GB2257974A - Hirudin analogues and process for their preparation - Google Patents

Description

231.

) '4 HIRUDIN ANALOGUES AND PROCESS FOR THEIR PREPARATION The present invention relates to hirudin derived polypeptides,,, to process for their preparation and to pharmaceutical compositions containing them.

Hirudin, isolated at first from the leech Hirudo medicinalis, is well known as a thrombin polypeptidic inhibitor (Markwardt, F., Methods in Enzymology, 19, pag. 924; Markwardt, F., 1985, Biomed. Biochim. Acta 44, pag. 1007).

In particular, hirudin performs its anticoagulant activity by binding thrombin through ionic interactions, Dreventing in such way, the fibrinogen conversion into fibrin and the consequent formation of the clot.

In studies effected on animals, hirudin has showed itself to be effective in preventing venous thromboses, vascular occlusions and the disseminated intravascular thrombin induced coagulation.

Besides, hirudin is characterized by low toxicity, scarce or absent antigenic property and also it is fastly removed from the circle (Markwardt, F. et al. 1982, Thromb. Haemostasis 47, pag. 226).

Three natural variants of hirudin are known. The sequence of a first variant called HVI was determined by Dodt et al. FEBS 165 (1984) 180-184.

The aminoacidic sequence of the HV1 variant is the 1 following (Eur. J. Biochem. 138, 9-37, 1984):

Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu 30 Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu r..

Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr so 60 Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro 65 Glu-Glu-Tyr-Leu-G1n.

b U 3 kt A second variant named HV2 has been described by Dodt et al., Biol. Chem. Hoppe-Seyler 367 (1986) 803-811 and a third variant named HV3 has been described by Harvey et al., Proc. Natl. Acad. Sci. USA (1986) 1084-1088.

The invention provides a compound which is a polypeptide of formula (I) P(Gly) n-X (I) wherein P is a HV1 or HV1(Val 65) polypeptidic moiety having an amino acid sequence as depicted in Figure 1; -(Gly)- is the bivalent radical of glycine, -NH-CH2-CO_; X is -OH or -NH2; and n is zero or 1; - 3 with the provisos that when P is HV1 and n is zero, X must be NH2, and that when n is 1, X must be -OH; or a pharmaceutically acceptable salt thereof.

The invention also provides a recombinant DNA 5 molecule and a recombinant expression vector encoding a polypeptide of formula (I) wherein X is -OH. A host cell transformed or transfected with a said vector and which is capable of expressing a polypeptide of formula (I) wherein X is OH is included within the scope of the invention.

Examples of pharmaceutically acceptable salts are salts with pharmaceutically acceptable inorganic acids, e.g. hydrochloric, hydrobromic, sulfuric and phosphoric acids, and salts with pharmaceutically acceptable organic acids, e.g. acetic, citric, maleic, malic, succinic, ascorbic and tartaric acids. Other examples of pharmaceutically acceptable salts are salts with pharmaceutically acceptable inorganic bases, e.g. sodium, potassium, calcium, magnesium or ammonium salts, or salts with organic bases which have a salifiable nitrogen, which are conventionally used in pharmaceuticals. since the polypeptides according to the invention contain both free carboxylic and amine groups, they can also form internal salts.

Compounds of the invention wherein X is -NH2 may be prepared by a process comprising amidating a polypeptide of formula (I) wherein n is 1 and X is -OH, and optionally converting the resulting polypeptide into a salt thereof. Compounds wherein X is -OH may be prepared by a process comprising causing expression of a polypeptide of formula (I) wherein X is -OH from a host cell capable of producing said polypeptide, isolating said polypeptide, and optionally - 4 converting said polypeptide into a pharmaceutically acceptable salt thereof. If desired, the compound obtained may then be purified.

The above processes preferably comprise (a) preparing a recombinant expression vector comprising a DNA molecule encoding a polypeptide of formula (I) wherein X is -OH, which vector is capable of expressing said polypeptide in a suitable host microorganism; b) introducing said expression vector into said suitable host microorganism; C) facilitating the expression, secretion and isolation of said polypeptide from said host microorganism, thus obtaining a polypeptide of formula (I) wherein X is -OH; and d) if desired, converting a polypeptide of formula (I) wherein n is 1 and X is -OH, into a corresponding polypeptide of formula (I) wherein n is zero and X is -NH2, by means of a suitable amidating enzyme; and e) if desired, the converting a polypeptide of formula (I) into a pharmaceutically acceptable salt thereof.

The invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and, as active principle, a compound of the invention. Compounds of the invention and pharmaceutical compositions containing compounds of the invention are suitable for use in treatment of the human or animal body by therapy, and are particularly suitable for use as antithrombotic or anticoagulation agents.

The compounds of the invention are HV1-NH2. HVlGly-OH, HV1(Val65)-NH2. Hvl(Val65)-OH, and HV1(Val65)-Gly-OH wherein HV1 and HV1(Val65) are polypeptidic moieties having the sequences depicted in Figure 1, and pharmaceutically acceptable salts thereof.

In the present invention, the term "primer" is used to mean primer nucleotide.

The polypeptidic derivatives of formula (I) wherein n is 1 and X is -OH (HV1(Gly 66) mutant and HV1(Val 65 Gly 66) mutant) have been made following the operative scheme hereinafter reported together with the respective technical 10 details and the different procedures used.

Svnthesis of svnthetic oligonucleotides, some of which contain the important mutations, to be used as primers during the amplification process.

Starting from the HV1 hirudin sequence, the following antisense oligonucleotides were synthesised:

HV1 (Gly 66) Mutant Gly 5'-GTC GAC GGA TCC TTA CTA ACC TTG CAG GTA WC WC 3' HV1 (Val 65 Gly 66) Mutant Gly Val 51-GTC GAC GGA TCC TTA CTA ACC AAC CAG GTA TTC TTC 31 The triplets coding for the amino acids which characterize the mutants are shown in bold type.

A second primer complementary to a region present in pFC HV1 (and pFC 85) plasmids was utilized for the obtaining 10 both mutants:

5'-CAC GAG GCC CTT TCG TCT TC 3' Plasmid pFC HV1 (also known as pFC 84) is described in WO-A-91/17250 and P. de Taxis du Poet, et al.: "Production of the HV1 variant of hirudin by recombinant DNA methodology"; Blood Coagulation and Fibrinolysis, 1991, vol. 2, pages 113120. Plasmid pFC 85 is identical to pFC 84 except for the hirudin variant gene inserted therein; pFC 84 carries the gene for HV1, whereas pFC 85 carries the gene for HV2. Plasmid pFC 85 can be readily obtained from pFC 84 by excising the HV1 gene and substituting it with the HV2 gene using the restriction sites located in critical positions in pFC 84.

Amplification of the different fragnents by Tag Polymerase Starting from the oligonucleotides described above, DNA fragments were amplified utilizing as the template the PFC HVl plasmid containing the HU hirudin gene (Fig. 2 and 3).

The amplification must lead to obtain a fragment of about 400 bp comprising the Ptrp promoter sequence, the RBS and the OmpA signal sequence and the HV1 complete genes modified at the terminal-C or HVI (Gly 66) and HVI (Val 65 Gly 66) genes. For such a purpose a standard method, hereinafter reported, has been followed.

Amnlification method with PCR The Stock Solution used are:

10x Buffer 1.25 mM dNTP mix MM M9C1 2 500 mM KC1 100 mM Tris-Cl pH 8.3 0.01 Gel (W/V) Primers 400 ng//jl Template: plasmidic DNA, pFC HV1, 0.1 ng/R1 Taq polymerase (Perkin Elmer:Cetus) 5 u/Wl The Reaction Scheme is the following:

Template 10 /11 10 x Buffer 10)ul d NTPS (nucleotides mixture) 16 P1 PRIMER A (HV1 Gly66 or HVl Va165 Gly66) 1 2 P] 2 PRIMER B - M9C1 2 p] 2 P1 Tao polymerase 0.5il H 0 to a final volume of 100 ul Cover the reaction mixture with a paraffin stopper (few drops) to avoid evaporation. Subject the samples to a series of suitable denaturation cycles, and elongation.

The cycle used by us comprise 1 x 213W at WC 30 X 1,30,' 213W 1 x 7' at 94C at 72'C at 72'C at 25'C (waiting time (denaturation) (couplinq and elongation) (waiting time) leave (maintainance) Purification of the amplified fragments, restriction and isolation thereof.

After efficiency prepared fol (Maniatis T. cloning, a Laboratory), according to Purification having verified the amplification process by electrophoretic analysis on agarose gel lowing the directions given by Maniatis et al Fritsch E. and Sambrook J., 9 laboratory manual - Cold Spring Harbor the amplified products have been purified 1982 Molecular the method hereinafter reported. of DNA fraqments after amplification with PCR Avoiding to remove the paraffin, collect the reaction mixture at the end of the amplification cycle.

1 Then proceed to an extraction with Phenol/Chloroform, adding in each Eppendorf the same Phenol/CHCL 3 volume.

Vortex and centrifugate for 5'.

Recover the supernatant phase (about 90 /jl), add Na acetate 3M pH 5 in a ratio 1/10 and 1.5 volumes of cool EtOH at 95%.

Admix and out at -80'C for 20'.

Centrifugate for 15' in Eppendorf at 40C. Discard supernatant and dry the pellet.

Re-suspend the pellet in sterile H 2 0.

Enzymatic restrictions have been effected on the so ouriflied fragments which restrictions have originated new shorter fragments, to be utilized in the following subclonings for the making of the mutagenised final genes.

The restrictions are effected utilizing the Hind III and Barr!Hi enzymes following the standard conditions suggested by the supplying firm itself (Boehringer).

The efficacy of the restriction cut has been verified analyzing the different DNAs by electrophoretic motion on agarose gel.

Always by electrophoretical motion, the specific fragments comprising the RBS, the OmpA signal sequence and the total aenes have been then isolated from the DNA extra portions which are present in the amplified original fragment (Ptrp).

1 Binding of the amplified fraqments in the pFC HU vector (Fig. 4 and 5) The binding is effected by T4 DNA ligase enzyme which is able to connect cohesive ends in the presence of ATP. The Vector/fragment ratio is generally 1/5 but it can be raised even at 1/10.

The different binding reactions are effected according to the optimal conditions suggested by the supplying firm (Boehrinaer). The pFC HV1 (Gly) (Fig. 4) and pFC HV1 (Val Gly) (Fig. 5) expression vectors are the result of the bindina.

Introduction expression, secretion and isolation of the HV1 (Glv 66) or the HO (Val 65 Gly 66) mutant.

The pFC HV1 (Gly) expression vector or the pFC HU (Val Gly) expression vector is introduced in a suitable host.

The receiving cells are transformed with the gene for HVI (Gly 66) mutant or for the HV1 (Val 65 Gly 66) mutant.

The cells transformed by the host are cultured, for instance, in such conditions as to help the expression of the HU (Gly 66) mutant or of the HV1 (Val 65 Gly 66) mutant.

Whatever host-vector compatible system can be utilized. The transformed host can be procaryotic, for instance a bacterium, e.g. E. coli or B. subtilis, or eucaryotic, for instance, a S. cerevisiae yeast.

One of the most preferred hosts of the bacterial type is - 11 a strain of E. coli of the B type.

As an alternative, insect cells can be utilized and, in such case, a "baculovirus" expression system is suitable.

The insect cells commonly utilized are Spodoptera Frugiperda cells.

As another possibility, cells coming from mammalian cellular lines can be utilized. A transgenic animal can be utilized, for instance, a non-human mammalian wherein hirudin is produced.

The HV1 (Gly 66) mutant or the HU (Val 65 Gly 66) mutant which is expressed in the above mentioned cellular systems, can be isolated and purified according well known techniques.

As already said above, the strains of the B type of the E. Coli bacterium, are the host cells preferred for the expression and the secretion in hirudin periplasma and in the derivatives thereof.

In fact, the insertion of the 9FC HVl (Gly) plasmid or of the pFC HV1 (Val Gly) plasmid into the strains of the B type of the E. Coli bacterium, leads to a high level of the production of the HV1 (Gly 66) mutant or of the HVI (Val 65 Gly 66) mutant, unlike the other E. coli strains which are less efficient.

A number of B type strains of E. coli are available and they can be utilized for the HVI (Gly 66) or HU (Val 65 Gly 66) production.

1 The preferred strains are: ATCC 12407, ATCC 11303 and NCTC 10537.

For instance, suitable cells of the NCTC 10537 strain have been prepared utilizing the process with calcium chloride by Mandel and Higa (Mandel and Higa, 1970, J. Mol. Biol., 53 pag. 154).

About 200)11 of a preparation of said cells at 1.10 g cells/ml, with 2 jil of DNA plasmid (approximative concen- tration 5 P g/ml) have been transformed.

The transforming cells have been selected on L-agar plates containing 100ligIml of ampicillin.

Two little colonies have been "dragged" by two toothpicks (three slips about I cm long have been obtained) on L-agar containinq the same antibiotic.

After 12 hours of incubation at 370C, some of the portions of the smear have been examined to verify the Droduction of the HV1 (Gly 66) and of the HVl (Val 65 Gly 66), inoculating 10 ml of LB medium (containing ampicillin at a concentration of 100 pglml) and incubating the cultures at 37C overnight.

The day after the cultures have been diluted 1:100 in M9 medium containing the same ampicillin concentration, and incubated for 6 hours at 370C.

ml of said cultures have been centrifugated a 12.000 xg, at 4'C for 10 minutes.

The bacterial pellet has been re-suspended in 2 ml of HC1 33 mM, Tris pH 8; an equal volume of a second solution of EDTA 33 mM, 40% of sucrose has been then added and the final mixture has been incubated under a moderate stirring at 370C for 10 minutes.

After the centrifugation, the permeabilized cells have been re-suspended in 2 ml of cold water and left for 10 minutes in the ice. The resulting supernatant has been isolated by centrifugation and it represents the periplasmic fraction of the bacterial cells.

Utilizing a chromogenic assay which bases itself on the inhibition of the thrombin capability to cleave a synthetic derivative S-2238 (Krstenanski, J.K. and Mao, S.J.T., 1987 FEBS Lett. 211, pag. 10) the presence of anti-throrn,bin activity in the periplasmic fraction of the HV1 (Gly66) and the HV1 (Val 65 Gly 66) producing cells has been measured.

No activity has been noticed in the control periplasmic fractions.

To extract and purify the HV1 (Gly 66) mutant and the HV1 (Val 65 gly 66) mutant, standard techniques commonly used by the skilled in the art have been utilized.

Said mutants can be used, if desired, as the ideal Substrate for the following amidation reaction which allows to convert a compound of formula (1) wherein, at the above 1 14 - mentioned conditions, n is 1 and X is -OH in a corresponding compound of formula M wherein n is zero and X is NH 2 Amiding enzymatic conversion Many biologically active peptides are protected at their carboxylic end by amiding and amiding being essential for the receptorial recognition and/or the biological activity W.F. Rehfeld, 1981, Am. J. Physiol. 240, 6251-6266).

The ami di ng at the terminal carboxylic moiety (or( -amidation) gives stability to mostly carboxyeptidases and likely, it prolongs the half life and helps the receptorial selectivity of said peptides. (G.S. Wand et al. 1985, Neuroendocrinol. 41, 482-489).

The CI\ - ani di ng represents one of post-translating changes of the secretion peptides in the eucaryotic organisms.

The -amiding can be carried out, for instance, by an enzyme called peptidyl-glycine -amiding monooxygenase (PAM), which converts the terminal carboxylic end, added with the glycine of a peptide, in the corresponding amide peotide deprived of the glycine at the Carboxylic end thereof, with the concomitant formation of the glyoxylic acid (A.F. Bradbury et al., 1982, Nature 289, 686,688).

The catalytic reaction occurs in the presence of oxygen and it may need the presence, as co-factors, of copper and ascorbate ions (B. EipQer et al., 1983, Proc. Natl. Acad Sci. USA, 80, 5144-5148).

In the present invention the polypeptidic analogues of hirudin of formula (I) wherein P is HU or HVI (Val 65), n is 1 and X is -NH 2 CHVl-NH 2 and HVI (Val65)-NH 221' can be prepared starting from the corresponding compounds of formula (I) wherein P is HVl or HVl (Val 65), n is 1 and X is -OH ZHVl (Gly 66) mutant and HV1 (Val 65 Gly 66) mutant7 obtained as above, by the action of a suitable amidating enzyme like for instance, a peptidyl-glycine 0-amiding nonooxygensase (PAM).

In Darticular the HV1 (Glv 66) mutant or the HU (Val 65 Gly 66) mutant has been incubated with a semi-purified preparation of the protease free PAM enzyme, for instance, with the prenration obtained from the rat Thyroid medullary carcinoma.

(N.M. Mehta et al. 1988, Arch. Biochem. Biophys. 261, 44-54) or from the conditioned medium of the cells deriving from saidtumour (J.P. Gilligan et al., 1989, Endocrinol. 124, 2729-2736).

The amiding enzyme can be also obtained by recombinant DNA techniques.

The enzymatic reaction has been carried out in an aqueous buffer as the TES at pH = 7, containing copper, ascorbate ions, catalase, potassium iodide, SDS and Tween 20.

The Issue of the amiding reaction has been controlled bv the Corbett and Corbett method (J. Org. Chem. 1980, 45, 2834- 2839), based on the glycoxylic acid determination, after derivativizina with nitrosobenzene.

The amidated product HVl-NH 2 or HU (Val 65)-NH 2 has been purified utilizing traditional techniques.

In alternative, the amiding reaction which allows to obtain HVl-NH 2 or HVI (Val 65)-NH 2 can be carried out via chemical way on the corresponding compounds HV1 and HV1 (Val 65) utilizing methods known in the art.

The hirudin analogues polipeptides according to the invention have the capability to inhibit all the proteolytic effects of thrombin, for instance the conversion of fibrinogen into fibrin and the activation of the factors V and VIII.

Therefore they can be utilized, for instance, as anticoagulating or antithrombotic drugs in the treatment of thromboembolythical pathologies as for instance the disseminated intravascular coagulation, the cerebral embolism, the deep venous thromboses and furthermore, in the anticoagulant and antithrombinic prophylaxis for instance, in the myocardium acute infarction therapy.

Another possible use of these hirudin derived polypeptides is outside of the human or animal body, in the Preparations which are utilized, for instance, as anticoagulant for-the blood subjected to a circle or to an extracorporeal treatment, for instance in the cases of renal dvalisis or of dyalisis for the elimination of the exceeding fats of the blood.

The activity of the compounds of the invention has been verified in the biological assays hereinafter reported accordinq to the advised procedures.

SPECIFIC ANTITHROMBIN ACTIVITY The specific antithrombin activity of the compounds object of the present invention has been determined basing or the specific and fast stoichiometrical reaction of hirudin with thrombin.

Said activity has been evaluated quantitatively by titration with a standardized solution of thrombin (Lundblad R.L., Kingdon H.S. e Hann K.G. , Methods in Enzymology, 45, 156-161, 1976).

The antithrombin activity of the compounds of the invention has been compared to the activity of the HVI (rHVI) recombinant hirudin which has been obtained with techniques of recombinant DNA in E. Coli.

The thrombin activity has been standardized at the "National Institute of Health Units (NIH units) utilizing a thrombin Standard International Preparation (00/15) supplied by the National Institute for Biological Standards and Controls "of London, (U.K.).

The activitv, neutralizing th thrombin of the tested compounds has been expressed in Antithrombin Units (U-AT); a U-AT is the amount of the tested compound which neutralizes a NIH unit of thrombin.

The test has been carried out as follows.

A standardized human fibrinogen solution (Kabi Vitrium, Sweden) has been incubated in the presence of known concentrations of the tested compounds.

The fibrinogen does not coagulate these conditions until the thrombin sufficient to neutralize all the present hirudin has been added.

The addition of e thrombin excess is made evident within some seconds with the appearance of the fibrin clot.

Aliquots from 0.01 to 0.1 ml of the tested compounds solution with 0.2 ml of a 0.05% fibrinogen solution in Tris. PCl buffer pH = 7.4 have been admixed.

A standardized thrombin Solution (bovine thrombin with an elevated purity degree, sigma), at a concentration of 100 NIH/ml units, in aliquots of 0. 005 ml (0.5 NIH units) has been then progressively added at one minute intervals and instantly admixed to the mixture containing the fibrinogen and the tested compound.

When fibrinogen coagulates in 1 minute, the titration final point has been reached.

The activity of the tested compounds has been expressed in U-AT/mg of protein.

The proteic content of the tested compounds has been determined by aminoacidic analysis.

CHROMOGENIC SUBSTRATE ASSAY The method is based on hirudin inhibiting activity in the reaction between thrombin and its synthetic chromogenic specific substrate S-2238 (Wiman et al. BBA, 579, 142-154, 1979).

The inhibiting activity of the compounds object of the present invention has been compared with the inhibiting activity of the HV1 (rHVl) recombinant hirudin reference compound.

The following procedure has been carried out.

630 pl of a S-2238 0.5 mM solution in Tris 0.05 M buffer pH 8.3 containing NaCl 0.1 M and aprotinin 100 KUI/ml have been incubated at 25'C for 10 minutes in a thermostatic cell.

After said pre-incubating period, 70 lAl of a thrombin solution 2 UI/ml in sodium-phosphate buffer 0.025 M pH 6.6 containing NaCl 0.3 M and bovine albumin 0.5% have been added to the mixture.

70/0 of buffer have been used as the control.

The kinetic readings have been started immediately after the thrombin addition.

The sDectrophotometer has been calibrated in such a way to read the absorption variations at a wavelength of 405 nm everv minute within a 6 minutes period. 7 pl of the solution of the tested compounds have been then added to the cuvettes and then the readings have been effected again every minute for 6 minutes.

The tested compounds final concentration was 1.25; 2.5; r, 7.5; 10 and 15 ng/ml.

The dilution buffer which had been used was sodium phosphate 0.025 M pH 6. 6 buffer containing NaCl 0.3 M.

The inhibiting percentage of the reaction between the thrombin and the chromogenic substrate has been calculated f6r each sample making the ratio between the A Abs/min mean obtained after having added the tested compounds and the Abs/min mean obtained before their addition.

A standard curve has been obtained plotting a graph with the inhibition percentage versus the inhibiting concentration.

The final results have been expressed as the final concentration of the tested compounds which induces the 50% inhibition (C.I. 50%) of the reaction between the thrombin and the chromogenic substrate.

ANTICOAGULATING ACTIVITY.

The anticoagulating activity of the compounds object of the present invention has been evaluated calculating the thrombin time (T.T.).

The test has been carried out using an automatic research coagulometer (ACL 300 Research, Instrumentation Laboratory, Italy).

The anticoagulating activity of the compounds of the invention has been compared to the anticoagulating activity of the HVI (rHVI) recombinant hirudin reference compound.

Increasing concentrations of the tested compounds have been added to normal citrate human plasma (0.38% final concentration).

The samples have been then settled in the automatic coagulometer which allows the thrombin time to be determined, by automatically adding to the plasma a human thrombin solution (Fibrindex; Ortho Diagnostics, Italy), until a final concentration of 5 UI/ml was reached.

The coagulation time, which has been determined for each tested compound, has been registered and plotted in a diagram versus the respective final concentration of each tested Compound.

The final results have been expressed as the final concentration of the tested compound which doubles the thrombin time normal value.

(Control sample only plasma).

In the hereabove assays the compounds of the invention resulted active, having an activity higher than that one of the rHVl reference compound.

For instance, in particular, as to the HV1 (Val 65 Gly 1 66) compound of the invention, the data reported in the following table have been obtained, showing for said compound an evident biological superiority with respect to the reference standard.

TABLE

COMPOUND U-AT/mg C.I. 50% T. T.

ratio=2 r HV1 11.000 5,47 ng/ml 0,132 M1 ratio=2 HVI mutant 19.000 0 / 57 ng/M1 0,058 f/M1 (Va165 Gly66) The hirudin analogous peptides according to the invention, can be administered in the form of pharmaceutical comoositions containing them as active principle, per se or in the form of pharmaceutically acceptable salts, together with one or more-excipients, for instance pharmaceutically acceptable carriers arid/or diluents and/or binders.

The administration of said pharmaceutical compositions can be carried out, for instance, by parenteral (intravenous, intramuscular or subcutaneous) route or by topical route, preferably by parenteral route.

In this case, further to the active principle, the injectable pharmaceutical composition will contain, for instance, sterile water, buffer, sodium chloride, mannitol or sorbitol.

Preferably, an endovenously injectable solution will contain as a carrier, for instance, sterile water or sterile isotonic saline solution.

The pharmaceutical compositions for topical application, such as for instance creams, lotions, gels, pastes, solutions or oily suspensions will contain as excipients, for instance, oily substances, eMUlsifiers or gelifiers, commonly used in the art.

Generally, the pharmaceutical compositions according to the invention can be prepared by well known techniques, according to usual procedures in the field of galenical preparations.

The effective dosage depends on the pathology to be treated, on the type of the formulation used, on the patient status and on the treatment period.

Claims (12)

1. A compound which is a polypeptide of formula (I) P- (Gly) n-X wherein P is a HV1 or HVl(Val 65) polypeptidic moiety having an amino acid sequence as depicted in Figure 1; -(Gly)- is the bivalent radical of glycine, -NH-CH2-CO-; X is -OH or -NH2; and n is zero or 1; with the provisos that when P is HV1 and n is zero, X must be NH2, and that when n is 1, X must be -OH; or a pharmaceutically acceptable salt thereof.

2. A recombinant DNA molecule encoding a polypeptide of formula (I) as defined in claim 1 wherein X is -OH.

(I)

3. A recombinant expression vector encoding a polypeptide of formula (I) as defined in claim 1 wherein X is -OH.

4. A host cell transformed or transfected with a vector as claimed in claim 3, and which is capable of expressing a polypeptide of formula (1) as defined in claim I wherein X is -OH.

5. A process for the preparation of a compound - 25 as claimed in claim 1 wherein X is -NH2J. which process comprises amidating a polypeptide of formula (I) as defined in claim 1 wherein n is 1 and X is -OH, and optionally converting the resulting polypeptide into a pharmaceutically acceptable 5 salt thereof.

6. A process for the preparation of a compound as claimed in claim 1 wherein X is -OH, which process comprises causing expression of a polypeptide of formula (I) as defined in claim 1 wherein X is -OH from a host cell as claimed in claim 4, isolating said polypeptide, and optionally converting said polypeptide into a pharmaceutically acceptable salt thereof.

7. A process according to claim 5 or 6, which process comprises a) preparing a recombinant expression vector comprising a DNA molecule encoding polypeptide of formula (I) as defined in claim 1 wherein X is -OH, which vector is capable of expressing said polypeptide in a suitable host microorganism; b) introducing said expression vector into said suitable host microorganism; C) facilitating the expression, secretion and isolation of said polypeptide from said host microorganism, thus obtaining a polypeptide of formula (I) as defined in claim 1 wherein X is -OH; and d) if desired, converting a polypeptide of formula (I) as defined in claim 1 wherein n is 1 and X is -OH, into a corresponding polypeptide of formula (I) as defied in claim 1 wherein n is zero and X is -NH2, by means of a suitable - 26 amidating enzyme; and e) if desired, the converting a polypeptide of formula (I) into a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and, as active principle, a compound as claimed in claim 1.

9. A compound as claimed in claim 1 or a pharmaceutical composition as claimed in claim 8, for use in the treatment of the human or animal body by therapy.

10. A compound according to claim 9 for use as an anticoagulation or antithrombotic agent.

11. A compound according to claim 1 specifically identified herein.

12. A process for preparing a compound as claimed in claim 1 substantially as described herein.