DK175116B1 - Tissue factor protein antagonist for pharmaceutical use, use of tissue factor protein antagonists for the manufacture of a drug for the treatment of hypercoagulative bleeding disorder, and therapeutic dosage form - Google Patents

Description

in DK 175116 B1

This invention relates to tissue factor protein antagonist for pharmaceutical use. In particular, the invention relates to the use of tissue factor protein antagonist for the manufacture of a medicament for treating an animal, preferably a human, with a hypercoagulative bleeding disorder and a therapeutic dosage form for administration to an animal with a bleeding disorder characterized by a hypercoagulative state.

BACKGROUND OF THE INVENTION

Bleeding is one of the most serious and significant manifestations of illness.

It can be from a local place or it can be generalized. Bleeding associated with a local lesion may be superimposed on either a normal or a defective hemostatic mechanism. Normal hemostasis includes mechanisms that act immediately after injury and those that work over a long period of time. Primary hemostasis consists mainly of two components of vaso-constriction and platelet formation. The retention mechanism consists of the fibrin clot produced on the coagulation system. Platelet formation is particularly important in capillary hemostasis, while vasoconstriction and fibrin clot formation are more important in larger vessels hemostasis. In the microcirculation, hemostasis consists of vasoconstriction and platelet formation. Platelet formation can be divided into several stages: adherence of platelets to subendothelial 20 surfaces exposed to trauma; platelet activation release reaction; platelet deaggregation resulting in sequestration of additional platelets on site and binding of fibrinogen and coagulation proteins to platelet surface, which includes thrombin formation; and fusion, which is the coalescence of fibrin and fused platelets to form a stable hemostatic plug.

25 Blood coagulation performs two functions: production of thrombin which induces platelet aggregation, and formation of fibrin which makes the platelet clot stable.

A number of distinct proenzymes and proco factors, referred to as "coagulation factors", participate in the coagulation process. The process consists of several stages and ends with fibrin formation. Fibrinogen is converted to fibrin through the action of thrombin. Thrombin is formed by the proteolytic cleavage of a proenzyme, prothrombin. This proteolysis is produced by activated factor X (referred to as factor Xa) which binds to the surface of activated platelets and in the presence of Va and calcium ion cleaves prothrombin.

2 DK 175116 B1

Factor X can be activated by one of two distinct pathways, the outer and the inner (Figure 1). The inner cascade consists of a series of reactions whereby a protein precursor is cleaved to form an active protease. At each step, the newly formed protease will catalyze the activation of the protease precursor in the subsequent cascade step. A lack of any of the proteins in the pathway blocks the activation process at that stage, thereby preventing clot formation and typically causing a tendency for bleeding. Failures of factor VIII or factor IX cause e.g. the severe bleeding syndromes respectively. Hemophilia A and B. In the outer pathway of blood coagulation, tissue factor, also referred to as tissue thromboplastin, is released from damaged cells and activates factor X in the presence of factor VII and calcium. Although activation of factor X was initially thought to be the only reaction catalyzed by tissue factor and factor VII, it is now known that a reinforcement loop exists between factor X, factor VII and factor IX (Osterud, B., and Sl Rapaport, Proc. Natl. Acad. Sci. USA 74: 15602-52-64.1977; Zur, M. et al., Blood 52: 198.1978). Each of the serine proteases in this scheme is capable of converting the other two into the activated form by proteolysis, thereby amplifying the signal at this stage in the coagulation process (Figure 2). It is now believed that the outer pathway may actually be the predominant physiological pathway for normal blood coagulation (Haemostasis 20 13: 120-155 1983). Since tissue factor is not normally found in the blood, the system does not coagulate continuously; therefore, the trigger for coagulation will be the release of tissue factor from damaged tissue.

Tissue factor is an integral membrane glycoprotein which, as discussed above, can trigger blood coagulation via the external reaction pathway; Bach, R. et al., J. Biol Chem.

256 (16), 8324-8331 (1981). Tissue factor consists of a protein component (early re termed tissue factor apoprotein II) and a phospholipid; Osterud, B. and Rapaport, S.I., PNAS 74, 5260-5264 (1977). The complex has been found on the membranes of monocytes and various cells in the blood vessel wall; Osterud, B., Scand. J. Haematol. 32, 337-345 (1984). Tissue factor from various organs and species has been reported to have a relative molecular mass of 42,000 - 53,000. Human tissue thromboplastin has been described as consisting of a tissue factor protein inserted into phospholipid bilayer at an optimal ratio of tissue factor protein to phospholipid of about 1:80. ; Lyberg, T. and Prydz, H. Nouv.

Rev. Fr. Hematol 25 (5), 291-293 (1983). Tissue factor purification has been reported from various tissues, such as: human brain (Guha, A. et al. PNAS 83, 3, 1986; Broze, GH et al., J. Biol. Chem. 260 [20], 10917-10920 [1985]; bovine brain (Bach, R. et al., J. Biol. Chem. 256. 8324-8331 [1981]; human placenta (Bom, VJJ et al., Thrombosis) Res. 42: 635-643 [1986]; and An-doh, K. et al., Thrombosis Res. 43: 275-286 [1986]); sheep brain (Carlsen, E. et al., Thromb. Haemostas. 48 [3], 315-319 [1982]), and lung (Glass, P. and

Astrup. T., Am. J. Physiol. 219. 1140-1146 [1970]. It has been shown that bovine and human tissue thromboplastin are identical in size and function; see e.g. Bronze, G.H. et al., J. Biol. Chem. 260 (20). 10917-10920 (1985). It is widely accepted that while there are differences in tissue factor protein structure between species, there are no functional differences, as measured by in vitro coagulation assays; Guha et al. above. Furthermore, tissue factor isolated from different tissues of an animal, e.g. canines, lung, arteries and veins in certain respects such as extinction coefficient, nitrogen and phosphorus content and optimal phospholipid / lipid ratio, but differed slightly with respect to molecular size, amino acid content, antibody reactivity and plasma half-life; Gonmori, H. and Takeda, Y. J. Physiol. 229 (3), 618-626 (1975). All the tissue factors from the various canine organs showed clotting activity in the presence of lipid; Ibid. It is widely accepted that to exhibit biological activity, tissue factor protein must be associated with phospholipids; Pitlick, F.A., and Menerson, Y., Biochemistry 9, 5105-5111 (1970) and Bach, R. et al. above, page 8324. It has been shown that the removal of the phospholipid component of tissue factor, e.g. using a phospholipase results in loss of its biological activity; Nemerson, Y.

J.C.I. 47, 72-80 (1968). Relipidation can restore in vitro tissue factor activity; Pitlick, F.A. and Nemerson, Y. Biochemistry 9, 5105-5113 (1970) and Freyssinet, J.M. et al., Thrombosis and Haemostasis 55, 112-118 [1986].

Tissue factor infusion has long been considered to compromise normal haemostasis. In 11834, the French physiologist de Blainville first determined that tissue factor contributed directly to blood coagulation; de Blainville, HI, Gazette Medicale Paris, Series 2, 524 (1834). de Blainville also observed that intravenous infusion of 30 brain tissue suspension caused immediate death, which he observed was correlated with a hypercoagulative condition which causes extensive disseminated blood clots found at autopsy. It is now well accepted that intravenous infusion of tissue thromboplastin induces intravascular coagulation and can cause death in various animals (dogs: Lewis, J. and Szeto I.F., J. Lab. Clin.

35 Med. 60, 261-273 (1962); rabbits: Fedder, G. et al., Thromb. Diath. Haemorrh.

4 DK 175116 B1 27, 365-376 (1972); rats: Giercksky, K.E. et al., Scand. J. Haematol. 17, 305-311 (1976); and sheep: Carlsen, E. et al., Thromb. Haemostas. 48, 315-319 [1982]).

In addition to intravascular coagulation or a hypercoagulatory state arising from the exogenous tissue factor administration, it has been suggested that intravascular release of tissue thromboplastin may initiate disseminated intravascular coagulation (DIC); Prentice, C.R., Clin. Haematol. ΛΑ (2), 423-442 (1985).

DIC can occur in various conditions, such as shock, septicemia, cardiac arrest, widespread trauma, bite tachycardia, acute liver disease, major surgical procedures, burns, septic abortion, heat stroke, disseminated malignancy, panereatic and ovarian carcinoma, promyelocytic leukemia, myocardial , systemic lupus erythematosus, kidney disease and eclampsia. The current treatment with DIC includes transfusion of blood and fresh frozen plasma, infusion of heparin, and removal of thrombi formed. The above clinical syndromes suggest that endogenous tissue factor release may result in serious clinical complications; Andoh, K. et al., Thromb. Res. 43, 275-286 (1986). Attempts have been made to overcome the thrombotic action of tissue thromboplastin using the enzyme thromboplastinase; Gollub, S. et al., Thromb. Diath. Haemorh. 7, 470-479 (1962). Thromboplastinase is a phospholipase and would probably cleave the phospholipid portion of tissue factor; Ibid.

Congenital coagulation disorders characteristically involve a single coagulation protein. Hemophilia is a haemorrhagic disease caused by inherited lack of a coagulation factor, e.g. the procoagulant activity of factor VIII. The basis of therapy for bleeding episodes is transfusion of material containing the missing coagulation protein, e.g. infusion of factor VIII procoagulant activity that temporarily corrects the specific deficiency of hemophilia A.

Von Willebrand's disease is another bleeding disorder characterized by a prolonged bleeding time associated with an abnormality or deficiency in the von Willebrand protein. The treatment is done by infusion of normal plasma or of a preparation rich in von Willebrandt protein. Congenital deficiencies in each of the other 30 coagulation factors occur and may be associated with a haemorrhagic tendency. The current therapies for the deficiencies are: Factor IX deficiency is treated using concentrates containing factor IX; plasma fusions are given for a factor XI deficiency; and plasma infusion is given for a factor XIII deficiency.

Acquired coagulation disorders occur in individuals with no prior bleeding history as a result of a disease process. Inhibitors of blood coagulation factors may be present in multitransfused individuals. Acquired coagulation factor deficiencies with unknown etiology also cause hemostatic problems. DIC describes a profound breakdown of the hemostasis mechanism.

It is an object of this invention to provide an anticoagulation therapeutic, i.e. an antagonist of tissue factor protein, to neutralize the thrombotic effects of endogenous tissue thromboplastin release, which may result in a hypercoagulative state. In particular, such an anticoagulant, i.e. an antagonist of tissue factor protein, neutralizing the hypercoagulant effects of endogenously released tissue thromboplastin by inactivating tissue factor protein. Such a tissue factor protein antagonist may be an antibody or other protein that specifically inactivates the protein component.

SUMMARY OF THE INVENTION

Tissue factor protein is the protein portion of tissue factor that lacks the normally occurring phospholipid, previously referred to as tissue factor apoprotein II and previously thought to be inactive. Tissue factor protein was found for the first time to correct the bleeding diet, i.e. a tendency for bleeding, associated with factor VIII deficiency in vivo. Furthermore, infusion of tissue factor protein would be expected to be ineffective in light of the articles describing tissue factor as having an absolute requirement for phospholipid. The observed effectiveness and lack of toxicity are in contrast to the results one would expect from the work of de Blainville and subsequent researchers over the past 152 years.

This invention is directed to an anticoagulant for neutralizing the coagulant effects of endogenously released tissue thromboplastin by activating tissue factor protein.

Accordingly, the invention relates to tissue factor protein antagonist for pharmaceutical use.

6 DK 175116 B1

In particular, the invention comprises the use of a tissue factor protein antagonist for the manufacture of a medicament for the treatment of an animal, preferably a human with a hypercoagulative bleeding disorder.

Furthermore, the invention comprises a therapeutic dosage form for administration to an animal, preferably a human with a bleeding disorder characterized by a hypercoagulative condition, which dosage form is peculiar in that it comprises a tissue factor protein antagonist and a pharmaceutically acceptable medium.

The invention also encompasses the use of tissue factor protein antagonist for the manufacture of a drug for the treatment of an animal, preferably a human 10 with disseminated vascular coagulation (DIC).

In a particular embodiment of the invention, the tissue factor protein antagonist is an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Diagram showing activation of blood coagulation via the internal reaction pathway.

Figure 2. Diagram showing amplification of the coagulation signal via the outer reaction path.

Figure 3. Cuticula bleeding times (CBT) in animals receiving tissue factor protein.

Arrows indicate the dose of tissue factor protein in units / kg. Prez refers to CBT before no-20 gene injection.

DETAILED DESCRIPTION

In this description of claims, "tissue factor protein" refers to a protein capable of correcting various bleeding disorders, especially those associated with deficiencies of coagulation factors. Tissue factor protein is different from tissue factor or tissue thromboplastin in that it lacks the naturally occurring lipid portion of the molecule. Infusion of tissue factor protein, as defined herein, does not result in disseminated intravascular coagulation. The ability of tissue factor proteins to correct various bleeding disorders is readily determined using different in vivo bleeding models, e.g. onset of coagulation in hemophilic dogs using cuticle bleeding time determination (Giles, A. R. et al., Blood 60: 727-730 [1982]).

The term "tissue factor protein antagonists" as used in this specification with claims refers to substances which can function in two ways. First, 5 tissue factor protein antagonists will bind to tissue factor protein with sufficient affinity and specificity to neutralize tissue factor protein so that it cannot bind to factor VII or VIIa or produce factor IX or X proteolysis when complexed with factor VII or VIIa. Alternatively, tissue factor protein antagonists will inactivate tissue factor protein or tissue factor / factor VIa complex by cleavage, e.g. a specific protease. Antagonists are useful, either alone or together, for the therapy of various coagulation disorders that appear at altered plasma fibrinogen levels as described in this specification, e.g. DIC, which occurs during severe infections and septicemia following surgery or trauma in place of or in combination with other anticoagulants, such as heparin.

An example of an antagonist that will neutralize tissue factor protein is a neutralizing antibody to tissue factor protein. Tissue factor protein-neutralizing antibodies are readily produced in animals, such as rabbits or mice, by immunization with tissue factor protein in "Freund's adjuvant" followed by enhancement injections as needed. Immunized mice are particularly useful in providing sources of B cells for the production of hybridomas, which in turn are grown to generate large amounts of inexpensive monoclonal anti-tissue factor protein antibodies. Such monoclonal tissue factor protein antibodies have been produced by Carson S.D. et al., Blood 66 (1), 152-156 (1985).

Tissue factor is released from damaged cells and activates factors IX and X in the presence of factor VII or VIIa and calcium (see Figure 2). The activation of factor X by the ydej

re coagulation pathway has an absolute requirement for tissue factor; Silverberg, S.A., et al., J.I.

Biol. Chem. 252. 8481-8488 (1977). Until the present invention, it was believed that the tissue factor lipid component was necessary for optimal tissue factor activity by factor VIII or Vila catalysis of factor X or factor IX. This invention encompasses the treatment of various acute and chronic bleeding disorders by circumventing these deficiencies via the administration of tissue factor protein.

More specifically, the invention can be applied to the bleeding disorders that occur in animals, preferably humans lacking different coagulation factors.

Tissue thromboplastin or tissue factor consists of a glycoprotein component (formerly referred to as tissue factor apoprotein II) that has been purified to apparent homogeneity (Bjorklid, E. et al., Biochem. Biophys. Res. Commun. 55, 969-976 [1973]), and a phospholipid fraction. Numerous reports have described the purification of tissue factor from many types of tissue, such as brain, lung and placenta. Sheep, cow, rabbit, dog, and human have been the source of tissue factor. The first step in the chemical purification has been to dissociate tissue factor from its native lipid using e.g. extraction with organic solvents. Examples of such organic solvents include pyridine, hepatane / butanol mixture and ethanol. Tissue factor protein has been purified by chemical means. Examples of such chemical agents are: treatment with detergents such as dioxycholate or "Triton® X-100"; gel filtration and preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; concanavalin A bound to a "Sepharose" column; and affinity columns using antibodies to the tissue factor protein or selective adsorption to factor VII. Included in the extent of tissue factor protein is tissue factor protein from recombinant or synthetic sources. 20 torprotein variants with amino acid replacements and / or deletions and / or additions, organic and inorganic salts and covalently modified derivatives of tissue factor protein Tissue factor protein produced by recombinant agents may include a naturally occurring pro form as well as a prep form tissue factor protein.

For use in the context of this invention, tissue factor protein antagonists may be formulated into an injectable composition. Parenteral compositions are suitable for use in the invention, preferably for intravenous administration. These compositions contain therapeutically effective amounts of tissue factor protein assay, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers and excipients. Typically, lyophilized compositions are reconstituted with suitable diluents, e.g. sterile water for injection, sterile saline and the like. wherein the biological activity is sufficient to induce hemostatic coagulation, observed in a rabbit infusion study.

9 DK 175116 B1 Tissue factor protein antagonists may be administered by intravascular injection at a dosage sufficient to correct a bleeding disorder, e.g. DIC. The dose will depend on various therapeutic variables known to those of ordinary skill in the art.

The tissue factor protein antagonist of the invention is preferably composed and administered as a sterile solution, although it is within the scope of the invention to use lyophilized tissue factor preparations. Sterile solutions are prepared by sterile filtration of the tissue factor protein antagonist or by other methods known in the art. The solutions are then lyophilized or filled into 10 pharmaceutical dosage containers. The pH of the solution should be in the range of 3.0-9.5, preferably 5.0-7.5. The tissue factor protein antagonist should be in a solution with a suitable pharmaceutically acceptable buffer such as phosphate, tris (hydroxymethyl) aminomethane HCl, citrate or the like. like. Buffer concentrations should be in the range of 1-100 mM. The tissue factor protein solution may also contain a salt, such as sodium chloride or potassium chloride, at a concentration of 50-750 mM. The compositions of the invention optionally include an effective amount of a stabilizing agent as needed, such as an albumin, globulin, gelatin, mono- or polysaccharide, amino acid or sugar. A stabilizing amount of detergent such as nonionic detergents (PRG or block copolymers), sodium deoxycholate, "Triton® X-100" or sodium dodecyl sulfate (SDS) may be added.

Tissue factor protein antagonist is preferably placed in a container having a sterile access port e.g. a bag or ampoule for intravenous solution with a plug which can be pierced by a hypodermic injection needle.

The invention is further illustrated by the following examples.

EXAMPLE 1

General materials and methods

Mature beef brains were obtained from Pel-Freeze, Rogers, AR, USA and stored at -20 ° C. Triton® X-100 "and α-D-methylglucoside were from Calbiochem, San Diego, CA, USA. Concanavalin-A" Sepharose "® resin (designated Con A "Sepharose" in Table 1) was from Pharmacia , and "Ultrogel AcA 44" from LKB, DK 10, 175116 B1

Gaithersburg, MD, USA. All chemicals and reagents for preparative and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad Laboratories, Richmond, CA, USA. Factor IXa / Factor X reagent and 2222/12581 were obtained from Helena Laboratories (Kabi Coatest kit, Helena 5 Laboratories, Beaumont, CA, USA. Catalog No. 5293). YM 10 ultrafiltration membranes were from Amicon. Factor VII was purified from bovine plasma (Broze, G. and Majerus, P., J. Biol. Chem. 255 (4): 1242-1247 [1980J) Factor VIII deficient and normally co-citrated plasma was from George King Bio-medicals , Overland Park, KS, USA. Raw phosphotidylcholine (lecithin granules from 10 soybeans) was obtained from Sigma, St. Louis, MO, USA. All other chemicals were of reagent grade or better.

Acetone delipidation of bovine brains

Two mature bovine homes were thawed at room temperature and rinsed free of coagulated blood with distilled water. The tissues were then homogenized in ice-cold ace-15 tone to a volume of 10 ml acetone per ml. grams of wet weight of bovine brain using an Ultra-Turrex tissue homogenizer. The homogenate was extracted at 4 ° C for 30 minutes and then filtered through "Whatman No. 1" filter paper onto the pumped out flask. The tissue slurry was resuspended in the original volume of ice-cold acetone, extracted and filtered 6 times. The final filter cake 20 was dried under a stream of nitrogen and stored at -20 ° C.

"Triton®X-100" Tissue Factor Solubilization

Acetone brain powder (145 g) was homogenized in 0.05 M Tris / 0.1 M NaCl, pH

7.5 (TBS) to a final volume of 20 ml buffer / g acetone brain powder. The homogenate was extracted at 4 ° C for 1 hour and then centrifuged at 10,000 x 25 G for 1 hour at 4 ° C. The supernatant was discarded and the pill rehomogenized in 3 liters of TBS / 0.1% Triton®X-100 ". The material was extracted and centrifuged as before. The thus obtained pill was then homogenized in 3 liters of TBS / 2% Triton®X The homogenate was extracted for 16 hours at 4 ° C and then centrifuged as before.

11 DK 175116 B1

Concanavalin A "Sepharose" ® Affinity Column

The supernatant from the 2% "Triton®X-100" extraction was made 1 mM h.t.

CaCb and MgCb and batch adsorbed with 100 ml of concanavalin A "Sepharose" resin for 16 hours at 4 ° C. After adsorption, the "Sepharose" resin 5 was poured into a 3 x 20 cm column and washed with 500 ml TBS 0.05% "Triton® X-100" at a flow rate of 2 ml / min. The absorbance was controlled at 280 nm. As no additional protein was washed from the column, the "Sepharose" was eluted isocratically with a buffer comprising 100 mg / ml aD-methylglucoside in TBS / 0.05% "Triton® X-100". 10 ml fractions were collected. at a flow rate of 2 ml / min. The fractions were precipitated and tested for tissue factor activity. Tissue factor protein was eluted in about 4 column volumes of eluent. The eluate was concentrated in an "Amicon" concentration cell using a YM 10 ultrafiltration membrane. .

Gel permeation chromatography 10 ml of concentrated concanavalin-A "Sepharose" eluate was dialyzed against TBS 0.1% "Triton® X-100", pH 7.4.1 liters volume with 4 buffer replacements. material placed on a 120 x 1.5 cm column of "AcA 44 Ultrogel" pre-equilibrated with TBS 0.1%, Triton®X-100 ". The column was developed isocratically at a flow rate of 6 ml / h. 1 ml fractions were collected. The fractions were precipitated and tested for tissue factor activity. Peak fractions were pooled to a final volume of 20 ml. This material was stored at -20 ° C before use.

Purification of Tissue Factor Protein Tissue factor protein was partially purified from bovine brain by a combination of acetone tone delipidation, Triton® X-100 extraction, lectin affinity chromatography and gel permeation chromatography. The highly purified tissue factor protein was purified 12,000 times from brain powder (Table 1). A sensitive chromogenic test for tissue factor protein was used to control the purification steps. After detergent extraction of acetone brain powder, the tissue factor protein activity could not be detected by the test with less tissue factor protein being relipidated. The material infused in the rabbits had no cofactor activity prior to replication by either the 1-step coagulation assay or the chromogenic 2- 2 step test described below (Table 2). This confirmed the well-known phospholipid dependence of the tissue factor; see Nemerson, Y., supra. Human placental tissue factor was isolated using known methods, see e.g.

Guha, A. et al. above. Human placental tissue factor protein was compared to bovine tissue factor protein. As shown in Table 5, both human placental tissue and bovine tissue factor have a lipid requirement for activity in an in vitro chromogenic assay. As discussed above, human placental tissue factor and bovine tissue factor are uniform in structure. Thus, human placental tissue factor would be expected to function similarly to beef tissue factor if infused in rabbits.

10 Tissue Factor Protein Test 1. Chromogenic Tissue Factor Test.

All samples extracted from bovine brain with nonionic detergent were precipitated prior to testing. As discussed above, tissue factor has an absolute requirement for phospholipid to exert activity in in vitro assay systems (Pitlick and Ne-15 meson, supra). Lecithin granules were homogenized in "Tris" 0.05 M, 0.1 M NaCl pH 7.4 (TBS) containing 0.25% sodium deoxycholate to a concentration of 1 mg / ml. This solution (PC / DOC) was used to relip tissue tissue as follows. Tissue factor protein was diluted in TBS containing 0.1% bovine serum albumin (TBSA). 50 μΙ was placed in a 12 x 75 mm polystyrene tube and 50 μΙ PC / DOC solution was added. Then, 350 μΙ TBSA together with 25 μ1100 mM CdCl 2 was added. This precipitation mixture was allowed to incubate at 37 ° C for 30 minutes.

For the chromogenic assay, samples of reciprocated tissue factor protein were diluted in TBSA. 10 μΙ was placed in a test tube containing 50 μΙ of the factor IXa / factor 25 X reagent and 2 μΙ of a purified factor VII solution, 30 units / ml. The tubes were heated to 37 ° C and 100 μΙ 25 mM CaCl 2 was added. Samples were incubated for 5 min at 37 ° C prior to the addition of 50 μΙ of chromogenic substrate S2222 containing the synthetic thrombin inhibitor 12581. The reaction was allowed to proceed for 10 min and was stopped by addition of 100 μΙ of 50% glacial acetic acid solution. Absorbance was detected at 405 nm. A standard curve was constructed using rabbit brain thromboplastin (commercially sold by Sigma, St. Louis, MO, USA, catalog No. T0263), this reagent being arbitrarily designated as containing 100 tissue factor units per ml.

13 DK 175116 B1

Dilutions were made from 1:10 to 1: 1000. The absorbance was deposited along the abscissa on semilog woven paper with dilution of the standard deposited along the ordinate.

2. One-step test for tissue factor activity.

5 100 µl of hemophilic plasma was added to 10 µΙ of lipid-free or lipid-free tissue factor or TBSA as control in a silicone-coated test tube to further nonspecific activation via the contact phase of coagulation. The reactants were heated to 37 ° C, 100 μΙ of 25 mM CaCl 2 was added and the clot formation was taken; Hvatum, Y. and Prydz, H., Thromb. Diath. Haemorrh. 21, 217-222 (1969).

EXAMPLE 2

Efficacy and lack of tissue factor protein toxicity in a rabbit model

Arterial and venous cannulae were inserted into the ears of two 1.8 kg white New Zealand rabbits. 0.8 ml of arterial blood was taken from each animal and anticoagulated with 0.2 ml of 0.13 M trisodium citrate. Both animals were then infused with 600 μΙ of protein A-purified, human, anti-human factor VIII antibody, 1700 Bethesda, and ml through the venous cannula. Thirty minutes after the infusion, the arterial cannulas were rinsed with 1 ml of saline solution and 1 ml of blood was taken and discarded. Then 0.8 ml of blood was anticoagulated for testing as described above.

20 300 μΙ TBS / 0.1% Triton® X-100 "was then infused in the first rabbit as a control, while the second rabbit received 300 μΙ tissue factor protein. After replication, this would represent a dose of 233 tissue factor units per kg.

(Enh./kg). 60 minutes after the antibody infusion, blood was taken from both rabbits for testing and the arterial cannula was removed. Blood was collected and the flow and duration of blood flow recorded.

Rabbit factor VIII cross-reacted with human anti-human factor VII1 antibodies in in vitro assay systems. These antibodies were then used to anticoagulate rabbits, allowing the detection of tissue factor protein factor VIII immediate activity in vivo. Thirty minutes after the infusion of anti-factor VIII-30 antibodies, factor VIII was not detected in the plasma by the chromogenic factor-VIII assay (Table 3). The control rabbit received an infusion of buffer (300 14 GB 175116 B1 μΙ) containing 0.1% "Triton®X-100M 30 minutes prior to removal of the arterial cannula. This resulted in severe bleeding which took eleven minutes to cease (Table 3 The animal that received tissue factor protein (Test # 2 in Table 3) bleed only slightly after the same treatment, and this flow stopped after 38 seconds, demonstrating that tissue factor protein bypasses factor VIII in vivo. received tissue factor protein, had no observable thrombi, as has been reported in the tissue factor literature and discussed above.

The toxicity of the tissue factor protein preparation was tested in 6 rabbits infused with 250 units. tissue factor protein per kg. After 3 days no adverse effects were observed (Table 4). It should be noted that this is the dose used in Table 3, whereby the bleeding defect was corrected. Two of the rabbits were then infused with a second dose of 250 units / kg, one received twice this dose and one rabbit received 5 times this dose. These animals, as well as the two who did not receive a second injection, were kept under surveillance for a further 2 days. All animals appeared normal after a total of 120 hours of observation, showing that the material is well tolerated and non-toxic. Therefore, similar preparations of human tissue factor protein are expected to be well tolerated when infused in patients (Table 4) and be able to correct bleeding disorders (Table 3).

EXAMPLE 3

Efficacy and Lack of Tissue Factor Protein Toxicity in a Dog Hemophilia Model Tissue factor protein is infused in hemophilic dogs using the procedure described by Giles, A.R. et al., Blood 60, 727-730 (1982).

Lack of tissue factor protein toxicity was first determined in a normal dog after bolus injection of doses of 50 tissue factor protein units. per. kg and 250 tissue factor protein unit. per. kg. A cuticle bleeding time determination (CBT) was performed (Giles above) before infusion and 30 min. after each injection. Blood was sampled and anticoagulated for coagulation tests at various times 30 during the experiment (Figure 3). To detect factor VIII bypass activity in vivo of tissue factor protein, experiments were conducted using hemophilic dogs. Fasting animals were anesthetized and a CBT was performed prior to no infusion. Tissue factor protein was then administered by bolus injection and CBTs were performed at various times up to 90 min after the infusion. Several doses of tissue factor protein were administered. Blood samples were taken for the duration of each experiment and tested for factor V, prothrombin time (PT) and 5 partial thromboplastin time (PTT). CBTs over 12 min. were considered highly abnormal. The claws were cauterized to prevent excessive blood loss.

An anesthetized normal dog was administered doses of tissue factor protein representing 50 and 250 units / kg of tissue factor protein after relipidation by the chromogenic assay. The CBT in this animal was about 3 min. before any infusion 10 (Figure 3). Factor V levels were normal for 30 min. after each infusion (Table 6). The prothrombin time (PT) and partial thromboplastin time (PTT) were unchanged at the end of the trial, and the CBTeme was also within the normal range. Thus, the tissue factor protein infusion was well tolerated in normal dogs and there was no evidence of disseminated intravascular coagulation.

A haemophilic dog with a prolonged CBT characteristic of haemophilia A was administered 50 units / kg tissue factor protein. The CBT was normalized 30 min after this infusion (Figure 3). This correction was not associated with a change in factor V levels, nor was the prothrombin time (PT) extended (Table 6). The procoagulant effect was not maintained for 90 min. after the infusion, as the 20 CBT effect was again abnormal at this time. A dose-response ratio was determined by infusion of 250 units. tissue factor protein per kg. At this dose, the CBT of the hemophilic dog was normalized at 30 and 90 min (Figure 3).

However, this increased dosage was associated with a decrease in factor V levels and a slight extension of the prothrombin time (PT) (Table 6). As a result, the experiments were repeated using a dose of 100 units. tissue factor protein per kg. to achieve the maximum duration of effectiveness while ensuring that other coagulation factor levels were unaffected. Thus, a hemophilic dog received 100 units. tissue factor protein per CBT was performed at 15, 30 and 45 min. Interestingly, at 15 min, the CBT was abnormal (Figure 3) and stasis was not achieved until 30 min after the infusion. This is an observation consistent with results obtained using conventional canine factor VIII preparations in non-inhibitor hemophilic dogs. At this dose, the CBT was normal after 45 min. Blood samples were taken and analyzed for evidence of consumptive coagulopathy (Table 16 DK 175116 B1 6). Factor V levels, prothrombin times, thrombin coagulation times, and platelet levels were unchanged from treatment. Thus, the efficacy of tissue factor protein in vivo was demonstrated at a dose that did not cause disseminated intravascular coagulation. Immediate activity was confirmed in a third hemophilic dog using a dose of 100 units. tissue factor protein per kg and CBTs completed after 30 and 45 min. While the efficacy was established at both times, some re-bleeding occurred at 45 min.

EXAMPLE 4

Functional homology between bovine and human tissue factor protein Functional homology between bovine and human tissue factor protein was shown using the chromogenic tissue factor test. Bovine factor protein was purified as described above. Human tissue factor protein was partially purified from placentae using the method described by Freyssinet et al., Thrombosis and Haemostasis 55 (1): 112-118 (1986), including affinity chromatography on concanavalin-A "Sepharose". The eluted material from this column was then subjected to gel filtration chromatography on an AcA 44 Ultroger column as described previously for the bovine protein.

Bovine and human tissue factor protein (designated BTFP and HTFP respectively in Table 5) were tested by the standard chromogen-20 tissue factor assay already described. Samples which had been precipitated prior to the test showed strong tissue factor cofactor activity (designated as BTFP + PI and HTFP + PI, respectively, in Table 5). Samples that had not been precipitated did not show cofactor activity in the test (BTFP - PI and HTFP - PI).

The protein concentrations in these samples were bovine factor protein 0.59 mg / ml and human tissue factor protein 13.55 mg / ml. The difference in protein concentration was a result of differences in purity. These results are evidence of the functional homology of the tissue factor proteins from human and bovine sources.

Purification of Bovine Heme Tissue Factor 17 TABLE 1

Volu-Protein Tissue Factor Activity Sp.act. Purification Sample but mg / ml total unit / ml total unit / mg times _ml_

Acetone Brain Powder 3500 7.35 25725 1.06 3675 0.14 TBS Washing Supernatant 3000 6.04 18120 0.16 480 0.1% "Triton" Supernatant 3000 1.42 4260 0.52 1560 2% Triton extrate 2750 3.00 8250 14.82 40761 4.94 35.2

Con A "Sepharose" supernatant 2750 2.4 6600 4.2 1133

Con A "Sepharose" elutes 420 0.2 71.4 53.5 22470 314.0 2242

Con A eluate after concentration 15 1.5 23 750.0 11250 489.0 3492 "Ultragel AcA44" cohesion 8 0.83 6.3 1400 10780 1711.0 12221

Characterization of partially purified tissue factor protein 18 TABLE 2

Chromogenic test Coagulation time Sample unit / ml_s._ TBS / 0.1% "Triton" buffer 0 250 Tissue factor protein 0 249

Released tissue factor_1400_66.2_ TABLE 3

Results of in vitro tissue factor protein mixin correction

Factor VIII

bleeding unit / ml

No. Rabbit Infusion before 30 min 60 min time (min) Vol.

T Control TBS / TX100 5Æ 0 0 ϊϊ ~ 0 15.2 2. Test TFP 233 units / kg 4.8 4.8 0 0.63 0.125 * 233 units / kg tissue factor activity after relipidation, as measured by the chromogenic test .

Survival after Tissue Factor Protein Infusion 19 TABLE 4

Time 0 72 hours 1201.

Infusion of TFP * Infusion of TFP * Survival No. Weight (kg) total unit unit / kg total unit unit / kg see (+/-) Ί: ΪΑ2 350 246 350 246 + 2 1.35 350 260 350 260 + 3 1.40 350 250 700 500 + 4 1.33 350 263 1750 1316 + 5 1.41 350 248 0 0 + 6 1.23 350 285 0 0 + Units were determined by chromogenic testing after tissue factor protein tests were precipitated.

TABLE 5

Functional homology between bovine and human tissue factor

Absorbent Tissue Activity

Sample Test Dilution 405 nm unit / ml BFTP + P1 500 0.785 800 BTFP + P1 1000 0.395 755 BTFP-P1 10,000 0 HTFP + P1 500 0.892 950 HTFP + P1 1000 0.491 910 HTFP-P1 10,000 0 TABLE 6 20 DK 175116 B1

Blood parameters in normal and haemophilic dogs following bolusinion of tissue factor protein

Dose Test time ef-tissue factor ter protein infusion PT PTT Factor V Platelets

Dog (unit / kg) (min) (s) (s) (unit / ml (106 / ml) N 50 Before 12 21 0.81 i.b.

67 12 22 0.96 i.b.

250 30 12 19 1.07 i.b.

60 12 16 1.22 i.b.

H1 50 Before 13 53 1.01 i.b.

150 13 54 1.03 i.b.

250 32 15 71 0.64 i.b.

H2 100 Before 13 51 1.24 205 15 13 51 1.23 169 57 13 51 1.17 223

The results of the coagulation test following bolus injection of tissue factor protein in normal and hemophilic dogs.

N = normal dog H1 and H2 = hemophilic dogs i.b. = not determined

Claims (8)

1. Tissue factor protein antagonist for pharmaceutical use. Tissue factor protein antagonist according to claim 1, characterized in that it is an antibody. Use of a tissue factor protein antagonist for the preparation of a drug moiety for treating an animal, preferably a human with a hypercoagulative bleeding disorder. 4. Therapeutic dosage form for administration to an animal, preferably a human with a bleeding disorder characterized by a hypercoagulative state, characterized in that it comprises a tissue factor protein antagonist and a pharmaceutically acceptable medium. Use of a tissue factor protein antagonist for the manufacture of a drug for the treatment of an animal, preferably a human with disseminated intra-vascular coagulation (DIC). A therapeutic dosage form for administration to an animal, preferably a human with DIC, characterized in that it comprises a tissue factor protein antagonist and a pharmaceutically acceptable medium. Use according to claim 3 or 5, characterized in that the tissue factor protein antagonist is an antibody. Therapeutic dosage form according to claim 4 or 6, characterized in that the tissue factor protein antagonist is an antibody.