MXPA97008513A - Inhibitors of fibrine and / or transglutamine degradation - Google Patents

Abstract

The present invention relates to inhibitors, which are obtained from tissues, or secretions of leeches typically of the order RHYNCHOBDELLIDA, have the following terminal sequence: NH2-Lys-Leu-Leu-Pro-Cys-Lys-Glu-Y- His-Gln-Gly-lle-Pro-Asn-Pro-Arg- where Y represents any amino acid sequence, or a pharmaceutically acceptable salt, derivative or bioprecursor of said sequence, or an analogue or homologue thereof. Due to its extreme efficacy in the nanomolar range it can be used to treat numerous diseases where protein degradation is important. They can be used for the treatment of Crohn's disease, tumor implantation, atherosclerosis, thrombotic microangiopathy, fibrous growth of the skin, acne, scar formation, membranous glomerulonephritis, cataracts, or infection with microfilarial nematodes. In particular they can be used to reduce thrombin stability so that they are more susceptible to lysis by thrombolytic agents

Description

INHIBITORS OF FIBRINE AND / OR TRANSGLUTAMINASE DEGRADATION Field of the Invention The present invention relates to a novel class of inhibitors of fibrin degradation and / or transglutaminase activity, and in particular, to such inhibitors which can be example, derived from leech tissue and / or leech secretions. BACKGROUND OF THE INVENTION Enzymes known as transglutaminases are primarily responsible for the stabilization of many protein aggregates, such as, for example, in the formation of blood clots. The degradation of proteins by the action of transglutaminases is the main way by which, for example, fibrin clots stabilize. In mammals, the stabilization of blood clots is caused by a transglutaminase known as Factor XIIla, which catalyzes the formation of degradation between fibrin fibers. Degraded blood clots are not as susceptible to the action of fibrinolytic enzymes and are virtually insoluble in denaturing solvents, such as 5M urea. Factor XlIIa is an atypical coagulation enzyme since it is not a serine protease but rather a transamidation enzyme containing cysteine, which catalyzes the reaction between the amino acid side chains of Usin and glutamine to form an amide bond with the elimination of the ammonia according to the following scheme: Factor Xllla R! -CONH2 + NH2-R2 > R! -CO-NH-R2 + NH3 When fibrin is the substrate, R? -CONH2 and R2-NH2 are side chains of glutamine and Usin respectively in different chains of the fibrin polypeptide. Factor Xllla can also catalyze the degradation of other proteins. For example, Factor Xllla is known to bind antiplasmin a2 to fibrin and increase resistance to fibrinolysis. In addition, it can cause degradations between a range of different contractile and structural proteins such as collagen, laminin, actin, myosin, thromboespondin, vinculin and vitronectin or the like. It is believed that this property is part of the wound healing process and may have a role in the pathology of various tissue remodeling diseases. Accordingly, it is desirable to provide a transglutaminase inhibitor whose inhibitor could be used, for example, in the treatment of various pathological or thromboembolic events. Transglutaminase inhibitors have previously been described and these generally fall into four main categories: (a) immunoglobulins directed to the enzyme; (b) low molecular weight substrates that compete with natural protein substrates; (c) reagents that react with the active site of the enzyme; and (d) peptide fragments of Factor Xllla itself. These inhibitors are not suitable for use, for example, in pharmaceutical formulations, for several reasons such as the following: Naturally circulating transglutaminase inhibitors have previously been identified as immunoglobulins targeting the subgroups of transglutaminase. Such inhibitors enhance a hemorrhagic condition caused by the reduction in circulation factor XIII. US Patent 5470957 discloses the use of such immunoglobulins in a therapeutic manner by raising the monoclonal antibodies to the subgroups of transglutaminase enzyme by known techniques. A disadvantage associated with such antibodies as the transglutaminase inhibitors is that they have high molecular weights and it is typically necessary to produce chimeric human analogs of the immunoglobulins before they can be used, for example, therapeutically in man. WO91 / 10427 discloses transglutaminase inhibitors which are amines, which act by binding the glutamine residues to a substrate to prevent degradation to another substrate. Such inhibitors are not very powerful because they need to be present at the same concentrations, or at higher concentrations, than the natural substrate in order to have any significant inhibitory effect. Accordingly, they are only effective at concentrations in the region of about 50 μM and higher. WO92 / 13530 discloses the use of various transglutaminase inhibitors that depend on the activity of the transglutaminase which is highly dependent on a reactive sulfhydryl group. Accordingly, any reagent that alkylates or oxidizes this sulfhydryl group must inhibit the activity of the transglutaminase. However, such reagents are very reactive and also very unstable and therefore are particularly unsuitable for use in, for example, pharmaceutical or therapeutic treatments. Attempts to provide peptide inhibitors that could be expected to be more specific and less toxic result only in low efficiency compounds. For example, such inhibitors are described in U.S. Patent 5328898 and Achyuthan KE, Slaughter TF, Santiago MA et al.; in J. Biol. Chem. 268: pp. 21284-21292, 1993, "Factor Xllla derived peptides inhibit transglutaminase activity: lozalisation of substrate recognition sites" ("Factor Xlla derived from peptide inhibitors of transglutaminase activity: substrate recognition localization sites"). SUMMARY OF THE INVENTION Accordingly, the purpose of an aspect of the present invention is to provide a potent inhibitor of transglutaminase enzymes and whose inhibitor can be used in, for example, pharmaceutical or therapeutic use. We have now isolated a novel polypeptide which inhibits the activity of transglutaminase and / or the degradation of fibrin, which polypeptide has the following amino acid sequence: 1 NH 2 -Lys-Leu-Leu-Pro-Cys-Lys-Glu-X_- His-Gln-Gly-20 Ile-Pro-Asn-Pro-Arg-Cys-X2-Cys-Gly-Ala-Asp-Leu 30 Glu-X3-Ala-Gln-Asp-Gln-Tyr-Cys-Ala- Phe-Ile-Pro- 40 Gln-Zi-Arg-Pro-Arg-Ser-Glu-Leu-Ile-Lys-Pro-Met-Asp-Asp-Ile-Tyr-Gln-Arg-Pro-Val-Z2-Phe -Pro-Asn- 60 66 Leu-Pro-Leu-Lys-Pro-Arg-Z3-COOH wherein Xi, X2 and X3 each represent any amino acid residue; Z_, Z2 and Z3 each represent, simultaneously or alternatively Cys or Glu; or a pharmaceutically acceptable salt, a derivative (such as a chimeric derivative) or a bioprecursor of said amino acid sequence, or a homologue or analogue thereof of substantially similar activity. By homologous, we understand a polypeptide in which no more than 23% of the amino acids in the polypeptide chain differ from those listed. The figure of 23% is based on the fact that many hirudin homologues that appear naturally in Hirudo medicinallis are described in the literature; the most diverse of these differ in 15 of the 65 amino acids in the polypeptide chain. By analogue, we understand that one or more additional amino acids can be interposed in the polypeptide chain, taking into account that they do not significantly interfere with the pharmacological activity of the polypeptide. The invention also encompasses truncated forms of the polypeptide having the amino acid sequence mentioned above. The polypeptides according to the invention are highly potent inhibitors of transglutaminase activity and / or fibrin degradation. The ability of the polypeptides according to the invention to prevent the formation of protein degradations has a dramatic effect on the instability of, for example, blood clots. The inhibitory effect of the polypeptides according to the invention on Factor Xllla can be measured by the increased solubility of fibrin clots in 5M urea. In addition, the inhibitory effect of the polypeptides can be measured by using the fact that the polypeptides inhibit the release of ammonia by the incorporation of ethylamine in the casein and also by the incorporation of biotinamidopentylamine into the casein. It is believed that the amino terminal domain is a particularly potent inhibitor of transglutaminase activity. Accordingly, the invention further comprises a polypeptide that specifically inhibits the activity of transglutaminase, which polypeptide comprises the following amino acid sequence: NH2-Lys-Leu-Leu-Pro-Cys-Lys-Glu-Y-His-Gln-Gly -Ile-Pro-Asn-Pro-Arg-wherein Y represents any amino acid sequence or a pharmaceutically acceptable salt, derivative or bioprecursor thereof, or a homologue or analogue thereof, of substantially similar activity.

The polypeptides according to the invention (hereinafter referred to as "Tridegins"), advantageously inhibit the activity of transglutaminase directly in concentrations in the nanomolar range of 1-50 (a difference of at least a factor of 1000 with relation to the known transglutaminase inhibitors of categories (b), (c) and (d) described above). The Tridegins can advantageously form pharmaceutically acceptable salts with any suitable, non-toxic, organic or inorganic acid. Examples of such inorganic acids include hydrochloric, hydrobromic, sulfuric or phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrosulfate. Examples of organic acids include mono, di and tri carboxylic acids such as acetic, glycolic, lactic, pyruvic and sulphonic acids or the like. Salts of the carboxy terminal amino acid residue include the non-toxic carboxylic acid salts formed with any suitable inorganic or organic base. The Tridegins according to the invention can be extracted from leech tissue or secretions by, for example, homogenization of substantially the whole leech, the salivary glands or the proboscis or the like, in a suitable regulator. The transglutaminase inhibitors had not previously been identified in, or extracted from, the leeches; therefore, the present invention comprises an inhibitor of transglutaminase activity derivable from leech tissue or from leech secretions. The term "derivable" as used herein encompasses the material that is derived directly, as well as the material that is derived indirectly or converted to a chemically modified derivative. Tridegins according to the invention are typically extracted or purified by the use of a combination of known techniques such as, for example, ion exchange, gel filtration and / or reverse phase chromatography. Leeches of the same genus or even the same species often have polypeptides in their saliva that have similar biochemical effects and are highly homologous in their amino acid structure. In the same species of leeches, there may be several different isoforms that differ in only a few amino acids. The Tridegins according to the invention are derivable from leech tissue or leech secretions, typically from leeches of the order Rhynchobdellida. However, because many of the components of the salivary gland or tissue secretions from leeches that have similar biochemical specificity are members of such homologous polypeptide families, the present invention also comprises such isoforms and analogs of the Trideginas according to the invention derivable from leeches. In addition, post-translational modification of the leech polypeptides is frequently observed, and in view of the fact that some of the residues in the Tridegins could not be assigned to a known amino acid structure, the present invention also includes such post-translationally modified polypeptides. corresponding to the polypeptides of the above mentioned sequences. According to a second aspect of the present invention, there is provided an inhibitor of fibrin degradation and / or transglutaminase activity, which inhibitor is derivable from leech tissue or from leech secretions, typically from leeches of the order Rhynchobdellida, more preferably of leeches of the genus Ifae-nenteria. The inhibitor according to the invention preferably has an apparent molecular weight in the range of between about 7,000 daltons and 8,000 daltons, as measured by polyacrylamide gel electrophoresis (PAGE), and has the ability to inhibit the release of ammonia catalyzed by the factor Xllla from the incorporation of amines in casein, and the incorporation of biotinamidopentilamina catalyzed by factor Xllla in casein. In addition to the effect of the Xllla factor, the Tridegins are inhibitors of many different transglutaminases since they inhibit the activity of both factor Xllla of plasma and platelet as well as tissue transglutaminase from guinea pig liver, albeit with different potency. Accordingly, they are also general inhibitors of transglutaminase and can be expected to inhibit many different types of this group of enzymes. The invention also comprises a diagnostic method for measuring the degree of inhibition of transglutaminase activity for an inhibitor according to the invention (as defined above), which method comprises measuring the amount of ammonia released from incorporation. of amines catalyzed by transglutaminase in casein, in the presence of the inhibitor, wherein the amount of ammonia released and / or the incorporation of the amine provides a measure of the level of inhibition of transglutaminase activity by the inhibitor. According to a further aspect of the present invention, there is provided a pharmaceutical formulation comprising an inhibitor according to the first or second aspect of the invention (as described above), and a pharmaceutically acceptable carrier, diluent or excipient therefor. Due to the low level of toxicity and the high level of inhibition of the transglutaminase activity associated with the Tridegins, they can be advantageously incorporated into pharmaceutical formulations, which formulations can be, for example, administered to a patient either orally or parenterally. . The term "parenteral" as used herein includes subcutaneous, intravenous, intra-articular and intratracheal injection and infusion techniques. Other means of administration such as oral administration or topical application can also be used. The parenteral compositions and the combinations are preferably administered intravenously either in a bolus form or as a constant infusion according to known procedures. The term "pharmaceutically acceptable carrier" as used herein is to be understood as any non-toxic, inert, solid or liquid encapsulating material, diluent or encapsulating material, which does not react negatively with the active compound or with a patient . Preferred liquid carriers that are well known include sterile water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants and wetting agents, etc. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs or the like, or they may be presented as a dry product for reconstitution with water or other suitable vehicle for use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Topical applications may be in the form of aqueous or oily suspensions, solutions, emulsions, jellies or, preferably, emulsion ointments. The unit doses of the pharmaceutical formulations according to the invention may contain daily amounts of Tridegina required, or submultiples thereof to make a desired dose. The therapeutically acceptable, optimal dose and dose rate for a given patient (which may be a mammal such as a human) depends on a variety of factors, such as the activity of the specific active material employed, age, body weight. , health in general, sex, diet, time and route of administration, speed of evacuation, the objective of treatment, that is, treatment or prophylaxis and the nature of the disease treated. It is expected that the systemic dose in the range of 0.05 to 50 mg / kg per body weight, preferably between 0.05 and 10 mg / kg and more preferably from 0.1 to 1 mg / kg will be effective. According to the nature of the disease to be treated, a single dose can contain from 0.05 to 10 mg / kg per body weight, whether applied systemically or topically. Tridegins can potentially be used to inhibit the stabilization of thrombus formation in, for example, acute coronary syndromes, venous thromboses or attacks and thereby improve the effect of thrombolytic therapy or even natural lytic processes. In this context, inhibition of the incorporation of fibrinolysis inhibitors such as antiplasmin a2 into fibrin clots could provide an additional benefit. The fact that Tridegins also inhibit other transglutaminases very effectively indicates that additional potential uses wherever transglutaminase activity is found causes a pathological event. Such a role for transglutaminases has been hypothetical in Crohn's disease, tumor implantation, thickening of the basal wall and atherosclerotic processes, thrombotic microangiopathy in, for example, the kidneys, fibrous growths of the skin such as scleroderm, membranous glomerulonephritis, repair or retinal damage, cataracts, acne, the formation of scar tissue and infection by various filarial nematodes. Not only can Tridegins be used for their therapeutic action against previous or related syndromes, but their high potency will allow lower doses. This possibility is illustrated very well in O93 / 18760, which describes the use of impotent inhibitor putrescine to treat hypertrophic scars with a preferred dose of 50 mM. The preferred concentration of a Tridegina in a similar circumstance is 1-100 μM. A formulation according to the second aspect of the invention can be advantageously administered in combination with an anticoagulant, a thrombolytic, fibrinolytic, or fibrinogenolytic agent, or the like, which can advantageously increase the ability of the formulation to digest or inhibit, example, blood clots. The anti-coagulant may comprise a polypeptide such as hirudin or heparin. Hirudin is disclosed in EP 0347376 and EP 0501821 and is a generic term for a family of homologous polypeptides found in a variety of leeches that specifically and potentially inhibit thrombin and subsequently inhibit blood coagulation. Similarly, a fibrinolytic / fibrinogenolytic agent such as hementin whose activity is found in the digestion of the fibrionegen can be used, making it non-coagulant. Hementin is a fibrinolytic agent found in various species of leeches, and is disclosed in, for example, US 4390630 and W091 / 15576. A particular effect of the Tridegins is to decrease the time of lysis of human plasma clots, both platelet-free and platelet-rich, when lysis is induced by any fibrinolytic enzyme. The combination of either the tissue plasminogen activator or the hementin with a Tridegina results in faster lysis than with Tridegina alone. Since Tridegina has no effect on its own, it shows a synergy between the two active materials. The Tridegins can be used in combination with fibrinolytic agents that pass directly by lysis to fibrin (such as hementin, plasmin or Eminase) or with plasminogen activators that act through plasmin (such as streptokinase tissue plasminogen activator, urokinase, staphylokinase or its derivatives) or truncated forms or hybrid molecules that have characteristics of two or more of these agents. Thrombolytic agents that can be included in the formulation according to the invention can comprise one or more tissue plasminogen activators, streptokinase, Eminase, urokinase and staphylokinase, as well as derivatives, truncated and hybrid forms thereof. Advantageously, when the formulation comprises, in addition to the Tridegins, the anticoagulant, thrombolytic or fibrinolytic agent, the time taken for the blood clots to be digested is markedly reduced. Accordingly, the Tridegins can potentially be used to inhibit the stabilization or formation of thrombi in, for example, acute coronary syndromes, venous thrombosis, or the like, and thereby improve the effect of thrombolytic therapy. Typically, the time required for 50% lysis of fibrin clots in the presence of plasmin is about half if the degradation is inhibited with one or more of the Tridegins. In addition, the time for 50% lysis of plasma clots in the presence of tissue plasminogen activator is reduced up to 40%, and similarly that of streptokinase in an amount greater than 25%. The term "in combination", as used throughout the specification, should be understood as the simultaneous or sequential administration of the Tridegins, according to the invention, together with any or all of the anticoagulant, fibrinolytic, fibrinogenolytic or thrombolytic agents. The Tridegins according to the invention can be used advantageously for the preparation of a medicament for the treatment of a thromboembolic disease. Other pathological events that can be treated using the Tridegins according to the invention include Crohn's disease, tumor implantation, thickening of the vasal walls in atherosclerotic processes, thrombotic microangiopathy, in for example the kidney, fibrous growths of the skin, Membranous glomerulonephritis, cataracts, acne and the formation of scar tissue, as well as infections with microfilarial nematodes. Advantageously, the Tridegins according to the invention should not only be useful in the treatment or therapeutic prevention of such syndromes, but the high potency of the Tridegins should allow lower doses to be used. The present invention further comprises a polypeptide produced by a recombinant DNA technique, which polypeptide is equivalent to the polypeptide defined above; the invention further comprises a synthetic equivalent or designed as a protein to the polypeptide according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS The exemplary processes for the isolation and characterization of the polypeptide according to the invention will now be described with reference to the accompanying drawings which are given by way of example only, wherein: Figure 1 is a graphic illustration of the elution of the inhibitory activity of the polypeptides according to the invention isolated according to Example 3 described below; Figure 2 is a graphic illustration of the results of Example 4 for the elution of the inhibitory activity of the polypeptide according to the invention as compared to hementin and ghilantin; Figure 3 is a graphic illustration of the results of Example 6 of the inhibitory activity of the polypeptide according to the invention; Figure 4 is a chromatography of the inhibitory activity of Figure 3; Figure 5 is a chromatography of the active fractions obtained from Figure 4; Figure 6 is an illustration of the results of the sodium dodecylsulfate polyacrylamide gel electrophoresis of Example 7; Figure 7 is an illustration of the results obtained from Example 22; Figure 8 is an illustration of the results obtained from Example 24; and Figure 9 is an illustration of the results obtained from Example 25. DETAILED DESCRIPTION OF THE PREFERRED MODALITY Example 1 In a first experiment A, the proboscis, the anterior and posterior salivary glands of a leech of the species Haementeria ghilianii were homogenized, together in a Potter homogenizer in 10 mM of Tris HCl, 0.85% w / v NaCl pH 7.0 (1 ml), and centrifuged at 13,000 rpm. The supernatant was tested in a clot solubility assay similar to that of Tymiak, Tuttle, Kimball, Wang and Lee "A simple and rapid screen for inhibitors of Factor Xllla" (A simple and rapid selection for factor Xllla inhibitors). Antibiotics 46 (1993) pp. 204-206. In a second experiment B, the proboscis, anterior and posterior salivary glands of a leech of the species Haementeria ghilianii were sectioned and homogenized separately in 0.2 ml aliquots of regulator. The effect was compared with the extracts of the proboscis, the anterior and posterior salivary glands of the leeches of the species Haementeria officinalis prepared in 0.2 ml of regulator.

The test samples (30 μl) were added to a solution * > of 10 mg / ml crude bovine fibrinogen containing factor XIII (30 μl). The reaction was started by adding 6.25 units / ml of bovine thrombin containing 9 mM CaCl 2 (40 μl). A clot formed in 15 min when 8M of urea (160 μl) were added and left in contact with the clot. After 30 min, the absorbance resulting from the opalescence of the clot was read at 405 nm. The decreased absorbency indicated solubility of the clot resulting from the inhibition of degradation. Table I shows the inhibitory effect (absorbance at 405nm) of the various extracts on the solubility of fibrin clots compared to iodoacetamide (a known factor Xllla inhibitor). The numerical values cited in the table are absorbance at 405 nm.

Table 1 Example 2 In order to confirm the presence of a factor Xllla inhibitor, the effect of the extracts on the ability of human factor Xllla to catalyze the incorporation of biotinamidopentylamine into casein was measured by the microtiter plate method described by Slaughter TF. Achyuthan KE, Lai T-S and Greenberg CS (1992). ("A microtitre tablet transglutaminase assay utilizing 5- (biotinamido) pentylamine as substrate" ("A microtiter plate assay of transglutaminase that uses 5- (biotinamide) pentylamine as a substrate") Anal Biochem 205: 166-171). Extracts of the proboscis, the anterior and posterior salivary glands of the leeches of the Haementeria species were prepared as in experiment A in Example 1. Those of Haementeria depressa were lyophilized. Since the salivary glands are not easily removed from the leeches of the species Hirudo medicinalis and Hirudinaria manillensis, the extracts were prepared by removing an anterior third of individual leeches and homogenizing them in 1 ml of 10 mM Tris HCl containing NaCl at 0.85%. The supernatant after centrifugation at 13000 rpm was used in the assay. N, N dimethylcasein was dissolved in 0.1M Tris HCl pH 8.5 when stirred at 85 ° C for 30 min 2000g for 20 min. A concentration of 10-20 mg / ml (0.2 ml) was used to cover the wells of a microtiter plate by incubation at 37 ° C for 1 h. Excess casein was discarded and the wells were blocked with 0.5% fat-free milk powder in 0.1M TRis HCl pH 8.5 for 30 min. The plate was then rinsed twice with 0.35 ml aliquots of the Tris buffer. Factor Xllla was prepared from citrated human plasma by defibrinogenation by the addition of bentonite * to solid (40 mg / ml), incubation for 10 min and centrifugation at 12000g for 2 min. The supernatant (0.5ml) was activated by the addition of 1000 U / ml of bovine thrombin (0.05ml) and 200mM of CaCl2 (0.025ml) and incubation at 37 ° C for 15 min. Thrombin was neutralized by the addition of 2000 ATU / ml of hirudin (0.5ml). The wells of the microtiter plate contained (total volume of 0.2ml) 5mM of CaCl2, 10mM of dithiothreitol, 0.5mM of biotinamidopentylamine, the test sample (0.05ml) and the activated plasma (0.05ml). After incubation at 37 ° C for 30 min, the liquid was discarded and the reaction was stopped by two rinses in 0.2M EDTA (0.35 ml each) followed by two rinses with 0.1M Tris HCl pH8.5 (0.35ml each). 0.25mg / ml of streptavidin-alkaline phosphatase was diluted at 1: 150 with 0.5% fat-free milk powder in the Tris buffer and 0.25 ml was added to each well and incubated for 1 h at 20 ° C. The plate was rinsed once with 0.1% Triton X-100 (0.35 ml) followed by three rinses with the Tris regulator (0.35 ml). The bound alkaline phosphatase was measured by the addition of lmg / ml p-nitrophenyl phosphate. 5mM MgCl2 in the Tris buffer (0.05ml) plus Tris buffer (0.2ml) and absorbance were measured after 30 min by using a Titertek Uniskan II microtiter plate reader at 405nm. Table 2 confirms, in a different and more sensitive assay, that the inhibitory activity of Factor Xllla (measured by the incorporation of biotinamidopentylamine in casein catalyzed by factor Xllla of human plasma) is found in the salivary organs of both Haementeria ghilianii and Haementeria offi cinalis. In addition, a significant but low inhibitory activity is detectable in the salivary glands of Haementeria depressa and in the previous portions -of Hirudo medicinali ^ s--, and the irudinaria manill ensis. ,, Table 2 * 1 unit is defined as twice the amount of transglutaminase inhibitor required to inhibit human factor Xllla in 1 ml of 50% normal human plasma. A plasma deposit from seven healthy donors was used for this standardization. Example 3 A homogenate in phosphate-buffered saline was prepared from five sets of the complete salivary complex (anterior, posterior and proboscis glands) of Haementeria ghilianii in the same manner as described in Example 1 and the supernatant was applied to a 1.6 x 80cm column of Superdex G-200 and passed in phosphate-buffered saline pH 7.2 at a flow rate of 1ml / min. The eluent was monitored at 280 nm and the inhibitory activity was determined by the same assay as described in Example 1. Figure 1 shows the separation and position where the inhibitory activity is eluted. The bar indicates the fractions that contain the activity of Tridegina. Example 4 A homogenate of the complete salivary complex of five Haementeria ghilianii in 20 mM Tris HCl pH 8.0 was prepared as in Example 1. The supernatant was applied to a 0.8 x 7.5 cm column of Express-Ion Exchanger Q (Whatman) and it was eluted with a linear gradient up to 20 mM Tris HCl pH 8.0 containing 0.3M NaCl. The eluent was monitored by absorption at 280 nm and the activity of Tridegina was determined by the clot solubility test as in Example 1 in addition, the activity of the hementin was measured by a fibrinogenolytic assay and the inhibitory activity of factor Xa by a chromogenic substrate assay. Hementin activity was assessed by incubation of 2mg / ml bovine fibrinogen (50μl) with 20mM of HEPES buffer containing lOmM of CaCl2 and 0.1% w / v of Brij 35 pH 7.5 (25μl) and serial dilutions of the column fractions (25μl) at 37 ° C for 60 min. Then lOOU / l of thrombin (lOμl) was added to cause coagulation and the clot was measured by turbidity at 405nm after 30 min.

The reduction in turbidity indicated the amount of digested fibrinogen. The chromogenic substrate assay of factor Xa was carried out by incubating 2mM of S2765 in 50mM Tris HCl pH 8.3 in a spectrophotometer and the speed of change of absorbance at 405 nm was measured. The reaction was initiated by the addition of human factor Xa. Figure 2 shows the elution profile on a SP Sepharose column eluted with a linear gradient up to 0.3M NaCl. The positions where Tridegina (T), hementin and factor Xa inhibitory activity are shown are shown; there is a very clear separation of Tridegina from the other two salivary components, namely hementin (H) and ghilanteno (G), which are known to be found in the salivary glands of these species of leeches, confirming that Tridegina differs from the known components. The fractions containing the inhibitory activity are marked with a bar and the respective letters T, G and H. Example 5 A homogenate of the complete salivary complex of the Haementeria ghilianii was prepared in 20mM ammonia format pH 3.5 (5ml) in a manner similar to that of Example 1 and applied to a 0.8 x 7.5cm column of Express-Ion Exchanger S (Whatman).

The fractions were eluted with a linear salt gradient up to 20 mM sodium format containing 1M NaCl pH 3.5. The eluent was monitored by absorbance at 280 nm and tested as in Example 1. The inhibitory fraction was eluted at approximately 0.6M. Example 6 By combining chromatography steps similar to those exemplified in Examples 3, 4 and 5, a huge batch was prepared. The complete salivary complex of fifty leeches of the Haementeria ghilianii species that were not fed for at least three months were homogenized in 20 mM Tris HCl pH 8.0 (50 ml) and centrifuged as in Example 1. The supernatant was applied to a column of 60 x lOcm of Q Sepharose Fast Flow (Pharmacia). The fractions were eluted with a linear gradient from the start buffer to one containing 0.1M NaCl. The eluent was monitored at 280 nm and the active fraction was found to levy at approximately 0.09M NaCl (see Figure 3 in which the fractions containing inhibitory activity are labeled with a bar). The active fractions (145 ml) were adjusted to pH 4 by the addition of formic acid and applied to a 5 x 12 cm column of SP Sepharose Fast Flow (Pharmacia) which had been equilibrated in 20 mM sodium formate buffer pH 3.5. The column was eluted with a linear gradient from the equilibrium regulator to the same regulator containing 1M NaCl. The active fraction was eluted in a peak at approximately 0.57M NaCl (see figure 4 in which the fractions containing inhibitory activity are marked again with a bar). This was lyophilized and reconstituted in a final volume of 2.4 ml of water and applied to a 1.6 x 60 cm column of Superdex G-75 which had been balanced in phosphate-buffered saline pH 7.2. The elution profile is shown in figure 5, in which the fractions containing the inhibitory activity are marked again with a bar.

The active fractions deposited contained 715 μg of protein and these were stored frozen. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and staining with either Coomasie blue or silver staining showed that the protein was substantially pure after this step and that by comparison with known molecular weight standards, the main band had a apparent molecular weight of approximately 7800 daltons with minor bands at a higher molecular weight, which were only detectable by the more sensitive silver dyeing method.

Example 7 In order to sequence the work, an additional purification step was carried out. 0.3ml of the active fraction from Example 6 was applied to a 0.5 x 10cm column of ProRPC equilibrated in 0.1% trifluoroacetic acid and eluted with a 0 to 100% gradient of acetonitrile containing 0.1% trifluoroacetic acid. A main peak was found that contained the inhibitory activity and was followed by, and widely separated from, two much smaller inactive peaks. The active fraction showed a single band in the sodium dodecylsulfate polyacrylamide gel electrophoresis with an apparent molecular weight of about 7800 compared to known molecular weight peptide standards that are illustrated in Figure 6. Figure 6 is an electrophoresis in polyacrylamide gel in sodium dodecyl sulfate of the pure polypeptide on a PhastGel high density gel (Pharmacia). The right side band (band 1) and band 7 are low molecular weight marker kits (Pharmacia) of 94, 67, 43, 30, 20.1 and 14.4 kD plus aprotinin (molecular weight 6.5kD). Bands 2 and 7: peptide marker kit (Pharmacia) of molecular weights 16.9, 14.4, 10.7, 8.2, and 6.2 kD plus aprotinin (6.5kD).

Band 3: water space. Band 4: Purified tridegina. Band 5 and 6: minor peaks of the reverse phase chromatography column. The lower molecular weight components migrated as close to the upper part of the gel. A single clean amino acid sequence was found from the amino terminus by an Applied Biosystems 473A automated protein sequencer that indicates that only one peptide was present. The amino acid sequence was found to be: NH2-Lys-Leu-Leu-Pro-X-Lys-Glu-Y-His-Gln-Gly-Ile-Pro-Asn-Pro-Arg-where X and Y did not identify in a positive way and therefore could be any amino acid. The cysteines in the sample were not derivatized and therefore could not be assigned. The sequence was repeated after pyridylethylation and this showed that residue X is a cysteine considering that Y did not give any peak and could not be assigned to any common amino acid. Example 8 In order to produce sufficient material for the amino acid sequence, a sample of transglutaminase inhibitor was prepared from the posterior salivary glands of only fifty leeches of the Haementeria ghilianii species by a method identical to that used in Examples 6 and 7. The aliquots were denatured, amidocarboxymethylated and digested either by trypsin or AspN endoprotease by the standard methods described in Matsudaira ("A practical guide to protein and peptide purification for microsequencing" "A practical guide for the purification of protein and peptides for microsequence" Academic Press, 2nd Edition pp. 45-67), and the fragments were separated on a column of 0.5 x lOcm of ProRPC equilibrated in 0.06% trifluoroacetic acid and eluted with sequential linear gradients of 2 to 38%, 38 to 75 % and 75 to 98% of elution buffer where the elution buffer was 80% acetonitrile in acid 0.0675% trifluoroacetic and was monitored at 210nm. The sequence of the isolated fragments was determined by an Applied Biosystems 473A automatic protein sequencer. The amino acid sequence of the complete polypeptide was deduced from this and the superimposed peptides that were found were: 1 NH 2 -Lys-Leu-Leu-Pro-Cys-Lys-Glu-X? -His-Gln-Gly- 20 Ile- Pro-Asn-Pro-Arg-Cys-X2-Cys-Gly-Ala-Asp-Leu 30 Glu-X3-Ala-Gln-Asp-Gln-Tyr-Cys-Ala-Phe-Ile-Pro-40 Gln- Zi-Arg-Pro-Arg-Ser-Glu-Leu-Ile-Lys-Pro-Met- 50 Asp-Asp-Ile-Tyr-Gln-Arg-Pro-Val-Z2-Phe-Pro-Asn- 60 66 Leu -Pro-Leu-Lys-Pro-Arg-Z3-COOH wherein the amino acids Xi, X2 and X3 were not identifiable and may represent residues that have been modified post translationally and Z_, Z2 and Z3 represent amino acids that could not be distinguished between Cys or Glu. The polypeptide having this sequence is designated as a variant of Tridegina 1. Example 9 Apart from the tests that demonstrated the ability of factor XIIla to incorporate amines in casein and the effects of Factor Xllla on the solubility of the clot, the specificity of the action Inhibitor can be shown by an assay that measures ammonia production from casein when the amines are incorporated.

The tranglutaminase activity of the human plasma Factor Xllla was measured spectrophotometrically by a modification of the method of Muszbek, Polgar and Fesus; "Kinetic determination of blood coagulation factor XIII in plasma" ("Kinetic determination of factor XIII of Blood coagulation in the plasma ") Clin Chem 31 (1985) pp. 35-40 This method measures the production of ammonia by binding it through the glutamate dehydrogenase reaction to the oxidation of NADH that can be monitored by the change in absorption at 340nm Factor XIII was activated by incubating defibrinised human plasma (2ml) and 200mM CaCl2 (O.lml) with lOOOunit / ml of bovine thrombin (O.lml) at 37 ° C. min, the reaction was stopped by the addition of 260 units of hirudin antithrombin The reaction cuvette contained: 2.5mM dithiothreitol (O.lml), 40mg / ml dephosphorylated β-casein (0.05ml), 70mM ethylamine (O.lml), 12 mM sodium 2-ketoglutarate (O.lml), 4mM NADH (O.lml), 1.2mM ADP (O.lml), 40 units / ml glutamate dehydrogenase (O.lml), 70mM regulator HEPES pH7.5 (0.25ml) and this was placed in a spectrophotometer at 20 ° C. All components were dissolved in 70 mM of HEPES pH7.5 buffer when possible. The reaction was initiated by the addition of activated factor XIII (0.2ml) and monitored at 340nm. The assay was validated with the use of plasma deficient in factor XIII (Sigma). The replacement of normal plasma with the deficient plasma resulted in a reaction rate that was 88% lower (1.87 versus 11.3 mAbs / min) demonstrating that the assay actually measures factor XIIla. The test samples were added in O.lml of the HEPES regulator to the reaction cuvette. The inhibitory effect of the variant of Tridegina 1 was compared with that of iodoacetamide, a known inhibitor of factor Xllla dependent on the sulfhydryl group and EGTA, an inhibitor of the activation and activity of factor XIII by virtue of its essential calcium chelation. as shown in Table 3. Tridegina decreased the proportion of ammonia production by approximately 93%, i.e., the inhibition was equivalent to that of iodoacetamide (as shown in Table 3). This is additional evidence showing that the Trideginas, as well as the clot solubilization inhibitors, are inhibitors of plasma transglutaminase or factor Xllla. Table 3 EXAMPLE 10 The effect of the variant of Tridegina 1, purified as in Example 6, on the ability of the human factor Xllla to catalyze the incorporation of biotinamidopentylamine into casein was measured by the microtiter plate method described by Slaughter TF, Achyuthan KE, Lai T-Sand Greenberg CS (1992). ("A microtitre tablet transglutaminase assay utilizing 5- (biotinamido) pentylamine as substrate" ("A microtiter plate transglutaminase assay using 5- (biotinamide) pentylamine as a substrate") Anal Biochem 205: (166-171). N, Ndimethylcasein was dissolved in 0.1M TrisHCl pH8.5 by shaking at 85 ° C for 30 min and then by centrifugation at 12000g for 20 min.A concentration of 10-20 mg / ml (0.2ml) was used to cover the wells of a microtiter plate by incubation at 37 ° C for 1 h.The excess casein was discarded and the wells were blocked with 0.5% fat-free milk powder in 0.1 M Tris HCl pH 8.5 for 30 min. it was then rinsed twice with aliquots of 0.35 ml of the Tris buffer, purified human platelet factor XIII (0.6 units / 0.12 ml) was activated by the addition of 150U / ml of thrombin in 15mM CaCl2 (0.18ml) and a incubation at 37 ° C for 15 min.Thrombin was then inhibited is by the addition of 140ATU / ml of natural hirudin (0.3ml). The microtiter plate wells (total volume 0.2 ml) contained 5 mM CaCl 2, 10 mM dithiothreitol, 0.5 mM biotinamidopentylamine, the Tridegina sample was prepared in a manner similar to that described in Example 6 and 0.25 units / ml factor. Xllla activated in 0.1M Tris HCl pH 8.5. After incubation at 37 ° C for 30 min, the liquid was discarded and the reaction was stopped by two rinses in 0.2M EDTA (0.35ml each) followed by two rinses with 0.1M Tris HCl pH8.5 (0.35ml each). 0.25mg / ml of streptavidin-alkaline phosphatase was diluted at 1: 150 with 0.5% fat-free milk powder in the Tris buffer and 0.25 ml was added to each well and incubated for 1 h at 20 ° C. The plate was rinsed once with 0.1% Triton X-100 (0.35 ml) followed by three rinses with the Tris regulator (0.35 ml). The bound alkaline phosphatase was measured by the addition of lmg / ml p-nitrophenyl phosphate, 5mM MgCl2 in the Tris buffer (0.05ml) plus Tris buffer (0.2ml) and the absorbance was measured after 30 min using a Titertek Uniskan II microtiter plate reader at 405 nm. The variant of Tridegina 1 clearly inhibited the incorporation of the amine with an IC50 of 0.026 + 0.002μg / ml (3.4nM) confirming its very potent inhibitory activity in platelet factor Xllla. In a separate experiment using an identical protocol except for the replacement of factor XIII purified with plasma from a healthy human volunteer and the variation of Tridegina concentration, the IC50 was determined to be 0.07 + 0.003μg / ml (9.2nM ) for the plasma form of factor Xllla.

Example 11 The effect of the purified Tridegina 1 variant as in Example 6 was examined in the enzyme coagulation, thrombin, with a chromogenic substrate assay. 1 mM of S2238 was incubated in 50 mM Tris HCl pH 8.3 and the reaction was initiated by the addition of thrombin to a final concentration of 0.15 U / ml. The reaction was monitored in a 405nm spectrophotometer. Table 4 shows that Tridegina had no effect on thrombin at a concentration of 4.6 μg / ml considering that hirudin at a concentration of 0.046 μg / ml had a marked inhibitory effect (95%). Table 4 Example 12 The effect of purified Tridegina as in Example 6 was examined on factor Xa. 2mM of S2765 was incubated in 50mM Tris HCl pH8.3 and the reaction was started by adding human factor Xa. The assay was carried out as in Example 11. The transglutaminase inhibitor at 4.6 μg / ml had no effect on factor Xa considering the recombinant password anticoagulant peptide (rTAP), a known factor Xa inhibitor (Waxman L, Smith DE, Acuri K et al. "Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa" "The password anticoagulant peptide (TAP) is a new inhibitor of the blood coagulation factor Xa" Science 248: 593 -596: 1990), was inhibited by 89.9% at a concentration of 0.046 μg / ml (Table 5). This confirms that, not only the transglutaminase inhibitor is different from the previously known inhibitors of factor Xa, but the methods exemplified to purify the transglutaminase inhibitor successfully remove the factor Xa inhibitors. Table 5 Example 13 In order to investigate whether the Tridegins have a fibrinogenolytic activity similar to hementin, the ability of the purified material of Example 6 to digest fibrinogen was evaluated by assessing the coagulation capacity of the fibrinogen after incubation of this with the inhibitor. 2mg / ml of bovine fibrinogen (50μl) were incubated with Tridegina, hementin or vehicle (50μl) and 20mM of HEPES buffer containing 10mM CaCl2 and 0.1% w / v of Brij 35 pH 7.5 (50μl) were purified. at 37 ° C for 60min. Then lOOU / ml thrombin (lOμl) was added to cause coagulation and the clot was measured by turbidity at 405nm after 30min. Table 6 shows that the transglutaminase inhibitor had no effect on clot formation considering that purified hementin clearly digested fibrinogen and resulted in a small clot. This indicates that the Tridegins have no proteolytic or fibrinogenic action and therefore are not hementin as described in WO 91/15576 ("Treatment of thrombotic events") and US Patent 4,390,630 ("Hementin - a fibrinolytic agent ") (Hementin - a fibrinolytic agent). In addition, this example further adds confirmation that the hementin found in the Haementeria ghilianii is separated from the transglutaminase inhibitor during the exemplified purification procedures.

Table 6 Example 14 The activity of the enzyme, destabilasa, can be measured by its effect in causing the release of p-nitroaniline from the chromogenic substrate, L-β-glutamyl p-nitroanilide. In order to investigate whether destabilasa and Trideginas have similar properties, the effect of the two agents on the chromogenic substrate was compared. The absorbance of the buckets containing 0.45mg / ml of L-β-glutamyl p-nitroanilide in 50mM Tris HCl, 10mM CaCl 2 pH 8.0 (0.9ml) were recorded continuously at 405nm in a spectrophotometer. Either 0.046mg / ml variant of Tridegina 1 (O.lml) or the supernatant of an extract of Hirudo medi cinalis (O.lml), a known source of destabilasa prepared as in Example 2 was added and the speed was measured of generation of nitroaniline. Table 7 shows the effect of Tridegina on the substrate of destabilasa, p-nitroaniluro of L -? - glutamyl, and indicates that although the extract of Hirudo medicinalis contains an activity that causes an increase in the speed of absorbance that indicates the separation of this substrate attributable to destabilasa, Tridegina has no such effect and in fact results in a slight increase in absorbency over time. Table 7 EXAMPLE 15 The effect of Tridegine on plasma coagulation was examined by comparing a sample of normal plasma to which 0.1 volumes of the purified inhibitor as in Example 6 (46 μg / ml) in phosphate buffered saline were added with one that I had only regulator. Standard coagulation tests were carried out on an automated analyzer. The results in Table 8 show that there is no difference in the two samples so that Tridegina has no effect on the coagulation time of normal human plasma. This property is expected since inhibitors of fibrin degradation have no effect on clot formation and only influence its physical and chemical properties after it has been formed. Furthermore, this confirms, in a different test, the absence of any other anticoagulant activity such as the inhibition of factor Xa or thrombin in Tridegina. The addition of platelets in platelet-rich, citrated, human plasma in a Bio / Data agometer was investigated in response to either 6.7μg / ml of collagen, 6.3μM of ADP or 0.4U / ml of thrombin. The Tridegina of Example 6, at a final concentration of 4.6 μg / ml, was compared to the regulator controls. Table 8 shows that Tridegina had no effect in the addition of platelets under these conditions. Table 8 Example 16 The effect of the inhibitor on guinea pig liver tissue transglutaminase was measured in an assay similar to that described in Example 10 where factor Xllla was replaced with tissue transglutaminase. Tridegina (4.5μg / ml) inhibited the incorporation of the amine in the casein catalyzed by this enzyme in 95.5%. When using different concentrations of Tridegina it was found that the IC50 is 1.55μg / ml. This assay indicates that Tridegina is a tissue transglutaminase inhibitor as well as plasma transglutaminase factor XIIla and is likely to be an inhibitor of many of the transglutaminase enzymes. Example 17 The transglutaminase inhibitor is measurable in the glands of the salivary system and in salivary secretions of Haementeria ghilianii by the amine incorporation assay of Slaughter TF, Achyutan KE, Lai T-S and nberg CS, (1992). ("A microtitre tablet transglutaminase assay utilizing 5- (biotinamido) pentilamine as substrate") ("A transglutaminase assay microtiter plate using 5- (biotinamide) pentylamine as a substrate"). Anal Biochem 205: pp. 166-171. The anterior and posterior salivary glands and the proboscis together with the rear sucker were removed from a hungry animal in the third feeding stage. The samples were homogenized in a glass homogenizer in lmM of Tris HCl pH8.0 (lml or 0.5ml in the case of the posterior glands) and centrifuged at 12,000rpm. The supernatant was used for the assay. For the collection of saliva secretion, the complete salivary apparatus (proboscis, anterior and posterior glands) was removed from each of the eight hungry Haemeria ghilianii in the third feeding stage, after cooling of the leeches at 5 ° C for 2 days. -3h. It was placed on a Sylgard base and bathed in physiological saline solution (65mM NaCl, 50mM NH4CI, 4mM KCl, lmM EGTA, lmM glucose, lOmM HEPES pH 7.4) at 20 ° C for 15 min. When the proboscis wall was cut longitudinally, the lumen was accessed and the secretions contained therein were collected by micropipette. Table 9 shows the inhibition of factor Xllla from human plasma by these extracts and secretions of the leech Haemeria ghilianii. The inhibitory activity is found in both salivary glands, in salivary secretion and in the proboscis. The very low detectable activity in the rear sucker is very low specific activity, being 0.35% that of the posterior salivary gland, and in fact the apparent activity detected may well result from the very high concentration of protein extracted from this large piece of tissue.

Table 9 1 unit is defined as twice the amount of the tranglutaminase inhibitor required to inhibit factor XIIla in 1 ml of normal human plasma by 50% in the amine incorporation assay described in Example 10. average of eight separate experiments. the protein concentration was too low to measure (the specific activity is very high).

Example 18 The potential effect of Tridegine on plasmin induced fibrinolysis was demonstrated by an absorbance method. LOmg / ml of bovine fibrinogen (O.lml) was incubated with 50U / ml of bovine thrombin (O.Olml) with either regulator or Tridegina of Example 6 (0.04ml) for 2h at 37 ° C in a microtiter plate. 2.56U / ml of plasmin (0.05ml) was added and the plate was incubated at 37 ° C.

The absorbance was measured every 15 min in a Titretek Uniskan II microtiter plate reader. The clot was observed every 15 minutes and the time taken for the clot to dissolve completely was recorded. At all concentrations tested, the transglutaminase inhibitor shortened the time for lysis to occur as illustrated in Table 10. Table 10 Example 19 The accelerating effect of Tridegine on fibrinolysis induced by tissue plasminogen activator is also shown in human plasma clots. Human plasma (O.lml) was incubated with 5U / ml of bovine thrombin in 0.18M CaCl2 containing 0.14M KCl (O.Olml) and either regulatory or Tridegina prepared as in Example 6 but from the posterior salivary glands of Haementeria ghilianii (0.04ml) for 2h at 37 ° C on a microtiter plate in replicas of six. The tissue plasminogen activator (0.05ml) was then added to a final concentration of 10 IU / ml and the plate was incubated at 37 ° C and absorbance readings were taken every 30 min at 405nm using a Titretek Uniskan microtiter plate reader II. The reduction in absorbency indicated the lysis of plasma clots. The time taken for 50% of the lysis in the control wells was 12.9 ± l.lh and in the wells containing the transglutaminase inhibitor, 7.9 ± 0.7h, a statistically significant reduction. This example confirms that the transglutaminase inhibitor dramatically accelerates tissue plasminogen activator action in human plasma clots. Example 20 Since platelets are invariably associated with thrombi in vivo, it is of interest to observe whether the transglutaminase inhibitors allow faster lysis of platelet-rich clots, a more rapid physiological examination. Platelets are a rich source of plasma transglutaminase, factor XIII, as well as inhibitors of fibrinolysis in order to severely reduce the efficacy of fibrinolytic agents. Platelet-rich or platelet-poor human plasma, prepared from the same donor (0.1 ml) was incubated with 5 U / ml of coil thrombin in 0.18 N of CaCl2 containing 0.14 M KCl (0.01 ml) and either regulatory or Tridegina (prepared as in Example 6 but from the posterior salivary glands of the species Haementeria ghilianii, 0.04 ml) for 2 h at 37 ° C on a microtiter plate in replicas of six. Tissue plasminogen activator (0.05 ml) was added to a final concentration of 10 IU / ml and the plate was incubated at 37 ° C. The absorbance readings were taken every 30min for 72h at 405nm by the use of a Molecular Devices Thermomax kinetic microtiter plate reader (decreased absorbance showed lysis of plasma clots). Table 11 shows that in the presence of platelets, clots did not achieve 50% lysis in an incubation period of 72 hours. Tridegina is even more effective in reducing the effect of platelets when they occur, since it reduces the time from > 72h to 24.9h in your presence and from 22.5h to 18. Oh in your absence in this Example.

Table 11 Example 21 The effect of Tridegina for Decreasing clot lysis times is a general effect and may show up when streptokinase is used as the lytic agent. Human plasma (0.1 ml) was incubated with 5 U / ml bovine thrombin in 0.18 M CaCl 2 containing 0.14 M KCl (0.01 ml) and either regulatory or Tridegina prepared as in Example 6 but from the salivary glands of the Haementeria ghilianii (0.04 ml) for 2 h at 37 ° C in a microtiter plate in triplicate. Streptokinase was then added to a final concentration of 30 U / ml and the plate was incubated in an IEMS kinetic microtiter plate reader at 37 ° C and absorbance readings were taken every 30 min for 47.5h at 405 nm. Although wells containing streptokinase alone did not undergo sufficient lysis to obtain a time of 50% lysis, all wells that contained Tridegina experienced lysis in 50% at 36.1 ± 1.6h demonstrating again the accelerating effect when used in combination with fibrinolytic agents such as streptokinase shown in Figure 7 (which shows the effect of Tridegina in plasma clot lysis induced by streptokinase). The results are ± SEM (n = 3). Example 22 The combination of Tridegina and hementin was investigated as described in Example 21, where the tissue plasminogen activator was replaced with 110 U / ml hementin. In this Example both platelet-free and platelet-rich plasma samples from the same donor were used in order to investigate if there was any difference. Table 12 shows the times taken for 50% lysis to occur in the samples. There is a clear effect of the platelets that increase the time required for the lysis from 34h up to > 56h, Tridegina still clearly reduces the time for 50% lysis to occur whether the platelets are absent or present. Tridegina seems to greatly overcome the effect of platelets to decrease the time of lysis close to that of the control. Table 12 Example 23 In order to investigate the properties of Tridegina derived from the Mexican leech, Haementeria officinalis, an extract was chromatographed on gel filtration followed by reverse phase high pressure liquid chromatography. The salivary glands and proboscises were sectioned from the five specimens of Haementeria officinalis that had been hungry to a point where blood was no longer found in the front of the digestive tract. These were homogenized in phosphate buffered saline pH 7.2 (1 ml) in a Teflon / glass homogenizer and centrifuged at 12000g for five minutes in order to obtain a clear supernatant. The supernatant was applied to a 1.6 x 60 cm column of Superdex G75 and the eluent was monitored at 280 nm and all fractions were collected and tested in a coagulum solubility test as in Example 1. The inhibitory activity was found in a only peak as indicated in figure 8 (in which the fractions containing the inhibitory activity are marked again with a bar). The active fraction was lyophilised and redissolved in water (1 ml). Part of this (0.3 ml) was applied to a 0.5 x 10 cm column of Pro-RPC which had been balanced in 0.1% trifluoroacetic acid (TFA). Elution with a linear gradient of 0.1% TFA to acetonitrile containing 0.1% TFA resulted in the elution of a large number of peaks that were absorbed at 280 nm. Each was tested in the coagulum solubility test as described in Example 1 and it was found that a single peak contained activity. The comparison of the elution positions in the two columns with that for similar extracts of Haementeria ghilianii indicated the exact similarity with the elution position of the Tridegina 1 variant. The inhibitory activity purified from the salivary glands of Haementeria offi cinalis it has very similar physical and chemical properties in terms of molecular weight, as determined by gel filtration, and partition coefficient, as determined by reverse phase high pressure liquid chromatography for those of the Tridegina 1 variant. Example 24 Para determine the behavior of the variant of Tridegina 1 in vivo, a dose of 0.207 mg / kg increased value formulated in 0.01 M sodium phosphate, 0.027 M KCl, 0.137 M NaCl pH 7.4 (4.7 ml), was administered in a manner intravenously to a group of four rats. Blood samples (approximately 0.3 ml) were taken from the tail vein both before and 2 or 5 and 10, 20, 30, 60 and 120 minutes after administration and immediately mixed with 0.04 ml of trisodium citrate at 3.8% w / v. The samples were centrifuged immediately at 12000g for 5 min and the supernatants were removed and quickly frozen on dry ice. No side effect of the administration of Tridegina was observed. Tridegine in the samples was tested by a modification of the amine incorporation assay used in Example 2, where intrinsic factor XIII was activated in each sample (0.097 ml) by adding 0.1M Tris HCl pH 8.5 (0.03 ml) and 1000 U / ml coil thrombin (0.01 ml) and incubation at 37 ° C for 15 minutes. The fibrin clot was removed by centrifugation and the serum was used for the assay. Samples for the standard curve were prepared by adding known concentrations of Tridegina pure 1 variant to the citrated rat plasma and activating factor XIIla identically. The concentration of Tridegine in each sample was then determined by the percentage inhibition of factor Xllla as in Example 2 by comparison with the standard curve. Figure 9 shows the pharmacokinetics of Tridegina in the rat. The course of time is clearly multiphase; the terminal half-life is 30-60 minutes, indicating a significant duration of action and that the pharmacokinetics of Tridegina makes it suitable for pharmaceutical use.

Claims (24)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. 1. A polypeptide having the following amino acid sequence NH2-Lys-Leu-Leu-Pro-Cys-Lys-Glu-Y-His-Gln-Gly-Ile-Pro-Asn-Pro-Arg-en, where Y represents any sequence amino acid or a pharmaceutically acceptable salt, derivative or bioprecursor of said sequence, or a homologue, analog or truncated form thereof, of substantially similar activity.
2. 2. A polypeptide having the following amino acid sequence 1 10 NH2-Lys-Leu-Leu-Pro-Cys-Lys-Glu-X? -His-Gln-Gly- 20 Ile-Pro-Asn-Pro-Arg-Cys- X2-Cys-Gly-Ala-Asp-Leu-30 Glu-X3-Ala-Gln-Asp-Gln-Tyr-Cys-Ala-Phe-Ile-Pro-40 Gln-Zi-Arg-Pro-Arg-Ser- Glu-Leu-Ile-Lys-Pro-Met- 50 Asp-Asp-Ile-Tyr-Gln-Arg-Pro-Val-Z2-Phe-Pro-Asn- 60 66 Leu-Pro-Leu-Lys-Pro-Arg -Z3-COOH wherein X_, X2 and X3 each represent any amino acid residue; Zi, Z2 and Z3 each represent simultaneously or alternatively Cys or Glu; or a pharmaceutically acceptable salt, derivative or bioprecursor of said amino acid sequence, or an homologous or analogous truncated form thereof, of substantially similar activity.
3. 3. A polypeptide according to claim 1 or 2, characterized in that it is derived from leech tissue or secretions.
4. 4. A polypeptide according to claim 3, characterized in that the leech is of the order Rhynchobdellida.
5. 5. A polypeptide according to any of claims 1 to 4, characterized in that it is derived from the tissue or secretions of leeches of the genus Haementeria.
6. 6. An inhibitor of transglutaminase activity, whose inhibitor is derivable from leech tissue or from leech secretions.
7. 7. An inhibitor according to claim 6, characterized in that the leeches are of the order Rhynchobdellida.
8. 8. An inhibitor according to claim 7, characterized in that the leeches are of the genus Ha emen t eri a.
9. 9. An inhibitor according to any of claims 6 to 8, characterized in that said inhibitor is a polypeptide with an apparent molecular weight of about 7000 to 8000 daltons as measured by polyacrylamide gel electrophoresis.
10. 10. An inhibitor according to any of claims 6 to 9, characterized in that said inhibitor has the ability to inhibit the factor Xllla catalyzed incorporation of amines in casein.
11. 11. An inhibitor according to any of claims 6 to 10, characterized in that said inhibitor has the ability to inhibit the Xllla factor-catalyzed incorporation of biotinamidopentylamine into casein with an IC50 of 0.026 ± 0.002 mg / ml.
12. 12. A diagnostic method for measuring the degree of inhibition of transglutaminase activity for a polypeptide according to any of claims 1 to 5 or an inhibitor according to any of claims 6 to 11, which method comprises measuring the amount of ammonia released from the transglutaminase catalyzed incorporation of amines in the casein in the presence of said polypeptide or extract, respectively, wherein the amount of ammonia released provides a measure of the level of inhibition of the transglutaminase.
13. 13. The use of a polypeptide according to any of claims 1 to 5, or of an inhibitor according to any of claims 6 to 11, characterized by the preparation of a medicament for the treatment of a thromboembolic disease.
14. The use of a polypeptide according to any of claims 1 to 5, or of an inhibitor according to any of claims 6 to 11, characterized by the preparation of a medicament for the treatment of Crohn's disease, the implantation of a tumor , the thickening of the vassal walls in atherosclerotic processes, thrombotic microangiopathy, fibrous growths of the skin, membranous glomerulonephritis, cataracts, acne or scar tissue formation or infection with microfilarial nematodes.
15. 15. A pharmaceutical formulation comprising a polypeptide according to any of claims 1 to 5, and / or an inhibitor according to any of claims 6 to 11, and a pharmaceutically acceptable carrier, diluent or excipient therefor.
16. 16. A pharmaceutical formulation according to claim 15, characterized by its administration in combination with an anticoagulant.
17. 17. A formulation according to claim 16, characterized in that the anticoagulant comprises hirudin or heparin.
18. 18. A formulation according to claim 15, characterized by its administration in combination with a fibrinolytic, fibrinogenolytic or thrombolytic agent.
19. 19. A formulation according to claim 18, characterized in that the thrombolytic agent comprises one or more tissue plasminogen activator, plasmin, streptokinase, eminase, urokinase, hementin and staphylokinase.
20. 20. A formulation according to claim 18, characterized in that the fibrinolytic or fibrinogenolytic agent comprises hementin.
21. 21. The use of a formulation according to any of claims 15 to 20, characterized by the preparation of a medicament for the treatment of a thromboembolic disease.
22. 22. The use of a formulation according to any of claims 15 to 20, characterized by the preparation of a medicament for the treatment of Crohn's disease, the implantation of a tumor, the thickening of the vasal walls in atherosclerotic processes, thrombotic microangiopathy , fibrous growths of the skin, membranous glomerulonephritis, cataracts, acne or formation of scar tissue or infection with microfilarial nematodes.
23. 23. An essentially purified polypeptide having transglutaminase inhibitory activity, said polypeptide being derivable from leech tissue or leech secretions by a process comprising ion exchange chromatographic purification and / or purification by gel filtration column chromatography.
24. 24. A process for the isolation of a polypeptide according to any of claims 1 to 5 or an inhibitor according to any of claims 6 to 11, which process comprises extraction of tissue or secretions of leeches of the order Rhynchobdellida and purification of the extracted material by one or more of ion exchange column chromatography, gel filtration column chromatography and reverse phase chromatography.