CN1187201A - Inhibitors of fibrin cross-linking and/or transglutaminases - Google Patents

Abstract

The inhibitors, obtainable from tissue or secretions of leeches typically of the order Rhynchobdellida, has the following terminal 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 of said sequence, or an analogue or homologue thereof. Because of their extreme potency in the nanomolar range, they can be used to treat a number of diseases where protein cross-linking is important. They can be used for the treatment of Crohn's disease, tumor implantation, atherosclerosis, thrombotic microangiophathy, fibrous growths of the skin, acne, scar formation, membranous glomerulonephritis, cataracts, or infection with microfilarial nematodes. In particular, they can be used to reduce the stability of thrombi so that they are more susceptible to lysis by thrombolytic agents.

Description

Inhibitors of fibrin cross-linking and/or transglutaminase

The present invention relates to a novel class of inhibitors of the cross-linking and/or transglutaminase activity of fibrin, in particular to such inhibitors as derived from leech tissue and/or from leech secretions.

Transglutaminase is mainly used to stabilize many protein aggregates such as in clot formation. Crosslinking of proteins by transglutaminase reactions is a major means of stabilizing fibrin clots, for example. In mammals, the stabilisation of blood clots is caused by transglutaminase called factor XIIIa, which catalyses the formation of interfibrous cross-links in fibrin. The cross-linked clot is not sensitive to the action of fibrinolytic enzymes and is practically insoluble in denaturing solvents such as 5M urea.

Factor XIIIa is an atypical coacervation enzyme in that it is not a serine protease but a cysteine-containing transamidase that catalyzes the reaction between the amino acid side chains of lysine and glutamine to form an amide bond and remove ammonia as follows;

when the substrate is fibrin, R₁-CONH₂And R₂-NH₂The side chains of glutamine and lysine, respectively, on different chains of the fibrin polypeptide.

Factor XIIIa may also catalyze cross-linking of other proteins, for example, it is known that factor XIIIa will α₂Anti-fibrinolytic enzymes bind to fibrin and enhance its anti-fibrinolysis. Moreover, it can cause cross-linking between a series of structurally disparate contractile proteins such as collagen, laminin, actin, myosin, thrombospondin, vinculin, vitronectin, and the like. This property is believed to be part of the wound healing process and is responsible for many tissue remodeling diseasesHas important function on pathology.

There is therefore a need to provide inhibitors of transglutaminase which are useful, for example, in the treatment of various pathological or thromboembolic events. Inhibitors of transglutaminase have been previously described and they are generally classified into four main groups:

(a) immunoglobulins directed against the enzyme;

(b) a low molecular weight substrate that competes with a native protein substrate;

(c) a reagent that reacts with an active site of an enzyme; and

(d) peptide fragments of factor XIII itself.

These inhibitors are not suitable for e.g. pharmaceutical formulations for various reasons, as follows:

naturally circulating transglutaminase inhibitors have previously been considered immunoglobulins directed against transglutaminase subunits. Such inhibitors cause bleeding states due to a reduction of circulating factor XIII. U.S. patent 5470957 discloses the use of such therapeutic immunoglobulins using prior art methods for generating monoclonal antibodies against transglutaminase subunits. One disadvantage of using such antibodies as transglutaminase inhibitors is that they have a high molecular weight and usually must produce chimeric human immunoglobulin analogues prior to use, such as prior to treatment of humans.

WO91/10427 discloses amines as inhibitors of transglutaminase, which amines react with glutamine residues on one substrate by cross-linking, thereby preventing cross-linking with another substrate. Such inhibitors are not very effective because they must have the same concentration as the natural substrate or a higher concentration than the natural substrate to have a significant inhibitory effect. Therefore, they are effective only at concentrations of about 50. mu.M and higher.

WO92/13530 discloses the use of various transglutaminase inhibitors which depend on the activity of the transglutaminase, mainly on the thiol group on which it is reacted. Thus, any agent which can alkylate or oxidize a sulfhydryl group can inhibit transglutaminase activity. However, these agents are very active and very unstable, and therefore they are not particularly suitable for e.g. pharmaceutical or medical treatment.

The desire to attempt to provide more specific, less toxic peptide inhibitors has resulted in compounds that currently produce lower efficiency. For example, in U.S. patent 5328898 and Achyuthan KE, slauguer TF, Santiago MA et al, journal of biochemistry (j.bio1.chem.) 268: page 21284-21292, 1993 states that "peptides derived from factor XIIIa inhibit transglutaminase activity: localization of substrate recognition sites ".

It is therefore an object of one aspect of the present invention to provide an effective inhibitor of transglutaminase, and the inhibitor may be used, for example, in pharmaceutical or medical applications.

We have now isolated a novel polypeptide which inhibits transglutaminase activity and/or fibrin cross-linking, the polypeptide having the following amino acid sequence:

NH₂-Lys-Leu-Leu-Pro-Cys-Lys-Glu-X₁-His-Gln-Gly-Ile-Pro-Asn-Pro-Arg-Cys-X₂-Cys-Gly-Ala-Asp-Leu-Glu-X₃-Ala-Gln-Asp-Gln-Tyr-Cys-Ala-Phe-Ile-Pro-Gln-Z₁-Arg-Pro-Arg-Ser-Glu-Leu-Ile-Lys-Pro-Met-Asp-Asp-Ile-Tyr-Gln-Arg-Pro-Val-Z₂-Phe-Pro-Asn-Leu-Pro-Leu-Lys-Pro-Arg-Z₃-COOH。

wherein X₁、X₂And X₃Each represents any amino acid residue; z₁、Z₂And Z₃Each simultaneously or non-simultaneously represents Cys or Glu; or a pharmaceutically acceptable salt, a derivative (such as a chimeric derivative) or bioprecursor of said amino acid sequence, or a homologue or analogue of an amino acid sequence having substantially similar activity. By homologue we mean a polypeptide in which no more than 23% of the amino acids in the polypeptide chain differ from the sequences listed above. The number 23% is based on the fact that many homologues of hirudin are described in the literature in naturally occurring Hirudo medicinalis (Hirudo medicinalos); the largest difference between them is that 15 of the 65 amino acids of the polypeptide chain are not identical. By analog we mean the insertion of one or more additional amino acids into a polypeptide chain, provided thatThey do not significantly interfere with the pharmaceutical activity of the polypeptide. The invention also includes truncated polypeptide forms having the above amino acid sequences.

The polypeptides of the invention are highly effective inhibitors of transglutaminase activity and/or fibrin cross-linking. The polypeptide of the present invention, which can prevent the formation of protein crosslinks, is very effective, for example, in unstable blood clots. The inhibitory effect of the polypeptides of the invention on factor XIIIa can be determined by the increased solubility of the fibrin clot in 5M urea. In addition, the inhibitory effect of the polypeptide can be measured by the polypeptide inhibiting the release of ammonia gas caused by the incorporation of ethylamine in casein and the incorporation of aminopentylamine biotin in casein.

The amino-terminal region is considered to be a particularly effective inhibitor of transglutaminase activity. The invention therefore also includes a polypeptide which specifically inhibits transglutaminase activity, the polypeptide comprising the amino acid sequence:

NH₂-Lys-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 of an amino acid sequence, or a homologue or analogue of an amino acid sequence having substantially similar activity.

The polypeptides of the invention (hereinafter referred to as "Tridegin") advantageously directly inhibit transglutaminase activity in the range of 1-50 nanomolar (at least 1000-fold difference compared to known transglutaminase inhibitors of the above-mentioned classes (b), (c) and (d)).

Tridegin can be formed into pharmaceutically acceptable salts with any suitable non-toxic organic or inorganic acid. Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid and metal salts such as sodium monohydrogen orthophosphate and potassium hydrosulfide. Examples of the organic acids include mono-, di-and tricarboxylic acids such as acetic acid, oxalic acid, lactic acid, pyruvic acid, sulfonic acid and the like. Carboxy-terminal amino acid salts include non-toxic carboxylic acid salts formed with any suitable inorganic or organic base.

The Tridegin of the present invention may be extracted from leech tissue or secretions, such as whole leeches, salivary glands or osculum, in a suitable buffer, in a substantially homogeneous slurry. Transglutaminase inhibitors have not been previously identified in leeches or have been extracted from leeches; the invention therefore encompasses an inhibitor of transglutaminase activity derived fromleech tissue or leech secretions. The term "derivatizable" as used herein includes substances that are directly derivable, as well as substances that are indirectly obtained or converted to chemically modified derivatives.

The Tridegin of the invention is typically extracted or purified using a combination of known techniques such as ion exchange, gel filtration and/or reverse phase chromatography.

Leeches of the same genus and even species generally have similar biochemical functions in their salivary polypeptides and a high degree of homology in their amino acid structures. There are several different isoforms of leeches that differ in only a few amino acids among the same species of leeches.

The Tridegin of the invention can be obtained from tissue or secretion of leeches, typically from leeches of the order Rhynchobdellida (Rhynchobhbellidal). However, since many components obtained from leech salivary gland or tissue secretions with similar biochemical specificity belong to members of this family of homologous polypeptides, the invention also includes isoforms and analogs of the leech-derived Tridegin of the invention. Furthermore, leech polypeptides are generally found to have post-transcriptional modifications, and since some residues of the Tridegin are not ascribed to known amino acid structures, the present invention also encompasses post-transcriptional modified polypeptides comprising the above polypeptide sequences.

In a second aspect of the invention there is provided an inhibitor of fibrin cross-linking and/or transglutaminase activity, the inhibitor being obtained from leech tissue or leech secretions, typically from leeches of the order of the sucking leeches, preferably leeches of the genus leech (Haementeria).

The inhibitors of the invention preferably have a molecular weight of about 7000 daltons to 8000 daltons, as measured by polyacrylamide gel electrophoresis (PAGE), and inhibit factor XIIIa-catalyzed release of ammonia gas by amine incorporation and aminopentylamine biotin incorporation into casein.

In addition to its effect on factor XIIIa, Tridegin is also an inhibitor of many different transglutaminase enzymes, and despite the different inhibitory effects, it inhibits the activity of plasma and platelet factor XIIIa and tissue transglutaminase obtained from guinea pig liver. They are therefore versatile transglutaminase inhibitors and are expected to inhibit many different types of such enzymes.

The invention also includes a diagnostic method for determining the extent to which an inhibitor of the invention (as defined above) inhibits transglutaminase activity, which method comprises determining the amount of ammonia evolved by the enzyme transglutaminase upon incorporation of amine into casein in the presence of the inhibitor, wherein the amount of ammonia produced and/or amine incorporated provides a means for determining the extent to which the inhibitor inhibits transglutaminase activity.

In a further aspect the invention provides a pharmaceutical formulation comprising the first and second aspects of the invention (as described above) and a pharmaceutically acceptable carrier, diluent or excipient therefor.

Due to the low level of toxicity and high level of inhibition of transglutaminase activity of Tridegin, they may advantageously be incorporated into pharmaceutical formulations which may be administered to patients, e.g., in parenteral or oral form.

The term "parenteral" as used herein includes subcutaneous injections, intravenous injections, intra-articular injections and intratracheal injections and infusion techniques. Other modes of administration such as oral or topical administration may also be employed. The compositions and combinations for parenteral administration are preferably administered intravenously in pills or continuous infusion using known techniques.

As used herein, "pharmaceutically acceptable carrier" refers to any inert, non-toxic, solid or liquid filler, diluent or encapsulating material that does not adversely react with the active compound or the patient. Known preferred liquid carriers include sterile water, saline solutions, dextrose solutions, sucrose solutions, ethanol, glycols and oils. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, wetting agents and the like. Liquid preparations for oral administration may be aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and the like, or may be dry products for reconstitution with water or other suitable vehicle.

These liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. The topical application may be in the form of an aqueous or oily suspension, solution, emulsion, jelly or, preferably, an ointment.

A unit dose of a pharmaceutical formulation of the invention may contain a daily dose of Tridegin, or a fraction thereof, to make up the required dose. The therapeutically acceptable dose and frequency of administration to a patient, which may be a mammal such as a human, is dependent on a number of factors, such as the activity of the particular active agent used, the age, body weight, health, sex, diet, time and route of administration, clearance rate, the subject being treated, i.e. the treatment or prophylaxis, and the characteristics of the condition being treated.

It is expected that a systemic dose will be effective at 0.05 to 50mg/kg body weight, preferably 0.05 to 10mg/kg body weight, more preferably 0.1 to 1mg/kg body weight. Depending on the nature of the disease to be treated, either systemically or locally, a single dose may contain from 0.05 to 10mg/kg body weight.

Tridegin can be effectively used to inhibit the stability of thrombosis in cases such as acute coronary syndrome, venous thrombosis or stroke, thus enhancing the effectiveness of thrombolytic therapy or natural lytic processes for this reason, fibrinolysis inhibitors such as α are added to the fibrin clot₂Inhibition against plasmin can provide other benefits.

The fact that Tridegin is also effective in inhibiting other transglutaminase suggests other potential uses in pathological situations caused by transglutaminase activity. This effect on transglutaminase is hypothesized to be useful in Crohn's disease, tumor transplantation, thickening of vessel walls during atherosclerosis, thrombolytic microangiopathy (e.g., in the kidney), fiber growth in the skin (e.g., scleroderma), membranous glomerulonephritis, repair of retinal damage, cataracts, acne, formation of wound tissue, and infection by various microfilaria nematodes. Tridegin can be used not only for its therapeutic effect on the above or related syndromes, but their high efficacy also reduces the dose.

This possibility is described in detail in WO93/18760, which describes the optimal dose of the non-functioning inhibitor butanediamine for the treatment of wound hypertrophy as 50 mM. In the same case, the preferred concentration of Tridegin is 1-100. mu.M.

The formulation according to the second aspect of the invention may advantageously be administered in admixture with an anticoagulant, thrombolytic, fibrinolytic or fibrinolytic agent or the like, which may advantageously increase digestion of the formulation or inhibit, for example, the viability of blood clots. The anticoagulant may comprise a polypeptide such as hirudin or heparin. Hirudin is disclosedin EP 0347376 and EP 0501821, which is one of a family of homologous polypeptides found in various leeches, which specifically and efficiently inhibit thrombin and thus inhibit clot aggregation. Likewise, fibrinolytic/fibrinolytic agents such as nuclear hormone (heme) may be used, the activity of which is to digest fibrinogen and render it non-clotting. The nucleus frostbite is a fibrinolytic agent found in a variety of leech species and is disclosed in, for example, us patent 4390630 and WO 91/15576.

A particular effect of Tridegin is that it reduces the lysis time of platelet-free and platelet-rich human plasma clots when any fibrinolytic enzyme induces lysis. Tissue type plasminogen activator or the combination of the nucleukin and the Tridegin can result in faster dissolution than Tridegin alone. Since Tridegin itself has no effect, this suggests a synergistic effect between the two active substances. Tridegin can be used in admixture with a fibrinolytic agent that directly hydrolyzes fibrin (e.g., mitogen, plasmin or Eminase), or with a plasminogen activator that acts through plasmin (e.g., streptokinase, urokinase, staphylokinase, tissue-type plasminogen activator or derivatives thereof), or with a truncated form or hybrid molecule having two or more of these agents.

The thrombolytic agent in the formulation of the invention may comprise one or more of tissue type plasminogen activator, streptokinase, Eminase, urokinase and staphylokinase, as well as derivatives, truncated forms and hybrids thereof. Advantageously, when the formulation includes an anticoagulant, thrombolytic or fibrinolytic agent in addition to the Tridegin, the formulation may greatly reduce the time required for clot digestion. Thus, Tridegin can be effectively used for inhibiting the stability of thrombus formation in, for example, acute coronary syndrome, venous thrombosis, etc., thereby enhancing the effect of thrombolytic therapy. If cross-linking is inhibited by one or more Tridegins, the time required for 50% hydrolysis of the fibrin clot in the presence of plasmin is typically reduced by about half.

Furthermore, the time required for 50% hydrolysis of the plasma clot in the presence of tissue type plasminogen activator is reduced to 40%, and likewise the time in the presence of streptokinase is reduced by more than 25%.

The term "combination" as used throughout the specification means that the Tridegin of the invention is administered simultaneously or sequentially with any or all of an anticoagulant, fibrinolytic agent or thrombolytic agent.

The Tridegin of the invention can be advantageously used for preparing a medicament for treating thromboembolic diseases. Other pathological conditions that can be treated with the Tridegin of the invention include Crohn's disease, tumor transplantation, vessel wall thickening during atherosclerosis, thrombolytic microangiopathy (e.g., in the kidney), fiber growth within the skin, membranous glomerulonephritis, cataracts, acne, formation of wound tissue and infection by various microfilaria nematodes. Advantageously, the trideegins of the invention are not only useful for therapeutic use or to prevent these syndromes, but also the high potency of the trideegins reduces the dose used.

The invention also encompasses a polypeptide prepared by recombinant DNA techniques, which corresponds to the polypeptide defined above: the invention also includes a synthetic or protein engineered polypeptide that is equivalent to the polypeptide of the invention.

Typical methods for isolating and identifying the polypeptides of the invention will be described by way of example with reference to the accompanying drawings given in which:

FIG. 1 illustrates the elution results of the inhibitory activity of the polypeptide of the present invention isolated according to example 3 described below;

FIG. 2 illustrates the elution results of the inhibitory activity of the polypeptide of the present invention in example 4 compared with those of nucleus hormone and knot hormone (ghilantin);

FIG. 3 illustrates the results of the inhibitory activity of the polypeptide of the present invention in example 6;

FIG. 4 is a chromatogram of the inhibitory activity of example 3;

FIG. 5 is a chromatogram of the active fragment obtained in example 4;

FIG. 6 illustrates the results of Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis in example 7;

FIG. 7 illustrates the results obtained from example 22;

FIG. 8 illustrates the results obtained from example 24; and

FIG. 9 shows the results obtained in example 25.

Example 1

In a first experiment A, the rostral, rostral and posterolateral salivary glands of a species of Haementeria ghilianii leeches were homogenized in 10mM Tris HCl and 0.85% w/vNaCl (pH7.0, 1ml) in a Potter homogenizer and centrifuged at 13000 rpm. The supernatant is determined by a clot solubility assay, which is performed withTymiak, Tuttle, Kimball, Wang and Lee in the J.antibiotics (J.antibiotics) volume 46 (1993) page 204-206 "a method for simple and rapid screening of inhibitors of factor XIIIa" is similar. In a second experiment B, the rostral, anterior and posterior salivary glands were secreted by Haementeria ghilianiii species of leeches, which were individually homogenized in 0.2ml aliquots of buffer. The results were comparedwith extracts obtained from the rostral, rostral and posterolateral salivary glands of two leeches of the species Haementeria officinalis, prepared in 0.2ml of buffer. The test sample (30. mu.l) was added to 10mg/ml crude bovine fibrinogen containing factor XIII (30. mu.l). Adding 9mM CaCl₂6.25 units/ml of bovine thrombin (40. mu.l) was startedShould be used. A clot formed within 15 minutes and 8M urea (160. mu.l) was added and allowed to contact the clot. After 30 minutes, the absorbance produced by the clot opalescence was read at 405 nm. The decrease in absorbance indicates the solubility of the clot due to inhibition of cross-linking. Table 1 shows the inhibitory effect (absorbance at 405 nm) of different extracts on the solubility of fibrin clots compared to iodoacetamide, a known factor XIIIa inhibitor. The values in the table are the absorbance at 405 nm.

TABLE 1

Test sample	Experiment A	Experiment B
			Tris buffer	0.74	0.83
Iodoacetamide (100. mu.M)	0.26	0.42
			Ghilianii Total saliva Mixed extracts	0.29	-
Ghilianii pro-salivary gland extract	-	0.43
			G Hilianii post salivary gland extract	-	0.43
Ghilianii lip extract	-	0.53
			Extract of the anterior salivary gland of officinalis	-	0.64
Retrosalivary gland extract of officnalis	-	0.55
			Extracts of the oscillales of Aficinalis	-	0.09

Example 2

To determine the presence of an inhibitor of factor XIIIa by an agent, the effect of extracts on the catalysis of the incorporation of aminopentanamine biotin into casein by human factor XIIIa was determined by microtiter plate methods described in Slaurighter TF, Achyuthan KE, Lai T-S and Grecnberg CS, volume 205 biochem (anal. biochem) 1992: page 166-171 in "method of measuring microtiter transglutaminase using 5- (Biotin amino) pentylamine as a substrate". Extracts of the rostral, anterior and posterior salivary glands of leeches of the species leech south american leech were prepared in the same manner as in experiment a in example 1. Those obtained from Haementeria depressa were lyophilized. Since salivary glands are not easily removed from leeches of the species Hirudinaria praecox and Hirudinaria manillensis, extracts were prepared by removing one third of the anterior salivary glands of the leeches and homogenized in 1ml of 10mM Tris HCl containing 0.85% NaCl. After centrifugation at 13000rpm, the supernatant was taken for the assay. N, N dimethyl casein was dissolved in 0.1M Tris HCl, pH8.5, and stirred at 85 ℃ for 30 minutes, and centrifuged at 2000g for 20 minutes. The wells of the microtiter plate were coated with this solution at a concentration of 10-20mg/ml (0.2ml) and incubated at 37 ℃ for 1 hour. Excess casein was removed and the wells were blocked with 0.1MHCl (pH8.5) containing 0.5% skim dry milk for 30 minutes. The plate was then washed twicewith 0.35ml aliquots of Tris buffer. Factor XIIIa was prepared by removing fibrinogen in human plasma containing citrate by adding solid bentonite (40mg/ml), incubating for 10 min and centrifuging at 12000g for 20 min. The supernatant was activated by adding 1000U/ml bovine thrombin (0.05ml) and 200mM calcium chloride (0.025ml) and incubating at 37 ℃ for 15 minutes. The thrombus was neutralized by the addition of 2000ATU/ml hirudin (0.5 ml). The wells of the microtiter plate (total volume 0.2ml) contained 5mM calcium chloride, 10mM dithiothreitol, 0.5mM biotin aminopentylamine, test sample (0.05ml) and activated plasma (0.05 ml). After incubation at 37 ℃ for 30 minutes, the liquid was removed and the reaction was stopped by washing twice with 0.2M EDTA (0.35ml each) and twice with 0.1M Tris HCl, pH8.5 (0.35ml each). 0.25mg/ml of streptavidin-alkaline phosphatase was diluted 1: 150 with Tris buffer containing 0.5% dry skim milk, and 0.25ml was added to each well and incubated at 20 ℃ for 1 hour. The plate was washed once more with 0.1% Triton X-100(0.35ml) and then three times with Tris buffer (0.35 ml).

Bound alkaline phosphatase was determined by adding 1mg/ml p-nitrophenylphosphate, Tris buffer (0.05ml) containing 5mM magnesium chloride and Tris buffer (0.2ml) and measuring the absorbance after 30 minutes at 405nm using a Titertek Uniskani microtiter plate reader. Table 2 demonstrates that factor XIIIa inhibitory activity was found in salivary organs of both Haementeria ghilianii and haementia officinalis in different, more sensitive assays (and was determined using human plasma factor XIIIa to catalyze the incorporation of biotin aminopentanamide into casein). Furthermore, a marked but lower inhibitory activity was detectable in both the salivary glands of hirudo continentalis and in the anterior part of hirudo continentalis and Hirudinaria mangllensis.

TABLE 2

Test sample	Inhibitory Activity of factor XIIIa (Unit/complete salivary gland mixture or unit/leech)*
		Buffer control	0.00
Haementeria ghilianii	128.7
		Haementeria officinalis	10.2
Haementeria depressa	0.5
		Leech of Odontoglossum	1.5
Hirudinaria manillensis	2.1

^*1 unit is determined by twice the amount of transglutaminase inhibitor required to inhibit 50% of human factor XIIIa in 1ml of normal human plasma. The standard used plasma from 7 healthy blood donors.

Example 3

Five groups of total salivary gland mixtures (anterior, posterior salivary glands and osculum) obtained from Haementeria ghilianii leeches were homogenized in phosphate buffer as described in example 1, and the supernatant was applied to a column of 1.6X 80cm Superdex G-200 and removed from the column at a flow rate of 1ml/min using phosphate buffer pH 7.2. The absorbance of the eluate was measured at 280nm, and the inhibitory activity was measured by the same measurement method as described in example 1. FIG. 1 shows the separation process and the location of elution of the inhibitory activity. The line indicates the fraction containing Tridegin activity.

Example 4

A homogenate of a mixture of five Haementeria ghilianii total salivary glands was prepared in 20mM Tris HCl, pH8.0 as in example 1. The supernatant was applied to a 0.8X 7.5cm column of Express-Ion exchange Q (Whatman) and eluted with a 20mM, pH8.0, Tris HCl linear gradient containing 0.3mM NaCl. The eluate was monitored by absorbance at 280nm and the activity of Tridegin was determined by the clot solubility assay of example 1. In addition, the activity of the nucleic acid is determined by the fibrinolysis assay and the inhibitory activity of factor Xa is determined by the chromogenic substrate assay. The activity of the nucleic acid molecules was determined by mixing 2mg/ml bovine fibrinogen (50. mu.l) with 10mM CaCl₂20mM HEPES buffer and 0.1% w/v, pH7.5 Brij35 (25. mu.l) and serial dilutions (25. mu.l) of the column components were incubated at 37 ℃ for 60 minutes. Then 100U/ml thrombin (10. mu.l) was added to form a clot and after 30 minutes the turbidity was measured at 405 nm. The reduction in turbidity represents the amount of fibrinogen digested. The chromogenic substrate method for factor Xa was performed by incubating 2mM S2765 in 50mM Tris HCl, pH8.3, in a spectrophotometer and measuring the rate of change of absorbance at 405 nm. The reaction was started after addition of human factor Xa.

FIG. 2 shows the elution profile on a SP Sepharose column with a linear gradient to 0.3M NaCl. Wherein the sites exhibiting inhibitory activity of Tridegin (T), Nuclear Meng and factor Xa are indicated; tridegin can be clearly separated from the other two salivary gland components, known as the Nuclear Mongolian (H) and ghilanten (G), thus demonstrating that Tridegin is different from the known components. The portions containing inhibitory activity are marked with a horizontal line and letters T, G and H, respectively.

Example 5

A homogenate of the whole salivary gland mixture obtained from Haementeriaghirianii was prepared in 20mM ammonia formate (5ml) at pH3.5 and loaded onto a 0.8X 7.5cm Express-Ion exchange S (Whatman) column as in example 1.

Fractions were eluted with a linear salt gradient to 20mM sodium formate pH3.5 containing 1M NaCl. The absorbance of the eluate at 280nm was monitored and determined as in example 1. Inhibiting the active ingredient from washing out at about 0.6M.

Example 6

A large batch was prepared by combining the chromatographic procedures exemplified in examples 3, 4 and 5. Homogenates of whole salivary gland mixtures obtained from fifty haemophilus Haementeria ghilianii, which had not been fed for at least 3 months, were prepared in 20mM Tris HCl (50ml) pH8.0 and centrifuged as in example 1. The supernatant was applied to a 60X 10cm Sepharose Fast Flow (Pharmacia) column. Fractions were eluted with a linear gradient from the starting buffer to a buffer containing 0.1M NaCl. The eluate was monitored at 280nm andfound to have active components in the eluate at about 0.09M NaCl (see FIG. 3, in which the portion containing inhibitory activity is marked by a horizontal line). Formic acid was added to the active fraction (145ml) to adjust to pH4 and the mixture was applied to a 5X 12cm SP Sepharose fast flow (Pharmacia) column, which was equilibrated with 20mM sodium formate buffer, pH 3.5. The column was eluted with a linear gradient from the equilibration buffer to the same buffer containing 1M NaCl. The active fraction had eluted bees at about 0.75M NaCl (see FIG. 4, in which the fraction containing inhibitory activity is marked with a horizontal line). It was lyophilized and then reconstituted in water to a final volume of 2.4ml and applied to a 1.6X 60cm Superdex G-75 column equilibrated beforehand with pH7.2 phosphate buffer. FIG. 5 shows the elution profile, in which the portions with inhibitory activity are marked with a horizontal line. The obtained protein contained 715. mu.g of protein and was cryopreserved.

SDS polyacrylamide gel electrophoresis using Coomassie blue or silver staining showed that the protein was substantially pure after this step, with the major band having an apparent molecular weight of about 7800 daltons compared to standards of known molecular weight, while the minor band with the higher molecular weight was only detectable by more sensitive silver staining.

Example 7

For sequencing, a further purification step is required. 0.3ml of the active ingredient obtained in example 6 was applied to a 0.5X 10cm ProRPC column equilibrated beforehand with 0.1% trifluoroacetic acid and then eluted with a gradient from 0% acetonitrile containing 0.1% trifluoroacetic acid to 100% acetonitrile. After two very small inactive peaks, a main peak containing inhibitory activity appears, completely separated from it. As described in example 6, the active fraction showed a single band in SDS polyacrylamide gel electrophoresis and had an apparent molecular weight of about 7800 daltons compared to peptide standards of known molecular weight.

FIG. 6 is a SDS polyacrylamide gel electrophoresis of pure polypeptide on PhastGel high density gel (Pharmacia).

The left lane (lane 1) and lane 7 are low molecular weight marker fractions (Pharmacia) with molecular weights of 94, 67, 43, 30, 20.1 and 14.4kD and aprotinin (molecular weight 6.5kD), respectively.

Lanes 2 and 7: peptide tag fractions (Pharmacia) with molecular weights of 16.9, 14.4, 10.7, 8.2 and 6.2kD and aprotinin (6.5 kD).

Lane 3: water blank. Lane 4: purified Tridegin.

Lanes 5 and 6: minor peaks on reverse phase chromatography column.

The lowest molecular weight component migrates to the nearest point of the gel.

A single, clear amino acid sequence was determined from the amino terminus using an Applied Biosystems 473A automated protein sequencer, indicating that only one polypeptide was present. The amino acid sequence found was:

NH₂-Lys-Leu-Leu-Pro-X-Lys-Glu-Y-His-Gly-Ile-Pro-Asn-Pro-Arg-

wherein X and Y are not defined and thus can be any amino acid. The cysteines in the sample were not derivatized and thus could not be included. Resequencing after pyridylethylation showed that X is cysteine, while Y did not show any peak and therefore could not be assigned to any common amino acid.

Example 8

To generate sufficient material for amino acid sequencing, transglutaminase inhibitors were prepared from the posterior salivary glands of fifty species of leeches of Haementeria ghilianii, as in examples 6 and 7. Aliquots were denatured using standard methods described in Matsudaira (a practical method for micro-sequencing of protein and peptide purification, "A systematic peptide-based and peptide purification for micro-sequencing", academic Press, second edition, pages 45-67), aminocarboxylated and digested with trypsin or AspN endoprotease, then fragments were separated on a 0.5X 10cm ProRPC column equilibrated beforehand with 0.06% trifluoroacetic acid, followed by linear gradient elution with from 2 to 38%, 38 to 75% and from 75 to 98% elution buffer, 0.0675% trifluoroacetic acid with 80% acetonitrile and detection at 210 nm. The sequence of the isolated fragments was sequenced using an Applied Biosystems 473A automated protein sequencer. From this the amino acid sequence of the entire polypeptide was deduced and overlapping polypeptides were found:

NH₂-Lys-Leu-Leu-Pro-Cys-Lys-Glu-X₁-His-Gln-Gly-Ile-Pro-Asn-Pro-Arg-Cys-X₂-Cys-Gly-Ala-Asp-Leu-Glu-X₃-Ala-Gln-Asp-Gln-Tyr-Cys-Ala-Phe-Ile-Pro-Gln-Z₁-Arg-Pro-Arg-Ser-Glu-Leu-Ile-Lys-Pro-Met-Asp-Asp-Ile-TyrGln-Arg-Pro-Val-Z₂-Phe-Pro-Asn-Leu-Pro-Leu-Lys-Pro-Arg-Z₃-COOH

wherein amino acid X₁、X₂And X₃Indeterminate, and may indicate a residue modified after transcription, Z₁、Z₂And Z₃Represents an amino acid that cannot be separated from Cys or Glu. The polypeptide having this sequence is defined as Tridegin variant 1.

Example 9

In addition to assays demonstrating that factor XIIIa is able to incorporate amines into casein and that factor XIIIa affects clot solubility, assays that measure the amount of ammonia gas generated from casein following amine addition can show specificity of inhibition. The transglutaminase activity of human plasma factor XIIIa can be determined spectrophotometrically by means of a modification of Muszbek, Polgar and Fesus (Kinetic determination of blood clotting factor XIII in plasma, "Clin Chem31(1985), pages 35-40). The ammonia gas is measuredThe method comprises connecting ammonia gas generation with NADH oxidation reaction via glutamate dehydrogenase reaction, wherein NADH oxidation reaction can be monitored by absorbance change at 340nmAnd (6) measuring. Defibrinated human plasma (2ml) and 200mM CaCl₂(0.1ml) was incubated with 1000 units/ml of bovine thrombin (0.1ml) at 37 ℃ to activate factor XIII.15 minutes before addition of 260 antithrombin units of hirudin stopped the reaction tank contained 2.5mM dithiothreitol (0.1ml), 40mg/ml dephosphorylated β -casein (0.05ml), 70mM ethylamine (0.1ml), 12mM 2-oxoglutarate (0.1ml), 4mM ADH (0.1ml), 1.2mM ADP (0.1ml), 40 units/ml of glutamate dehydrogenase (0.1ml), 70mM, pH7.5 HEPES buffer (0.25ml) at 20 ℃ in a spectrophotometer, possibly all components were dissolved in 70mM HEPES buffer pH 7.5. the reaction was started with factor XIII (0.2ml) added and monitored at 340 nm. the method achieves a factor XIII deficiency (Sigma deficiency) which resulted in the determination of factor XIII at a rate of less than normal factor XIII.88 min; the rate of the reaction was determined by the method from a deficiency of Sigma 3. this factor XIII was found to be accurate.

The test sample was added to 0.1ml of HEPES buffer solution in the reaction vessel. The inhibitory effect of Tridegin variant 1 is shown in table 3 in comparison to iodoacetamide (a known thiol-dependent factor XIIIa inhibitor) and ETGA (an inhibitor that inhibits factor XIII activation and activity by chelating with basic calcium). Tridegin reduced ammonia production by about 93%, i.e., the inhibitory effect was comparable to that of iodoacetamide (shown in Table 3). There is also evidence that Tridegin is an inhibitor of clot solubilization, or an inhibitor of plasma transglutaminase or factor XIIIa.

TABLE 3

Test sample	Change in Absorbance (mAbs/min)
		Control	4.14
EGTA(77mM)	0.024
		Iodoacetamide (0.077mM)	0.356
Tridegin(3.8μg/ml)	0.28

Example 10

The inhibitory effect of Tridegin variant 1 purified in example 6 on the catalysis of biotin aminopentylamine incorporation into casein by human factor XIIIa was determined by the microtiter plate method described by Slaurighter TF, Achyuthan KE, Lai T-Sand Greenberg CS. (1992) (microtiter plate transglutaminase assay using 5-biotin aminopentylamine as substrate, Anal. biochem. vol. 205: 166-171 p.). N, N dimethyl casein was dissolved in 0.1M TrisHCl pH8.5 and stirred at 85 ℃ for 30 minutes, then centrifuged at 12000g for 20 minutes. Solutions with concentrations of 10-20mg/ml (0.2ml) were used to coat the wells of a microtiter plate and incubated at 37 ℃ for 1 hour. The remaining casein was discarded and the wells were blocked with 0.5% of 0.1M skim milk for 30 min, ph8.5 TrisHCl. The plate was then washed twice with 0.35ml portions of Tris buffer. Purified human platelet factor XIII (0.6 units/0.12 ml) was activated by addition of 15mM calcium chloride (0.18ml) containing 150U/ml thrombin and incubated at 37 ℃ for 15 minutes. Then, natural hirudin (0.3ml) at 140ATU/ml was added to inhibit thrombin. The wells of the microtiter plate (total volume 0.2ml) contained 5mM calcium chloride, 10mM dithiothreitol, 0.5mM biotin aminopentanamide, Tridegin samples prepared as described in example 6 and 0.25 units/ml activated factor XIIIa in 0.1M Tris HCl, pH 8.5. After incubation at 37 ℃ for 30 minutes, the liquid was removed and the reaction was stopped by washing twice with 0.2M EDTA (0.35ml each) and then twice with 0.1M Tris HCl, pH8.5 (0.35ml each). 0.25mg/ml of streptavidin-alkaline phosphatase was diluted 1: 150 with Tris buffer containing 0.5% dry skim milk and 0.25ml was added to each well and incubated at 20 ℃ for 1 hour. The plate was washed once more with 0.1% Triton X-100(0.35ml) and then three times with Tris buffer (0.35 ml). Bound alkaline phosphatase was determined by adding 1mg/ml of p-nitrophenylphosphate, Tris buffer (0.05ml) containing 5mM magnesium chloride and Tris buffer (0.2ml), 30 minutes later the absorbance was determined at 405nm with a Titertek Uniskan II microtiter plate reader.

Tridegin variant 1 significantly inhibited the amine incorporation reaction with an IC50 of 0.026. + -. 0.002. mu.g/ml (3.4nM), demonstrating that it was effective in inhibiting platelet factor XIIIa activity.

In a separate experiment, the same procedure was performed except that the purified factor XIII was replaced by plasma from healthy donors and the concentrations of Tridegin varied, resulting in a factor XIIIa IC50 of 0.07. + -. 0.003. mu.g/ml (9.2nM) for plasma.

Example 11

The effect of the trinegin variant 1 purified in example 6 on coagulase and thrombin was determined by the chromogenic substrate method. The reaction was started by incubating 1mM S2238 in 50mM Tris HCl, pH8.3, and adding thrombin to a final concentration of 0.15U/ml. The reaction was detected spectrophotometrically at 405 nm. Table 4 shows that Tridegin had no effect on thrombin at a concentration of 4.6. mu.g/ml, whereas hirudin had a significant inhibitory effect (95%) at a concentration of 0.046. mu.g/ml.

TABLE 4

Test sample	Concentration (μ g/ml)	Reaction Rate (mAbs/min)
			Control	-	49.8
Tridegin	4.6	51.8
			Hirudin	0.046	2.63

Example 12

The effect of trinegin purified in example 6 on factor Xa was determined. The reaction was initiated by incubation of 2mM S2765 in 50mM TrisHCl, pH8.3, and addition of human factor Xa. The measurement was carried out as in example 11. The transglutaminase inhibitor had no effect on factor Xa at a concentration of 4.6. mu.g/ml, whereas the inhibition rate of recombinant tick anticoagulant peptide (rTAP), a known inhibitor of factor Xa (Waxman L, Smith DE, Arcuri K et al, "tick anticoagulant peptide, a novel inhibitor of blood coagulation factor Xa", science 248: 593-. This not only demonstrates that this transglutaminase inhibitor is different from known inhibitors of factor Xa, but that the exemplified method of purifying transglutaminase inhibitors also successfully removes the factor Xa inhibitor.

TABLE 5

Test sample	Concentration (μ g/ml)	Reaction Rate (mAbs/min)
			Control		49.8
Tridegin	4.6	56.1
			rTAP	0.046	53.5

Example 13

To determine whether Tridegin has a mitogen-like fibrinolytic activity, the ability of the purified material of example 6 to digest fibrinogen was assessed by measuring the coagulability of fibrinogen after incubation of the fibrinogen with inhibitors. 2mg/ml bovine fibrinogen (50. mu.l) and Tridegin, purified Nuclear Mengdin or vector (50. mu.l), 20mM HEPES buffer containing 10mM calcium chloride, and 0.1% w/v, pH7.5 Brij35 (50. mu.l) were incubated at 37 ℃ for 60 minutes. 100U/ml thrombin (10. mu.l) was then added to form a clot, and after 30 minutes the clot was assayed for turbidity at 405 nm. Table 6 shows that transglutaminase inhibitors have no effect on clot formation, whereas purified nuclear hormone stings significantly digest fibrinogen and thus form less clots. This indicates that Tridegin is not proteolytic on fibrinogen and therefore is not a mitogen as described in WO 91/15576 ("Treatment of thrombotic events") and us patent 4,390,630 ("mitogen-a fibrinolytic agent"). In addition, this example further demonstrates that the nuclear hormone stings found in Haementeria ghilianii are separated from transglutaminase inhibitors during the exemplified purification process.

TABLE 6

Test sample	Absorbance of the solution	Absorbance of the solution
			Buffer control	0.238	0.224
Tridegin(35μg/ml)	0.226	-
			Nuclear Mongolian Ding (30 units/ml)	-	0.007

Example 14

The activity of the destabilizing enzyme can be determined by its effect of dissociating p-nitroaniline from the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. To determine whether the destabilizing enzyme and Tridegin have the same properties, the effect of the two reagents on the chromogenic substrate was compared. The absorbance was read separately in a spectrophotometer at 405nm in 50mM TrisHCl, 10mM, pH8.0 calcium chloride (0.9ml) cuvette containing 0.45mg/ml L- γ -glutamyl-p-nitroanilide. The rate of nitroaniline production was determined by adding 0.046mg/ml Tridegin variant 1(0.1ml) or winter leech extract supernatant (0.1ml) which was prepared as in example 2, a known source of destabilising enzyme). Table 7 shows the effect of Tridegin on L-gamma-glutaminyl-p-nitroanilide, a substrate for the destabilising enzyme, and shows that although extracts from Whitmania pigra contain an activity which increases the absorbance (which means that the substrate is dissociated by the destabilising enzyme), Tridegin does not so, thus resulting in a slight decrease in absorbance over time.

TABLE 7

	Reaction Rate (mAbs/min)
		Extract of leech of Aster continentalis	2.04
Tridegin(4.6μg/ml)	-1.17

Example 15

The effect of Tridegin on clotting in plasma was determined by adding 0.1 volume of the inhibitor purified in example 6 (46. mu.g/ml) in phosphate buffer to normal plasma, comparing normal plasma samples with samples added to buffer only. Standard clot assays were performed in an automated analyzer. The results in table 8 show that there is no difference between the two samples, and therefore the Tridegin has no effect on the clotting time of normal plasma. This property is expected since inhibitors of fibrin cross-linking have no effect on clot formation, and only affect the physical and chemical properties after formation. Furthermore, this demonstrates that in different assays, there are other anticoagulant activities such as inhibition of factor Xa or thrombin in the Tridegin.

Platelet aggregation of human citrate-containing platelet-rich plasma was assessed in a Bio/Data aggregometer (Bio/Data aggregometer) under 6.7 μ g/ml collagen, 6.3 μ M ADP, or 0.4U/ml thrombin. Example 6 Tridegin at a final concentration of 4.6. mu.g/ml was compared to a buffer control. Table 8 shows that trinegin has apparently no effect on platelet aggregation under these conditions.

TABLE 8

Parameter(s)	Control	Tridegin(4.6μg/ml)
			Thrombin clotting time (seconds)	9.8	9.8
One step prothrombin time (sec)	15.2	14.7
			Kaolin cephalin clotting time (%/minute)	28.5	27.9
Collagen induced coagulation (%/min)	23	28
			ADP-induced coagulation (%/min)	11	14
Coagulation induced by Thrombin (%/min)	73	82

Example 16

The effect of the inhibitors on guinea pig liver tissue-type transglutaminase was determined as described in example 10, with tissue-type transglutaminase replacing activated factor XIIIa. Tridegin (4.5. mu.g/ml) inhibited 95.5% of the amine incorporation reaction in casein catalyzed by this enzyme. IC50 was found to be 1.55. mu.g/ml with varying concentrations of Tridegin. This assay indicates that Tridegin is an inhibitor of tissue-type transglutaminase and plasma transglutaminase factor XIIIa, which can also be inhibitors of many transglutaminase enzymes.

Example 17

Transglutaminase inhibitors in the salivary gland system and in salivary secretions of Haementeria ghilianii were determined by Slaughter TF, Achyuthan KE, Lai T-Sand Greenberg CS. (1992) ((microtiter transglutaminase assay with 5-Biotin Aminopentylamine as substrate), amine incorporation assay in Biochemical Anal biochem (volume 205: 166-171). The anterior and posterior salivary glands and lips and the posterior sucker were removed from leeches starved for three times of feeding. The samples were homogenized in a glass homogenizer with 1mM, pH8.0 TrisHCl (1ml or 0.5ml in the case of the posterior gland) and centrifuged at 12000 rpm. The supernatant was used for the assay. To collect salivary gland secretions, eight three-time starved Haementeria ghilianii leeches were frozen at 5 ℃ for 2-3 hours and their intact salivary gland organs (rostral, antero-lateral and postero-lateral glands) were removed. It was pinned to a Sylgard substrate and soaked in physiological saline (65mM NaCl, 50mM NH) at 20 deg.C₄Cl, 4mM KGl, 1mM EGTA, 11mM glucose, 10mM, pH7.4 HEPES) for 15 minutes. The wall of the osculum was cut longitudinally, the body cavity was entered and the secretions contained therein were collected with a micropipette.

Table 9 shows the inhibition of human plasma factor XIIIa by extracts and secretions of Haementeria ghilianiii leeches. Inhibitory activity was found in both salivary glands, salivary secretions and lips. A small amount of activity was found to be detectable in the posterior sucker, which was very low in specific activity, 0.35% of that of the posterior salivary gland, and indeed a clearly detectable activity was produced by the high concentration of protein extracted from this large piece of tissue.

TABLE 9

Tissue of	Specific activity (unit/mg) #	% inhibition
			Anterior salivary gland	19.0	99.7
Posterior salivary gland	93.3	100
			Osculum part	11.7	95.5
Rear suction cup	0.33	48.5
			Intraluminal secretions of the lips*	-+	61.9

# As described in example 10, one unit in the amine incorporation assay was determined as twice the amount of transglutaminase inhibitor required to inhibit 50% of factor XIIIa in 1ml of normal human plasma.

^*Average of 8 individual experiments.

⁺The protein concentration was too low to be measured (specific activity was very high).

Example 18

The absorbance method can prove that Tridegin has the enhancement effect on fibrinolysis induced by plasmin. 10mg/ml bovine fibrinogen was incubated with 50U/ml bovine thrombin (0.01ml) and either buffer or Tridegin from example 6 in microtiter plates for 2 hours at 37 ℃. 2.56U/ml plasmin (0.05ml) was added and the plates were incubated at 37 ℃. The absorbance was measured every 15 minutes in a Titretek Uniskan II microtiter plate reader. The clot was observed every 15 minutes and the time to complete dissolution of the clot was recorded. As shown in table 10, transglutaminase inhibitors reduced the time to dissolution at all concentrations tested.

Watch 10

Tridegin(μg/ml)	Dissolution time (hours)
		0	3.0
2.0	1.0
		1.0	1.25
0.2	1.25
		0.1	2.5
0.04	1.5

Example 19

The enhanced effect of Tridegin on fibrinolysis induced by tissue-type plasminogen activator is also manifested on human plasma clots. Human plasma (0.1ml) and 5U/ml bovine thrombin in 0.18M CaCl containing 0.14MKCl₂(0.01ml), and buffer or TriDegin (0.4ml) prepared as in example 6 were incubated in microtiter plates in six replicates at 37 ℃ for 2 hours, except that the TriDegin was obtained from the lateral salivary gland of Haementeriagiliianii leech. Tissue type plasminogen activator (0.05ml) was then added to a final concentration of 10IU/ml, the plates were incubated at 37 ℃ and absorbance read at 405nm every 30 minutes using a Titretek Uniskan II microtiter plate reader. A decrease in absorbance indicates dissolution of the plasma clot. The time for 50% dissolution of the control wells was 12.9 ± 1.1 hours, and 7.9 ± 0.7 hours in the wells containing transglutaminase inhibitor, which was statistically significantly reduced. This example demonstrates that transglutaminase inhibitors can greatly accelerate the reversal of human plasma clots by tissue-type plasminogen activatorsShould be used.

Example 20

Since platelets are associated with thrombi in vivo, it is of interest to also perform physiological tests if transglutaminase inhibitors can dissolve platelet rich clots more rapidly. Platelets are an abundant source of plasma transglutaminase, factor XIII and fibrinolysis inhibitors, and therefore they greatly reduce the potency of fibrinolytic agents. Human platelet-rich or platelet-poor plasma (0.1ml) was prepared from the same donor and incubated with 5U/ml bovine thrombin in 0.18M calcium chloride (0.01ml) containing 0.14M KCl and buffer or Tridegin (prepared as described in example 6 except prepared from the posterior salivary gland of Haementeria ghilianii leech, 0.04ml) at 37 ℃ for 2 hours with samples in six replicates in microtiter plates. Tissue type plasminogen activator (0.5ml) was added to a final concentration of 10IU/ml and the plates were incubated at 37 ℃. The absorbance at 405nm was recorded every 30 minutes over 72 hours using a Molecular Devices Thermomax kinetic microtiter plate reader (a decrease in absorbance indicates lysis of the plasma clot). Table 11 shows that in the presence of platelets, the clot did not dissolve more than 50% over a 72 hour incubation period. Tridegin is more effective in reducing the effects of platelets in the presence of platelets, in this example, Tridegin reduces time from greater than 72 hours to 24.9 hours in the presence of platelets and from 22.5 hours to 18.0 hours in the absence of platelets.

TABLE 11

	Control buffer (expressed in hours 50) % time of dissolution)	Tridegin (expressed in hours as 50% soluble) Time of solution)
			Platelet-deficient plasma	22.5±1.99	18.0±1.03
Platelet rich plasma	＞72	24.9±5.57

Example 21

The effect of Tridegin on decreasing clot lysis time is a general effect that can be exhibited when streptokinase is used as a lytic agent. Human plasma (0.1ml) was incubated with 5U/ml bovine thrombin in 0.18M calcium chloride (0.01ml) containing 0.14M KCl and buffer or Tridengin (prepared as described in example 6, but prepared from the posterior salivary gland of Haementeria ghilianii leech, 0.04ml) at 37 ℃ for 2 hours with samples placed in microtiter plates in triplicate. Streptokinase was added to a final concentration of 30U/mland the plates were incubated at 37 ℃ in an iEMS kinetic microtiter plate reader and the absorbance at 405nm was recorded every 30 minutes over 47.5 hours. Although streptokinase-only wells failed to adequately dissolve the clot to achieve a 50% dissolution time, the 50% dissolution time for all Tridegin-containing wells was 36.1 + -1.6 hours, again demonstrating the greatly increased effect when used with fibrinolytic agents such as streptokinase, the results are shown in FIG. 7 (which represents the effect of Tridegin on streptokinase-induced plasma clot lysis reactions). The result is ± SEM (n ═ 3).

Example 22

The effect of mixed use of Tridegin and Nuclear Meng was tested as described in example 21, where 110U/ml Nuclear Meng was used instead of tissue type plasminogen activator. Platelet-free and platelet-rich plasma from the same donor are used in this example to determine if there is a difference between them. Table 12 shows the time to 50% dissolution of the recorded samples. Platelets have the effect of significantly increasing the time required for lysis, which increases the time from 34 hours to greater than 56 hours, while it is clear that Tridegin can reduce the time to 50% lysis, regardless of the presence or absence of platelets. Tridegin can reduce the lysis time to close to the control, so Tridegin appears to largely overcome the effects of platelets.

TABLE 12

	Buffer (50% solution in hours) Time of solution)	Tridegin (expressed in hours as 50% soluble) Time of solution)
			Platelet-free plasma	34±3.5	24±0.8
Platelet rich plasma	＞56±7.8	36±2.2

Example 23

To investigate the properties of Tridegein obtained from Haementa officinalis leech in Mexico, the extracts were subjected to reverse phase high pressure liquid chromatography after gel filtration chromatography. Salivary glands and rostral regions were obtained from five Haementeria officinalis leeches, and the leeches were starved until no blood was found in the foregut. They were homogenized in a Teflon/glass homogenizer at pH7.2 in phosphate buffer (1ml) and centrifuged at 12000g for 5 minutes to obtain a clear supernatant. The supernatant was applied to a 1.6X 60cm Superdex G75 column, monitored at 280nm,and all fractions were collected and clot solubility determined as in example 1. As shown in FIG. 8, the inhibitory activity was found in only one peak (wherein the components containing the inhibitory activity were marked with a horizontal line).

The active fraction was lyophilized and then redissolved in water (1 ml). Wherein a portion (0.3ml) was loaded onto a 0.5X 10cm Pro-RPC column previously equilibrated with 0.1% trifluoroacetic acid (TFA). Elution was performed with a linear gradient from 0.1% TFA to acetonitrile containing 0.1% TFA, and the eluate had multiple absorption peaks at 280 nm. Each absorption peak was determined using the clot solubility assay as described in example 1 and was found to contain activity within only one peak.

The elution sites on both columns were compared to those of a similar Haementeria ghilianii extract, which showed a close proximity to TriDegin variant 1. The inhibitory activity obtained from salivary gland purification of Haementeria officinalis has physiochemical properties very close to those of Tridegin variant 1 in terms of molecular weight (as determined by gel filtration) and partition coefficient (as determined by reversed phase high pressure liquid chromatography variant 1).

Example 24

To determine the in vivo behavior of TriDegin variant 1, a group of four rats was injected intravenously with an intravenous formulation (4.7ml) containing 0.01M sodium phosphate, 0.027M KCl, 0.137M NaCl, pH7.4 containing 0.207mg/kg TriDegin variant 1. Bleeding samples (approximately 0.3ml) were taken from the tail vein at 2 or 5 and 10, 20, 30, 60 and 120 minutes of administration and immediately mixed with 0.04ml of 3.8% trisodium citrate. The samples were immediately centrifuged at 12000g for 5 minutes and the supernatant removed and snap frozen on dry ice. No adverse effects of trinegin administration were found.

Tridegin in the samples was determined using a modification of the amine incorporation assay used in example 2, wherein activation of internal factor XIII was determined in each sample (0.097ml) by adding 0.1M Tris HCl pH8.5 (0.03ml) and 1000U/ml bovine thrombin (0.01ml) and incubating for 15 min at 37 ℃. The fibrin clot was removed by centrifugation and the serum was used for the assay. Samples of the standard curve were prepared by adding pure Tridegin variant 1 at known concentrations to citrated rat plasma and activating factor XIIIa in the same way. The percentage inhibition of factor XIIIa as in example 2 was then compared to a standard curve to obtain the concentration of Tridegin in each sample.

Figure 9 shows the pharmacokinetics of Tridegin in rats. The time course is obviously multi-phase; the terminal half-life of 30-60 minutes represents its significant duration of action, and the pharmacokinetics of Tridegin make it suitable for pharmaceutical use.

Claims (24)

1. A polypeptide having the following amino acid sequence

NH₂-Lys-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 of said sequence, or a homologue, analogue or truncated form of an amino acid sequence having substantially similar activity.

2. A polypeptide having the following amino acid sequence,

1 10

NH₂-Lys-Leu-Leu-Pro-Cys-Lys-Glu-X₁-His-Gln-Gly-Ile-Pro-Asn-Pro-Arg-Cys-

2030

X₂-Cys-Gly-Ala-Asp-Leu-Glu-X₃-Ala-Gln-Asp-Gln-Tyr-Cys-Ala-Phe-Ile-Pro-

40 50

Gln-Z₁-Arg-Pro-Arg-Ser-Glu-Leu-Ile-Lys-Pro-Met-Asp-Asp-Ile-Tyr-Gln-Arg-

60 66

Pro-Val-Z₂-Phe-Pro-Asn-Leu-Pro-Leu-Lys-Pro-Arg-Z₃-COOH

wherein X₁，X₂And X₃Each represents any amino acid residue; z₁，Z₂And Z₃Each simultaneously or non-simultaneously represents Cys or Glu; or a pharmaceutically acceptable salt, derivative or bioprecursor of said amino acid sequence, or a truncated form, homologue or analogue of an amino acid sequence having substantially the same activity.

3. The polypeptide of claim 1 or 2, which is obtained from leech tissue or secretions.

4. The polypeptide of claim 3, wherein the leech belongs to the order of the oscrudiniformes.

5. The polypeptide according to any one of claims 1 to 4, which is obtained from the tissue or secretion of leeches of the genus leech.

6. An inhibitor of transglutaminase activity is obtained from leech tissue or leech secretion.

7. The inhibitor according to claim 6, wherein the leech belongs to the order of the oscrudiniformes.

8. The inhibitor according to claim 7, wherein the leech belongs to thegenus hirudinaria.

9. The inhibitor according to any one of claims 6 to 8, wherein the inhibitor is a polypeptide having an apparent molecular weight of about 7000 to 8000 daltons as measured by polyacrylamide gel electrophoresis.

10. The inhibitor according to any one of claims 6 to 9, wherein the inhibitor has activity in inhibiting factor XIIIa-catalysed incorporation of an amine into casein.

11. The inhibitor according to any one of claims 6 to 10, wherein the inhibitor has an IC50 of 0.026 ± 0.002mg/ml against factor XIIIa activity in catalyzing amine incorporation into casein.

12. A diagnostic method for determining the level of inhibition of transglutaminase activity by a polypeptide according to any of claims 1 to 5 or by an inhibitor according to any of claims 6 to 11, comprising determining the amount of ammonia produced by the transglutaminase, after amine incorporation in the casein, respectively, in the presence of said polypeptide or extract, wherein the amount of ammonia produced provides a criterion for determining the level of inhibition of transglutaminase.

13. Use of a polypeptide according to any one of claims 1 to 5 or an inhibitor according to any one of claims 6 to 1 i in the manufacture of a medicament for the treatment of a thrombotic disorder.

14. Use of a polypeptide according to any one of claims 1 to 5 or an inhibitor according to any one of claims 6 to 11 for the manufacture of a medicament for the treatment of Crohn's disease, tumor transplantation, thickening of the vessel wallduring atherosclerosis, thrombolytic microangiopathy, fibrogrowth in the skin, membranous glomerulonephritis, cataracts, acne or wound tissue formation or infections by various microfilaria nematodes.

15. A pharmaceutical formulation comprising a polypeptide according to any one of claims 1 to 5, and/or an inhibitor according to any one of claims 6 to 11, and a pharmaceutically acceptable carrier, diluent or excipient.

16. The pharmaceutical formulation of claim 15, which is administered in combination with an anticoagulant.

17. The formulation of claim 16, wherein the anticoagulant comprises hirudin or heparin.

18. The formulation of claim 15, which is administered in admixture with a fibrinolytic agent, or a thrombolytic agent.

19. The formulation of claim 18, wherein the thrombolytic agent comprises one or more of tissue-type plasminogen activator, plasmin, streptokinase, eminase, urokinase, nuclear hormone and staphylokinase.

20. The formulation of claim 18, wherein the fibrinolytic or fibrinolytic agent comprises a mitogen.

21. Use of a formulation according to any one of claims 15 to 20 for the manufacture of a medicament for the treatment of thrombotic disorders.

22. Use of a preparation according to any one of claims 15 to 20 for the preparation of a medicament for the treatment of Crohn's disease, tumor transplantation, thickening of vessel walls during atherosclerosis, thrombolytic microangiopathic larvae, fibrogenetic growth in the skin, membranous glomerulonephritis, cataracts, acne or wound tissue formation or infections by various microfilaria nematodes.

23. A substantially purified polypeptide that inhibits transglutaminase activity, wherein the polypeptide is obtained from leech tissue or leech secretions by a method comprising ion exchange chromatography purification and/or gel filtration column chromatography purification.

24. A method for isolating a polypeptide according to any one of claims 1 to 5 or an inhibitor according to any one of claims 6 to 11, the method comprising extracting a tissue or secretion from leeches of the order oscdellida, purifying the extract by ion exchange column chromatography, gel filtration column chromatography and reverse phase chromatography.

Images