MXPA01003292A - Contortrostatin (cn) and methods for its use in preventing metastasis and other conditions - Google Patents

Abstract

The amino acid sequence of native contortrostatin was used in a cloning strategy to obtain full-length cDNA and deduced amino acid sequnces for a contortrostatin precursor. The precursor includes pro-protein, metalloproteinase, and disintegrin (contortrostatin) regions of the multidomain protein. The sequences can be used to produce recombinant DNA molecules which code on expression for contortrostatin proteins, including biologically active variants and fragments. When formulated as a pharmaceutically acceptable composition, the proteins can be used to treat patients by inhibiting disease processes associated with an integrin binding to an integrin receptor.

Description

CONTOUROSTATINE AND METHODS FOR ITS USE TO PREVENT METASTASIS AND OTHER CONDITIONS FIELD OF THE INVENTION The present invention relates generally to the field of biochemistry and medicine in particular to the cloning, sequencing and production of contortrostatin and its precursor.

BACKGROUND OF THE INVENTION Breast cancer is one of the leading causes of death among nonsmoking women and the spread of the disease from the breast to different sites is a leading cause of death in patients with breast cancer. At the time of diagnosis, more than 60% of patients with breast cancer will have disease that has spread from a primary site in the breast to other distant sites. The spread of cancer to remote sites, for example bone, lungs, liver, brain, a process called metastasis, is a characteristic of malignancy and frequently leads to an inoperable disease. Metastasis is the most common factor that leads to death from breast cancer. The control of metastasis offers an important avenue for the treatment of breast cancer. Cancer cells metastasize through the blood or lymphoid vessels. The first step of metastasis involves the attachment of cancer cells to the tissues around the primary site, ie, to the extracellular matrix (ECM) via cell surface integrins and other adhesion receptors. Integrins mediate cell-cell and cell-substrate interactions and are involved in the signaling and targeting that binds the ECM with cytoskeletal proteins. The integrins play an important role in the interaction of breast carcinoma cells with the ECM. In the second step, the cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade these tissues. Finally, the tumor cells enter the blood or lymphatic system where they repeat the steps of adhesion and invasion at a distant (metastatic) site. At this remote site, tumor cells induce the formation of new blood vessels (a process called neovascularization), in and around the growing tumor. These new blood vessels supplement the nutrients for the metastatic tumor and allow its growth. Treatments that block any of these steps should act to inhibit metastasis. The integrins on cancer cells play an important role in the invasion of the tumor and in its extension. You are a family of proteins found on the cell surface of many cell types that mediate the interactions between cells, and between cells and their surroundings. CN binds to a specific integrity on the surface of blood platelets, and blocks the ability of platelets to adhere to one another (a process called platelet aggregation). Platelets are small fragments of bone marrow cells that are found in the bloodstream. You have both beneficial and harmful activities. Its useful action is to stop the bleeding after an injury by facilitating the formation of a blood clot. But under certain conditions they are involved in the blockage of arteries that maintain nutrition to the heart - an action that can lead to a heart attack. The integrins are heterodimers, composed of subunits a and β involved in cell-cell and cell-substrate interactions. Integrins serve as receptors for extracellular matrix proteins such as fibronectin, fibrinogen, vitronectin, collagen and laminin. Some of these interactions have been found to be mediated via an Arg-Gly-Asp (RGD) sequence present in the matrix proteins. Both subunits a and ß are required for binding to fibrogen. For example, one member of the superfamily of cell surface receptors by integrins is the platelet membrane glycoprotein (GP) llb / llla which interacts with plasma fibrinogen in platelet aggregation. The integrin-like cell surface receptors have been investigated in the role of platelets in mediating coronary artery thrombosis and rethrombosis in the genesis of acute myocardial infarction [Zucker, M.B., Sci. American 242: 86 (1990)]. For platelet aggregation an RGD sequence present in fibrinogen is essential for the interaction with (GP) llb / Illa (Ginsberg, MH et al., Thrombos, Haemostas, 59: 1 (1988).] Because of its inhibition of platelet aggregation Snake venom has been the target of several investigations and it was found that a number of proteins purified from snake venom of the families Crotalidae and Viperidae inhibit the glycoprotein (GP) IIb / llla mediating platelet aggregation (see , for example, Huang, TF et al., J. Biol. Chem. 262: 16157 (1987); Gan, Z.R. et al., J. Biol. Chem. 263: 19827 (1988); Yasuda, T. Et al., J. Am. Coil. Cardiol 16: 714 (1990); Trinkha, M. Et al., Fibrinolysis 4 (Suppl 1): 105 (1990); Trinkha, M. et al., Blood 76 (Suppl 1): 479a (1990); Holahan, M.A. et al., Pharmacology 42: 340 (1991); Shebuski, R.J. et al., Circulation 82: 169 (1990); Yasuda T. Et al., Circulation 83: 1038 (1991)]. These proteins, classified as disintegrins, are typically rich in disulfide. In addition, all disintegrins isolated so far, with the exception of barburine (Scarborough, RM et al., J. Biol. Chem. 266: 9359 (1991)] contain an RGD sequence (Arg-Gly-Asp) that has been implicated As a participant in integrin-mediated inhibition interactions, in particular, the RGD sequence of disintegrins can compete for fibrinogen-binding sites on the platelet membrane, thereby inhibiting platelet aggregation induced by ADP or other agents. , the evidence that disintegrins may have a unique surface geometry that facilitates interaction with integrins by mechanisms other than those based only on the RGD site seems to be increased, for example, the finding that a mutation, chemically synthesized and derived from the echistatin (in which alanine is replaced by arginine in the RGD sequence) still possesses similar biological activity, suggesting that other regions in the protein may participate in the binding and that there may be some flexibility in the RGD binding site [Connolly, T.M. et al., Circulation 82 (Suppl Ill): 660 (1990)]. The synthetic peptides RGD due to their small size, generally do not possess the molecular topography of the disintegrins and therefore can not interact via the multiplicity of mechanisms that are probably involved in the disintegrin binding. In other investigations, prevention of reocclusion after thrombolysis using tissue-type plasminogen activator in a canine model system using 30 μg / kg plus 3 μg / kg / min of bitistatin, an 83 amino acid disintegrin derived from the venom, has been reported. of Bitis arietans [Shebuski et al., previously mentioned], or 15 μg / kg / min iv of echistatin, a disintegrin of 49 amino acids derived from the venom of Echis carinatus [Holahan et al., previously mentioned]. In the reported methods, an initial bolus of heparin (100 U / kg i.v.) and subsequent boluses of 50 U / kg per hour were used to increase the activated partial thromboplastin times at least 1.5 times over the control. Although it had previously been observed that heparin in combination with tissue-type plasminogen activator (PA) does not affect the incidence of acute reocclusion in this model system, the addition of equistatin or bitistatin led to a dramatic reduction in the incidence of thrombotic reocclusion. acute The administration of heparin was, however, apparently necessary for the prevention of acute thrombotic reocclusion. Similarly, kisrina (a 68 amino acid disintegrin derived from the venom of Agkistrodon rhodostoma) was evaluated in conjunction with the recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis with a high degree of overlap stenosis (Yasuda et al. (1991), previously mentioned). An effective dose of 4 μg / kg / min was determined to be sufficient to prevent reocclusion. Simultaneously the systemic anticoagulant therapeutic heparin was used; the dose of heparin was selected to maintain the partial thromboplastin time level more than double through the experimental observation period. The patent of E.U.A. 5,066,592 to Huang et al. describes the use of trigamin, a 72-amino-disintegrin isolated from the venom of Trimeresurus gramineus, to inhibit the binding of fibrinogen to human platelets and thereby inhibiting the fibrinogen-induced aggregation of human platelets. It is also reported that trigeramines inhibit the binding of von Willebrand factor. It is reported that trigramines inhibit the binding of 125I-fibrinogen to platelets stimulated by ADP (10 μmolar) in a concentration-dependent manner with an IC50 of 2.8-5.6 x 10"8 M. The isolation of a factor has also been reported. antiplatelet called aplagin from the venom of Agkistrodon piscivorus piscivorus [Chao, BH et al., Proc. Nati, Acad. Sci. USA 86: 8050 (1989); Savage, B. et. A, J. Biol. Chem. 265: 11766 (1990)]. It is reported that aplagin, unlike trigramine, inhibits the secretion of dense granules in conjunction with the inhibition of platelet aggregation in a dose-dependent manner. Although initially described as a homodimer with at least two interchain disulfide bridges [Chao et al. (1989), previously mentioned], a subsequent report indicates that the analysis of purified lamellae by mass spectroscopy shows the presence of monomers of aplagin with a mass of 7,666 Daltones and shows no evidence of dimerization [Wencel-Drake, J.D. et al., Blood 81: 62 (1993)]. A disintegrin is of particular interest is CN which has been isolated from the venom of Agkistrodon contotrix contotrix (the snake head of copper from the Southeast). The purification procedure originally reported included a molecular sieve chromatography on Sephadex G-100 SF, the elimination of salt on Sephadex G-25F and a reverse phase HPLC. The improved aggregation to ADP was monitored on the agitated plasma rich in human platelets and the inhibition thereof by CN at 37 ° C. It was found that preincubation for one minute of the platelet-rich plasma (3 x 105 / mm3) with 5μl of the low molecular weight peak after Sephadex G-100 SF resulted in 76% inhibition of platelet aggregation induced by 10 μM ADP [Trikha et al, (1990), previously mentioned]. In a subsequent report it was pointed out that in crude venom, inhibition was not easily detected due to the presence of a platelet aggregation activity; However, following the first purification step (hydrophobic interaction HPL), the inhibition activity was separated in the aggregation activity and in the fibrinolytic enzyme degrading the chain present in the venom. The inhibitory activity was added after HPLC and applied to a HPLC column of hydroxylapatite. In the final step of the purification, reverse phase C4 HPLC chromatography was used. The yield of the homogeneous protein was 3-5 mg per gram of venom. It is reported that CN has a molecular weight of 18-21 kDa under non-reducing conditions and 9 kDa under reducing conditions; thus, it is believed that the molecule is a homodimer with the two subunits held together by disulfide bond (s). The isoelectric point showed that the protein had an acidic pl. CN was reported not to exhibit fibrinolytic activity and that it was not a 5 'nucleotidase or a phospholipase based on the molecular weight and the kinetics of the inhibition of platelet aggregation. After pre-incubation for one minute, it was reported that CN at approximately 100 nM completely inhibits platelet aggregation induced by ADP [Trikha et al. (1990), previously mentioned]. It has also been reported that CN has 70 amino acids with five or six disulfide bonds, and that the CN sequence seems to start with 10 amino acids towards the 3 'end of the aplagin (an inhibitor of platelet aggregation of Agkistrodon piscivorus venom). It is speculated that the CN may have an insertion and / or C-terminal extension of 9 amino acids. It is also reported that 50% of the inhibition (IC50) of human platelet aggregation in platelet-rich plasma is observed at 0.8 μg / ml CN, and at 2.2 μg / ml with canine platelets fTrikha, M. et al., Journal of Cellular Biochem. 16F: 180 (1992)]. It is reported that CN inhibits the binding of human fibrosarcoma (HT-1080) and rat embryo cells transfected with c-Ha-ras (4R) to fibronectin-coated plates but not to plates covered with Matrigel. Inhibition of 4R cells for fibronectin binding in the presence of CN at 1 μg / ml and 5 μg / ml was 46% and 88% respectively, and for HT1080 cells the inhibition was 89% and 85%, respectively [ Trikha M. et al., Proceedings of the American Association for Cancer Research 33:34 (1992)]. Since it appears that CN can inhibit interactions between integrins and their receptors, and may be useful in the management of diseases associated with these interactions, there is a need for improved methods that produce a greater amount of purified contortrostatin, substantially free of other components. of the snake venom.

BRIEF DESCRIPTION OF THE INVENTION The present invention satisfies the need for greater amounts of contortrostatin which can be used to inhibit biological processes such as platelet aggregation, cell growth, adhesion, metastasis and neovascularization. The native contortrostatin was purified and a partial amino acid was determined by Edman degradation. This information enables the cDNA cloning strategy, which results in a full-length cDNA sequence and an amino acid sequence deduced from a contortrostatin precursor protein. The contortrostatin precursor includes a pro-protein region, a metalloproteinase region, and a disintegrin region. The metalloproteinase region includes a metal binding motif and the disintegrin region includes an RGD loop, which can act as an integrin antagonist. The sequences for native contortrostatin are contained in the disintegrin region. The present invention includes purified contortrostatin proteins, including the contortrostatin precursor, biologically active variants, and fragments thereof. The contortrostatin protein preferably includes an amino acid sequence that binds to the native contortrostatin monomer (amino acids numbers 419 to 483 of SEQ ID NO: 2), the metalloproteinase region (amino acids numbers 191 to 410 of SEQ ID NO: 2), the region pro-protein (amino acids numbers 1 to 190 of SEQ ID NO: 2), or to the precursor of contortrostatin as a whole (SEQ ID NO: 2). A most preferred purified protein comprises contortrostatin monomers, each having a molecular mass of about 5 to about 7 kDa, which forms a homodimer. A purified contortrostatin, which can act as an integrin antagonist will generally include in each monomer a reduced Arg-Gly-Asp (RGD) sequence at the end of a flexible peptide loop of approximately 13 amino acid residues flanked by two Cys residues, The amino acid sequence comprises amino acids numbers 457 to 469 of SEQ ID NO: 2. Biologically active variants can include amino acid substitutions, deletions and insertions, but will generally have an amino acid sequence that is at least 90% homologous to the amino acids. pro-protein, metalloproteinase, disintegrin and / or contortrostatin regions of the precursor protein. Variants may also include a peptide recognized by an antibody to contortrostatin. The proteins of the present invention can be made using synthetic methods. The synthetic process may include transcribing and translating a contortrostatin cDNA molecule as described herein, preferably into a transformed host cell. Alternatively, the process involves the synthesis of a polypeptide having the amino acid sequence of contortrostatin, as described herein. Proteins prepared by recombinant DNA methodology will generally include the use of a recombinant DNA molecule comprising a DNA sequence encoding expression for contortrostatin, such as SEQ ID NO: 1. preferably the recombinant DNA molecule encodes sequences having the less a biological activity such as metalloproteinase (for example nucleotides numbers 657 to 1316 of SEQ ID NO: 1), or disintegrin (for example, nucleotides numbers 1341 to 1535 of SEQ ID NO: 1). In addition, the recombinant DNA molecule can also include the sequences coding for pro-protein (eg, nucleotides numbers 87 to 656 of SEQ ID NO: 1), the complete precursor protein (nucleotides numbers 87 to 1535 of SEQ ID NO: 1), or simply the coding sequences of the native contortrostatin monomer (nucleotides numbers 1341 to 1535 of SEQ ID NO: 1). The present invention further provides a vector, which includes the recombinant DNA molecule, which can be used to transform prokaryotic or host eukaryotic cells. The host cells can be mammalian cells, plant cells, insect cells, yeast and other fungi or bacteria. Processes for producing recombinant contortrostatin will generally include steps, such as culturing the host cell and recovering the contortrostatin expressed by the host cell. The contortrostatin proteins of the present invention can be formulated as pharmaceutically acceptable compositions, comprising a pharmaceutically acceptable carrier and the purified protein. The pharmaceutical composition can be used in a method for treating a patient having a disease associated with the binding of a ligand to an integrin receptor. The treatment generally involves the administration of the composition to the patient so that the binding of the integrin to the integrin receptor is substantially inhibited and the patient is treated. The treatment method can be used to inhibit platelet aggregation, tumor metastasis, angiogenesis, neovascularization, cell adhesion, invasion, or growth.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is also better understood with reference to the accompanying drawings, which include: Figure 1 illustrates the strategy for cloning contortrostatin cDNA, wherein Figure 1A shows the partial amino acid sequence of CN (CN) based on the test of Edman degradation compared to other disintegrins illustrated with common RGD sequences and highly conserved sequences, Figure 1B shows primers for PCR, Figure 1C shows the principle of the overlap extension reaction used to generate the full-length cDNA of contortrostatin; Figure 2 shows the electrophoresis for the PCR products, where a major band of about 1,300 bp was amplified with the PCR primer by the? Gt10 forward and PCR-2 (line 1), a band of approximately 700 pb that resulted from the PCR initiator by PCR-2 and the reverse? gt10 (line 2), and the overlapping products of the two fragments are shown in line 3, with a molecular size of approximately 2,000 bp; Figure 3 shows the nucleotide sequence of total length of the CN cDNA and deduced amino acids, including an untranslated region at the 5 'end of 86 nucleotides (NTR), and an open reading frame between nucleotides 87 and 1535, a stop codon at nucleotides 1536 to 1538, and a 3'-NTR, which includes an AATAAA site and a term with a poly-A tail; Figure 4 shows the structure of the multidomain of the contortrostatin precursor, in comparison with the precursor of trigramin and of other hemorrhagic proteins of the snake venom; Figure 5 illustrates the formation of a contortrostatin homodimer, wherein Figure 5A shows the amino acid sequence and the disulfide bond pattern of the kistrin disintegrin compared to contortrostatin, which has 6 amino acids truncated at the N-terminus. terminal, including two cysteine residues that result in two cysteine residues that are unpaired, and Figure 5B shows the unpaired cysteines that can participate in the formation of the two intermolecular disulfide bonds to form a unique homodimeric structure, wherein the two monomers can be joined in parallel or in antiparallel pattern; Figure 6 illustrates the results of determinations of CN inhibition of platelet aggregation in rabbit, human and dog; Figures 7A and 7B illustrate the results of the binding studies of CN to (GP) llb / llla (Figure 7A) and canine (Figure 7B) in the presence of a fixed saturating concentration of the murine monoclonal antibody 7E3; Figure 8 is a schematic representation of an instrumented dog carotid artery showing the placement of an ultrasonic flow probe, or a mechanical constrictor (stenosis) and an intracarotid anodal electrode to induce intimal injury to the blood vessel wall to initiate the formation of thrombi; Figure 9 illustrates platelet count and platelet aggregability as a percentage of the zero time value in the dog treated with the anisoylated plasminogen streptokinase activator (APSAC) and CN complex; Figure 10 shows that CN inhibits the adhesion of MDA-MB-435 to fibronectin; Figure 11 shows that CN inhibits adhesion of MDA-MB-435 to vitronectin; Figure 12 shows the inhibition of binding of human mammary carcinoma cells to CN immobilized with GRG-DSP; Figure 13 shows the inhibition of binding of human mammary carcinoma cells to CN immobilized with EDTA; Figure 14 shows the inhibition of cell invasion MDA-MB-435 through an invasion chamber covered with Matrigel. Figure 15 shows the effect of CN on tumor growth MDA-MB-435 in experimental nude mice; Figure 16 is a photograph demonstrating tumor-induced angiogenesis in a chorioallantoic membrane of the control chicken embryo (CAM) (figure 16A), treated CAM with 20 μg of CN (figure 16B), and treated with CAM 150 μg of CN (Figure 16B); and Figure 17 shows the effect of CN on the proliferation of MD.A-MB-435 cells in vitro.

DETAILED DESCRIPTION OF THE INVENTION Characterization, cloning and expression of CN The inventors have purified and characterized a disintegrin: contortrostatin (CN) from the venom of A. c. contortrix CN is a homodimer with a mass of 13.505 of the intact protein and 6.956 for the reduced and pyridylethylated protein. To evaluate the binding affinity to (GP) llb / llla (platelet fibrinogen receptor), CN competition was analyzed with [125] 7E3, an antibody directed to (GP) llb / IIIA, using plasma rich in human platelets ( PRP). CN exhibited an IC50 of 25 nM. Thus, CN is a potent antagonist of β3 integrin.

The CN cDNA has been amplified from a genomic library of the A.c. contortrix built in? gtlO. The amino acid composition and partial amino acid sequence of CN have been determined by Edman degradation: see figure 1. Using this information a full-length cDNA of 2.027 nucleotides (SEQ ID NO: 1) has been cloned and sequenced, which encodes a contortrostatin precursor protein.

As a member of the disintegrin superfamily, CN shows high similarity with other disintegrins including, trigramin whose nucleotide sequence is known. Figure 1A shows the partial amino acid sequence of CN based on the Edman degradation assay. The partial sequence is also compared to other disintegrins as indicated. The RGD sequence is in bold. The highly conserved PCCDAATCKL sequences on which the PCR primers were designed are underlined. Figures 1A and 1B show how the CN cDNA has been cloned by PCR using primers based on the highly homologous sequences between the disintegrin family as well as the known sequences of? Gt10 flanking the cDNA inserts. The pair of primers for PCR are: SEQ ID NO: 5 (forward? Gt10 primer) and SEQ ID NO: 3 (PCR-1) 5'-GATTTACAGGTTGCAGCATCGC-3 \ which is antisense of the trigramin cDNA encoding part of the conserved sequence underlined (figure 1A and 1 B). SEQ ID NO: 4 (PCR-2), which is complementary to PCR-1 and SEQ ID NO: 6 (? Gt10 reverse primer). SEQ ID NO: 5 and 3 amplify the DNA encoding the amino acids for the 5 'end of the underlined part. SEQ ID Nos. 4 and 6 amplify those coding for the part towards the 3 'end of CN. The full-length cDNA has been obtained by the overlapping extensions of the two pieces of the PCR product (see Figures 1 C and 2). The cDNA sequence and deduced amino acid sequence of the contortrostatin precursor are shown in Figure 3. The full length sequence is comprised of 2,029 nucleotides (SEQ ID NO: 1). It is composed of 86 nucleotides from a non-translatable region at the 5 'end, and an open reading frame that encodes 483 amino acids (SEQ ID NO: 2), and a region that does not encode at the 3' end. Figure 4 shows the multidomain structure of contortrostatin compared to the other four hemorrhagic snake venom proteins: trigramin; Cat (catrocolastatin from Crotalus atrox venom); jararagina (from Bothrops jararaca venom); and Ht-e (from C. Atrox's poison). In accordance with the structural division of the snake venom metalloproteinases, the contortrostatin precursor can be divided into a pro-protein (amino acid residues 1 to 190 of (SEQ ID NO: 1 or 2). Metalloproteinase (residues 191 to 410 of (SEQ ID NO: 1 or 2) and disintegrin (residues 419 to 483 of (SEQ ID NO: 1 or 2) domains The mature monomer of the native disintegrin starts at D419, ie, the residue of aspartic acid in the position 419. The non-underlined portions of Figure 4 show the RGD sequences of both contortrostatin and trigramin, as well as the conserved HEMGHNLGIHH sequences of the zinc binding motifs in the metalloproteinase domains for each molecule In accordance with the present invention, therefore, a protein consisting essentially of purified contortrostatin or purified contortrostatin variants which retains the properties of a destinary grin, or a contortrostatin precursor having pro-protein, metalloproteinase, and disintegrin domains is provided. These proteins can be purified from natural sources such as snake venom or they can be made by recombinant techniques as understood by those skilled in the art with reference to what is described herein. In addition, the present application claims both the native sequences and the synthetic amino acid and nucleotide sequences. Unless otherwise modified, the term "protein" as used herein encompasses both polypeptides and native and synthetic peptides. The synthetic proteins include recombinant protein and chemically synthesized. Unless otherwise indicated, the term "contortrostatin" includes both the native and synthetic versions of the proteins. The term "nucleotide sequence" includes both the DNA and RNA sequences. For example, the nucleotide sequence for the contortrostatin protein ("contortrostatin nucleotide sequence") includes the gene ("contortrostatin gene") that encodes the native protein and precursor, its complementary DNA, and the RNA that corresponds to it.; also included are the messenger RNAs that code for the contortrostatin protein, its complementary RNA, and the DNA corresponding to the previous one. In addition, as used in this application, the nucleotide sequence includes: (1) the DNA sequence encoding the contortrostatin proteins, (2) the nucleotide sequence (which may be DNA or RNA) complementary to the preceding sequences, ( 3) the RNA sequences corresponding to the DNA sequences wherein thymidine ("T") in the described DNA sequences is replaced with uracil ("U"), (4) nucleotide sequences wherein other nucleotides known in the art such as nucleotide analogs, replace those in the original sequence, for example, 5-methyl-cytokine which replaces cytokine, and (5) nucleotide sequences which are for example, within 20% and preferably 10% variability of the previous nucleotide sequences. Since the codons of the nucleotides are redundant, equivalent nucleotide sequences which include: the nucleotide sequences encoding or that can be translated into contortrostatin proteins, their protein variants, functional equivalents, or derivatives are also included within the scope of this invention. These nucleotide sequences can also be used in the practice of the invention. In addition to the above, the nucleotide sequences of contortrostatin also include: (1) nucleotide sequences which are capable of hybridizing to the coding sequences of the respective nucleotide sequences, under severe hybridization conditions, and (2) fragments or mutated nucleotide sequences of those described herein that may encode or be translated into proteins having substantially the same characteristics / biological activities of the respective contortrostatin proteins, eg antagonists and integrin, zinc-binding agents, proteinases, anti-angiogenic factor, and other activities as described herein in modalities and additional examples.

The terms "contortrostatin proteins", as used in connection with proteins, include the respective proteins described in the Examples section, below, and protein precursors obtained by the methods of the present invention, more preferably proteins that exhibit the properties of a protein. disintegrin that are obtained from the isolation methods of the examples section below, and: (1) the protein variants of these proteins; for example these protein variants may contain amino acid sequences having for example at least 90% or more preferably at least 95% of their amino acid sequence concordant with that of the pro-protein, metalloproteinase, disintegrin and / or native contortrostatin in the regions of the protein; (2) the functions equivalent to these proteins and their variants, respectively; and (3) the derivatives, including fragments, of the contortrostatin proteins and their variants, respectively. Variants may result, for example, in substitution, insertion, or deletion of the amino acid sequence of the contortrostatin proteins. The derivatives of the protein and its variants include fragments of these proteins and immunoreactive peptides that specifically bind with antibodies to contortrostatin. Two amino acid sequences are functionally equivalent if they have substantially the same biological activity as the ability to bind to an integrin receptor. The proteins can be fused to other proteins, for example, fusions of the signal sequence can be used in order to more rapidly direct the secretion of recombinant contortrostatin proteins. Substitution variants of the proteins described herein are those in which at least one residue in the described sequence has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Thus, modifications of the primary amino acid sequences of the contortrostatin proteins also include conservative variations. The term "conservative variation", as used herein, denotes the replacement of one amino acid residue by another, biologically similar. Examples of conservative variations include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the replacement of arginine with lysine, glutamic acid with aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of substituted amino acids in place of a parent non-substituted amino acid that provides for the polypeptide to retain its biological activity, for example, the antibodies generated by the substituted polypeptides also react with the unsubstituted polypeptide. Also, as is the case for all proteins, the precise chemical structure depends on a number of factors. Since groups of ionizable amino acids and carboxyl groups are present in the molecule, a particular protein can be obtained in the form of acidic or basic salt, or in neutral form. All preparations that retain their activity when placed under suitable environmental conditions are included in the definition. Additionally, the primary amino acid sequence can be increased by derivatization using sugar portions (glycosylation) or by other complementary molecules such as lipids, phosphate, acetyl groups and the like, most commonly by conjugation with saccharides. Certain aspects of this increase are accompanied by a post-translational processing system of the producer host; other such modifications can be introduced in vitro. In any event, such modifications are included in the definition as long as the activity of the protein is not destroyed. It is expected that such modifications may affect the activity quantitatively or qualitatively, either by improving or decreasing the activity of the protein in several trials. The individual amino acid residues in the chain can also be modified by oxidation, reduction, or other derivatization, and the protein can be broken to obtain fragments which retain their activity. These alterations that do not destroy the activity do not remove the protein sequence from the definition. Below we discuss some of the modifications in greater detail as an example. Thus, glycosylation variants are included within the scope of the contortrostatin proteins. These include variants lacking completely glycosylation (non-glycosylated) and variants that have at least one glycosylation less than in the native form (deglucosylated) as well as variants in which the glycosylation has changed.

As illustrated in the examples, native CN can be isolated from the venom of Agkistrodon contortrix contortrix in a relatively direct manner. Alternatively, CN can also be prepared by exploiting a variety of commonly used biochemical methods, such as recombinant DNA technology or the like. In addition, the sequence information reported here can be used to make probes to identify variants, fragments, conserved domains or pro-proteins that have substantial homology to CN and its precursor (s). Once identified, the genes can be isolated, further manipulated, and cloned into expression vectors. Also provided is a vector containing a DNA molecule encoding a contortrostatin protein made in accordance with techniques understood by those skilled in the art with reference to those described herein. In the present invention, the nucleotide sequence of contortrostatin can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that can be manipulated by insertion or incorporation of a genetic sequence of contortrostatin. Said expression vectors contain a promoter sequence which facilitates the efficient transcription of the genetic sequence inserted in the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cell. These vectors can be used to transform competent hosts to produce transformants that are capable of producing the same protein from snake venom. In addition, a prokaryotic or eukaryotic host cell stably transformed or transfected by the vector is provided, as well as a method for making the contortrostatin protein or its biological variants. The method includes the steps of, first culturing a prokaryotic or eukaryotic host cell transformed with DNA encoding contortrostatin protein; and then, recover the protein contortrostatin. The host cell can be a mammalian cell, plant cells, insect cells, yeast and other fungi or bacteria. Sources of the DNA sequence encoding the proteins include DNA isolated from the appropriate cells or cell lines, DNA cloned from the snake venom genomic library or cloned DNA from a complementary DNA library, wherein the total complementary DNA is it is retrotranscribed to DNA and cloned. Once the DNA sequence has been identified which codes for the protein of interest, the base sequence can be determined by known means [eg, Maxam and Gilbert, Proc. Nati Acad. Sci USA 74: 560 (1977)]. In addition, hybrid DNA technology can be used to obtain expression. The DNA sequence can be mapped by restriction and the appropriate sites for breaking defined. In this sense, the sequence can be cleaved and introduced into a vector having the appropriate regulatory signals. A more detailed discussion of suitable techniques for the identification and expression of the disintegrin genes is provided, for example, U.S. Pat. No. 5,182,260 and 5,196,403 to Maraganore et al., The full description of which is incorporated herein by reference. In addition, the sequence encoding the native protein can be manipulated (eg, by single or multiple deletions or mutations) in a manner well known in the art to provide modified proteins, in which changes of one or more amino acids have been introduced. Following the procedures described herein, the determination of whether a particular polypeptide exhibits an activity profile characteristic of CN could be a matter of routine experimentation. Accordingly, the present invention contemplates both native CN and mutations thereof which exhibit the characteristic activity of the profile defined herein. further, CN can also be employed in the form of a fusion protein with a suitable thrombolytic agent. Fusion proteins of this type can be prepared analogously to those described for the formation of platelet aggregation inhibitor / antithrombin polypeptide fusion proteins in the aforementioned U.S.A. No. 5,182,260 and 5,196,403 of Maraganore et al. Despite the high frequency of homology with other disintegrins (shown in Figure 1A) [Niewiarowski, S., McLanc, MA, Kloezewiak, M., and Stewart, GJ Disitegrins and other Naturally Occurring Antagonists of Platelet Fibrinogen Receptors, Seminars in Hematology 31: 289-300 (1994)], contortrostatin exhibits a unique amino terminal sequence with a truncation of about 8 amino acids. In comparison with another disintegrin, the two cysteine residues are missing in the amino terminus of contortrostatin. It is well established that monomeric disintegrins possess an even number of cysteines, all of which are involved in the formation of the disulfide bond. The loss of two cysteine in the precursor of contortrostatin can lead to the alteration of the pairing of the disulfide bond. In this sense it could lead to the formation of two intermolecular disulfide bonds of two identical chains to form the homodimeric structure of contortrostatin. Figure 5 presents a hypothetical model of the structure of the dimeric disintegrin compared with a disintegrin, kistrin, whose position of the disulfide bond is known [Adler. M., Cárter, P., Lazarus, R.A., and Wagner, G. Cysteine pairing in the glycoprotein llb / llla antagonist kistrin using NMR, chemical analysis, and structure calculations, Biochemistry 32: 282-9 (1993)]. In figure 5A, the sequences of the two disintegrins are compared. It can be easily seen that kistrin, with 12 halves of cysteines, forms 6 disulfide bonds. By comparison, contortrostatin, which has two unpaired cysteine residues, can form the homodimeric structure present in Figure 5B. The inventors are not sure so far which alignment is preferred, but they , based on the analysis of the mass spectrum and on SDS-PAGE of the native and reduced contortrostatin, that a homodimeric structure is formed more likely to be identical to one of the two shown CN is clearly very different from aplagin, since the latter has undoubtedly proved to be a monomer [Wencel-Drake et al. (1993), previously mentioned]. In addition, CN does not inhibit platelet release reactions, as is shown to be the case with the aplagin in the aforementioned U. A. Patent numbers 5,182,260 and 5,196,403 from Maraganore et al. Finally, despite the similarities in the sequence, there are no significant differences between the sequences with respect to both the starting site and the following sequences.

Methods of use It has been found that CN is a potent inhibitor of platelet aggregation in humans, rabbit and dog in vitro. Unlike the aplagin, however, CN does not inhibit platelet release reactions. The platelets comprise a plurality of different granules, including alpha granules and dense granules, whose content is released when the platelets are added. The finding that CN does not inhibit the release of platelets from granular content (including ATP from dense granules) means that platelets can still release their contents (and therefore maintain some resemblance to normal physiological activity) no matter the inhibition of aggregation. By contrast, when aplagin inhibits platelet aggregation it also inhibits platelet release (as measured by, for example, the inhibition of serotonin release from dense granules). Thus, the normal physiological processes of platelets are necessarily disturbed with the administration of aplagin. Several lines of evidence indicate that CN inhibits platelet aggregation by specifically inhibiting the integrin receptor (GP) llb / llla. For example, in a fibrinogen- (GP) IIb / llla ELISA [Dennis, M.S. et al., Proc. Nati Acad. Sci. (USA) 87: 2471 (1990)], in which the degree of purification of (GP) IIb / IIIa bound to immobilized fibrinogen can be quantified, CN effectively blocked binding to (GP) IIb / IIIa. Additionally, the partial amino acid sequence of CN indicates considerable similarity with other known disintegrins that bind to (GP) llb / Illa. Finally, CN blocks the binding of 7E3 with (GP) llb / llla. 7E3 is a murine monoclonal antibody that specifically binds to (GP) llb / Illa, thereby inhibiting human and canine platelet aggregation [Coller, B. S. et al., J. Clin. Invest. 72: 325 (1983)]. In the presence of a low concentration of CN, the binding of 7E3 to platelets is significantly inhibited. The disintegrins of snake venom, kistrina [Yasuda et al. (1990), previously mentioned], echistatin [Holahan et al. (1991), previously mentioned] and bitistatin [Shebuski, R. J. et al. (1990), previously mentioned], have demonstrated a potential role as antithrombotic agents for use in thrombolytic therapy by improving and sustaining arterial thrombolysis in conjunction with a recombinant tissue plasminogen activator. Based on the low IC50 values of CN, its efficacy in vivo as an antithrombotic agent was examined. Using a model of the carotid artery thrombosis dog for reocclusion, it was found that CN efficiently maintains the carotid artery opening in conjunction with the anisoylated plasminogen streptokinase activator complex (APSAC).

• APSAC was found to be insufficient to prevent rapid reocclusion of the carotid artery. Heparin was not needed to maintain the opening when CN was administered with APSAC. This is a significant distinction over the other disintegrins (eg, echistatin, bitistatin, and kistrin) which have been evaluated in models of coronary artery thrombosis. The compositions of the present invention are particularly useful for treatment of thrombotic diseases in mammals, alone or in conjunction with one or more thrombolytic agents. In particular, the compositions of the present invention have utility for treating or preventing venous and microvascular thrombosis and thromboembolism. Thus, the compositions have utility to treat shock, ischemic attacks transient arteriosclerosis, atherosclerosis, pulmonary embolism, aneurysm and angina. In particular, the compositions have utility for preventing or treating myocardial infarcts. The compositions of the present invention also have utility for inhibiting metastasis in melanomas, carcinomas and patients with sarcoma. It has been observed that CN binds to at least two sites in human melanoma M24met cells: a high affinity site with a dissociation constant (Kd) of 1.1 (± 0.7) nM and 96,000 (± 39,000) sites per cell; and a low affinity site with Kd of 41 (± 13) nM and 480,000 (± 90,000) sites per cell. In addition, it has been found that CN inhibits the adhesion of M24met cells from human melanoma to fibronectin and vitronectin, and to a lesser extent to collagen and laminin. Thus, methods and compositions for preventing metastasis in patients with melanoma, carcinoma and sarcoma are also contemplated within the scope of the present invention. In accordance with the present invention, the unique properties of the disintegrins are exploited in methods and compositions to prevent metastasis in patients with carcinoma, sarcoma and melanoma of patients. In particular modalities the positions and methods are provided to prevent metastasis in patients with breast cancer. In additional embodiments, the inventors provide the contortrostatin protein from the venom of the Southeastern copperhead snake which possesses potent antitumor activity. A sophisticated technique has been developed to purify this protein from the complex mixture of protein found in the poison of the Southeastern copper head. As indicated above, originally CN was characterized as an inhibitor for platelet aggregation. The inventors have purified several disintegrins from snake venoms. The contortrostatin (CN) was purified from the poison of the copper head of the Southeast. The disintegrins contain a constant Arg-Gly-Asp sequence (RGD) at the end of a flexible peptide loop, of about 13 amino acid residues flanked by Cys residues, leaving the main protein core. See, for example, amino acid residues 457 to 469 of SEQ ID NO: I or 2. This exposed RGD sequence enables disintegrins to bind to integrins with high affinity. The inventors have developed a metastatic breast cancer model to implant human breast cancer cells within mouse mammary fat pads. The mice that are used are generally genetically engineered so that their immune system is deficient and they are unable to reject the implanted human cancer cells. The inventors observed that the palpable tumor masses develop in the mammary areas two weeks after the implantation of the cancer cells, and that the tumor cells extend into the lungs in the untreated animals within 12 weeks. Daily CN or placebo is injected into tumors in several different groups of mice. Following the treatment the inventors found that the size of the tumor masses in the mice treated with CN was significantly smaller than those treated with placebo. Significantly, the group treated with CN showed > 90% inhibition of tumor spread to other sites in the body (metastasis), compared to the placebo group. Studies indicate that CN blocks the binding of breast cancer cells to proteins that are found as essential components of the walls of blood vessels. CN also inhibits the formation of new blood vessels (neovascularization) induced by breast cancer cells after incubation on the respiratory organ of the embryonic chicken membrane called chorioallantoic membrane while placebo treatment did not. Since neovascularization is critical to continue the proliferation of a growing tumor, the ability to inhibit the growth of new vessels is an important action of CN against cancer. Based on these studies, it seems that disintegrins such as the contortrostatin protein of snake venom possess antimetastatic activity. The inventors' findings suggest that CN blocks several critical steps in metastasis and therefore, is more potent than other agents that only block a single step. The compositions of the present invention containing disintegrin are also useful in the treatment of osteoporosis. Osteoclasts are multinucleated cells of more than 400 μm in diameter which reabsorb mineralized tissue in vertebrates. The bone resorption seems to proceed by a combination of processes involved in bone binding, polarized secretion of acid and proteases, and active mobility of osteoclasts on the bone substrate.; osteoclasts bind to bone and to an RGD sequence as a mandatory step in bone resorption, and this RGD-binding integrin is in the adhesion structures [Sato, M. et al., J. Cell Biol. 111: 1713 ( 1990)]. The molecular mechanisms by which osteoclasts bind to bone are not well understood; however, by analogy to other cells, it is believed that members of the integrin superfamily of divalent cation-dependent adhesion molecules mediate this interaction. Disintegrins, such as echistatin [Sato et al. (1990), previously mentioned] and presumably CN, inhibit bone resorption by isolated osteoclasts; The mechanism of action presumably is by altering adhesion structures. Accordingly, the compositions and methods for the treatment of osteoporosis employ an amount of CN effective to inhibit the bone resorption by osteoclasts which is also contemplated within the scope of the present invention. Finally, CN have a utility in the promotion of healing. It is known that the events involved in healing include alterations in integrin expression or functional activity and suggest that integrin receptor modulations play a central role in healing and inflammation. It is also known that fibronectin plays a number of roles in the healing process. Although the functions of fibronectin are thought to be critical for effective healing, there are reports that at least one of its activities (binding to bacteria) can be counterproductive [Grinnell, F., J. Cell. Biochem. 26: 107 (1984); Clark, R.A.F., Arch. Dermatol. 124: 201 (1988)]; The presence of fibronectin in the lesion can promote bacterial union and infection. Fibronectin also seems to be intimately involved in the formation of keloids. Keloids are pathological consequences of scarring that affect a significant proportion of non-Caucasian patients. Keloids are benign connective tissue tumors that grow over the boundaries of the original lesion and are rich in fibronectin and type I collagen [Sible, J.C. and Oliver, N., J. Cell. Biochem. Suppl. 16F: 170 (1992)]. By virtue of their cell-cell inhibition-cell-extracellular matrix interactions (including the interaction with fibronectin), disintegrins such as CN could be expected to have a profound effect on processes involving scarring, including keloid formation. A major problem that follows obstetric and gynecological surgery is the formation of adhesions. This widespread phenomenon is observed in the healing of peritoneal lesions and is a continuous cause of pain, intestinal obstruction and infertility. Adhesion formation seems to imply an imbalance in fibrinolytic and fibroproliferative inflammatory responses and may also involve a modulation of cell-cell or cell-extracellular matrix interactions. There is strong evidence for an important role of fibrin during the early stages of adhesion formation [diZerega, G.S., Prog. Clin. Biol. Res 381: 1 (1993)]. The presence of cellular elements, including platelets, also exacerbates the role of fibrin. In view of the role of platelets and fibrin in the formation of adhesions, the use of disintegrins such as CN as a potential therapeutic agent is very attractive. In preliminary studies in the rabbit model of adhesion formation, abrasion and devascularization of the uterine horns of rabbits was used to induce adhesion formation during the healing process in untreated animals [Rodgers, K et al., Int. J. Fertile. :40 (1990)]. Alzer pumps were used to continuously send CN at a rate of 10 μl / hr (36 μg / ml). In this model system, decreased adhesion formation was observed in treated animals compared to controls. Therefore, compositions and methods for preventing adhesion formation wherein an amount of CN effective to prevent adhesion formation is administered to a patient in need of such treatment are also contemplated within the scope of the present invention. The compositions of the present invention comprise a minimum amount of CN effective to achieve the desired effect (i.e., prevent the formation of thrombi, prevent metastasis in patients with carcinoma, prevent adhesion formation, etc.) and a suitable vehicle or excipient. Generally, in these compositions, CN is present in an amount sufficient to provide from about 0.01 mg / kg to about 50 mg / kg per day, preferably from about 0.1 mg / kg to about 5.0 mg / kg per day, and more preferably from about 0.1 mg / kg to about 0.5 mg / kg per day. Said compositions have particular utility in the prevention of thrombus formation. Alternatively, CN is administered in combination with at least one thrombolytic agent present in an amount effective to achieve thrombolysis. Suitable thrombolytic agents include, but are not limited to, the following: anisoylated plasminogen streptokinase activator complex (APSAC); tissue-type plasminogen activator (tPA); urokinase-type plasminogen activator (uPA); and fibrolase, a fibrinolytic agent of snake venom described in U.S. Pat. No. 4,610,879 to Markland, Jr. et al. CN can be administered in a variety of suitable means known hereafter to administer it into the bloodstream in substantial amounts. Intravenous administration of CN in a suitable liquid carrier or excipient is currently contemplated as the preferred route of administration. CN is soluble in water, and can therefore be effectively administered in a suitable aqueous solution (eg, phosphate buffer). Alternatively, CN can be administered orally (in the form of tablets or capsules formulated with a suitable binder or excipient material, or in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs) or as a parenteral suspension. As is well known in the art, adjuvants such as local anesthetics, preservatives, lubricating buffers, wetting agents, colorants, sweeteners, fillers and diluents can be suitably included in any of these formulations.

EXAMPLES These additional embodiments may be better understood with reference to the appended examples, which are intended only for purposes of illustration and should not be taken in any way as limiting the scope of the invention as defined in the appended claims. To conduct the following examples, lyophilized venom was obtained from Agkistrodon contortrix contortrix from Biotoxins, Inc., St. Cloud, FL.

All the chemicals were of the highest possible degree. The Pierce protein assay kit was used using bicinchoninic acid to determine protein concentrations [Smith, P.K. et al., Anal. Biochem. 150: 76 (1985)]. For the hydrophobic interaction using HPLC- (HIC) a Perkin Elmer 410 LC pump with LC-95 UV / VIS detector was used. For reverse phase HPLC, a Spectra Physics LC 8810 pump was used with an SP 8450 UV / VIS detector. The absorbance for HPLC-HIC was monitored at 280 nm and for HPLC-RP at 215 nm. A column of polypropyl aspartamide (250 x 21 mm) (Poly LC, Columbia, MD) was used for the hydrophobic interaction by HPLC. Columns C18 (218TP54 and 218 TP510) were used for reverse phase HPLC (RP) (Vydac, Hesperia, CA). A CM (carboxymethyl) 300 column (SynChrom, Inc., Lafayette, IN) was used for cation exchange chromatography.

EXAMPLE 1 Purification and characterization of CN CN was purified from the venom of Agkistradon contortrix contortrix (Southeastern copper head) using a four-step HPLC procedure. For the first purification step the crude poison (1 g) was dissolved in 0.1 M phosphate buffer containing 1 M ammonium sulfate, pH 6.8 (buffer A) and applied to an HPLC-HIC column of polypropylpartamide. The elution was achieved as follows: 50 minutes isocratically with 100% buffer A; a linear gradient for 90 minutes of 0.1 M phosphate, pH 6.8 (buffer B); 40 minutes Socratic in 100% buffer B. The 10 ml fractions were collected in a Pharmacia Frac 100 fraction collector at 4 ° C using a flow rate of 5 ml / min. The fractions containing the platelet aggregation inhibiting activity were pooled and concentrated by ultrafiltration using an Amicon agitation cell with a YM3 membrane. The proteins were detected at 280 nm; the inhibitory activity of platelet aggregation was observed during the flow. Further purification was achieved by C18RP-HPLC. Fractions containing platelet aggregation inhibitory activity were pooled and concentrated for this second step. Column C18 (218TP510) was equilibrated with 95% 0.1% TFA in water (solvent A) and 5% 80% acetonitrile in. % TFA in water (solvent B). The elution was achieved as follows: isocratic at 95% solvent A and 5% solvent B for 10 minutes; a linear gradient to 40% of solvent B in 65 minutes; linear gradient at 100% solvent B for 20 minutes; isocratic to 100% solvent B for 25 minutes. The fractions were collected manually every minute at a flow rate of 7 ml / minute. CN was eluted with 28% acetonitrile (66 minutes). The fractions containing the inhibitory activity of the platelet aggregation were pooled and run again on the same C18 RP-HPLC column using a shallower gradient. Elution was achieved as follows: Socratic to 30% of solvent A and 20% of solvent B for 20 minutes; a linear gradient to 30% of solvent B for 90 minutes; and a linear gradient for 25 minutes at 100% of the solvent B. CN was eluted as a defined peak with 22% acetonitrile (82 minutes). The minor elution peak just before CN also contained platelet aggregation inhibitory activity and had a molecular weight similar to that of CN. Due to the low performance, this peak was not characterized. A final purification step was carried out using pooled fractions from the previous steps. These pooled fractions were applied to a cation exchange column, CM300, HPLC and elution was achieved by increasing the sodium chloride gradient. CN elutes at 52.5 minutes (160 mM NaCl). This step achieved the separation of CN from the isoform of it. A product of 1-2 mg of purified CN was obtained in four steps per gram of crude venom.

For the polyacrylamide-SDS gel electrophoresis (SDS-PAGE), a Tris-Tricine gel of 16.5% was used in accordance with the protocols published under reducing and nonreducing conditions [Schagger, H. and Von Jagow, G., Anal . Biochem. 166: 368 (1987)]. The gel was run using a BioRad minigel system and stained with silver [Morrisey, J. H., Anal. Biochem. 117: 307 (1981)] or Coomassie blue R250. The SDS-PAGE analysis of CN revealed that it has a molecular mass of approximately 15,000 Daltones under non-reducing conditions and 5,000-7,000 Daltones under reducing conditions. These strongly suggest that CN is composed of two subunits. Another possibility, although unlikely, is that the large difference in migration can be attributed to differential SDS intakes under non-reducing and reducing conditions. The molecular weight of CN was confirmed by mass spectrometry using a triple quadrupole instrument with an ion source of electrospray. A mass of 13,507 Daltones was determined for the intact CN; The analysis also indicates a high degree of purity. The mass spectrometry of the reduced and pyridylethylated protein gave a mass of 7, 996 Daltones. This is the expected value for the individual chains of a homodimer of this molecular weight, taking into account the incorporation of 1, 248 mass units for the 12 pyridylethyl groups incorporated in the 6 disulfide bonds (based on the homology with the known disintegrins, they should be 6 disulfide bonds). These findings place CN in a unique position among all the disintegrins reported so far as it exists as a dimer. The Scatchard analysis of CN bound to inactive human platelets reveals a unique class of binding sites with a dissociation constant (K) of 37 nM and a number of binding sites (Bm) equal to 100,000. The reduction of disulfide bonds completely eliminates the inhibitory activity of platelet aggregation, even at concentrations ten times the IC50, suggesting that structural parameters are critical for the maintenance of activity.

EXAMPLE 2 Cloning of contortrostatin cDNA using the polymerase chain reaction Analysis of the partial amino acid sequence of contortrostatin using the Edman degradation method suggested that the contortrostatin subunit is homologous with other disintegrins (Figure 1A) with aligned cysteine residues, as well as the RGD sequences [Niewiarowski, S., McLane, MA, Kloczewiak, M. and Stewart, GJ Disintegrins and Other Naturally Occurring Antagonists of Platelet Fibrinogen Receptors, Seminars in Hematology 31: 289-300 (1994)]. The strategy for the cloning of contortrostatin cDNA with PCR is based on the structural homology between the disintegrin family. The design of the PCR primers is illustrated schematically in Figure 1. The sequences underlined in Figure 1A are highly conserved among the disintegrin family. PCR primers were synthesized based on this region. The sequence of nucleotides encoding this region in the trigramin cDNA from • Trimeresurus gramineus were used to synthesize the PCR-1 and 5 PCR-2 primers (Figure 1B). PCR-1 and PCR-2 are complementary primers that correspond to the coding sequence of a consensus sequence PCCDAATCKL between disintegrins. The nucleotide sequences are: PCR-1: 5'-GTTTACAGGTTGCAGCATCGC-3 '(SEQ ID NO: 3) PCR-2: 5'-GCGATGCTGCAACCTGTAAAC-3' (SEQ ID NO: 4) 10? Gt10 forward and the opposite initiators which flank the EcoRI site of the vector that was used for PCR. The nucleotide sequences of the primers are listed below:? Gt10 forward primer: 5'-AGCAAGTTCAGCCTGGTTAAG-3 '(SEQ ID NO: 5) 15? Gtl 0 reverse primer: 5'-CTTATGAGTATTTCTTCCAGGGTA-3' (SEQ ID NO: 6) • Oligonucleotide primers were synthesized at the microchemical core facilities of the University of Southern California Comprehensive Cancer Center. The initiators were provided in a way lyophilized, unprotected, and resuspended and diluted to the appropriate concentration with water before use.

EXAMPLE 3 PCR amplification of contortrostatin cDNA The inventors used the cDNA library of the venom gland of Agkistrodon contortrix contortix constructed in the vector gt10 at the EcoRI site. The estimated title of the library was 1010 plaque-forming units (pfu) / ml and the complexity was 50,000. 500 μl of the cDNA library of the phage solution was mixed with 500 μl of 20% polyethylene glycol (PEG) / 1 M NaCl solution in an Eppendorf tube - the Eppendorf tube was inverted twice and was incubated at room temperature 30 minutes. The solution was centrifuged at 14,000 rpm for 10 minutes. The supernatant was discarded and the concentrate was resuspended in 10μl of sterile water. The suspension was incubated with 10μl of proteinase K (10 mg / ml) at 50 ° C for 1 hour. The suspension of phage particles was extracted with phenol / chloroform twice and the DNA was precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of absolute ethanol followed by washes with 80% ethanol. The DNA was resuspended in 10μl of sterile water in preparation for PCR. The PCR reaction was established as follows: 5 μl of DNA solution was mixed with 1 μl of 25 mM dNTP (Pharmacia), 1 μl (100 ng / μl) each of the primers for forward and reverse PCR, and 5 μl 10x PCR buffer The final volume of the reaction was brought to 50 μl with water. After mixing, a drop of mineral oil was applied on the surface of the liquid. The Eppendorf tubes were preheated to 98 ° C for 5 minutes, then incubated at 70 ° C and 60 ° C for one minute respectively before cooling to room temperature. 2.5 units of Taq DNA polymerase (Pharmacia) were added to each mixture. The thermal cycler was programmed as follows: 96 ° C for 15 sec, 55 ° C for 30 sec, 72 ° C for 1 min. The PCR amplification was carried out for 30 cycles. Following the last cycle, a final extension step was allowed at 72 ° C for 7 minutes before the samples were cooled to 30 ° C. The PCR product was extracted with chloroform before it was analyzed by agarose gel electrophoresis. Bands resolved by electrophoresis were recovered from the agarose gel using a Geneclean kit (Bio 101, Inc.) according to the manufacturers manual for direct DNA sequencing. The forward primers? Gt10 and PCR-1 were used to amplify the region towards the 5 'end of the adhesion site (Fig. 1 B). Similarly, the reverse primers PCR-2 and? Gt10 were coupled to amplify the part towards the 3 'end of the cDNA (Figure 1B). A main band of about 1300 bp (designated CN-N) was obtained with the first pair of primers (figure 2, line 1), and a main band of approximately 700 bp (designated CN-C) resulted from the PCR using the last pair of initiators (figure 2, line 2). CN-N and CN-C were subjected to nucleotide sequencing analysis before the extension of overlap. As expected, CN-N showed high similarity to the trigramin cDNA which encodes its N-terminal signal peptide. The deduced amino acid sequence of the nucleotide CN-C was very similar to that of the COOH site encoding the RGD terminal sequence. Since PCR-1 and PCR-2 were complementary, CN-N and CN-C overlap at this site, and therefore, can be assembled into a full-length cDNA. To achieve this objective, an overlap extension method was used as shown in Figure 1C. Briefly, equal molar amounts of the double-stranded PCR products of CN-N and CN-C were mixed with? Gt10 forward and the reverse primer. After the denaturing of both double strands, the subsequent reattachment resulted in two types of molecules. One is coupled to CN-N and CN-C in the adhesion site with the recessive ends in the term 3. This molecule can be automatically elongated into double strands of full length using PCR.The other molecule is similarly coupled, but with the recessive ends at the 5 'terminus Although these molecules are not self-assembling, the respective parts can be filled with primer by the? gt10 primers at each end.Figure 2, line 3 shows the products of the superimposed extension as solved by electrophoresis at Agarose gel The size of the main band is estimated to be 2,000 bp which is equal to the sum of CN-N and CN-C, and is therefore designated "total length." The total length band is Removed from the gel and treated with EcoRI Subsequently, this piece of DNA was subcloned into the plasmid vector pcDNA3.1 (+).

Example 4 Subcloning of PCR product into the plasmid vector Plasmid pcDNA3.1 (+) was digested with EcoRI (Pharmacia) followed by dephosphorylation using T4 phosphatase (Boehringer.Mannheim). The extension product that overlaps the PCR was also digested with EcoRI. The PCR product was inserted into the linearized vector by ligation reaction using T4 ligase (Pharmacia) at 16 ° C overnight. All reactions were established and carried out in accordance with standard protocols. Successful ligation was selected when seeding transformed E. coli (DH5a) on plates containing ampicillin. The plasmids containing the insert were amplified in E. coli. The purified DNA plasmid was obtained with Quiagene DNA miniprep columns.

EXAMPLE 5 cDNA sequencing Automated DNA sequencing was carried out at the microchemical core facilities. The PCR primers were used as sequence primers to direct the sequencing of the PCR products. For the sequence analysis of the insert in plasmid pcDNA3.1 (+), the initiator promoter T7 and the reverse primer BGH were used to initiate the assay, which flanked the multiple cloning sequences (MCS). Typical reactions gave readable sequences of 400 to 600 bp. The sequencing reactions were carried out on double-stranded DNA in the case of the DNA plasmid. With the synthesis of new sequence primers, additional sequences were obtained, these were assembled into a contiguous sequence superimposed using the DNAsis computer program. Figure 3 shows the total length nucleotide sequence inserted between the EcoRI sites. It is composed of 2,029 nucleotides, which is the size of the total length band of the superimposed extension (Figure 2, line 3). After an untranslated region at the 5 'end of 86 nucleotides (5'-NTR), an open reading frame is found between nucleotides numbers 87 and 1535. Nucleotide 1536 to 1538 is the stop codon. 3'-NRT possesses an AAT AAA site in the non-coding region of the 3 'end, and ends with a poly (A) tail, suggesting that the cDNA that the inventors obtained with the extension of overlap was in fact a complete cDNA ( figure 3). The open reading frame encodes 485 amino acids. The cDNA structure deduced from the amino acid sequence can be divided into three domains. The first 190 amino acids, starting with methionine, are highly similar to the pro-protein of many cloned snake venom proteins (the comparison is shown in Figure 4). Of amino acids 191 to 418 is the metalloproteinase domain that includes a zinc binding motif HEMGHNLGISH (aa 334 to 344). The remaining 65 amino acids belong to the contortrostatin monomer which is identical to the partial amino acid sequence known for contortrostatin determined by the Edman methodology. This sequence is very similar to that of many disintegrins whose sequences have been • determined (figure 4 and 1A). The calculated molecular weight of the disintegrin is 5. 6.77 kDa, which is equal to the CN monomer. The sequence RGD (aa 461 to 463) is in bold. The structure of the three domains is coupled to the precursor model of the snake venom metalloproteinase and the disintegrin proposed by Kini et al. [Structural Domains in Venom Proteins: Evidence that Metalloproteinases and Nonenzymatic Platelet Aggregation Inhibitors 10 (Disintegrins) from Snake Venoms are Derived by Proteolysis from a Common Precursor, Toxicon 30: 265-296, (1992)]. There is evidence that disintegrins are synthesized in snake venom glandular cells as multidomain precursors which leads to post-translational proteolysis and folding to generate a mature disintegrin. 15 EXAMPLE 6 Platelet aggregation inhibitory activity assay Fractions of the column obtained during purification are tested for activity using fresh plasma rich in human platelets (PRP) prepared from blood obtained from human volunteers who had no medication for at least two weeks. The blood (36 ml) was added to 4 ml of 0.1 M citrate and centrifuged at 150 x g for 20 minutes. The supernatant, PRP, was removed and the remaining blood was centrifuged at 10,000 RPM to obtain a poor plasma concentration (PPP). The platelet count was adjusted to 250,000 platelets / μL using a Coulter counter. A Helena four-channel aggregometer was used to monitor platelet aggregation. The inhibition of ADP-induced platelet aggregation was monitored at 37 ° C by adding venom fractions one minute before the addition of ADP (final concentration 10-20 μM). The fractions exhibiting platelet aggregation inhibitory activity were pooled and subsequently purified. The rabbit and dog PRP were prepared by the same procedures and used in the studies described below. CN inhibits the platelet aggregation induced by ADP in PRP of human, dog and rabbit (figure 6). Empty circles represent plasma rich in human platelets, solid circles represent PRP, from dog and empty triangles represent rabbit PRP. The CN variant concentrations were pre-incubated for one minute with PRP before the addition of ADP. CN (0.73 μg / ml) inhibited human platelet aggregation induced by 10 μM ADP for 50% (IC50). The IC5o for 20 μM of canine platelet aggregation induced by ADP was 1.8 μg / ml for CN. Interestingly, the IC5o for CN mediated the inhibition of rabbit platelet aggregation that was considerably higher; IC5o for 20 μM of rabbit platelet aggregation induced by ADP was 17.3 μg / ml for CN.

EXAMPLE 7 Measurement of specific binding (GP) llb / llla The measurement of CN bound to the platelet receptor (GP) llb / llla was carried out using PRP prepared from blood obtained from human volunteers or male mongrel dogs. The PRP was prepared as described above and the platelet count was determined with an H-10 cell counter (Texas International Laboratories, Inc., Houston, TX). The PRP (180 μl) was incubated with 20 μl of varying concentrations of CN at room temperature. The radiolabelled antibody (125 I-7E3 IgG, 20 μl, 18 mg / ml, 80,000 cpm), specific for (GP) IIb / Illa, was added and the mixture was incubated for 30 minutes. To establish the binding equilibrium, aliquots of 50 μl of the binding assay mixture were maintained on 200 μl of 30% sucrose in 0.4 ml microcentrifuge tubes and centrifuged at 10,000 RPM for 4 minutes in a bucket rotor. rolling to separate the antibody bound to the platelet from the free antibody. The pellet and the supernatant were separated and counted in a Packard Minaxi 5000 series gamma counter. The number of 125I-7E3 molecules bound per platelet in the presence and absence of CN were calculated using the following formula: (4) x 0.9 ug 7E3 x 3.76 x 1012 molecules! The I ug (5) where (1) = platelet count; (2) = supernatant count; (3) = total CPM (1) + (2); (4) = joined fraction (1) / (3); and (5) = platelet count per μl x 45 μl. Competitive binding studies using 7E3 demonstrated a binding of the platelet-specific receptor (GP) llb / Illa for CN for both human platelets (Figure 7A) and for dogs (Figure 7B). The concentration of CN to inhibit 50% of the binding 7E3 to (GP) llb / llla human (IC50) is 0.4 μg / ml. The IC50 for dog CN is (GP) llb / llla 0.24 μg / ml. These studies confirm that CN inhibits platelet aggregation by binding to (GP) llb / Illa.

EXAMPLE 8 In vivo thrombolytic efficacy of CN CN has been studied in the canine model of reocclusion of arterial thrombosis. The protein was initially studied by systemic infusion at different doses to determine its relative potency. This data has allowed an evaluation of the systemic doses necessary for an effective antithrombotic (antiplatelet) activity. Effects on physiological parameters and circulating coagulation factors have also been monitored.

The model of carotid artery thrombosis described in the anesthetized dog is a modification of a development for the study of experimentally induced coronary arterial thrombosis [Romson, J. L. et al., Thromb. Res. 17: 841 (1980)]. The experimental procedure results in the formation of platelet-rich intravascular thrombi at the site of an electrolytically induced endothelial lesion in the vicinity of a distal arterial stenosis. The carotid artery is selected for the experimental model, since it allows one vessel to be used as a control and the other to be used after the administration of thrombolytic and antithrombotic therapy. APSAC (anxiolytic plasminogen streptokinase activator complex has been successfully used as the thrombolytic agent in this model.) The response of the carotid artery to the electrolyte lesion is similar to that seen in the dog's coronary artery but has the advantage that Each dog demonstrates the ability to form bilateral occlusive thrombi The combination of lytic-antithrombotic agents can then be administered to only one of the occluded vessels, this allows internal control and eliminates those animals that probably do not form thrombi due to causes unrelated to the vascular wall lesion and the subsequent formation of occlusive thrombi, that is, low platelet count in circulation, improved spontaneous thrombolysis, presence of heart dirofilaria, etc. The parameters which are recorded include repeated measurements of: blood flow velocity arterial blood in the phasic stage and medium us An ultrasonic flow probe, thrombotic occlusion time, recanution time, ex vivo platelet aggregation, prothrombin time, thrombin time, activated partial thromboplastin time, red cell and white cell count, hematocrit, EKG profile and body temperature, before and after the administration of APSAC or APSAC plus CN to separate groups of animals. Male Mongrel conditioned dogs (8-10 kg) have been used for all in vivo studies. Dogs are anesthetized with sodium pentobarbital, intubated, and allowed to aspirate with positive pressure breathing. All determinations of arterial blood gases and pH are made every 45 minutes and appropriate adjustments are made to maintain blood gas and arterial pH limits within normal limits. Both common carotid arteries and the right internal jugular vein are exposed. A catheter is inserted into the jugular vein to collect blood samples and to administer the test drug. Arterial blood pressure is monitored from the cannulated femoral artery with the use of a blood pressure transducer. A Doppler flow probe is placed over each proximal carotid artery to both the insertion point of the intraarterial electrode and the mechanical constrictor. The constrictor is adjusted until the pulsatile flow of the pattern is reduced by 50% without altering the mean blood flow. The blood flow velocity in the carotid vessels is continuously monitored. Figure 8 is a schematic representation of the instrumentation of the carotid artery.

The electrolytic lesion to the intimal surface of each carotid vessel is accompanied by the use of an intravascular electrode. Each intraarterial electrode is connected to the positive pole (anode) of a dual channel stimulator. The cathode is connected to a distant subcutaneous site. The current sent to each vessel is continuously monitored and maintained at 300 μA. The anodal electrode is positioned so that it has the non-isolated portion in intimate contact with the endothelial surface of the vessel. The proper position of the electrode in each of the carotid arteries is confirmed by visual inspection at the end of each experiment. The anodal current is applied for a maximum period of 3 hours or 30 minutes after blood flow is terminated in the vessels involved that remain stable at a flow rate of 0 to verify that verification of a stable occlusive thrombus has been achieved. The right carotid artery serves as a control vessel, where the left carotid vein serves as the test vessel. The walls of damaged vessels are simultaneously induced in each carotid artery. The APSAC (0.05 U / kg) is inserted as a bolus proximal to the thrombus in the left carotid artery only. The dose of APSAC has been determined as that which will consistently lyse the locally injected carotid thrombus without producing a systemic lytic effect. Thus, lysis should not occur in the right carotid not injected. CN is given intravenously in a 10% bolus immediately after APSAC and the remaining 90% is incorporated after one hour. The doses of CN are in a range of 0.155 to 0.40 mg / kg; the agent was dissolved in the appropriate dose in a volume of 20 ml of sterile saline for infusion. Reperfusion is defined as the restoration of the blood flow velocity of the carotid artery to 20% of baseline values. The pattern is defined as the measurable carotid arterial flow velocity. Blood pressure, heart rate, speed of carotid arterial flow are monitored for 6 hours or until re-thrombosis occurs. Blood (20 ml) was taken for platelet studies from the jugular cannula into a plastic syringe containing 3.2% sodium citrate as an anticoagulant (1/10 citrate / blood, vol / vol). Blood was taken for platelet aggregation and total blood cell count at baseline 60,120, 180, 240 and 300 minutes after CN administration. The platelet count was determined with a cell counter. The platelet-rich plasma, the supernatant present after centrifugation of the anticoagulated total blood at 140 x g for 5 minutes, was diluted with platelet-poor plasma to achieve a platelet count of 200,000 / mm3. The platelet-poor plasma was prepared after removing the platelet-rich plasma, by centrifuging the remaining blood at 12,000 x g for 10 minutes and discarding the bottom cell layer. Platelet aggregation ex v / Vo was measured to establish spectrophotometric methods with a four-channel aggregometer to record the increase in light transmission through the agitated suspension of platelet-rich plasma maintained at 37 ° C. Aggregation was induced with arachidonic acid (0.65 mM and 0.3252 mM) and ADP (20 μM and 5 μM). An adrenaline dose (550 nM) was used for subagregation to prepare the platelets before stimulation. The values expressed as percentages of aggregation represent the percentage of standardized light transmission for platelet-rich samples and platelet-poor plasma, producing 0% and 100% light transmission, respectively. At the conclusion of the study protocol, each segment of the vessel was ligated, proximally and distally to the point of injury, and removed without disturbing the intravascular thrombus. The segment of the vessel opens and the intact thrombus is removed and weighed. So far we have studied five animals with CN plus APSAC, six with APSAC alone, and a positive control group of six dogs with APSAC plus the anti-monoclonal antibody (GP) llb / llla 7E3. These were essentially unchanged in mean arterial blood pressure or heart blood flow following infusion of CN. In addition, the velocity of carotid arterial flow remained at high levels following the infusion of APSAC plus CN as compared to the infusion alone of APSAC. In the group of animals perfused with APSAC alone, the carotid artery was opened for a few minutes following the infusion of the lytic agent but was then reoccluded and remained closed for the duration of the study. In the positive control group, the animals were perfused with APSAC (0.1 U / kg) intra-arterially and this was followed by a bolus of anti- (GP) llb / llla F (ab ') 2 7E3 (0.8 mg / kg ). In these three animals, the carotid artery remained open following the infusion of the combination of APSAC and 7E3 and remained open until the conclusion of the experimental protocol. In the group of five animals perfused with APSAC plus CN the results were essentially the same as with the combination of 7E3 and APSAC. However, Table 1 reveals that there were significant advantages to the combination of ASPSAC with CN in terms of weight of residual thrombus. In Table 1, CTTX = CN and RCA = right carotid artery. In the group of five animals treated with this combination of agents, the weight of the residual thrombus per kg of dog weight was 1.5, versus 2.4 in the six animals in the APSAC plus 7E3 group, and 4.1 in the APSAC group alone (six animals ). Finally, in one of the dogs treated with APSAC plus CN (0.155 mg / kg) the platelet aggregation and the platelet count were as follows (figure 9); CN infusion started at time 0 and continued for 60 minutes.

TABLE 1 WEIGHT OF RESIDUAL THROMBO IN THE THROMBOSIS MODEL IN • CAROTID DOG ARTERY • 10 These results are typical of those dogs in this group. It can be seen that the platelet aggregation was compromised by the treatment with the venom protein, but that it seemed to have a return of aggregation at the conclusion of the experiment. Similarly, the platelet count also was decreased during the course of the experiment. It is suspected that platelets are sequestered in a sanctuary such as the spleen and then released after a short period of residence; Platelets return to circulation and appear to be functional. There is a drop in the platelet count of 10-20% of the base value, with fluctuations of aggregability in some way due to the low platelet count; however, it can be seen that the platelet aggregability in the residual platelets seems to return to normal at the conclusion of the experimental procedure.

EXAMPLE 9 Effects of CN on adhesion and invasion of breast carcinoma The effect of CN on the binding of highly metastatic human breast cancer cells, cell line MDA-MB-435, to ECM proteins was examined. Human fibronectin and vitronectin were immobilized in wells of 96-well microtiter boxes. Referring to Figures 3 and 4, CN inhibited the adhesion of MDA-MB-435 to both ECM proteins in a dose-dependent manner. The IC50 for adhesion of fibronectin is 18 nM (figure 10) and for vitronectin the IC50 is 1.5 nM (figure 11). CN had a minimal effect on the weak adhesion seen for MDA-MB-435 cells to human collagen type I or to rat collagen type I whereby MDA cells have a relatively strong affinity. In a variation of the previous experiments, CN was immobilized. It was found that CN can support the binding of MDA-MB-435 cells in a dose-dependent manner. The binding of the MDA-MB-435 cells to immobilized CN is blocked by the peptide RGD, GRGDSP (IC5α or = 0.4 mM), and EDTA (IC50 = 0.8 mM). Since integrin receptors require metal ions for the non-covalent association of their subunits, the inventors' findings indicate that the binding of CN to integrin receptors on the • Surface of MDA-MR-435 cells is via a mechanism mediated by 5 RGD. The finding that immobilized CN can support adhesion of MD.A-MB-435 cells suggests that this binding involves cell surface receptors on tumor cells. Referring to Figures 12 and 13, the variable concentrations of GRGDSP (Figure 12) or EDTA (Figure 13) were used to inhibit the binding of the human mammary carcinoma cell to CN • 10 immobilized. CN was at 0.1 μg / well. The vertical line at each data point indicates the error bar on the y-axis. All experiments were conducted as groups of three in triplicate for each data point. Since the adhesion of the MDA-MB-435 cells to immobilized CN is completely blocked by GRGDSP and by EDTA (FIGS. and 13), CN binds only to the integrin receptors of MDA-MB-435 cells via an RGD-dependent mechanism. Referring to Figure 14, the inventors have also demonstrated the inhibitory effect of CN on the invasion of a synthetic basement membrane by MDA-MB-435 cells using an invasion chamber covered with matrigel. 2.5 x 103 MDA-MB-435 cells treated with various concentrations of CN were allowed to migrate through the Matrigel layer for 48 hours. The assays for each CN concentration were carried out in triplicate. The cells that invaded through the Matrigel filter were fixed and stained. The invading cells were quantified under a microscope by means of the cell number in three randomly selected fields of vision.

EXAMPLE 10 CN inhibits the growth and metastasis of breast cancer MDA-MB-435 in the nude mouse experimental model A spontaneous (orthotopic) metastatic model of nude mice has been established by implantation of MDA-MB-435 cells (5x105 in 0.1 ml) in the mouse breast fat pads (mfp). The palpable tumors appear on the 10th day post-implantation. Daily CN injections were carried out within the tumor masses of each of the groups, starting on the 14th day by implantation. By the 8th week postimplantation, the tumors should be removed. The animals were allowed to survive for two more weeks without CN administration. Animals were sacrificed and lung metastases were carefully examined. Referring to Figure 15, the inventors' findings indicate that local injection of CN substantially inhibits the rate of tumor growth. The volumes of the tumor masses (mean ± SD) of the control (dark bars), group treated with CN at low dose (0.5 μg / day, gray bars), and at high dose (5 μ / day, slightly gray bars) are shown . The seven groups of bars from left to right represent the pre-injection data (PI, day 14 ° post-implantation) and in the 1st to the 6th week of injection. Student's t-tests were used to evaluate the meaning of the differences. * and ** indicate PO.05 and p < 0.01, respectively. The weight medium of tumors treated with high doses of CN (5 μg / day) is • significantly lower than the control group (P <0.05). Table 1 shows the 5 incidence of pulmonary metastases based on gross examination and the count of superficial nodules. The extended metastases in the control group is much more extensive than in the group with high doses of CN which show > 90% inhibition of metastasis. These data demonstrate the potential therapeutic role of CN in the treatment of breast cancer • 10 human.

TABLE 2 Effect of contortrostatin on the incidence of metastatic breast cancer MDA-MB-435 in the nude mouse experimental model 15 Incidence of metastasis Groups Recurrence Recurrence # of nodules in Others In situ of lung size organs1 medium (median) tumor (mm3) Control 4/5 66.7 ± 51.7 47.5 5/5 CN (5μg / day) 2/6 48.7 ± 10. 5 4.5 2/6 20 1 Organs include: pulmonary wall, mediastinum, diaphragm, and pleura 2In 2/5 of animals, the lungs are directly invaded by cancer cells from the pleura and mediastinum EXAMPLE 11 CN inhibits tumor-induced angiogenesis MDA-MB-435 in CAM The hypothesis that the inhibitory effect on tumor growth is likely to result at least in part from the blockage of angiogenesis by CN has been preliminarily verified by observing the effect of CN on tumor-induced angiogenesis on the chorioallantoic membrane of the chicken embryo ( CAM) The tumor masses of MDA-MB-435 were inoculated on CAM of 10-day-old chicken embryos. CN was injected at several doses intravenously within CAM at day 2 postinoculation. Tumor induced angiogenesis induced angiogenesis and the inhibitory effect of CN on angiogenesis can easily be observed in CAM after 3 days of incubation. As shown in Figure 16, the vessels are distributed in a convergent manner with the mass of the tumor in the center in the control embryo. The chicken embryo is immunodeficient, and therefore allows the growth of the implanted tumor. The embryos are incubated to MDA-MB-435. The embryos are incubated at 37 ° C with 60% humidity. CN was injected intravenously at several doses within CAM at 2 days post-inoculation. The tumor-induced angiogenesis on the third day is demonstrated in the photographs. In the upper part (figure 16A) is the control embryo. The vessels are distributed in a convergent manner with the tumor mass in the center. In the middle (Figure 16B) is the CAM treated with 20 μg of CN. In the lower part (figure 16C) is the CAM treated with 150 μg of CN. The blood vessels of the embryo treated with 20 μg CN are thinner and less dense than the control; the tumor mass is smaller than in the control CAM. In CAMs treated with 150 μg of CN the blood vessels are even thinner and the convergent distribution of the pattern disappears completely; there is a necrotic tumor mass with a volume significantly lower than the control and low doses of CN, presumably due to the loss of blood complement (Figure 16).

EXAMPLE 12 CN has no effect on the growth of MDA-MB-435 cells in vitro MDA-MB-435 cells (0.3x103 / m)) were added to each well on 6-well culture dishes covered with a 1/100 dilution of Matrigel. Then the cells were treated with CN at various concentrations. The growth curves of MDA-MB-435 cells are shown in vivo without CN (circles), and with CN at 100 nM (triangles), and 500 nM (diamonds). Six culture boxes covered with matrigel (1/100 dilution) were seeded with 3 ml of a cell suspension (0.3x10d / ml) of MDA-MB-435. The cell density was determined every 24 hours. Referring to Figure 17, the cells in the presence of CN proliferated equally well as the control cells. The result indicates that CN did not have direct cytotoxicity during the in vitro culture of the MDA-MB-435 cells.

EXAMPLE 13 CN is effective and well tolerated in vivo It can be concluded from the chronic experiment with nude mice mentioned above that CN is not toxic. Despite its inhibitory activity on platelet aggregation, non-spontaneous hemorrhages were observed during the experiment. However, some bleeding was observed at the sites of the injection in the animals treated with CN. CN is a new antimetastatic agent. The inventors hypothesize that CN blocks several critical steps (eg, adhesion, invasion, angiogenesis) in the metastasis of cancer and progression. Therefore it is more powerful than other agents that block a single step. From the foregoing description, one skilled in the art can easily determine the essential characteristics of the invention and can adapt the invention, without departing from the spirit and scope thereof, to various uses and conditions. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or provide the record, and although the specific terms have been used here, they are intended in a descriptive sense and not for purposes of limitation. < 110 > Markland, Francis S. Zhou, Qing < 120 > Contortrostatin (CN) and methods for its use to prevent metastasis and other conditions. < 130 > 1920-338C3 < 140 > N / A < 141 > 1999-09-29 < 150 > US 09 / 163,047 < 151 > 1998-09-29 < 160 > 6 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 2029 < 212 > DNA < 213 > Agkistrodon contortrix < 400 > Q3sr.tcaggg ccaatagagg aagagctcae g tggcttgß aagcaag ooattgcc g ^ ag 60 tcttccagcc asacccagcc gccaaaat to cccaggctct cttggtgact cca ^ gcttag 12th cagcttttcc ttatcaaggg agccccat 's tcctggaatc TGGG &? Aegtt aataattatg 180 aotaccg ^ a ccacaaaaa gtcactgcat tgcccaaagg agcagtrcag ccaaagratg 240 aagacaccat gcaatatgaa tttaaagtga atggagagcc ag- ggtcctt cacctsgaa = 300? AAAS aaß9g ßcttttttii aaagattaca gcgagectca ttattectct gatggsagaa 3S0 eaattacaac aaaccctccg gttgaggatc actgctatta tcatggacgc atccagaacg 420 atgctgactc aactgeaagc stcagtgesic gciacggttt gaaaggacat ttcaagctec ISO aaggggagac gtaccttacc gaaecctfcga agctttccga cagtgßßgcc cacgcagtct 540 aaacgcagaa acaaatatga aaagaagatg aggcccccaa aatgtgtggg gtaacccaga 500 ctßattggga atcagatgag cccateaaaa aggceeceea gttaaatctt aetectgasc 6G0 aacaa? gatt cccccaaaga tacattgagc ttgttgtagt tgcagatcac agaatgttca 720 cgaaatacaa cggcaartcs aatacratca gaatapgggt acatgaactt gt.csacacea 750 tgastgtgtat tta? agacct ctgaarat c gtgtctcact gactgacct? g «t,» gtt g €; t &0 0 cßgßccaaga tttgatcaac gtgcagccsg cagcggctga tactttggao gcatctggag 900 aerggagaga gacagtcttr? etgaatc? ca taagtcatga taatgctcag ccactcacsg 960 - | Q ccattgagct tgatggagaa actatsggat tggctaacag gggcaccacg tgcgacccsa 1020 agctttceac aggaattgtt caggatcata gtgcaataaa tctttgggrr gcagttscßs 1080 • cgccccatgí »g ^ tgggtcat aatctgggta ttagtcacgs tggaaateag tgtcattgcg 1140 atgctaactc otgcottatg agtgaagaac taagagaaco actttecttt gagttcagcg 120C arcstagcca gaatcaacat cagacacatc taacccacaa tcactgarca cgcatgctca 1260 acgaaccctt gagaacagac attgtttcaa ccccagtttc cttttggana tggaaatgaa 1320 cuyoagaaga aagtsacfctc gacoctccrg caaacccßtQ ctgeaatQGt acaacat ^ ta 1380 aíictgacaac agggtcac ^ g tgtgcagatg gac-cgtgctg tgaccagtgc ? aaLttatja 1440 aaga < iggaac ßgtatgccgg agagcaaggg gtgatgacct ggatgattac tgcoatggca lí > 00 eacctgctgg ctgtcccaga aatcccttcc atgcctaacc aacaatggag atggaatggt 1S60 ctgcagcaac aggcagtgtg ttgatctgaa tacagcctaa taatcaacct ctggcttctc 1620 ? 15 tca tcatggagat atttga gaaaatttca cctccttcca cttccctcaa atccaaagag 1680 ctgcatccta aeceatctgc ctagtaaaee aeocctagßt teeagatggt atccaaat.cc 1740 tgtaataect cttctccata tttaatctat ttacctcttg ctgtaacaaß acc: ccc tc 1800 tgtcacaaag ctccatgggc atgtacagct cacctgctgt caagaaaaaa aatggccaeu 2860 ttaccgtttg ccagttacaa sgcacattta atgcaacaag tertect t tgagctgatg 1920 tattcaaage caatgcttcc tctcccaaaa tttcatgctg gcttcccsag atgragctgc 1980 ttccgtcaat aaacaaacea? tacatrca aaaaaa aaa cccgaarrc 2029 0 < 210 > 2 < 211 > 483 < 212 > PRT < 213 > Agkistrodon contortrix Mer lie Glrs Val Leu Leu Val Thr Leu Cys Leu? The Wing Pne Pro Tyr 1 5 10 15 Gl i Gly Ser Ser lie lie Leu Glu Ser Gly? Sn Val? Sn As Tyr Glu 20 25 30 Vaol Leu Tyr Pro Gln Lys Val Thr? Leu Pro Lys Gly Val Val 35n .0 .5 Pro Lys Tyr Glu Asp Thr Met Gln Tyr Giu Phe Lys Val Asn Gly Glu SO 55 dO Pro to Val Leu Hio Leu Glu Lys A3n Ly3 Gly Leu Phe Ser Lys As 65 70 75 80 Tyr Ser Giu Thr His Tyr Ser Ser Asp Gly Arg Lys He Thr Thr Asn 35 90? S Pro Pro Val Giu Asp His Cys Tyr Tyr His Gly Arg He Gln Asp Asp 100 105 110 Wing Asp Being Thr Wing Being Be Wing Cys Asn Gly Leu Lys Gly His US 12C 12S Phe Lys Leu Gln Gly Glu Thr Tyr Leu He Gl? Pro Leu Lys Leu Ser 130 135 140 ASP Ser Glu Ala His Wing Val Tyr Lys Tyr Gl? Asn V * l Glu Ly = Glu 1-55 150 155 1S0 Asp Glu Wing Pro Lys Met Cys Gly val Thr Gln Thr Asn Trp Glu Ser 165 170 175 Asp Glu Pro lie Lys Lys Wing Being Gln Leu? Sn Leu Thr Pro Glu Gln 18? 1S5 190 Gln Gly P # P or Gln Arg Tyr lie Glu Leu Val Val Val Wing Asp His 195 200 205 Arg Mee Phe Thr Lys Tyr Aan Gly Asn Leu Asn Thr lie, Axg lie Trp 210 215 220 Val His Glu Leu Val? Sn Thr M € t ASn Val Phe Tyr Ar? Pro l.Jßu Aen 225 230 235 240 I le? Rg Val Ser Leu Thr Asp Leu Glu Val Trp Ser Asp Gln Asp Leu Z45 250 255 lie Asn Val Glp £ rs Wing Wing Asp Thr Leu Glu Wing Phß Cly Asp 2S0 2 € 5 270 Trp Arg Glu Thr v * ? Leu Leu Asn Aro lie Be HAs Asp Aen Ala Gln 275 280 285 Leu Leu Thr Ala lie Glu Leu i \ s¡ > Giy Glu Thr lie Gly Leu Wing Asn 230 295 300 Arg Cly Thr Met Cys hsp Pro Lys Leu Ser Thr Gly lie Val Gln Asp 305 310 315 320 Hie Ser Ala? Lc? Sn Leu Trp Val Wing Val Thr Met Wing His Ciu Mßt 325 330 335 Gly Hi * Asn Leu Sly He Ser His Asp Gly Asn Cln Cys His Cys Asp 3 < 0 345 350 Wing Asn Ser Cys He Met Ser Glu Glu Leu Arg Gly Gln Leu Ser Phe 355 360 365 Ciu Pñe S r Asp Cys Ser Gin Asn Gin Tyr Gln Thr Tyr Leu Thr Asp 370 375 380 His Asn Pro Gln Cys Mßt Leu Asn Glu Pro Leu Arg Thr Asp lie Vßi 385 390 395 400 Be Thr Pro Val Ser Gl Asn Glu Leu L «u Clu Thr Gly Glu Glu 3er« 03 410 415 hsp Phe Asp Wing Pro Wing Asn Pro Cys Cys Asp Ala. lz Thr Cys Lys 420 425 430 Leu Thr Thr Giy Ser Cln Cys Ala? sp Gly Leu Cys Cys? sp Gin Cys 435 440 445 Lye Phe Mee Lys Glu Gly Thr Val Cys Arg Arg Wing Arg Gly Asp Asp 450 455 460 L «au Asp Asp Tyir Cy * Asn Giy He S r Ala Gly Cys Pro Arg Asn Pro 465 470 475 48C Phe HÍS Ms < 210 > 3 < 211 > 21 < 212 > DNA < 213 > Trimeresurus gramineus < 400 > 3 gtttacaggt tgcagcartcg c < 210 > 4 < 21 1 > 21 < 212 > DNA < 213 > Trimeresurus gramineus < 400 > 4 gcgatgctgc aacctgtaaa c < 210 > 5 < 21 1 > 21 < 212 > DNA < 213 > Lambda bacteriophage gt10 < 400 > 5 gcgatgctgc aacctgtaaa c < 210 > 6 < 21 1 > 24 < 212 > DNA < 213 > Lambda bacteriophage gt10 < 400 > 6 cttatgagta tttcttccag ggta

Claims (24)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A protein consisting essentially of purified contortrostatin, the precursor of contortrostatin or the biologically active variants thereof.
2. 2. A recombinant protein according to claim 1.
3. 3. A protein according to claim 1, further characterized in that it has an amino acid sequence selected from the group consisting of: a) amino acids numbers 419 to 483 of SEQ ID NO: 2; b) amino acids numbers 191 to 410 of SEQ ID NO: 2, c) amino acids numbers 1 to 190 of SEQ ID NO: 2, and d) SEQ ID NO: 2.
4. 4. A purified protein in accordance with the claim 1, further characterized in that the contortrostatin comprises a monomer having a molecular mass of about 5 to about 7kDa.
5. 5. A purified protein according to claim 4, further characterized in that the contortrostatin monomer forms a homodimer with another contortrostatin monomer.
6. 6. A purified protein according to claim 1, further characterized in that it comprises a sequence of contortrostatin Arg-Gly-Asp (RGD) at the end of a flexible peptide loop of about 13 amino acid residues flanked by two Cys residues, wherein the peptide loop is an integrin antagonist which has an amino acid sequence comprising amino acids numbers 457 to 469 of SEQ ID NO: 2.
7. 7. A protein according to claim 1, further characterized in that said biologically active variants have an amino acid sequence with at least 90% homology to: a) amino acids numbers 419 to 483 of SEQ ID NO: 2; b) amino acids numbers 191 to 410 of SEQ ID NO: 2; c) amino acids numbers 1 to 190 of SEQ ID NO: 2; and d) SEQ ID NO: 2. 8.- A peptide recognized by an antibody to contortrostatin. 9. A protein preparation with contortrostatin encoded by the nucleotide sequence of SEQ ID NO: 1. 10. A contortrostatin protein preparation according to claim 9, further characterized in that said preparation is substantially free of other components of the snake venom, wherein said contortrostatin is made by the process of transcribing and translating a cDNA molecule having the nucleotide sequence of SEQ ID NO: 1. 11. The preparation according to claim 10, further characterized in that said transcription and translation are carried out in a host cell containing recombinant DNA. 12. A preparation of contortrostatin substantially free of other components of the snake venom, wherein said contortrostatin is made by a process of synthesizing a polypeptide having an amino acid sequence in accordance with SEQ ID NO: 2 13.- A recombinant DNA molecule comprising a DNA sequence coding for the expression for contortrostatin. 14. A recombinant DNA molecule according to claim 13, further characterized in that it encodes the expression of a contortrostatin protein having at least one biological activity selected from the group consisting of metal binding, proteinase, or disintegrin. 15. A purified and isolated DNA molecule consisting essentially of a nucleotide sequence encoding contortrostatin, wherein said nucleotide sequence is selected from the group consisting of; a) nucleotides numbers 1341 to 1535 of SEQ ID NO: 1; b) nucleotides numbers 657 to 1316 of SEQ ID NO: 1; c) nucleotides numbers 87 to 356 of SEQ ID NO: 1; d) nucleotides numbers 87 to 1535 of SEQ ID NO: 1, and e) SEQ ID NO: 1. 16. A vector comprising a recombinant DNA molecule according to claim 13. 17. A host cell transformed with the vector according to claim 16, further characterized in that said host cell is selected from the group consisting of cells animals, plant cells, insect cells, yeast and other fungi and bacteria. 18. - A process for the production of contortrostatin comprising the step of cultivating the host cell according to claim 17. 19. The process according to claim 18, further characterized in that it also comprises the recovery step of the contortrostatin expressed by the host cell. 20. A recombinant protein produced by a process comprising the steps of: a) culturing a host cell, said host cell is selected from the group consisting of mammalian cells, plant cells, insect cells, yeast and other fungi and bacteria said host cell is transformed with a vector, said vector comprises a DNA sequence that codes for the expression of contortrostatin, and b) recovering the contortrostatin expressed by the host cell. 21. A pharmaceutically acceptable composition comprising a pharmaceutically acceptable carrier and a purified protein according to claim 1. 22. The use of the composition according to claim 21, for the manufacture of a medicament for inhibiting an associated disease. with integrin binding to an integrin receptor in a patient. 23. The use according to claim 22, wherein the disease to be inhibited is platelet aggregation, tumor metastasis, angiogenesis, neovascularization, cell adhesion, invasiveness or growth. 24. - The recombinant protein according to claim 20, further characterized in that said DNA sequence is selected from the group consisting of: a) nucleotides numbers 1341 to 1535 of SEQ ID N0: 1, B) nucleotides numbers 657 to 1316 of SEQ ID N0: 1, c) nucleotides number 87 to 656 of SEQ ID NO: 1; d) nucleotides numbers at 87 to 1535 of SEQ ID NO: 1; and e) SEQ ID NO: 1.