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Title:  Use of tissue factor agonist or tissue factor antagonist for treatment of conditions related to apoptosis

United States Patent:  6,858,587

Issued:  February 22, 2005

Inventors:  Sorensen; Brit Binow (Birkerod, DK); Petersen; Lars Christian (Horsholm, DK)

Assignee:  Novo Nordisk Pharmaceuticals, Inc. (Princeton, NJ)

Appl. No.:  285970

Filed:  November 1, 2002

Abstract

The present invention relates to use of FVII and/or FVIIa and/or another TF agonist and/or FVIIai and/or another TF antagonist in therapeutic treatment of pathological conditions increased or decreased cell apoptosis is required.

Description of the Invention

FIELD OF THE INVENTION

A novel cell regulating activity of a tissue factor (TF) agonist such as, for example, coagulation factor VII (FVII) or a tissue factor antagonist such as, for example, inactivated coagulation factor VIIa (FVIIai) on cells expressing tissue factor (TF) has been described. The present invention relates to a method for regulating cell apoptosis by contacting the cell with a TF agonist, e.g. FVIIa, or a TF antagonist, e.g. FVIIai. The invention also relates to the use of FVIIa or another TF agonist, or FVIIai or another TF antagonist for the preparation of a medicament for regulation of conditions related to apoptosis in a patient. Moreover the present invention relates to a method of treating conditions in a patient, where a decrease or an increase of apoptosis is required.

BACKGROUND OF THE INVENTION

The extrinsic pathway of blood coagulation is initiated when FVIIa circulating in plasma binds to the integral-membrane protein, tissue factor (TF). The involvement of FVIIa as a proteolytic enzyme in the blood coagulation cascade is believed to be confined to the extracellular leaflet of TF expressing cells. Studies of a putative intracellular signaling capacity of FVIIa have shown that it induce mobilization of intracellular free calcium (Ca2+) in a human bladder carcinoma cell line, which constitutively express TF and in umbilical vein endothelial cells which were pre-treated with interleukin-1 to express TF, but have failed to show any cytokine-like activation of intracellular tyrosine kinases. Recent reports indicate that TF may influence important biological functions other than coagulation, such as angiogenesis, embryo vascularization and tumor metastasis. At present, however, it is unclear how TF contributes to these biological processes.

A potential role for the TF cytoplasmic domain in signal transduction is indicated in studies that showed prometastatic function of TF is critically dependent on the TF cytoplasmic domain. Further, TF cytoplasmic domain is shown to interact with actin-binding protein 280 (ABP-280) and supports cell adhesion and migration through recruitment of ABP-280 to TF-mediated adhesion contacts.

However, TF has also been shown to participate certain types of cell signaling by serving as a cofactor for its physiological ligand FVIIa in an extracellular signaling by a putative proteolytic mechanism. For example, binding of FVIIa to cell surface TF is shown to induce intracellular Ca2+ oscillations in a number of TF expressing cells, transient phosphorylation of tyrosine in monocytes, activation of MAP kinase, alteration in gene expression in fibroblasts and enhanced expression of urokinase receptor in tumor cells. Catalytically inactive FVIIa (FVIIai) fails to induce many of the above signaling responses, from Ca2+ oscillations to MAP kinase activation and gene reduction, and it appears that the catalytic activity of FVIIa may be required for at least some TF-FVIIa-mediated signal transduction. At present, not much is known about signaling pathway(s) that are induced by proteolytically active FVIIa.

Normal tissues in the body are formed either by cells that have reached a terminally differentiated state and no longer divide or by cells that die after a period of time and are replaced from a pool of dividing cells. For example, nervous tissue is formed early in development and the cells of the nervous system reach a terminally differentiated state soon after birth. In general, when nervous tissue is damaged, the nerve cells are incapable of dividing and, therefore, the loss of function due to the damaged nerve cells is not repaired.

In comparison to the nervous system, the skin is composed of stratified layers of epithelial cells, in which the upper (outer) layer of cells constantly is sloughed off and the lower layer of cells divides so as to replace the lost cells. Thus, the skin is an example of a tissue that is maintained in a steady-state, where the number of cells that are lost is equivalent to the number of new cells produced.

In some tissues such as skin, the steady-state is maintained, in part, due to a process of programmed cell death, in which the cells are genetically "programmed" to die after a certain period of time. A cell experiencing programmed cell death undergoes morphologic changes characteristic of apoptosis, including, for example, fragmentation of its DNA and collapse of its nucleus.

Apoptosis is particularly prominent during the development of an organism, where cells that perform transitory functions are programmed to die after their function no longer is required. In addition, apoptosis can occur in cells that have undergone major genetic alterations, thus providing the organism with a means to rid itself of defective and potentially cancer forming cells. Apoptosis also can be induced due to exposure of an organism to various external stimuli, including, for example, bacterial toxins, ethanol and ultraviolet radiation. Chemotherapeutic agents for treating cancer also are potent inducers of apoptosis.

DESCRIPTION OF THE INVENTION

The present invention relates to usage of FVII and/or FVIIa and/or another TF agonist and/or FVIIai and/or another TF antagonist in therapeutic treatment of pathological conditions that can be related to apoptosis.

Generally, the blood components, which participate in what has been referred to as the coagulation "cascade" are proenzymes or zymogens, enzymatically inactive proteins, which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors", and are designated by the addition of the letter "a" to the name of the coagulation factor (e.g. factor VIIa).

The term "FVII" or "factor VII" means "single chain" (zymogenic) coagulation factor VII. The term "Factor VIIa", or "FVIIa" means "two chain" activated coagulation factor VII cleaved by specific cleavage at the Arg152-IIe153 peptide bond. FVII and FVIIa may be purified from blood or produced by recombinant means. It is evident that the practice of the methods described herein is independent of how the purified factor VIIa is derived and, therefore, the present invention is contemplated to cover use of any factor FVII or FVIIa preparations suitable for use herein. Preferred are human FVIIa.

The term "modified factor VII", "inactivated FVII", or "F VIIai" is intended to mean FVIIa having at least one modification, which modification substantially inhibits the ability of modified FVIIa to activate FX and/or FIX. This modification may be in the catalytic centre of FVIIa. The terms may be used interchangeably. Such modification includes amino acid substitution (or replacement) of one or more of the catalytic triad residues Ser344, Asp142 and His193, and also includes modification of catalytic triad residues with wine protease inhibitors such as organo-phosphor compounds, sulfanylfluoride, peptide halomethyl ketone or azapeptide. Modifications also includes amino acid deletions and insertions. FFR-FVIIa is one example of a FVIIai derivative obtained by blocking of the active centre of FVIIa with the irreversible inhibitor, D-phenylalanine-L-phenylalanine-L-arginine chloromethyl ketone (FFR cmk). Other suitable FVIIai derivates are inactivated FVIIa obtained or obtainable by blocking the active centre with L-phenylalanine-L-phenylalanine-L-arginine chloromethyl ketone, dansyl-L-phenylalanine-L-phenylalanine-L-arginine chloromethyl ketone, or dansyl-D-phenylalanine-L-phenylalanine-L-arginine chloromethyl ketone. Preferred is FFR-FVIIa (FVIIa inactivated by FFR cmk).

The term "TF agonist", as used herein is intended to mean any compound reducing or inhibiting apoptosis of a cell population as determined in the apoptosis assay described in example 3 by direct binding to TF (e.g. FVIIa) or other TF dependent mechanism.

In one embodiment of the invention, the TF agonist is recombinant factor VIIa. In a further embodiment the TF agonist is a factor VIIa equivalents. In one embodiment, the factor VII equivalents are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild-type factor VII (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Pat. No. 4,784,950). In another embodiment, the factor VIIa variants have no more than 15 amino acids replaced, deleted or inserted; in another embodiment, the factor VII variants have no more than 10 amino acids replaced, deleted or inserted; in another embodiment, the factor VII variants have no more than 8 amino acids replaced, deleted or inserted; in another embodiment, the factor VII variants have no more than 6 amino acids replaced, deleted or inserted; in another embodiment, the factor VII variants have no more than 5 amino acids replaced, deleted or inserted; in another embodiment, the factor VIIa variants have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor VII. In one embodiment, the factor VIIa variants are selected from the list of L305V-FVIIa, L305V/M306D/D309S-FVIIa, L305I-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T/M298Q-FVIIa, V158D/E296V/M298Q-FVIIa, K337A-FVIIa, M298Q-FVIIa, V158D/M298Q-FVIIa, L305V/K337A-FVIIa, V158D/E296V/M298Q/L305V-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, V158D/E296V/M298Q/L305V/K337A-FVIIa, K157A-FVIIa, E296V-FVIIa, E296V/M298Q-FVIIa, V158D/E296V-FVIIa, V158D/M298K-FVIIa, and S336G-FVIIa.

In the present context the three-letter or one-letter indications of the amino acids have been used in their conventional meaning as indicated in table 1. Unless indicated explicitly, the amino acids mentioned herein are L-amino acids. The terminology for factor VIIa variants with amino acid substitutions are as follows. The first letter represent by the one-letter code the amino acid naturally present at a position of human wild-type factor VIIa. The following number represent the position in human wild-type factor VIIa. The second letter represent by the one-letter code the different amino acid substituting for the natural amino acid. An example is L305V/K337A-FVII, the leucine at position 305 of wild-type factor VIIa is replaced by a valine and the Lysine at position 337 of human wild-type factor VIIa is replaced by an alanine, both mutations in the same Factor VII variant.

                             TABLE 1
                  Abbreviations for amino acids:
          Amino acid        Tree-letter code    One-letter code
          Glycine                Gly                G
          Proline                Pro                P
          Alanine                Ala                A
          Valine                 Val                V
          Leucine                Leu                L
          Isoleucine             Ile                I
          Methionine             Met                M
          Cysteine               Cys                C
          Phenylalanine          Phe                F
          Tyrosine               Tyr                Y
          Tryptophan             Trp                W
          Histidine              His                H
          Lysine                 Lys                K
          Arginine               Arg                R
          Glutamine              Gln                Q
          Asparagine             Asn                N
          Glutamic Acid          Glu                E
          Aspartic Acid          Asp                D
          Serine                 Ser                S
          Threonine              Thr                T

The term "TF antagonist", as used herein is intended to mean any compound binding directly to TF without reducing or inhibiting apoptosis of a cell population as determined in the apoptosis assay described in example 3 (e.g. FVIIai).

In one embodiment of the invention, the TF antagonist is an antibody against human TF. In one embodiment the antibody is a human antibody. In a further embodiment the antibody is monoclonal antibody. In a further embodiment the antibody is a Fab fragment, F(ab)2 fragment, F(ab')2 fragment, or a single chain Fv fragment

In this context, the term "treatment" is meant to include both prevention of an adverse condition and regulation of an already occurring condition with the purpose of inhibiting or minimising the condition. Prophylactic administration of FVIIa or another TF agonist, or FVIIai or another TF antagonist is thus included in the term "treatment". In this context, the term "patient" is defined as any animal, in particular mammals, such as humans. The term "subject" is used interchangeably with "patient".

Conditions, which may be treated, comprises pathological conditions such as, for example, various cancers, various degenerative neurological disorders, neuropathologies including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS). In tissues such as skin and intestine, which are turned-over continually during the life of an organism, the cells forming these tissues undergo programmed cell death throughout the life of the organism. Normally, this process is tightly regulated and the number of cells produced due to cell division is balanced by the number of cells undergoing programmed cell death. However, the regulation of programmed cell death is a complex process involving numerous pathways and, on occasion, defects occur in the regulation of programmed cell death. Given the critical role of this process in maintaining a steady-state number of cells in a tissue or in maintaining the appropriate cells during development of an organism, defects in programmed cell death often are associated with pathologic conditions.

Various disease states occur due to aberrant regulation of programmed cell death in an organism. For example, defects that result in a decreased level of apoptosis in a tissue as compared to the normal level required to maintain the steady-state of the tissue can result in an increased number of cells in the tissue. Such a mechanism of increasing cell numbers has been identified in various cancers, where the formation of a tumor occurs not because the cancer cells necessarily are dividing more rapidly than their normal counterparts, but because the cells are not dying at their normal rate. In comparison to cancer, where the likelihood of a cell undergoing apoptosis is decreased, various pathologies are associated with tissues containing cells undergoing a higher than normal amount of apoptosis. For example, increased levels of apoptosis are observed in various neuropathologies, including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS). Since nerve cells generally do not divide in adults and, therefore, new cells are not available to replace the dying cells, the nerve cell death occurring in such diseases results in the progressively deteriorating condition of patients suffering from the disease. Other conditions associated with a higher than normal level of apoptosis, which may be treated with a TF agonist, comprises myopathies and muscular dystrophies, glomerulosclerosis, Monckeberg's medial sclerosis, inflammatory bowel disease, Crohn disease, autoimmune hepatitis, hemochromatosis and Wilson disease, viral hepatitis, alcoholic hepatitis, acute hepatic failure of different etiology, diseases of the bile ducts, atherosclerosis, hypertension and apoptosis associated with the use of chemotherapeutic drugs.

In a first aspect, the invention relates to a method for reducing or inhibiting apoptosis of a cell population, comprising the step of contacting said cells with a tissue factor agonist. In one embodiment the cells are human cells expressing tissue factor, including fibroblasts, smooth muscle cells, tumour cells, haematopoietic cells, monocytes, macrophages, epithelial cells, keratinocytes, nerve cells and endothelial cells.

In a second aspect, the invention relates to a method for inducing or enhancing apoptosis of a cell population, comprising the step of contacting said cells with a tissue factor antagonist. In one embodiment the cells are human cells expressing tissue factor, including fibroblasts, smooth muscle cells, tumour cells, haematopoietic cells, monocytes, macrophages, epithelial cells, keratinocytes, nerve cells and endothelial cells.

In a further aspect, the invention relates to a method of reducing the severity of a condition in an individual characterized by an elevated level of apoptosis by inhibiting or reducing the level of apoptosis of a cell population, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising Factor VIIa or factor VII or another tissue factor agonist. In one embodiment of the invention the condition characterized by an elevated level of apoptosis is a neurodegenerative disease. In a further embodiment of the invention the disease or condition characterized by an elevated level of apoptosis is selected from the group consisting of Parkinson's disease, Alzheimer's disease. amyotrophic lateral sclerosis (ALS), denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS), myopathies and muscular dystrophies, glomerulosclerosis, Monckeberg's medial sclerosis, inflammatory bowel disease, Crohn's disease, autoimmune hepatitis, hemochromatosis and Wilson disease, viral hepatitis, alcoholic hepatitis, acute hepatic failure of different etiology, diseases of the bile ducts, atherosclerosis, hypertension, apoptosis induced hair loss and apoptosis associated with the use of chemotherapeutic drugs. In one embodiment the apoptosis associated with the use of chemotherapeutic drugs results in hair loss.

In a further aspect, the invention relates to a method of reducing the severity of a condition in an individual characterized by a reduced level of apoptosis by inducing or enhancing the level of apoptosis of a cell population, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising a tissue factor antagonist. In one embodiment of the invention the disease or condition characterized by a reduced level of apoptosis is selected from the group consisting of primary tumour growth, tumour invasion, metastasis, psoriasis, an autoimmune disease and restenosis.

In a further aspect, the invention relates to the use of a tissue factor agonist for the manufacture of a medicament for treatment of disease or condition associated with undesired apoptosis of a cell population.

In a further aspect, the invention relates to the use of a tissue factor antagonist for the manufacture of a medicament for treatment of disease or condition, where induction or enhancement of apoptosis of a cell population is desired.

In a further aspect, the invention relates to a method of regulating apoptosis of a cell population, comprising the step of either contacting said cells with a tissue factor agonist or contacting said cells with a tissue factor antagonist.

In one embodiment of the invention the tissue factor agonist is FVII or FVIIa.

In a further embodiment of the invention the tissue factor antagonist is modified FVII. In one embodiment the modified factor VII is selected from factor VII modified with Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone, D-Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg chloromethylketone Phe-Pro-Arg chloromethylketone, D-Phe-Pro-Arg chloromethylketone, Phe-Pro-Arg chloromethylketone, D-Phe-Pro-Arg chloromethylketone, L-Glu-Gly-Arg chloromethylketone and D-Glu-Gly-Arg chloromethylketone, Dansyl-Phe-Phe-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethyl ketone, Dansyl-D-Phe-Phe-Arg chloromethylketone, Dansyl-Phe-Pro-Arg chloromethylketone, Dansyl-D-Phe-Pro-Arg chloromethylketone, Dansyl-Phe-Pro-Arg chloromethylketone, Dansyl-D-Phe-Pro-Arg chloromethylketone, Dansyl-L-Glu-Gly-Arg chloromethylketone and Dansyl-D-Glu-Gly-Arg chloromethylketone.

In another aspect, the present invention relates to a method of detecting drug candidates that regulate apoptosis of a cell population, which method comprise

a) culturing a TF expressing cells;

b) measuring the apoptosis of a cell population;

c) incubating the cells with a drug candidate, and

d) measuring the apoptosis of the incubated cells and determining any change in the level of apoptosis compared to the level of apoptosis measured in step b, such change being indicative of biologically active drug candidate in said cell.

The term "TF expressing cell" means any mammalian cell that expresses TF.

The term "drug candidate" is intended to indicate any sample, which has a biological function or exerts a biological effect in a cellular system. The sample may be a sample of a biological material such as a microbial or plant extract, or it may be a sample containing a compound or mixture of compounds prepared by organic synthesis or genetic techniques.

In a further aspect, the invention relates to the use of tissue factor for the protection of cells from apoptosis. As can be seen from the experiments disclosed by the present inventors only cells expressing TF are protected from apoptosis. Thus, in a further aspect, the present invention relates to a method of producing a recombinant protein, the method comprising:

a) transfection of a cell with a polynucleotide constructs encoding TF;

b) transfection of the same cell with a polynucleotide constructs encoding the recombinant protein to be produced;

c) cultivating the cell in an appropriate growth medium under conditions allowing expression of the polynucleotide constructs and recovering the resulting polypeptide from the culture medium.

In one embodiment of the invention, the recombinant protein to be produced is human FVII. In a further embodiment the appropriate growth medium comprises FVIIa.

In a further aspect, the invention relates to a method for reducing or inhibiting apoptosis of a cell population, comprising the step of contacting said cells with an activated coagulation factor.

In a further aspect, the invention relates to a method for reducing or inhibiting apoptosis of a cell population, comprising the step of contacting said cells with thrombin or coagulation factor Xa.

In a further aspect, the invention relates to a method for inducing or enhancing apoptosis of a cell population, comprising the step of contacting said cells with a thrombin inhibitor or a coagulation factor Xa inhibitor.

In a further aspect, the invention relates to a method of reducing the severity of a condition in an individual characterized by an elevated level of apoptosis by inhibiting or reducing the level of apoptosis of a cell population, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising thrombin or coagulation factor Xa.

In a further aspect, the invention relates to a method of reducing the severity of a condition in an individual characterized by a reduced level of apoptosis by inducing or enhancing the level of apoptosis of a cell population, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising a thrombin inhibitor or a coagulation factor Xa inhibitor.

In a further aspect, the invention relates to the use of thrombin or coagulation factor Xa for the manufacture of a medicament for treatment of disease or condition associated with undesired apoptosis of a cell population.

In a further aspect, the invention relates to the use of a thrombin inhibitor or a coagulation factor Xa inhibitor for the manufacture of a medicament for treatment of disease or condition, where induction or enhancement of apoptosis of a cell population is desired.

In a further aspect, the invention relates to a method of regulating apoptosis of a cell population, comprising the step of either contacting said cells with thrombin or coagulation factor Xa or contacting said cells with a thrombin inhibitor or a coagulation factor Xa inhibitor.

In one embodiment of the present invention the thrombin inhibitor is hirudin. In another embodiment of the present invention the coagulation factor Xa inhibitor is Tick Anticoagulant Protein (TAP). In a specific embodiment the Tick Anticoagulant Protein is a recombinant human protein.

                          Abbreviations:
 TF        tissue factor
 FVII      factor VII in its single-chain, unactivated form
 FVIIa     factor VII in its activated form
 rFVIIa    recombinant factor VII in its activated form
 FVIIai    modified (inactivated) factor VII
 FFR-      factor VII inactivated by reaction with 
           D-Phe-L-Phe-L-Arg
 FVIIai    chloromethyl ketone

Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa) and the complex is principal initiator of blood coagulation. We have studied the effects of FVIIa binding to TF on cell apoptosis of cells that express high amounts of TF. TF expressing cells incubated with FVIIa is shown to be less sensitive to apoptosis.

Below we show for the first time a clear connection between FVIIa binding to TF and the cell apoptosis. We present data that FVIIa stimulation of TF expressing cells leads to a reduction or inhibition of apoptosis of a cell population. Furthermore, active site-inhibited FVIIa (FFR-FVIIa) is shown to induce or enhance apoptosis of a cell population. TF is constitutively expressed on the plasma membrane of many extravascular cells, such as stromal fibroblasts in vascular adventitia and in fibrous capsules of liver, spleen and kidney. Thus, expression of TF is found at sites physically separated from the circulating blood and providing a haemostatic envelope. Upon injury this barrier is thought to protect the organism against bleeding. TF can, however, be induced in monocytes/macrophages, vascular smooth muscle cells, endothelial cells and in a number of tumour cells by a variety of agents, including cytokines and growth factors. Induction at the transcriptional level occurs rapidly after stimulation, identifying TF as a growth-related immediate early gene.

Not only binding to TF, but also the catalytic activity of TF/FVIIa seem to be mandatory, since active-site inhibited FVIIa did not elicit the protection from apoptosis. We excluded that the protection from apoptosis by FVIIa occurred due to FXa or thrombin, since TAP and Hirudin did not abolish the effect of FVIIa in the apoptosis assay. A dose-response of the anti-apoptotic effect of FVIIa was seen in BHK cells transfected with TF.

The regimen for any patient to be treated with FVIIa or another TF agonist or FVIIai or another TF antagonist as mentioned herein should be determined by those skilled in the art. The daily dose to be administered in therapy can be determined by a physician and will depend on the particular compound employed, on the route of administration and on the weight and the condition of the patient. An effective amount is suitably a daily dosage from about 5 .mu.g/kg/day to about 500 .mu.g/kg/day, preferably from about 10 .mu.g/kg/day to 300 .mu.g/kg/day, more preferred from about 15 .mu.g/kg/day to 200 .mu.g/kg/day, most preferred from about 20 .mu.g/kg/day to 100 .mu.g/kg/day.

The FVIIa or another TF agonist or FVIIai or another TF antagonist should be administered in one single dose, but it can also be given in multiple doses preferably with intervals of 4-6-12 hours depending on the dose given and the condition of the patient.

In a particular embodiment the effective amount is a daily dosage from about 5 .mu.g/kg/day to about 500 .mu.g/kg/day of FVIIa or another TF agonist or FVIIai or another TF antagonist.

The FVIIa or another TF agonist or FVIIai or another TF antagonist may be administered intravenously or it may be administered by continuous or pulsatile infusion or it may be administered directly to the relevant site such as, for example, injected directly into a turnout. FVIIa or another TF agonist or FVIIai or another TF antagonist is preferably administered by intravenous injections and in an amount of about 100-100,000 units per kg body weight, and preferably in an amount of about 250-25,000 units per kg body weight corresponding to about 5-500 .mu.g/kg, a dose that may have to be repeated 2-4 times per 24 hours.

Conventional techniques for preparing pharmaceutical compositions, which can be used according to the present invention are, for example, described in Remington's Pharmaceutical Sciences, 1985.

The compositions used according to this invention are prepared by methods known per se by the skilled artisan.

In short, pharmaceutical preparations suitable for use according to the present invention is made by mixing FVII, FVIIa or another TF agonist or FVIIai or another TF antagonist, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. Suitable physiological acceptable carriers or diluents include sterile water and saline. Suitable adjuvants, in this regard, include calcium, proteins (e.g. albumins), or other inert peptides (e.g. glycylglycine) or amino acids (e.g. glycine, or histidine) to stabilise the purified factor VIIa. Other physiological acceptable adjuvants are non-reducing sugars, polyalcohols (e.g. sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextrins, detergents (e.g. polysorbate) and antioxidants (e.g. bisulfite and ascorbate). The adjuvants are generally present in a concentration of from 0.001 to 4% w/v. The pharmaceutical preparation may also contain protease inhibitors, e.g. apronitin, and preserving agents.

The preparations may be sterilised by, for example, filtration through a bacteria-retaining filter, by incorporating sterilising agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile medium suitable for injection prior to or immediately before use.

Claim 1 of 10 Claims

What is claimed is:

1. A method of treating an autoimmune disease characterized by a reduced level of apoptosis comprising administering to an individual in need of such treatment an amount of a composition comprising a tissue factor antagonist and a pharmaceutically acceptable carrier effective to treat the autoimmune disease.


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