MXPA97001720A - Process for the manufacture of a modified palidipine, inhibitor of platelet aggregation induced by colag - Google Patents

Abstract

The present invention relates to a process for the manufacture of a recombinant protein called Asp-Palidipine. Asp-Palidipine inhibits the platelet aggregation induced by the collagen of mammalian platelets. Asp-Palidipine comprises: (i) a protein (Palidipine) selected from the group of Palidipine proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of Palidipine. The process comprises the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises: (i) a DNA or cDNA encoding recombinant Asp-Palidipine, (ii) a peptide sequence of signal, appropriate whose signal sequence is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence observed from the position of Palidipine and (iii) an appropriate promoter, bb) the expression of the preprotein comprising Asp -Palidipine and the signal sequence, cc) the transport of Asp-Palidipine from the cytoplasm of the bacteria to the periplasm, the excision of the preprotein by at least one protease during transport, producing Asp-Palidipine, dd) Isolation of Asp-Palidipine by extraction of the periplasm, and ee) purification of Asp-Palidipine. The protein is used as a drug for the inhibition of human platelet aggregation induced by collagen, or cancer with metastatic tumor cells

Description

PROCESS FOR THE MANUFACTURE OF A MODIFIED PALIDIPINE, INHIBITOR OF PLAQUETARY AGGREGATION, INDUCED BY COLLAGEN The invention relates to a process for the manufacture of a platelet aggregation inhibitor, induced by collagen, called modified Palidipine. In addition, the invention comprises the substance Palidipine which is modified. Collagen is the most potent inducer known from human platelet aggregation. For example, after damage to the vessel wall and exposure to collagen, blood platelets adhere quickly and become active (HR Baumgartner (1977) Thro b Haemostas 37: 1-16, J. Hawiger (1987) Human Pathol 18: 111-112). The collagen-induced platelet aggregation of human platelets thus represents a risk factor for patients suffering from procedures affecting the blood vessels, for example, angioplasty or sepsis, for those suffering from myocardial infarction, for those who recover from treatment for myocardial infarction, among others.

In some cases, it is necessary to inhibit platelet aggregation induced by collagen. It is known that some compounds are inhibited to such aggregation. For example, synthetic oligopeptides inhibit platelet aggregation induced by collagen; by joining the platelets. See for example, Bevers et al. (1985) "Octapeptide Derived from Collagen Inhibits Platelet Procoagulant Activity Induced by the Combined Action of Collagen and Thrombin", Thrombosis Research, 37: 365-370; Karniguian et al. (1983) "Effect of an Octapeptide Derived from Collagen on the Different Steps of the Platelet / Collagen Interaction" Thrombosis Research 32: 593-604; Caen et al. (1981) "Oligopeptides with specific properties of inhibition of aggregation induced by collagen, process for the preparation thereof and pharmaceutical compositions containing them", and European Patent EPA 0 040 149. Another source of inhibitor of the platelet aggregation induced by collagen, is an inhibitor identified in a snake venom that has an unknown structure. See Smith et al., "Identification of 50 kDalton snake venom proteins, which specifically inhibit platelet adhesion to collagen" (1991) FEBS 283: 307-310. A third inhibitor of this type, derived from the saliva of a medicinal leech, is described by Munro et al. (1991) "Calin - an inhibitor of platelet aggregation from medicinal leech saliva", Blood Coagulation and Fibrinolysis 2: 179-184. The publication of European Patent Application EP 0,480,651 (Merck &Co., Inc. published on April 15, 1992) discloses a protein having a molecular weight of approximately 16 kDalton (kD) and an ability to inhibit aggregation induced by Collagen of human platelets, whose protein is derived from the salivary glands of the leech Haemaen teri a offi cinali s. LAPP is a 16 kDa protein from the leech Haemaen teri a offi cinali s described in Connolly et al. (1992) J. Biol. Chem. 267: 6893-6898. See also Moubatin, described in Waxman and Connolly (1993) J. Biol. Chem. 268: 5445-5449. Yet another type of platelet aggregation induced by collagen is insulated from insects, as described in Patent Publication No. EP 0,530,937. These proteins are designated "Palidipins". Alkaline phosphatase (APase) is an E protein. coli which is secreted into the periplasmic space. APase is synthesized as a precursor protein, which has a guiding sequence of 21 amino acids in length which is cloned or held by the guiding peptidase in the course of translocation through the bacterial inner membrane within the periplasmic space (Y. Kikuchi et al. (1981) Nucí Acids Res. 9: 5671-5678). Its biosynthesis is regulated by the phosphate concentration of the culture medium, and the export of heterologous gene products placed downstream of the APase promoter is achieved by the use of low phosphate concentrations (C. Monteilhet et al. (1993) Gene 125: 223-228). The natural source of Palidipine protein is limited. The processes that use biotechnological methods are a logical solution for the manufacture of Palidipine. The expression of Palidipine in baby hamster kidney cells (EP 0.530.937) occurs at a production rate that should be increased in order to express Palidipine in industrial quantities. Therefore, an expression system was necessary. Thus, there was a need for an improved process, for the manufacture of recombinant Palidipine, which inhibits platelet aggregation induced by collagen, and whose process has a high yield and allows the reproducible isolation of the protein with a high degree of purification . The new process should not adversely affect the biological activity of the resulting Palidipine protein. It has now been found that the problem can be solved by a process of manufacturing a recombinant Palidipine protein (Asp-Palidipine), wherein Asp-Palidipine inhibits platelet aggregation induced by collagen, of mammalian platelets, and where the Asp-Palidipine comprises: (i) a protein (Palidipine) selected from the group of Palidipine proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of the Palidipine; whereby the Asp-Palidipine has the following amino acid sequences: a) the sequences indicated in aa) SEQ ID NO: 1 bb) SEQ ID NO: 2; or ce) SEQ ID NO: 3; ob) allelic variants or modifications, or muteins of the sequences in any of SEQ ID NOS: 1 to 3, whose allelic variations or modifications or muteins do not substantially affect the activity of the protein, or c) a protein according to any of SEQ ID NOS: 1 to 3 or its variants or muteins mentioned under subparagraph b) having post-translational modifications, which do not substantially affect the activity of the mature protein; comprising the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises: (i) a DNA or cDNA encoding recombinant Asp-Palidipine (ii) a signal peptide sequence appropriate, whose signal sequence is cleaved such that, the amino acid aspartic acid is in the +1 position of the amino acid sequence observed from the Palidipine position and (iii) an appropriate promoter; bb) the expression of the preprotein comprising the Asp-Palidipine and the signal sequence; ce) the transport of Asp-Palidipine from the cytoplasm of the bacterium to the periplasitia, the cleavage of the preprotein by at least one protease during the transport and production of Asp-Palidipine, dd) the isolation of Asp-Palidipine by the extraction of the periplasm, and ee) the purification of Asp-Palidipine.

E. col i is a rapid expression system for the production of heterologous proteins, e.g., eukaryotic. Incorrect folding of proteins is frequently a problem during the expression of eukaryotic gene products by E. coli, which can result in decreased activity of the expression products. The probability of correct folding of the protein is much higher when the protein is transported to the periplasm of the E. coli cells than when the protein is maintained in the cytoplasm. The transport of proteins to the periplasm is induced by peptide signal sequences linked to mature proteins; these signal peptide sequences together with the mature protein sequences are designated "preproteins". The correct cleavage of the preprotein between the signal peptide sequence and the mature protein is necessary for the expression of mature eukaryotic proteins in E. col i; the sequence of the signal sequence and the sequence of the mature protein have an influence on the correct excision. Therefore, not all signal peptide sequences are compatible with all coding sequences. The signal sequence of the alkaline phosphatase of E. coli is known to be effective for the export of preproteins. But it is known that the combination of a given signal peptide sequence and a sequence of a mature protein will not necessarily result in the proper processing of the preprotein. Surprisingly, it has been found that the performance of the manufacturing process of the invention is 15 times greater than the performance of the expression system using the baby hamster kidney cells. These results are shown in the Examples. The function and activity of Asp-Palidipine, in comparison with the eukaryotic system of hamster kidney, is not adversely affected by the process of the invention. An additional advantage is that the protease (for example, the guiding peptidase) necessary for the cleavage of the preprotein, is produced by E. Coli himself. The purification of mature Asp-Palidipine is much easier using the process of the invention than it is the purification of the proteins expressed, produced and stored within the cytoplasm of the bacteria. The osmotic shock is sufficient to release the accumulated Asp-Palidipine in the periplasm. In a preferred embodiment, the DNA encoding the signal peptide sequence encodes the alkaline phosphatase (APase) signal sequence, preferably the E. coli APase. The invention comprises a process wherein the vector of the Asp-Palidipine of the invention is derived from the vector pSB94 (U. Boidol et al. (1982) Mol. Gen. Genet, 185: 510-512). A further aspect of the invention is a vector as mentioned above and additionally a suitable signal peptide, a suitable promoter and, if necessary, a suitable enhancer. The vectors are described in detail in the literature of the Examples, and also in European Publications EP 0,480,651; 0.462.632 and 0.173.177. The bacterium E. Coli is the preferred guest. Other microorganisms are also suitable, for example, Bacill us s? Btil i s. In addition, the invention comprises a recombinant protein Asp-Palidipine, wherein Asp-Palidipine inhibits collagen-induced platelet aggregation of mammalian platelets, and wherein Asp-Palidipin comprises (i) a protein (Palidipine) selected from the group of proteins Palidipine, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of Palidipine; with which Asp-Palidipine has the amino acid sequences selected from: a) a sequence selected from: aa) SEQ ID NO. 1, bb) SEQ ID NO. 2; or ce) SEQ ID NO. 3; or b) a variant or allelic modification, or mutein of a sequence of SEQ ID NOS: 1 to 3, whose variant or allelic modification or mutein has substantially the same activity as the Asp-Palidipine of SEQ ID NO: 1 to 3; or c) a protein according to SEQ ID NOS: 1 to 3 or its variants or muteins mentioned in b), which have post-translational modifications, which do not substantially affect the activity of the mature protein.

The invention also comprises a recombinant protein Asp-Palidipine, wherein Asp-Palidipine, inhibits platelet aggregation induced by collagen from mammalian platelets, and wherein Asp-Palidipine comprises: (i) a protein (Palidipine) selected from the group of proteins Palidipine, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of Palidipine; whereby Asp-Palidipine has the following amino acid sequences: a) the sequences indicated in aa) SEQ ID NO: 1; bb) SEQ ID NO: 2; or ce) SEQ ID NO: 3; or b) the allelic variants or muteins of the sequences in any of SEQ ID NOS: 1 to 3, whose allelic variants or muteins do not substantially affect the activity of the protein, or c) a protein according to any of the SEQ ID NOS : 1 to 3, or its variants or muteins mentioned under subsection b) that have post-translational modifications which do not substantially affect the activity of the mature protein; wherein Asp-Palidipine is produced by a process comprising the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises an operable linkage of: (i) a first DNA molecule or CDNA, which codes for recombinant Asp-Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) an appropriate promoter; whereby, after the expression, the preprotein comprising the signal peptide and the Asp-Palidipine, is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the mature Asp-Palidipine, bb) the expression of the preprotein comprising the Asp-Palidipine and the signal peptide sequence; ce) the transport of Asp-Palidipine from the cytoplasm of the bacterium to the periplasm, whereby cleavage of the preprotein by at least one protease during transport produces the mature Asp-Palidipine, dd) the isolation of the Asp- Palidipine by the extraction of the periplasm, and ee) the purification of Asp-Palidipine.

In a further preferred embodiment, Asp-Palidipine is produced by a process comprising the steps of: cultivating a bacterium transfected with an appropriate vector wherein the vector comprises an operable linkage of: (i) a first DNA molecule or CDNA, which codes for a recombinant Asp-Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) a suitable promoter; whereby, after the expression, the preprotein comprising the signal peptide and the Asp-Palidipine is cleaved so that the amino acid aspartic acid which is in the +1 position of the amino acid sequence of the mature Asp-Palidipine, under conditions whereby the preprotein comprising the Asp-Palidipine and the signal peptide sequence is expressed, and the Asp-Palidipine is transported through the cytoplasm of the bacterium to the periplasm, in which the cleavage of the preprotein by less a protease during transport produces the mature Asp-Palidipine, and the purification of the Asp-Palidipine from the periplasm.

A protein is preferred wherein the signal peptide sequence is the signal sequence of the alkaline phosphatase (APase), preferably Easease. coli The industrial application of the proteins of the invention is the use of the proteins as a pharmaceutical composition comprising a protein according to the invention in association with a pharmaceutically acceptable diluent or carrier. Variants or allelic modifications as mentioned above, include the alteration in the sequence of the nucleotides or amino acids, the alteration of the genotype or phenotype. At least one nucleotide or an amino acid may be substituted, deleted or inserted. Most deletions, insertions and substitutions in particular, are not expected to produce radical changes in the characteristics of the protein of the invention. Modified or mutated proteins according to the invention can be routinely elaborated and selected in order to determine the exact effect of substitution, deletion, or insertion, by comparing the functions of the mutated or modified protein, with the characteristic functions of the protein of the invention, for example, the proteins of SEQ ID NOS: 1 to 3, or with the native Palidipine, whereby it is determined whether the altered protein has comparable activity, for example, biological activity. The genetic code is degenerative; that is, most amino acids are encoded by more than one codon of three nucleotides. Consequently, modification or allelic variation in the nucleotide sequence may or may not alter the amino acid sequence. Therefore, allelic variations are mainly at the DNA level and may also exist secondarily at the level of the amino acid sequence. The DNA sequence encoding the protein of the invention can be modified by conventional techniques to produce variations in the final protein of the invention, which still has substantially the same activity as the protein of the invention, for example, Asp- Palidipine of SEQ ID NOS: 1 to 3, or compared to the native Palidipin protein. The activity is measured according to the Examples. In this way, one or two amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ... up to 15 amino acids, can be added, replaced or removed without substantially affecting the activity of the protein of the invention. Substitutions can generally be made with the following Table 1, when it is desired to finely modulate the amino acid sequence of the protein of the invention. Substantial changes in immune function or identity are made by selecting substitutions that are less conservative than those in Table 1, for example the selection of residues that differ most significantly in their effect on maintenance (a) of the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule, or c) the volume of the side chains.

TABLE 1 NORMAL SUBSTITUTIONS OF AMINO ACIDS IN A PROTEIN ORIGINAL RESIDUES EXEMPLARY SUBSTITUTIONS Ala Gly, Ser Arg Lys Asn Gln, His Asp Glu Cys Ser TABLE 1 (continued) NORMAL SUBSTITUTIONS OF AMINO ACIDS IN A PROTEIN ORIGINAL RESIDUES EXEMPLARY SUBSTITUTIONS Gln Asn Glu Asp Gly Ala, Pro His Asn, Gln He Leu, Val Leu He, Val Lys Arg, Gln, Glu Met Leu, Tyr, He Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val He , Leu Muteins are defined by the homology between two compared proteins. The expression "homology" comprises similarities of the amino acids and empty spaces in the sequences of both compared sequences. The amino acid similarity is defined for example in Table 1. Preferably, the muteins of this invention comprise an amino acid sequence having a homology of at least 60%, more preferably at least 80%, and even more preferably at least 90%, and still more preferably at least 95% with the sequence of one of the proteins of SEQ ID NOS: 1 through 3. By "post-translational variations" as mentioned above, variations are understood during or after of translation, such as the formation of disulfide bridges and chemical modifications of amino acids. Proteins frequently form intravalent, covalent bonds. These disulfide bonds are formed between the amino acids cysteine-SH in the folded protein or in the protein that folds during translation. The bonds stabilize the three-dimensional structure of the protein. Such disulfide bonds are rarely formed in protein molecules that are still in the cytosol of the cell, because the high intracellular concentration of the reducing agent -SS- (disulfide) in glutathione breaks down most such bonds. Once the proteins are outside the cytoplasm, they are secreted or are on the cell surface; these frequently form additional covalent intrachain links. In addition, amino acids can be altered as described in PCT Application WO 91/10684. Other alterations of the side chains of the amino acids are possible. The protein of the invention has at least a purity of 40%, preferably of at least 60%, more preferably of at least 80%, and still more preferably of at least 90%. The purity is defined by the amount of the protein of the invention in relation to the total amount of protein. Using the purification methods described in the Examples, other proteins than the proteins of the invention are not detectable. Using the purified protein of the invention, monoclonal antibodies can be produced according to the well known method of Koehler and Milstein, which in particular, conventionally comprises the immunization of mice or rabbits with the purified protein of the invention as an immunogen, followed by the production of hybridomas from the cells that produce antibodies, mouse or rabbit.

The preferred embodiment of the invention is the protein mentioned in SEQ ID NO: 1, expressed in E. coli strain E 15. The Asp-Palidipine shows pharmacological activity and can, therefore, be useful as a pharmaceutical product. Asp-Palidipine can be used in a pharmaceutical composition comprising Asp-Palidipine in association with a pharmaceutically acceptable carrier or diluent. In addition, the invention comprises a pharmaceutical composition containing a pharmaceutically active Asp-Palidipine, according to the invention, and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier. In particular, Asp-Palidipine inhibits platelet aggregation induced by collagen and inhibits the adhesion of tumor cells, preferably from metastatic tumor cells, to collagen. Asp-Palidipine inhibits platelet aggregation. The test system is described in the Examples. Asp-Palidipine significantly inhibits platelet aggregation at a concentration of 0.5 to 50 μg of protein. The most preferred Asp-Palidipine, the protein of SEQ ID NO: 1, has an IC50 of 50 nmol / L of highly purified protein according to the Examples. Asp-Palidipine inhibits platelet aggregation at concentrations from 5 nmol / L to approximately 1,000 nmol / L. The results of the in vi tro test systems indicate that the proteins of the invention can be used as a medicament or can be used for medical treatment. The test results for the in vi tro system can be correlated with the in vi vo system, because this is a system established in this field. R.J. Shebuski et al. (1990) Thrombosis and Haemostasis, 64: 576-581. Asp-Palidipine can be administered by intraperitoneal injections, which can be given daily or 2 to 3 times a week. When animals receive daily injections to achieve a blood concentration of 100 nmol / L, their platelet aggregation is reduced. No serious side effects are verified periodically under these conditions. Asp-Palidipine produces this inhibition of platelet aggregation in mice at daily doses that achieve a blood concentration of approximately 10 nmol / L to 1,000 nmol / L.

Asp-Palidipine is, therefore, useful for the treatment of lesions due to atherosclerotic or thrombotic disease, or for the prevention of reocclusion after the treatment of myocardial infarction. Asp-Palidipine can be used as an antiatherosclerotic and antithrombotic agent in mammals, including humans, for example, to treat atherosclerotic / thrombotic lesions, for example due to rupture of atherosclerotic plaques or those due to endothelial disruption or removal, for example in sepsis or transplants, or to treat unstable angina. This can also be used to prevent reocclusion after treatment of myocardial infarction by fibrinolysis or by angioplasty (PTCA). If fibrinolytic therapy (with estre, ptocinasa, t-PA or other plasminogen activators) is applied to treat myocardial infarction, Asp-Palidipine can be used as an adjuvant agent to prevent reocclusion of blood vessels. The treatment of myocardial infarction with a balloon catheter (PCTA) also damages the wall of the vessels and this can lead to the formation of a new thrombus. This can be prevented by the administration of Asp-Palidipine during and after the procedure. Asp-Palidipine can be used in coronary angioplasty as well as in other angioplasty applications.

The invention provides a) the use of a protein of the invention for the manufacture of a medicament for the treatment of atherosclerotic or thrombotic disease or for the prevention of reocclusion after the treatment of myocardial infarction (in this way, proteins are useful as prophylactically effective medications for the treatment of patients known to be at risk of developing a disease or condition); b) a method for the treatment of atherosclerotic or thrombotic disease or for the prevention of reocclusion after treatment of myocardial infarction, which comprises the administration of an effective amount that suppresses the disease, of the protein of the invention to a patient in need of such treatment; c) a pharmaceutical composition for the treatment of atherosclerotic or thrombotic disease or for the prevention of reocclusion after treatment of myocardial infarction, which comprises a protein of the invention and a pharmaceutically acceptable carrier or diluent. For these indications the appropriate dose will vary, of course, depending on, for example, the compound of the invention employed, the host, the mode of administration and the nature and severity of the condition being treated. However, in general, it is indicated that satisfactory results are obtained in animals at daily doses to achieve a blood concentration from 10 to 1,000 nmol / L, preferably at daily doses from 30 to 300 nmol / L. The proteins of the invention can be administered by any conventional route, in particular enterally or parenterally, for example in the form of injectable solutions or suspensions. Intraperitoneal injection is preferred. The protein of SEQ ID NO: 1 is the preferred compound. The present invention provides pharmaceutical compositions comprising compounds of the invention in association with at least one pharmaceutically acceptable carrier or diluent. Such compositions can be manufactured in a conventional manner. See Remington's Pharmaceutical Science, 15th edition of Mack Publishig Company, Easton Pennsylvania (1980). The proteins of the invention also inhibit the adhesion of metastatic tumor cells to collagen. The test system is described in the Examples. The proteins of the invention show a significant inhibition of the adhesion of the metastatic tumor cells to the collagen at a concentration of 1 to 100 μm of protein. The most preferred protein test, the protein of SEQ ID NO: l, shows a IC50 value of 10 nmol / L of the highly purified protein according to Examples 2 and 15. The proteins of the invention show the inhibition of the adhesion of the metastatic tumor cells to the collagen, at a concentration of 10 to 2,000 nmol / L. The results of the in vi tro test systems indicate that the proteins of the invention can be used as a medicament, or can be used for medical treatment. The test results can be transferred from the in vi tro system to the in vi ve system, because this is a system established in this field. Chan and collaborators. (1990), Science, 2: 1600-1602.

The proteins of the invention can be administered during and after the surgical operations of the primary tumor, to prevent the formation of metastases by the loose tumor cells, which can enter the bloodstream during the operation. These antimetastatic effects can be demonstrated in an "experimental" and "spontaneous" animal model, as described by Chan et al. (1990), Science, 2: 1600-1602. The proteins of the invention can be administered by intraperitoneal injections which are given daily or 2 to 3 times a week. When the animals receive daily injections to achieve a blood concentration of 200 nmol / L, they have a reduced adhesion of the metastatic tumor cells, measured by counting the value of the established metastatic cell centers. No serious side effects are noted under these conditions. The proteins of the invention show this inhibition of the adhesion of metastatic tumor cells to collagen, in mice at daily doses to achieve a blood concentration from 20 to 2,000 nmol / L, preferably concentrations of 60 to 600 nmol / L.

The proteins of the invention are, therefore, useful for the treatment of cancer, especially cancer with metastatic tumor cells, more preferably cancer with highly metastatic tumor cells. The invention thus provides a) the use of a protein of the invention for the manufacture of a medicament for the treatment of cancer with metastatic tumor cells (the proteins are thus useful for prophylactically effective medicaments, administered before, for example, of surgical removal of tumors); b) a method for the treatment of cancer with metastatic tumor cells, which comprises administering an effective amount that suppresses the disease, of the protein of the invention, to a patient in need of such treatment; c) a pharmaceutical composition for the treatment of cancer with metastatic tumor cells, which comprises a protein of the invention and a pharmaceutically acceptable carrier or diluent.

For these indications the appropriate dose will vary, of course, depending on, for example, the compound of the invention employed, the host, the mode of administration and the nature and severity of the condition being treated. However, in general, it is indicated that satisfactory results are obtained in animals at daily doses to achieve a blood concentration from 20 to 2,000 nmol / L, preferably at daily doses of 60 to 600 nmol / L. The proteins of the invention can be administered by any conventional route, in particular enterally or parenterally, for example in the form of suspensions or injectable solutions. The protein of SEQ ID NO: 1 is the preferred compound. The present invention provides the pharmaceutical compositions comprising compounds of the invention in association with at least one pharmaceutical carrier or diluent. Such compositions can be manufactured in a conventional manner. See Remington's Pharmaceutical Science, 15th edition. Mack Publishing Company, Easton Pennsylvania (1980). In another aspect of this invention, the DNA sequences, the vectors containing these sequences, the cells containing said vectors, the methods for the recombinant production of proteins and antibodies for the proteins of this invention are provided. Also provided are the isolated and / or recombinant DNA sequences (e.g., genomic or cDNA) that encode a protein that inhibits the aggregation induced by collagen from human platelets. In a further aspect, the invention provides the recombinantly produced proteins of this invention, for example having the sequences described herein. By the term "isolated" is meant that the inhibitor of this invention or another entity is present in a form separated from (purified from) the components by which it is produced recombinantly or synthetically. All degrees of such isolation or purification are generically included. The degrees of isolation or purification by which the inhibitor is useful for pharmaceutical purposes are preferred. For example, such degrees of isolation (for example activities or purities) can be routinely achieved by chromatographic techniques such as those used in the examples. Further purifications, for example, up to homogeneity, can be routinely achieved using conventional methods, such as those described in the following texts: Methods of Enzymology, Volume 182, Guide to Protein Purification, ed. Murray P. Deutscher, Academic Press 1990; Protein Purification Applications -. A Practical Approach. ed. E.L.V. Harris and S. Angel, IRL-Press 1990; Protein Purification, Principies and Practice, Robert Scopes, Springer-Verlag 1982; and Protein Purification, Principies, High Resolution Methods and Applications, ed. J.-C. Janson and L. Ryden, VCH publishers 1989.

Purity can be determined by any of a number of routine methods, for example, SDS-polyacrylamide gel electrophoresis, analytical HPLC (high-performance liquid chromatography), etc. The purified inhibitor can be used to determine the amino acid sequence of the protein according to completely routine methods for someone of ordinary skill in the art. He ick, R.M. et al. (1981) J. Biol. Chem. 256, 7990-7997. The amino acid sequence of the inhibitor of the present invention can be used to determine the sequence of the appropriate DNA probes, and which can be used to find new inhibitors, for example, in other species. Such probes can be routinely synthesized, for example, using automated DNA synthesizers, and the selection of genomic or cDNA libraries is uniquely routine for someone of ordinary skill in the art. (See International Publication WO 90/07861, dated July 26, 1990). Therefore, the present invention also includes the DNA sequence corresponding to (coding for) the DNA sequence (gene) for Asp-Palidipine, when it is isolated from the natural environment, for example in solution or on a vector, as well as the muteins of it. Methods for the production of muteins are also routine and conventional to one of ordinary skill in the art, such as screening methods for testing the efficacy of such novel proteins, for example, as described herein.

Suitable muteins are those which have at least one fraction, for example, at least 5%, preferably at least 50%, more preferably at least 90% of the biological activity, for example, the inhibition of platelet aggregation induced by the collagen, of the inhibitor as described herein. In addition, the invention comprises a method for purification, wherein the purification comprises the following consecutive steps: (i) purification by cation exchange chromatography; (ii) purification by anion exchange and (iii) purification by size exclusion.

Without further elaboration, it is believed that one of skill in the art, using the foregoing description, may utilize the present invention to its fullest extent. The following specific and preferred modalities, therefore, should be considered as merely illustrative and not limiting of the rest of the description in any way whatsoever.

In the above and in the following examples, all temperatures are described as uncorrected in degrees Celsius; and, unless stated otherwise, all parts and percentages are by weight. Full descriptions of all applications, patents and publications, cited above and below, if any, including European Patent EP 94250224.6 filed on September 2, 1994, are incorporated by reference herein.

EXAMPLES Example 1: Construction of the expression vector For the construction of the vector of the invention, coding for Asp-Palidipine, the following sense and antisense primers are employed: p3: 5 '-GCGATATCGCGACGAAGAATGCGAACTCATG-3' (Nrul) (SEQ ID NO: 4); and p4: 5'-GCGATAGGATCCAAGCTTATTACTTCATGTTATC-3 '(BamHI) (SEQ ID NO: 5) PCR is for 8 cycles of 2 minutes at 94 ° C, 1 minute and 30 seconds at 42 ° C and 2 minutes and 30 seconds at 72 ° C using p3 and p4, as primer pairs and 1 μg of template DNA. After gel purification and digestion with NruI and BamHI, the fragment is subcloned into pSB / pho. The plasmid is prepared by digestion with Xmal followed by treatment with Mung bean to blunt the 5 'overhang and digestion with BamHI. The construction is verified by complete DNA sequencing of the inserted fragments using the dideoxy chain termination method (F. Sanger et al. (1977) Proc. Nat'l. Acad. Sci. USA 74: 5463-5467) and a sequencing equipment with [35S] dATP. The transformation of E. competent E15 coli with the expression construct of Palidipine and the empty plasmid (simulated transformation) is carried out using standard methods (J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratoy Press, Cold Spring Harbor, NY).

Example 2: Expression and Extraction of Asp-Palidipine For the plasmid pSB / pho (derived from pSB94, see J. Daum et al (1989) Eur. J. Biochem. 185: 347-354), E15 cultures overnight (S.J. Hayashi et al. (1964) J. Biol. Chem. 239: 3091-3106) are diluted to 8% (v / v) in medium with low phosphate concentration and developed for 6 hours at 37 ° C. (To Becker et al. (1994) Protein Expression and Purification 5: 50-56). Bacteria are harvested after induction by centrifugation and resuspended in 1/10 volumes of Tris-HCl at 50 mmol / L (pH 8.0) / 100 mmol / L NaCl. This preparation is frozen, thawed, and centrifuged, thereby giving a first supernatant fraction (SN1). The bacterial button is resuspended and equilibrated for 10 minutes at room temperature in 0.5 mol / L of sucrose before centrifugation. The supernatant (SN2) is maintained for analysis, while the button is resuspended in ice-cold water supplemented with 1 mmol / L of PMSF (phenylmethylsulfonyl fluoride) and incubate for 10 minutes on ice. After this osmotic shock, the cells are centrifuged and the supernatant (SN3) is collected. The button containing the cytoplasmic fraction (CF) is resuspended in 50 mmol / L of Tris-HCl (pH 8.0) / 100 mmol / L of NaCl.

Example 3: Purification of Asp-Palidipine Recombinates The supernatant fraction containing the button of the Asp-Palidipine Recombinant (SN1) is adjusted to pH 4.0 with acetic acid and applied to a cation exchange column (Mono S, Pharmacia) using an FPLC system. After washing with a gradient of 0 to 500 mmol / L of NaCl in sodium acetate pH 4.0, the elution of Asp-Palidipine is achieved with 20 mmol / L of NaPi, pH 7.0. The eluted fractions containing Asp-Palidipine, as judged by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting, are combined, adjusted to pH 8.0 and applied to an anion exchange column (Fractogel-EMD-TMAE 650 , Merck). Elution of Asp-Palidipine is achieved with a gradient of 0 to 1 mol / L of NaCl in 20 mmol / L of sodium acetate pH 8.4. For final purification, the eluting fractions containing Asp-Palidipine are combined, concentrated in Seed-Vac (Bachofer) and subjected to size exclusion chromatography using SuperScope 12 (Pharmacia).

Example 4: Platelet Aggregation Assay The assay is carried out essentially as described in the publication of C. Noeske-Jungblut (C. Noeske-Jungblut (1994) J. Biol. Chem. 269: 5050-5053). In summary, human blood is collected in a volume of 1/6 of 71 mmol / L citric acid / 85 mmol / L trisodium citrate / 111 mmol / L glucose. Platelet-rich plasma is obtained by centrifugation at 135 g for 20 min. The Asp-Palidipine is cooled with 500 μl of platelet-rich plasma for 1 minute at 37 ° C before the addition of the collagen (2 μg / ml). The aggregation is checked periodically using a Micron aggregometer and the maximum value is determined. The IC5o has a value of approximately 50 nM. A significant difference between the compounds with or without Asp in the first position can not be observed. The biological activity and the yield of purified Asp-Palidipine are determined from the periplasmic space of E. col i. The platelet-rich plasma is incubated with Asp-Palidipine and controls.

Aggregation is induced by the addition of collagen. The wild-type Palidipin purified from saliva is used as a positive control. In addition, some other constructions are used as controls, showing the advantage of the Asp-Palidipine of the invention. The protein of the invention and the controls show different yields (Table 2).

TABLE 2 Performance Strain Recombinant Palidipine 461 μg Recombinant Arginyl-Palidipine 298 μg Recombinant Aspartyl-Palidipine 864 μg Example 5: The adhesion of tumor cells to collagen is decreased in the presence of the protein of the invention The protein of the invention inhibits the adhesion of tumor cells to a collagen matrix. Therefore, it can be partially or completely prevented that the migrating tumor cells are established in organs or blood vessels, when the protein of the invention is within the blood or plasma of the patient. The MTLn3 cells (rat mammary tumor cells) are labeled with 31 Cr. A plate with wells is coated with collagen (type III) at 4 ° C overnight. 2 • 104 cells labeled in 500 μl of F12 DMEM medium, 20 mmol / l of Hepes, 1 mmol / L of bicarbonate, 1% of BSA are first incubated with 0, 2, 5 or 10 μl of protein of the invention (" Combined Superóse ", 0.5 mg protein / ml) respectively for 10 minutes at 37 ° C. Subsequently this suspension is transferred to a well coated with collagen and incubated for 2 hours at 37 ° C. After this, the wells are washed and the adherent cells are removed with 1 mol / L of NaOH. The radioactivity of the adherent cells is counted.

TABLE 3 Quantity of the aggregated inhibitor Cell coupling μl (cpm) 0 2215 2 2071 5 1608 10 1081 Example 6: Production of antibodies Approximately 100 μg of the purified inhibitor according to the examples are added to 0.5 ml of Freund's complete adjuvant, and the emulsion is injected subcutaneously into a rabbit. After 2 weeks a second injection consisting of approximately 80 μg of purified inhibitor and 0.5 ml of incomplete Freund's adjuvant is given. After the injection, several serum samples are taken to verify the production of specific antibodies. These are tested in a Western blot. 20 ng of the purified inhibitor is applied on a 12.5% SDS-polyacrylamide gel and the electrophoresis, staining and detection are performed according to the standard methods described by E. Harlowe, D. Lane, (1988) Antibodies: a laboratory manual, Cold Spring Harbor Laboratory (dilution of the test serum 1: 500, IgG conjugated with goat anti-rabbit peroxidase as second antibody, detection with the ECL team Amersham International, Amersham, Reino United) . The spotting shows that the antiserum reacts specifically with the purified inhibitor. The preceding examples can be repeated with similar success by substituting the generic or specifically described reagents and / or the operating conditions of this invention for those used in the preceding examples. From the foregoing description, one skilled in the art can readily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to the various uses and terms.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: SCHERING AKTIENGESELLSCHAFT (B) STREET: MUELLERSTRASSE 172 - 178 (C) CITY: BERLIN (D) COUNTRY: GERMANY (E) ZIP CODE: 13353 (F) TELEPHONE: (030 ) - 468-2085 (G) TELEFAX: (030) - 468-2058 (ii) TITLE OF THE INVENTION: PROCESS FOR THE MANUFACTURE OF A MODIFIED PALIDIPINE, INHIBITOR OF PLATELET AGGREGATION INDUCED BY COLLAGEN (iii) SEQUENCE NUMBER: 5 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: floppy disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: patentln Relay # 1.0, Version 1.30 (EPO) (v) DATA OF THE CURRENT APPLICATION: NUMBER OF THE APPLICATION: EP 94250224.6 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 172 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: no ( v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Asp Glu Glu Cys Glu Leu Met Pro Pro Gly Asp Asn Phe Asp Leu Glu 1 5 10 15 Lys Tyr Phe Ser He Pro His Val Tyr Val Thr His Ser Arg Asn Gly 20 25 30 Pro Lys Glu Gln Val Cys Arg Glu Tyr Lys Thr Thr Lys Asn Ser Asp 35 40 45 Gly Thr Thr Thr Thr Thu Leu Val Thr Ser Asp Tyr Lys Thr Gly Gly 50 55 60 Lys Pro Tyr His Ser Glu Leu Lys Cys Thr Asn Thr Pro Lys Ser Gly 65 70 75 80 Gly Lys Gly Gln Phe Ser Val Glu Cys Glu Val Pro Asn Gly Asn Gly 85 90 95 Gly Lys Lys Lys He His Val Glu Thr Ser Val He Wing Thr Asp Tyr 100 105 110 Lys Asn Tyr Ala Leu Leu Gln Ser Cys Thr Lys Thr Glu Ser Gly He 115 120 125 Wing Asp Asp Val Leu Leu Leu Gln Thr Lys Lys Glu Gly Val Asp Pro 130 135 140 Gly Val Thr Ser Val Leu Lys Ser Val Asn Trp Ser Leu Asp Asp Trp 145 150 155 160 Phe Ser Arg Ser Lys Val Asn Cys Asp Asn Met Lys 165 170 ) INFORMATION FOR SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 171 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Asp Glu Glu Cys Glu Leu Met Pro Pro Gly Asp Asn Phe Asp Leu Glu 1 5 10 15 Lys Tyr Phe Ser He Pro His Val Tyr Val Thr His Ser Arg Asn Gly 20 25 30 Pro Lys Glu Gln Val Cys Arg Glu Tyr Lys Thr Thr Lys Asn Ser Asp 35 40 45 Gly Thr Thr Thr Thr Leu Val Thr Ser Asp Tyr Lys Thr Gly Gly Lys 50 55 60 Pro Tyr His Ser Glu Leu Lys Cys Thr Asn Thr Pro Lys Ser Gly Val 65 70 75 80 Lys Gly Gln Phe Ser Val Glu Cys Glu Val Pro Asn Gly Asn Gly Gly 85 90 95 Lys Lys Lys He His Val Glu Thr Ser Val He Wing Thr Asp Tyr Lys 100 105 110 Asn Tyr Ala Leu Leu Gln Ser Cys Thr Lys Thr Glu Ser Gly He Ala 115 120 125 Asp Asp Val Leu Leu Leu Gln Thr Lys Lys Glu Gly Val Asp Pro Gly 130 135 140 Val Thr Ser Val Leu Lys Ser Val Asn Trp Ser Leu Asp Asp Trp Phe 145 150 155 160 Ser Arg Ser Lys Val Asn Cys Asp Asn Met Lys 165 170 ) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 172 amino acids (B) TI PO: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: l ineal (ii) ) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: no (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Asp Glu Glu Cys Glu Leu Met Pro Pro Gly Asp Asn Phe Asp Leu Glu 1 5 10 15 Lys Tyr Phe Ser He Pro His Val Tyr Val Thr His Ser Arg Asn Gly 20 25 30 Pro Lys Glu Gln Val Cys Arg Glu Tyr Lys Thr Thr Lys Asn Ser Asp 35 40 45 Gly Thr Thr Thr Thr Thu Leu Val Thr Ser Asp Tyr Lys Thr Gly Gly 50 55 60 Lys Pro Tyr His Ser Glu Leu Lys Cys Thr Asn Thr Gln Lys Ser Gly 65 70 75 80 Gly Lys Gly Gln Phe Ser Val Glu Cys Glu Val Pro Asn Gly Asn Gly 85 90 95 Gly Lys Lys Lys He His Val Glu Thr Ser Val He Wing Thr Asp Tyr 100 105 110 Lys Asn Tyr Ala Leu Leu Gln Ser Cys Thr Lys Thr Glu Ser Gly He 115 120 125 Wing Asp Asp Val Leu Leu Leu Gln Thr Lys Lys Glu Gly Val Asp Pro 130 135 140 Gly Val Thr Ser Val Leu Lys Ser Val Asn Trp Ser Leu Asp Asp Trp 145 150 155 160 Phe Ser Arg Ser Lys Val Asn Cys Asp Asn Met Lys 165 170 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "primer in sense" (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GCGATATCGC GACGAAGAAT GCGAACTCAT G 31 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "antisense primer" (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GCGATAGGAT CCAAGCTTAT TACTTCATGT TATC 34

Claims (16)

RE IVINDICATIONS

1. A process for the manufacture of a recombinant Palidipine protein (Asp-Palidipine), wherein Asp-Palidipine inhibits platelet aggregation induced by collagen, of mammalian platelets, and wherein Asp-Palidipine comprises: (i) a protein , (Palidipine) selected from the group of proteins Palidipine, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of the Palidipine; whereby Asp-Palidipine has the following amino acid sequences; a) the sequences indicated in aa) SEQ ID NO: l; bb) SEQ ID NO: 2; or ce) SEQ ID NO: 3; or b) an allelic variant or mutein of the sequences, in any of the SEQ ID NOS: la 3, whose allelic variation or mutein has the activity of the mature protein, except those sequences that do not have the aspartic acid connected by a peptide bond with the N-terminal end of Palidipine or c) a protein according to any of SEQ ID NOS: la 3 or a variant or mutein mentioned under subparagraph b), which has a post-translational modification which does not have the activity of the mature protein; comprising the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises an operable linkage of: (i) a first DNA or cDNA molecule, encoding recombinant Asp-Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) an appropriate promoter; whereby, after expression and transport to the periplasm, the preprotein comprising the signal peptide and the Asp-Palidipine is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the Asp. - Mature palidipine, bb) the expression of the preprotein comprising the Asp-Palidipine and the signal peptide sequence; ce) the transport of Asp-Palidipine from the cytoplasm of the bacterium to the periplasm, whereby the cleavage of the preprotein by at least one protease during transport produces the mature Asp-Palidipine, dd) the isolation of the Asp- Palidipine by extracting the periplasm, and ee) the purification of Asp-Palidipine

2. A process according to claim 1, wherein the bacterium that is transfected with an appropriate vector is cultured, wherein the vector comprises an operable linkage of: (i) a first DNA or cDNA molecule, encoding an Asp- Recombinant palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) an appropriate promoter; whereby, after expression and transport to the periplasm, the preprotein comprising the signal peptide and the Asp-Palidipine is cleaved such that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the Asp. - Mature lipidipin, under conditions whereby: (A) the preprotein comprising the Asp-Palidipine and the signal peptide sequence is expressed, and (B) the Asp-Palidipine is transported from the cytoplasm of the bacterium to the periplasm, whereby the cleavage of the protein by at least one protease during transport, produces the mature Asp-Palidipine, and the purification of the Asp-Palidipine produced in this way, coming from the periplasm.

3. A process according to claim 1 or 2, wherein the DNA encoding the signal sequence codes for the alkaline phosphatase signal sequence (APase).

4. A process according to claim 1 to 3, wherein the bacterium is E. coli

5. A recombinant protein of Asp-Palidipine, wherein Asp-Palidipine inhibits the platelet aggregation induced by collagen, of human platelets, and wherein Asp-Palidipine comprises: (i) a protein, (Palidipine) selected from the group of Palidipine proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminus of the Palidipine; whereby Asp-Palidipine has the following amino acid sequences: a) the sequences indicated in: (aa) SEQ ID NO: 1; (bb) SEQ ID NO: 2; or (ce) SEQ ID NO: 3; or b) an allelic or mutein variant of the sequences according to any of SEQ ID NOS: 1 to 3, whose allelic variation or mutein has the activity of the mature protein, except those sequences which do not have aspartic acid connected by a link peptide with the N-terminal end of Palidipine. or c) a protein according to any one of SEQ ID NOS: 1 to 3 or a variant or mutein mentioned under subparagraph b) having a post-translational modification which does not substantially affect the activity of the mature protein.

6. A recombinant protein according to claim 5, wherein Asp-Palidipine is produced by a process comprising the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises an operable link of : (i) a first DNA or cDNA molecule, which codes for recombinant Asp-Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) an appropriate promoter; whereby, after expression and transport of the periplasm, the preprotein comprising the signal peptide and the Asp-Palidipine, is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the Asp. Mature-lipidipine, bb) the expression of the preprotein comprising the Asp-Palidipine and the signal peptide sequence; ce) the transport of Asp-Palidipine from the cytoplasm of the bacterium to the periplasm, by which the cleavage of the preprotein by at least one protease during transport, produces the mature Asp-Palidipine, dd) the isolation of the Asp. -Palidipine by extracting the periplasm, and ee) the purification of Asp-Palidipine.

7. A recombinant protein according to claim 5, wherein the Asp-Palidipine is produced by a process comprising: cultivating a bacterium transfected with an appropriate vector, wherein the vector comprises an operable link of: (i) a first DNA or cDNA molecule, which codes for a recombinant Asp-Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iii) an appropriate promoter; whereby, after expression and transport to the periplasm, the preprotein comprising the signal peptide and the Asp-Palidipine is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the Asp. - Mature lipidipin, under conditions whereby (A) the preprotein comprising the Asp-Palidipine and the signal peptide sequence is expressed, and (B) the Asp-Palidipine is transported from the cytoplasm of the bacterium to the periplasm, with which cleavage of the protein by at least one protease during transport produces the mature Asp-Palidipine, and the purification of the Asp-Palidipine from the periplasm.

8. An Asp-Palidipine according to claim 6 or 7, wherein the DNA encoding the signal peptide sequence encodes an alkaline phosphatase signal sequence (APase).

9. An Asp-Palidipine according to any of claims 5 to 8, which is to be used as a medicine.

10. A pharmaceutical composition, comprising the Asp-Palidipine according to any of claims 5 to 8 in association with a pharmaceutically acceptable diluent or carrier.

11. The use of an Asp-Palidipine according to any of claims 5 to 8, for the manufacture of a medicament for the treatment of atherosclerotic or thrombotic disease or for the prevention of reocclusion after the treatment of myocardial infarction.

12. The use of Asp-Palidipine according to any of claims 5 to 8, for the manufacture of a medicament for the treatment of cancer with metastatic tumor cells.

13. A process for the purification of Asp-Palidipine, wherein the purification comprises the following consecutive steps: (i) purification by cation exchange chromatography; (ii) purification by anion exchange; and (iii) purification by size exclusion.

14. A recombinant vector, comprising a linkage of: (i) a first DNA molecule or cDNA, encoding a recombinant Asp-Palidipine, having an amino acid sequence selected from: a) the sequences indicated in aa) SEQ ID NO: 1; bb) SEQ ID NO: 2; or ce) SEQ ID NO: 3; or b) an allelic or mutein variant of the sequences according to any of SEQ ID NOS: 1 to 3, whose allelic variation or mutein has the activity of the mature protein, except those sequences which do not have the aspartic acid connd by a peptide bond to the N-terminus of Palidipine, (ii) a second DNA molecule, which codes for an appropriate signal peptide sequence, and (iü) an appropriate promoter; whereby, after expression in an appropriate bacterial host, the preprotein comprising the signal peptide and the Asp-Palidipine is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence of the Asp. -Palidipina matures.

15. A bacterial host, transformed with a vector according to claim 14.

16. A bacterial host, according to claim 15, which is an E. col i. SUMMARY The invention provides a process for the manufacture of a recombinant protein called Asp-Palidipine. Asp-Palidipine inhibits the platelet aggregation induced by the collagen of mammalian platelets. Asp-Palidipine comprises: (i) a protein (Palidipine) selected from the group of Palidipine proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond to the N-terminal end of the Palidipine . The process comprises the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises: (i) a DNA or cDNA encoding recombinant Asp-Palidipine, (ii) a peptide sequence of signal, appropriate whose signal sequence is cleaved so that the amino acid aspartic acid is in the +1 position of the amino acid sequence observed from the position of Palidipine and (iii) an appropriate promoter, bb) the expression of the preprotein that comprises Asp-Palidipine and the signal sequence; ce) the transport of Asp-Palidipine from the cytoplasm of the bacteria to the periplasm, the excision of the preprotein by at least one protease during transport, producing Asp-Palidipine, dd) the isolation of Asp-Palidipine by extraction of the periplasm, and ee) the purification of Asp-Palidipine. The protein is used as a drug for the inhibition of human platelet aggregation induced by collagen, or cancer with metastatic tumor cells.