; solvent A: 0.1 % trifluoroacetic acid in water; solvent B: 0.1 % trifluoroacetic acid in acetonitrile; the gradient is 10 % B to 48 % B in 30 min]. After 8 hours about 80 % of desulphatohirudin is cleaved. The reaction is complete after 24 hours.When a higher enzyme concentration (or lower substrate/enzyme ratio) is applied, the reaction is complete considerably earlier: at a ratio of 500 after about 8 hours, at a ratio of 100 at about 4 hours.

Two products are observed in the HPLC one eluting at 13.95 min (product A) the other at 22.35 min (product B) while the starting desulphatohirudin elutes at 21.15 min.

Products A and B are isolated from the column and subjected to amino acid sequence analysis using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate method [J-Y. Chang, Methods Enzymol. 91, 455-466 (1983); Anal. Biochem. 170, 542-556 (1988)]. The following sequences are determined: Product A: Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Product B: Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln Accordingly, product A is hirudin HVll-52 and product B is desulphatohirudin HV153-65.

The quantitative N-terminal sequence analysis [J-Y. Chang, Anal. Biochem. 170, 542-556 (1988)] of the HPLC fraction containing hirudin HVll-52 gives only Val ( > 99.5 %) as N-terminal amino acid. Not even trace amounts of Asp are detectable showing that the ThP-AspS and Ser32-Asp33 bonds of desulphatohirudin HV1 are not cleaved by Asp-N. The amino acid composition of the two reaction products is determined by the dimethylaminoazobenzene sulphonyl chloride precolumn derivatization method [R. Knecht and J-Y. Chang, Anal. Chem. 158, 2375-2379 (1986)]. Approximately 1 ,eg of peptide is hydrolysed and derivatized and 50 ng are injected for analysis. The results are summarized in Table 1.

Example 2: Hirudin HV11-56 and Hirudin HV11-53 Recombinant desulphatohirudin at a concentration of 5 mglml in 0.01 N HC1 is digested at room temperature with pepsin (Merck, Darmstadt, FRG) at a substrate/enzyme ratio (by weight) of 2500. Time-course analysis and separation by HPLC is done as described in Example 1. After 3 hours about 90 % of desulphatohirudin is cleaved. The reaction is complete after about 24 hours. At a substrate/enzyme ratio of 500 quantitative digestion of the substrate is achieved within 1 hour.

Two N-terminal core fragments are observed in HPLC one eluting at 16.70 min (main product, product C), the second eluting at 14.86 min (minor product, product D).

Products C and D are isolated from the column and subjected to amino acid sequence analysis using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate method [J-Y. Chang, Methods Enzymol. 91, 455-466 (1983); Anal. Biochem. 170, 542-556 (1988)]. The following sequences are determined: Product C Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys lIe Leu Gly Ser Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Product D Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Accordingly, product C is hirudin HVl l-56 and product D is hirudin HVll-53.

The amino acid composition of products C and D is determined as described in Example 1. The results are summarized in Table 1.

It is noteworth that hirudin HV11-56 is not further converted to hirudin HV1l-53 even after prolonged digestion with high concentration of pepsin. This suggests that any cleavage of the Asp53-Gly54 bond must precede the breakdown of the Phe56-G1u57 bond.

Example 3: Hirudin HVll49 In an analogous manner as described in Example 1 desulphatohirudin HV1 is digested with chymotrypsin (Sigma), elastase (Fluka) and thermolysin (Calbiochem), respectively, at substrate/enzyme ratios of 20 (chymotrypsin), 500 (elastase) or 100 (thermolysin). The reactions are complete after about 24 hours.

Hirudin HV1149 is obtained by HPLC separation using the conditions detailed in Example 1 and elutes at 14.28 min. The following sequence is determined by amino acid sequence analysis: Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln.

The amino acid composition of the product is determined as described in Example 1. The result is detailed in Table 1.

Table 1: Amino acid compositions of hirudins HVl1-49, HVll-52, HVll-53, HVll-56 and desulphatohirudin HVl53-65 Hir1-49 Hir152 Hirl-53 Hir1-56 Hir53-65 Asp 5.85 (6) 6.66 (7) 8.05 (8) 8.87 (9) 2.02 (2) Glu 7.80 (8) 7.68 (8) 7.95 (8) 8.05 (8) 4.99 (5) Ser 2.95 (3) 3.98 (4) 4.09 (4) 4.09 (4) 0 (0) Thr 3.90 (4) 4.02 (4) 3.93 (4) 4.17 (4) 0 (0) Gly 7.85 (8) 7.75 (8) 7.72 (8) 9.10 (9) 1.00 (1) Ala 0.27 (0) 0 (0) 0 (0) 0.24 (0) 0 (0) Arg 0(0) 0(0) 0(0) 0 t0)0(0) 0 (0)0(0) Pro 1.98 (2) 1.81(2) 2.29 (2) 2.03 (2) 0.98 (1) Val 3.55 (4) 3.39 (4) 3.89 (4) 3.60 (4) 0 (0) Met 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Ile 1.00 (1) 1.13 (1) 1.25 (1) 0.97 (1) 0.84 (1) Leu 3.63 (3) 3.36 (3) 3.89 (3) 3.20 (3) 0.95 (1) Phe 0(0) 0.10(0) 0(0) 1.05(1) 0.92 (1) Cys 6.04 (6) 6.31(6) 5.80 (6) 6.10 (6) 0 (0) Lys 3.35 (3) 3.58 (3) 3.23 (3) 3.17 (3) 0 (0) His 0(0) 0.93 (1) 1.03 (1) 0.89(1) 0(0) Tyr 0.87 (1) 0.96 (1) 0.85 (1) 1.00 (1) 0.99 (1) Example 4: Anti-amidolytic and anticoagulant activities of hirudin HVl N-terminal core fragments The anti-amidolytic activities of hirudin HV1149, HV11-52, HV11-53 and HVl1-56 toward thrombin, urokinase, plasmin, trypsin, factor Xa and chymotrypsin are measured by their ability to inhibit the target enzyme from digesting para-nitro aniline based chromozym.

The reaction is carried out at 230C in 67 mM Tris-HCl buffer, pH 8.0, containing 133 mM NaCl and 0.13 % poly(ethylene glycol) 6000. The digestion is followed at 405 nm for a period of 2 min. The concentration of chromogenic substrate is 200 I1M. The concentration of enzyme is adjusted in between 2.5 to 25 nM. The hirudin fragments bind to the active site of thrombin and blocks thrombin's proteolytic activity toward both fibrinogen and non-fibrinogen substrates. The binding is highly specific: Based on various chromozym as says, the dissociation constants of binding to trypsin, chymotrypsin, urokinase, plasmin and factor Xa are at least greater than 10 KLM while the dissociation constants of hirudin fragment-thrombin complexes are in the nM range: HV 11-52 35 nM, He 1149 72 nM and HV11-56 19nM.Furthermore, the hirudin fragments do not bind to the fibrinogen recognition site of thrombin as evaluated by the method of chemical modification [J.Y. Chang, J. Biol. Chem. 264, 7141-7146 (1989)].

The anticoagulant activity of the hirudin fragments is measured by Coagulometer KC1 (from Amelung GmbH, FRG) using fibrinogen (Kabi Vitrum) as the substrate. Fibrinogen (2 mg/ml dissolved in the same Tris-HCl buffer as for anti-amidolytic assay) is pre-incubated at 230C for 10 min before adding thrombin or hirudin fragment/thrombin mixture. Thrombin is fixed at 50 nM.

Applying 0.5 I1M of hirudin fragments clotting times of about 64 sec (hirudin Hull 49), 90 sec. (hirudin HV11-52) and 114 sec. (hirudin HV11-56), respectively, are observed while the control sample (without inhibitor) gives a clotting time of about 18 sec. The hirudin fragments are thus effecitve anticoagulants at nanomolar concentration.

Example 5: Stability of hirudin HV1 N-terminal core fragment a. Buffer, acidic and alkaline conditions Hirudin fragments (1 nmole) are dissolved in 25 1 of Tris-HCl buffer (67 mM, pH 8.0), 0.1 N HC1 (pH 1.47) or 0.1 N NaOH (pH 12.6) and incubated at different temperatures for various periods of time. The treated samples are diluted with 200 1ll of 67 mM Tris-HCl buffer and their anticoagulant activities are measured with the Coagulometer KG1 as described in Example 4. Hirudin fragments dissolved in Tris-HCl buffer alone without heating are used as control samples (their anticoagulant activity are taken as 100 %).

The stability of hirudin N-terminal core fragments HV1lA9, HV1l-52, HVll-53 and HV11-56 is tested against various extreme conditions. The molecules prove stable at high temperature (950C) in Tris-HCl buffer (pH 8.0), and in acidic (pH 1.47) or alkaline (pH 12.6) solutions. The anticoagulant activity of the hirudin fragments also remained essentially intact with simultaneous treatment of low pH (1.47) and high temperature (700C). One condition which inactivates the hirudin fragments is the combination of high pH and high temperature. Under pH 12.6 alone, there is no significant loss of anticoagulant activity after two hours of incubation at 230C. Drastic decrease of the activity is, however, observed when the alkaline solution is heated up to 500C-700C, a temperature range which appears to induce the melting of the molecules.

b. Enzymatic stability For analysis of enzymatic stability, the hirudin fragments (5 Rg) are incubated with 1 ttg of various enzymes in 25 cell of ammonium bicarbonate solution (pH 8.0), acetate buffer (pH 5.4, for carboxypeptidase Y) or 0.01 N HCl (pepsin). Incubation is performed at 370C for 7 h. The samples are directly diluted with 200 cell of Tris-HCl buffer and analysed for their surviving anticoagulant activities, or freeze-dried and evaluated by quantitative NH2-terminal analysis and amino acid analysis. For each hirudin fragment, two kinds of control samples are processed in parallel, one is the hirudin fragment alone without addition of enzyme, the other is the enzyme alone without the hirudin fragment.All control samples with enzyme alone are found to have no effect on thrombin's coagulation activity.

The hirudin fragments prove resistant to various endo- and exo-proteinases. The results are summarized in Table 2. Hirudin HVl156and hirudin HV11-52 are further converted to His149 by chymotrypsin and elastase. The hirudin fragments HVll49, HVll-52 and HVl1-56 are cleaved by V8 protease at Glu43-Gly44, leading to the much less active core fragment Hire~43. However, V8 protease is isolated from Staphylococcus aureus cells and there is no evidence that enzymes with similar specificity exist in the mammalian system.

It is notable that all three hirudin fragments are completely resistant to pepsin, a major digestive enzyme of the stomach. The core fragments also withstand carboxypeptidases A, B and Y. The only core fragment responding to carboxypeptidase is hirudin HVl156 which is converted to hirudin HV155 by carboxypeptidase A.Hirudin HV11-55 has an anticoagulant activity indistinguishable from that of hirudin HV1 1.56 Table 2: Stability of hirudin N-terrninal core fragments against various proteinases hirudin fragment proteinase stabilitya evaluated byb HV11A9 trypsin stable QNA & anticoagulation pepsin stable QNA Asp-N stable QNA chymotrypsin stable QNA & anticoagulation elastase stable QNA & anticoagulation thermolysin stable QNA & anticoagulation CPA stable AAA & anticoagulation CPB stable AAA & anticoagulation CPY stable AAA V8 protease to Hir1-43 QNA & sequencing Hvll-52 trypsin stable QNA & anticoagulation pepsin stable QNA & anticoagulation Asp-N stable QNA & anticoagulation chymotrypsin to Hir149 QNA & sequencing elastase to Hir149 QNA & sequencing thermolysin to Hir1-49 QNA & sequencing CPA stable AAA & anticoagulation CPB stable AAA & anticoagulation CPY stable AAA V8 protease to Hir1-43 QNA & sequencing HV1156 trypsin stable QNA & anticoagulation pepsin stable QNA & anticoagulation Asp-N to Hir1-52 QNA & sequencing chymotrypsin to Hir1-49 QNA & sequencing elastase to Hir1-49 QNA & sequencing thermolysin to Hirl 49 QNA & sequencing CPA toHirl~55 AAA & nticoagulation CPB stable AAA & anticoagulation CPY stable AAA V8 protease to Hir143 QNA & sequencing a. Stability is defined by the observation that at least 90 % of the fragment remains intact after 7 h incubation in a system with the enzyme/substrate weight ratio of 20:100.

b. The intactness of the fragments following digestion is evaluated by quantitative NH2-terminal analysis (QNA) and three cycles of sequencing to determine the cleaved position, by assay of anticoagulation, and by amino acid analysis (AAA) in case of carboxypeptidases A, B und Y (CPA, CPB and CPY) digestion.

Example 6: Pharmaceutical composition for parenteral administration A solution containing hirudin HVl1-52 is dialysed against a 0.9 % NaCl solution. The concentration of the solution is then adjusted by diluting with the same NaCl solution to 0.2 mg/ml or 2 mg/ml. These solutions are sterilized by ultrafiltration (membranes with 0.22 llm pores).

The sterilized solutions can be used directly, for example for intravenous administration.

Instead of hirudin HV 11.52 any other N-terminal core fragment of hirudin mentioned in the preceding examples, such as hirudin HV1l-56, can be applied in an analogous manner.

Claims (21)

Claims

1. A polypeptide consisting of amino acids 1-52, 1-53 or 1-56 of hirudin, mutants thereof in which the antithrombotic activity is retained, or a salt thereof.

2. A polypeptide according to claim 1 having the formula X1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Gln Gly Asn X2 Cys Ile Leu Gly Ser X3 Gly Glu X4 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Xs Pro Gln Ser His X6 (I) in which X1 represents the dipeptide radical Val-Val-, Ile-Thr- or Leu-Thr-, X2 represents Lys or Gln, X3 represents Asp or Asn, X4 represents Lys or Gln, Xs represents Lys or Arg, and X6 represents Asn, the dipeptide radical -Asn-Asp or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe, or a salt thereof.

3. A polypeptide according to claim 1 having the formula Y1 Y2 Y3 Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Y4 Asp Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Y5 Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Y6 (11 > in which Y1 represents Gly, Leu, Iie or Val, Y2 represents Thr, Arg, Lys or Val, Y3 represents Phe, Trp or Tyr, Y4 represents Ser or Asp, Y5 represents the hexapeptidyl radical -Gly-Ser-Asp-Gly-Glu-Lys- or the dipeptidyl radical -Asp-Gly-, and Y6 represents Asn, the dipeptide radical -As n-Asp or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe, or a salt thereof.

4. A polypeptide according to claim 1 having the formula Z1 Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gy Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Z2 Pro Glu Ser His 5 (Ill) in which Z1 represents the dipeptide radical Ile-Thr- or Val-Val-, Z2 represents Asn or Lys, and Z3 represents Asn, the dipeptide radical -Asn-Asn or the pentapeptide radical -Asn-Asn-Gly-Asp-Phe, or a salt thereof.

5. A polypeptide according to claim 1 having the formula Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Z4 (IV) in which Z4 represents Asn, or the dipeptide radical -Asn-Gln, or a salt thereof.

6. A polypeptide of the formula I according to claim 2 in which X1 represents the dipeptide radical Val-Val- or Leu-Thr, X2, X4 and Xs each represent Lys, X3 represents Asp, and X6 represents Asn or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe.

7. A polypeptide of the formula II according to claim 3 in which Y1 and Y2 each represent Val, Y3 represents Tyr or Phe, Y4 is Ser or Asp, Y5 represents the hexapeptidyl radical -Gly-Ser-Asp-Gly-Glu-Lys-, and Y6 represents Asn or the pentapeptide radical -Asn-Asp-Gly-Asp-Phe, or a salt thereof.

8. A polypeptide of the formula III according to claim 4 in which Z1 represents the dipeptide radical Ile-Thr-, Z2 represents Lys and 5 represents Asn, or a salt thereof.

9. A polypeptide of the formula IV according to claim 5 in which Z4 represents Asn, or a salt thereof.

10. Hirudin HVll-52 according to claim 6.

11. Hirudin HV11-53 according to claim 6.

12. Hirudin HVll-56 according to claim 6.

13. A pharmaceutical composition comprising a polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptabe carrier.

14. A polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof for use in a method for the therapeutic or prophylactic treatment of the human or animal body.

15. Method for the preparation of a polypeptide according to claim 1 comprising culturing a microbial host strain which has been transformed with a hybrid vector comprising a DNA sequence coding for said polypeptide which DNA sequence is controlled by a promoter, and isolating said polypeptide, and, if desired, converting an obtained polypeptide having free carboxy and/or amino groups into a salt or converting a salt obtained into the free compound.

16. A polypeptide obtainable by the method according to claim 15.

17. A hybrid vector comprising a DNA sequence coding for a polypeptide according to claim 1, which DNA sequence is controlled by a promoter.

18. A microbial host strain which has been transformed with a hybrid vector comprising a DNA sequence coding for a polypeptide according to claim 1, which DNA sequence is controlled by a promoter.

19. A method for the preparation of a polypeptide consisting of amino acids 1-49, 1-52, 1-53 or 1-56 comprising digesting hirudin or a mutant thereof with an endopeptidase selected from the group consisting of endopeptidase Asp-N, pepsin, chymotrypsin, elastase and thermolysin, amino acid 53 of the hirudin being aspartic acid in the case of Asp-N digestion, and isolating said polypeptide, and, if desired, converting an obtained polypeptide having free carboxy and/or amino groups into a salt or converting a salt obtained into the free compound.

20. A method for the preparation of a polypeptide according to claim 1 comprising digesting hirudin or a mutant thereof with an endopeptidase selected from the group consisting of endopeptidase Asp-N and pepsin, amino acid 53 of the hirudin being aspartic acid in the case of Asp-N digestion, and isolating said polypeptide, and, if desired, converting an obtained polypeptide having free carboxy and/or amino groups into a salt or converting a salt obtained into the free compound.

21. A polypeptide obtainable by the method according to claim 20.