EP1443956A2 - Utilisation d'un agoniste ou d'un antagoniste du facteur tissulaire pour le traitement d'etats associes a l'apoptose - Google Patents

Utilisation d'un agoniste ou d'un antagoniste du facteur tissulaire pour le traitement d'etats associes a l'apoptose

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Publication number
EP1443956A2
EP1443956A2 EP02772090A EP02772090A EP1443956A2 EP 1443956 A2 EP1443956 A2 EP 1443956A2 EP 02772090 A EP02772090 A EP 02772090A EP 02772090 A EP02772090 A EP 02772090A EP 1443956 A2 EP1443956 A2 EP 1443956A2
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Prior art keywords
phe
cells
apoptosis
arg
fviia
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EP02772090A
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German (de)
English (en)
Inventor
Brit Binow Sorensen
Lars Christian Petersen
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Novo Nordisk Health Care AG
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Novo Nordisk Health Care AG
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Publication of EP1443956A2 publication Critical patent/EP1443956A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P27/00—Drugs for disorders of the senses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • 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 Vila (FVIIai) on cells expressing tissue factor (TF) has been described.
  • the present invention relates to a method for regulating cell apdptosis 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.
  • the present invention relates to a method of treating conditions in a patient, where a decrease or an increase of apoptosis is required.
  • FVIIa circulating in plasma binds to the integral-membrane protein, tissue factor (TF).
  • TF tissue factor
  • TF cytoplasmic domain 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.
  • ABS280 actin-binding protein 280
  • 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.
  • FVIIa physiological ligand FVIIa
  • binding of FVIIa to cell surface TF is shown to induce intracellular Ca 2+ 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 fails to induce many of the above signaling responses, from Ca 2+ 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-FVI la-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 re- placed from a pool of dividing cells.
  • nervous tissue is formed early in development and the cells of the nervous system reach a terminally differentiated state soon after birth.
  • the nerve cells are incapable of dividing and, therefore, the loss of function due to the damaged nerve cells is not repaired.
  • the skin 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Vila).
  • FVII or "factor VII” means "single chain” (zymogenic) coagulation factor VII.
  • Factor Vila or “FVIIa” means "two chain” activated coagulation factor VII cleaved by specific cleavage at the Arg152-lle153 peptide bond.
  • FVII and FVIIa may be puri- tied 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 Vila 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.
  • modified factor VII 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.
  • 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 serine protease inhibi- tors such as organo-phosphor compounds, sulfanylfluoride, peptide halomethyl ketone or azapeptide.
  • 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-argininine chloromethyl ketone (FFR cmk).
  • FVIIai derivates are inactivated FVIIa obtained or obtainable by blocking the active centre with L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, dansyl-L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, or dansyl-D- phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone, Preferred is FFR-FVIIa (FVIIa inactivated by FFR cmk).
  • TF agonist as used herein is intendend 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.
  • the TF agonist is recombinant factor Vila. In a further embodiment the TF agonist is recombinant human factor Vila. In a further embodiment the TF agonist is a factor Vila equivalents.
  • 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. Patent No. 4,784,950).
  • the factor Vila 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 Vila variants have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor VII.
  • the factor Vila variants are selected from the list of L305V-FVIIa, L305V/M306D/D309S-FVIIa, L305l-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, V158D/E296V/M298Q/L305V/K337A-FVIIa, K157
  • the amino acids mentioned herein are L-amino acids.
  • the terminology for factor Vila 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 Vila. The following number represent the position in human wild-type factor Vila. The second letter represent by the one-letter code the different amino acid substituting for the natural amino acid.
  • L305V/K337A-FVII the leucine at position 305 of wild-type factor Vila is replaced by a valine and the Lysine at position 337 of human wild-type factor Vila is replaced by an alanine, both mutations in the same Factor VII variant.
  • TF antagonist as used herein is intendend 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).
  • the TF antagonist is an antibody againts human TF.
  • the antibody is a human antibody.
  • the antibody is monoclonal antibody.
  • the antibody is a Fab fragment, F(ab) 2 fragment, F(ab') 2 fragment, or a single chain Fv fragment.
  • 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”.
  • the term "patient” is defined as any animal, in particular mammals, such as humans.
  • the term “subject” is used in- terchangeably 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), den- ervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS).
  • pathological conditions such as, for example, various cancers, various degenerative neurological disorders, neuropathologies including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), den- ervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS).
  • pathological conditions such as, for example, various cancers, various degenerative neurological disorders, neuropathologies including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), den- ervation atrophy
  • 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.
  • various pathologies are associated with tissues containing cells undergoing a higher than normal amount of apoptosis.
  • 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 encephalopa- thy 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.
  • ALS amyotrophic lateral sclerosis
  • AIDS acquired immunodeficiency disease
  • apoptosis comprises myopathies and muscular dystrophies, glomerulosderosis, 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 chemo- therapeutic drugs.
  • 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.
  • 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.
  • 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.
  • the cells are human cells expressing tissue factor, including fi- broblasts, smooth muscle cells, tumour cells, haematopoietic cells, monocytes, macro- phages, epithelial cells, keratinocytes, nerve cells and endothelial cells.
  • tissue factor including fi- broblasts, smooth muscle cells, tumour cells, haematopoietic cells, monocytes, macro- phages, epithelial cells, keratinocytes, nerve cells and endothelial cells.
  • 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 Vila or factor VII or another tissue factor agonist.
  • the condition characterized by an elevated level of apoptosis is a neurodegenerative disease.
  • 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, mul- tiple sclerosis, Huntington's disease and the encephalopathy associated with acquired immunodeficiency disease (AIDS), myopathies and muscular dystrophies, glomerulosderosis, 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, apoptosis induced hair loss and apoptosis associated with the use of chemotherapeutic drugs.
  • 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.
  • 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.
  • 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 unde- sired apoptosis of a cell population.
  • 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 en- hancement of apoptosis of a cell population is desired.
  • 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.
  • tissue factor agonist is FVII or FVIIa.
  • tissue factor antagonist is modified FVII.
  • 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 chloromethylke- tone, 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 chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe
  • 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.
  • TF expressing cell means any mammalian cell that expresses TF.
  • 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.
  • 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.
  • 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.
  • the recombinant protein to be produced is human FVII.
  • the appropriate growth medium comprises FVIIa.
  • 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 co- agulation factor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the thrombin inhibitor is hirudin.
  • the coagulation factor Xa inhibitor is Tick Anticoagulant Protein (TAP).
  • Tick Anticoagulant Protein is a recombinant human protein.
  • Tissue factor is the cellular receptor for factor FVIIa (FVIIa) and the complex is principal initiator of blood coagulation.
  • FVIIa factor FVIIa
  • TF expressing cells incubated with FVIIa is shown to be less sensitive to apoptosis.
  • FVIIa binding to TF and the cell apoptosis.
  • FVIIa stimulation of TF expressing cells leads to a reduction or inhibition of apoptosis of a cell population.
  • active site-inhibited FVIIa FFR-FVIIa
  • 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.
  • FFR-FVIIa active site-inhibited FVIIa
  • 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.
  • expression of TF is found at sites physically separated from the circulating blood and providing a haemostatic envelope.
  • 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.
  • 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 ⁇ g/kg/day to about 500 ⁇ g/kg/day, preferably from about 10 ⁇ g/kg/day to 300 ⁇ g/kg/day, more preferred from about 15 ⁇ g/kg/day to 200 ⁇ g/kg/day, most preferred from about 20 ⁇ g/kg/day to 100 ⁇ g/kg/day.
  • the FVIIa or another TF agonist or FVIIai or another TF antagonist should be adminis- tered 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.
  • the effective amount is a daily dosage from about 5 ⁇ g/kg/day to about 500 ⁇ 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 tumour.
  • FVIIa or another TF agonist or FVIIai or another TF antagonist is preferably administered by intraveneous 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 ⁇ g/kg, a dose that may have to be repeated 2-4 times per 24 hours.
  • compositions used according to this 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.
  • compositions 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 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 Vila.
  • 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.
  • Figure 1 Apoptosis illustrated by FITC-dUTP staining of BHK(+TF). Dose-response of FVIIa.
  • Figure 2 Apoptosis illustrated by FITC-dUTP staining of BHK wt. Dose-response of FVIIa.
  • Figure 3 Apoptosis illustrated by FITC-dUTP staining of BHK(+TF). Involvement of FXa and thrombin inhibitors.
  • Figure 4 Apoptosis illustrated by FITC-dUTP staining of BHK wt. Involvement of FXa and thrombin inhibitors.
  • Figure 5 Apoptosis illustrated by FITC-dUTP staining of BHK(+TF). Effect of FVIIa and FFR- FVIIa on serum-starvation induced apoptosis.
  • Figure 6 Apoptosis illustrated by FITC-dUTP staining of BHK(+TF). Time-dependency of serum-starvation induced apoptosis.
  • Figure 7 Apoptosis illustrated by Hoechst staining of condensed nuclei (marked with an arrow) in BHK(+TF) cells.
  • Figure 8 Apoptosis illustrated by FITC-dUTP staining. Dose-dependent anti-apoptotic effect of FVIIa in cells expressing TF. TUNEL analysis by flow cytometry.
  • Figure 9 Apoptosis illustrated by FITC-dUTP staining of BHK(+TF) analyzed by TUNEL and flow cytometry. Involvement of FFR-FVIIa. Involvement of FXa and thrombin inhibitors
  • Figure 10 Time-dependent activation of caspase 3 upon serum deprivation of BHK(+TF) cells.
  • Western blot analysis using anti-caspase 3 ab's.
  • FVIIa show anti-apoptotic effect at all times tested.
  • Figure 11 Apoptosis illustrated by activation of caspase 3.
  • Western blot anlysis using anti- caspase 3 ab's.
  • Anti-apoptotic effect of FVIIa only in cells expressing TF.
  • Figure 12 Apoptosis illustrated by Caspase 3 activation in CHO cells. Western blot analysis using anti-caspase 3 ab's. Effect of FVIIa only in cells expressing TF.
  • Figure 13 The anti-apoptotic effect of FVIIa is dose-dependent and correlates with the ability of FVIIa to activate p44/42 MAPK and Akt in BHK(+TF) cells. Western blot analysis.
  • Figure 14 Apoptosis illustrated by wb analysis of activated caspase 3 in BHK(+TF) cells. Preventing FVIIa from binding to TF neutralizes the anti-apoptotic effect of FVIIa.
  • Human purified factor Vila suitable for use in the present invention is preferably made by DNA recombinant technology, e.g. as described by Hagen et al., Proc.Natl.Acad.Sd. USA 83: 2412-2416, 1986 or as described in European Patent No. 200.421 (ZymoGenetics).
  • Factor Vila produced by recombinant technology may be authentic factor Vila or a more or less modified factor Vila provided that such factor Vila has substantially the same biological activity for blood coagulation as authentic factor Vila.
  • modified factor Vila may be produced by modifying the nucleic acid sequence encoding factor VII either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural FVII by known means, e.g. by site-specific mutagenesis.
  • Factor VII may also be produced by the methods described by Braze and Majerus,
  • factor VII without detectable amounts of other blood coagulation factors.
  • An even further purified factor VII preparation may be obtained by including an additional gel filtration as the final purification step.
  • Factor VII is then converted into activated FVIIa by known means, e.g. by several different plasma proteins, such as factor XI la, IX a or Xa.
  • factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like.
  • Modified factor VII suitable for use in the present invention is made, e.g. as described in International Publications Nos. 92/15686, 94/27631 , 96/12800 and 97/47651 ZymoGenet- ics/Novo Nordisk).
  • apoptosis of cells was induced by serum-starvation for 24 hr and 48 hr. Apoptosis was detected by flow cytometry after Terminal deoxynucleotidyl-transferase dUTP Nick End Labelling (TUNEL).
  • TUNEL Terminal deoxynucleotidyl-transferase dUTP Nick End Labelling
  • One of the later steps in apoptosis is DNA fragmentation, a process which results from the activation of endonucleases during the apoptotic program. The nucleases degrade the higher order chromatin structure into fragments of approximately 300 kb and subsequently into smaller DNA pieces about 50 bp length.
  • TdT catalyses a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3'-hydroxyl termini of double- and single-stranded DNA in paraformalde- hyde and ethanol fixed cells. After incorporation these sites are identified by flow cytomertic means by staining the cells with a FITC-labelled anti-BrdU mAb. To follow cell-cycle pro- gress, the cells are in the final step labelled with Propidium Iodine (PI). Proteins - Human recombinant FVII and FVIIa was expressed and purified as described by Thim, L. et al. Biochem 27: 7785-7793, 1988.
  • PI Propidium Iodine
  • FVIIai was obtained by blocking of FVIIa in the active site with D-Phe-L-Phe-L-Arg chloro- methyl ketone (FFR-FVIIa) as previously described by Sorensen B. B. et al. J.Biol.Chem. 272: 11863-11868, 1997.
  • Thrombin was from Enzyme Research Lab.
  • the specific thrombin inhibitor hirudin may be purchased from Sigma-Aldrich and the specific FXa inhibitor Recombinant TAP (Tick Anticoagulant Protein) was kindly provided by Dr. G. P. Vlasuk, Corvas (San Diego, CA).
  • Cell Cul- ture The baby hamster kidney cell line BHK-21 tk- ts13 (ATCC CRL 1632) was cultured in Dulbecco's modified Eagle's medium containing 10% FCS, 100 lU/ml penicillin, 100 ⁇ g/ml streptomycin. All cell lines were grown in T80 or T175 flasks and subcultured into 10 cm single culture dishes (78 cm 2 ).
  • BHK cells were transfected with the TF expression plasmid using the calcium phosphate coprecipitation standard technique. Cells with stably integrated constructs were selected with 1 ⁇ M Methotrexate.
  • the cells were seeded (800.000 cells/dish) in DMEM supplemented with 10% FCS,
  • DMEM+/+ 100 lU/ml penicillin, 100 ⁇ g/ml streptomycin (DMEM+/+). After two days the cells were approximately 80% confluent and ready for serum-starvation. The cells were washed two times with DMEM -/- media (before use DMEM -/- was left in the incubator over night for right pH stabilization and temperature) and after the final wash cells were left with 6 ml DMEM-/- and the indicated compounds for 24 hr and/or 48 hr. At the end of the incubation period the loose cells in the media was pooled with the trypsin-treated adhered cells and centrifugated at 300xg for 5 min.
  • BHK(+TF) cells were serum-starved in the presence of increasing concentrations of FVIIa (from 12.5 nM to 100 nM).
  • FVIIa concentration of FVIIa
  • Fig. 1 A a clear increase in the number of apoptotic cells
  • the remaining four curves in Fig. 1A represent increasing concentration of FVIIa, and the results show that as low concentration as 12.5 nM FVIIa was able to rescue the cell population from apoptosis.
  • B BHK(+TF) cells were keep in serum-deprived media for 48 hr with increasing concentration of FVIIa.
  • BHK wt cells were also investigated in the same experiment exploring the effect of specific inhibitors of down-stream coagulation products on the anti-apoptotic effect of FVIIa.
  • BHK wt cells were serum-starved for 24 hr there was no increase in apoptotic cells (Fig. 4A) compared to 10%-treated cells and this correlates well with the experiment shown in Fig. 2A.
  • Serum-starvation for 48 hr of BHK wt cells (Fig. 4B) clearly resulted in an increase in apoptotic cells compare to heathy cells (Fig. 4B).
  • FFR-FVIIa active site-inactivated variant of FVIIa
  • Fig. 5 depicts an experiment employing BHK(+TF) cells where FVIIa at both 24 hr (Fig. 5A) and 48 hr (Fig. 5B) serum-starvation had a clear apoptosis- suppressive effect.
  • FFR-FVIIa (Fig. 5C) was not able to rescue any cells and the population of cells was identical to non-treated cells both at 24 hr and 48 hr of serum- deprivation.
  • Factors X, FXa, thrombin and hirudin were obtained from Enzyme Research Laboratories (South Bend, IN). The specific FXa inhibitor, recombinant tick anticoagulant protein (TAP), was a generous gift from Dr. Vlasuk (Corvas, La Jolla, CA). Antibodies against caspase 3, phosphorylated p44/42 MAPK, and phosphorylated Akt were from Cell Signaling Technology (Boston, MA), ⁇ -actin antibody was from Abeam (Cambridge, UK). LY294002 and U0126 were from Promega (Madison, Wl).
  • the Baby Hamster Kidney cell line BHK-21 tk " (ATCC CRL1632) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, 100 lU/ml penicillin, 100 ⁇ g/ml streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • the complete human TF cDNA was cloned into the mammalian Zem219b expression vector and transfected into BHK cells using the calcium phosphate coprecipitation procedure and stably integrated constructs were selected using 1 ⁇ M methotrexate (Sorensen, B. B., Freskgard, P.-O., Nielsen, L. S., Rao, L. V.
  • CHO-K1 Chinese hamster ovary (CHO-K1 , ATCC CCL-61) cells were cultured in Ham-F12 medium supplemented with 10% FCS and 1% non-essential amino acids. CHO cells were transfected with the complete human TF in the pcDNA3 (Invitrogen, Carlsbad, CA) using FuGeneTM (Roche Diagnostics, Indianapolis, IN). Stable cell lines were selected by resistance to geneticin (0.7 mg/ml). Clonal cell lines were tested for TF expression in a FXa generation assay employing intact monolayers of cells. The expression of functional TF in the two transfected cell lines BHK(+TF) and CHO(+TF) were comparable as judged by the FXa generation assay.
  • TUNEUflow cytometry The cells were seeded in 9 cm dishes (800,000 cells/dish) in DMEM supplemented with 10% FCS, 100 lU/ml penicillin, 100 ⁇ g/ml streptomycin. When cells were approximately 80% confluent, they were subjected to serum deprivation in the presence and absence of experimental compounds. Briefly, the monolayers were washed twice with DMEM (before use the DMEM was left in the incubator over night for pH stabilization) and after the final wash the cells were overlaid with 6 ml DMEM containing the experimental test compounds for 24 or 48 h.
  • the loose cells in the media were collected and pooled with the trypsin-detached adhered cells, and centri- fuged at 300 x g for 5 min.
  • the cells were washed with PBS before fixation in 1 % parafor- maldehyde in PBS on ice for 15 min. After the fixation, the cells were washed in PBS and resuspended in 500 ⁇ l PBS before adding 5 ml of 70% EtOH.
  • the cell suspension was left for at least 18 h at -20°C before they were analyzed by TUNEL assay (Apo-BRDU, BD Bio- sciences Pharmingen, San Diego, CA) following the procedure described in manufacturer's technical bulletin. Stained cells were analyzed by a FACScanTM flow cytometer (Becton Dick- inson).
  • Detection of caspase 3 activation - Cells were cultured in 9-cm dishes and serum deprived in the presence or absence of experimental test compound as described under TUNEL. At the end of specific time interval, as indicated in Results, the cultured dish was placed on ice and the overlaying medium containing loose cells was collected. The adhered cells were scraped from the bottom of the dish and pooled with the conditioned medium and centrifuged at 300 x g for 5 min at 4°C to obtain the cell pellet. The supernatant was discarded and the tubes were drained carefully (by inverting the tubes) to obtain the cell pellet.
  • the cells were lysed by adding 50 ⁇ l of ice-cold Chaps Cell Extract Buffer (50 mM Pipes/KOH, pH 6.5, 2 mM EDTA, 0.1% Chaps, supplemented immediately prior to use with 20 ⁇ g/ml Leupeptin, 10 ⁇ g/ml aprotinin, 5 mM DTT, 0.1 mM AEBSF (4-(2-Aminoethyl)- bezenesulfonylfluoride)).
  • the lysates were frozen and thawed three times, centrifuged at 15,000 x g for 5 min at 4°C. The supernatant was collected and total protein concentration was determined by Bio-Rad protein assay kit.
  • Lysis buffer (20 mM Tris, 0.1% (v/w) Triton X- 100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium-fluoride, 10 mM sodium ⁇ -glycerophosphate, 5 mM sodium pyrophosphate, 0.1 mM AEBSF (4-(2-Aminoethyl)-bezenesulfonylfluoride), 1 mM benzamidine, 150 mM NaCI, pH 7.5.
  • Chemiluminescence substrate (Supersignal, Pierce) was added for 5 min and the chemiluminescence was detected by a cooled CCD-camera (LAS1000, Fujifilm). These digital images were used to quantify the intensity of the bands using Image Gauge v. 4.0 (Fujifilm).
  • Immunocytochemistry - BHK(+TF) cells were seeded in 8-chamber glass slides (Nalge Nunc International, Rochester, NY) and grown to 30% confluence. Cells were washed and incubated with serum free medium supplemented with none, 20 nM FVIIa, 20 nM FFR- FVIIa, or 10%) FCS for 6 hr. At the end of the experiment cells were washed, fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. at 4°C, briefly rinsed in PBS, post-fixed in 70% (v/v) ethanol for 15 min at RT, and air-dried.
  • PBS phosphate-buffered saline
  • Antigen retrieval was achieved by microwave oven (Polar Patent, Umea, Sweden) pre-treatment in 10 mM citrate buffer pH 6.0 preheated to 60°C for 3 x 5 min at 80% effect.
  • the cells were cooled for 10 min in the citrate buffer at room temperature, rinsed in Tris-buffered saline (TBS), pre-incubated for 15 minutes in 5% donkey serum in TBS, followed by incubation with goat anti-human TF IgG (American Diagnostica Incorporation, Greenwich, Ct) overnight at 4 °C.
  • TBS Tris-buffered saline
  • goat anti-human TF IgG American Diagnostica Incorporation, Greenwich, Ct
  • the cells were then incubated with biotinylated donkey anti-goat (Jackson ImmunoReseach Laboratory,
  • FVIIa prevents nuclei chromatin condensation under serum-free conditions.
  • the inventors of the present invention have found out, that upon serum-deprivation, BHK(+TF) cells tend to round up, loosely adhere to culture dishes (detach easily from the dish) and undergo subtle morphological changes. These changes were diminished, when FVIIa was included in serum-free medium.
  • the present invention is based on the examined effect of FVIIa and FFR-FVIIa on cell viability and apoptosis, where cells were examined for apoptotic morphol- ogy changes. Under serum-starvation conditions (Fig.
  • the cells were rescued from apoptosis in a dose dependent manner upon addition of 1 pM to 100 nM FVIIa to the serum-deprived medium.
  • the anti-apoptotic effect of FVIIa was evident at 1 nM with substantially increased cell survival at 10 nM FVIIa; that is at a concentration equivalent to the FVII plasma level.
  • An increase to 100 nM FVIIa slightly improved cell survival further.
  • a similar profile of anti-apoptotic effect of FVIIa was observed in cells exposed to FVIIa for 48 h (Fig. 8B).
  • Wild-type, non-transfected BHK cells do not express detectable levels of TF. Like TF-transfected cells, culture of these cells under serum free conditions for 48 hr resulted in extensive apoptosis as demonstrated by the TUNEL assay. However, Fig. 8C shows that in contrast to BHK(+TF) cells, wild-type BHK cells could not be rescued from apoptosis by FVIIa suggesting that binding to TF is essential for its anti-apoptotic effect.
  • FVIIa the down-stream proteases of the TF coagulation pathway, FXa and thrombin, are known to induce intracellular signaling producing various cellular responses. It was therefore important to rule out that FVIIa exerted its effect through generation of small amounts of down-stream coagulation factors.
  • FVIIa suppresses caspase 3 activation -
  • Caspases a family of cysteine proteases, are central regulators of apoptosis. Caspases are synthesized as latent zymogens organized in cascade systems that upon activation stimulate apoptosis. Inhibition of apoptosis is accomplished either by inhibiting the activity of caspases or by preventing their activation.
  • FVIIa inhibits apoptosis
  • caspase 3 we investigated the activation of the central apoptosis effector, caspase 3, by Western blot analysis using an antibody that recognizes the zymogen as well as the activated enzyme form.
  • Serum removal induced a time-dependent appearance of a 18-20 kDa caspase 3 band in BHK(+TF) cells (Fig. 10).
  • Caspase 3 activation was evident 2 hr after serum deprivation and reached a maximum at 3 hr. The activation was maintained for at least 5 hr (the duration of the experiment).
  • the presence of 100 nM FVIIa in the serum free medium clearly attenuated caspase 3 activa- tion with 70-80% at 3, 4, and 5 hr.
  • FVIIa induced a dose-dependent inhibition of caspase 3 activation, which correlated with the dose- dependent inhibition of apoptosis measured in TUNEL assay.
  • FVIIa failed to inhibit caspase 3 activation in wild type BHK cells that do not express TF (Fig. 1 1 B). Serum abolished the activation of caspase 3 in both BHK(+TF) and wild-type BHK cells. Similar data were obtained with wild type CHO-K1 and TF-transfected CHO cells (Fig. 12).
  • FVIIa/TF was shown in quiescent BHK(+TF) cells to activate p44/42 MAPK (Fig. 13A) as well as Akt (Fig. 13B), and both are considered important players in survival signaling.
  • Akt Fig. 13B
  • kinase is responsible for the anti-apoptotic effect of FVIIa.
  • Experiments with the specific MEK inhibitor U0126 and the specific PI3-kinase inhibitor LY294003 indicates that both kinases might be involved in the anti-apoptotic effect of FVIIa (data not shown).
  • FVIIa antagonists of FVIIa - TF interaction
  • FFR-FVIIa and mAb 1 F44A1 antagonists of FVIIa - TF interaction
  • FFR-FVIIa and mAb 1 F44A1 were preincubated with the cells before addition of FVIIa in serum-free medium.
  • Apoptosis was detected by caspases-3 acti- vation.
  • FFR-FVIIa and the monoclonal anti-human TF antibody 1F44A1 were able to attenuate the anti-apoptotic effect of FVIIa (Fig. 14).

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Abstract

La présente invention concerne l'utilisation de FVII et/ou de FVIIa et/ou d'un autre agoniste du facteur tissulaire et/ou de FVIIai et/ou d'un autre antagoniste du facteur tissulaire dans le traitement thérapeutique d'états pathologiques dans lesquels une augmentation ou une diminution de l'apoptose cellulaire est nécessaire.
EP02772090A 2001-11-02 2002-11-01 Utilisation d'un agoniste ou d'un antagoniste du facteur tissulaire pour le traitement d'etats associes a l'apoptose Withdrawn EP1443956A2 (fr)

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US5833982A (en) * 1991-02-28 1998-11-10 Zymogenetics, Inc. Modified factor VII
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