WO2004041302A1 - Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie - Google Patents

Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie Download PDF

Info

Publication number
WO2004041302A1
WO2004041302A1 PCT/DK2003/000751 DK0300751W WO2004041302A1 WO 2004041302 A1 WO2004041302 A1 WO 2004041302A1 DK 0300751 W DK0300751 W DK 0300751W WO 2004041302 A1 WO2004041302 A1 WO 2004041302A1
Authority
WO
WIPO (PCT)
Prior art keywords
phe
antagonist
arthritis
insulin
blood glucose
Prior art date
Application number
PCT/DK2003/000751
Other languages
English (en)
Inventor
Lars Christian Petersen
Jakob Michael Back
Christian Meyer
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU2003275947A priority Critical patent/AU2003275947A1/en
Publication of WO2004041302A1 publication Critical patent/WO2004041302A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a pharmaceutical composition comprising a TF antagonist and a blood glucose regulator.
  • the invention also relates to the use of a combination of a TF antagonist and a blood glucose regulator for the manufacture of a medicament for treatment of Thrombotic or Coagulopathic related diseases, Respiratory diseases and Inflammatory diseases, or prevention hereof.
  • Tissue Factor is a cellular transmembrane receptor for plasma coagulation factor Vila and formation of TF/FVlla complexes on the cell surface triggers the coagulation cascade in vivo,
  • the TF/FVlla complex efficiently activates coagulation factors IX and X.
  • the resultant prote- ase factor Xa (Xa) activates prothrombin to thrombin, which in turn converts fibrinogen into a fibrin matrix.
  • TF is constitutively expressed on the surface of many extravascular cell types that are not in contact with the blood, such as fibroblasts, pericytes, smooth muscle cells and epithelial cells, but not on the surface of cells that come in contact with blood, such as endothe- lial cells and monocytes.
  • TF is also expressed in various pathophysiological conditions where it is believed to be involved in progression of disease states within cancer, inflammation, atherosclerosis and ischemia/reperfusion.
  • tissue factor is now recognised as a target for therapeutic intervention in conditions associated with increased expression.
  • Factor Vila is a two-chain, 50 kilodalton (kDa) vitamin-K dependent, plasma ser- ine protease which participates in the complex regulation of in vivo haemostasis.
  • FVIIa is generated from proteolysis of a single peptide bond from its single chain zymogen, Factor VII (FVII), which is present at approximately 0.5 pg/ml in plasma.
  • the zymogen is catalytically inactive.
  • the conversion of zymogen FVII into the activated two-chain molecule occurs by cleavage of an internal peptide bond.
  • FVIIa binds with high affinity to exposed TF, which acts as a cofactor for FVIIa, enhancing the proteolytic activation of its substrates FVII, Factor IX and FX.
  • TF In addition to its established role as an initiator of the coagulation process, TF was recently shown to function as a mediator of intracellular activities either by interactions of the cytoplasmic domain of TF with the cytoskeleton or by supporting the Vila-protease dependent signaling. Such activities may be responsible, at least partly, for the implicated role of TF in tumour development, metastasis and angiogenesis. Cellular exposure of TF activity is advantageous in a crisis of vascular damage but may be fatal when exposure is sustained as it is in these various diseased states. Thus, it is critical to regulate the expression of TF activity in maintaining the health.
  • FVIIai Inactivated FVII is FVIIa modified in such a way that it is catalytically inactive. FVIia is thus not able to catalyse the conversion of FX to FXa, but still able to bind to TF in com- petition with active endogenous FVIIa and thereby inhibit the TF activity.
  • Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, or other agents may be used, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders.
  • DIC disseminated intravascular coagulation
  • heparin and other anticoagulants may, however, have undesirable side effects.
  • Available anticoagulants generally act throughout the body, rather than acting specifically at a clot site. Heparin, for example, may cause heavy bleeding. Because heparin acts as a cofactor for antithrombin III (AT III), and AT III is rapidly depleted in DIC treatment, it is often difficult to maintain the proper heparin dosage, necessitating continuous monitoring of AT III and heparin levels. Heparin is also ineffective if AT III depletion is extreme. Further, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of osteoporosis. Indandione derivatives may also have toxic side effects.
  • anticoagulant activity such as, for example, anticoagulant proteins isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries and human placenta-derived anticoagulant proteins.
  • Inhibitors of tissue factor may act as antagonists for tissue factor-mediated induction of coagulation, thus blocking the production of thrombin and the subsequent deposition of fibrin.
  • TF antagonists may be useful for inhibiting tissue factor activity resulting in, for example, the inhibition of blood coagulation, thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), platelet deposition, fibrin deposition in lungs and kidneys associated with gram-negative endotoxemia, myocardial infarction, and inflammatory responses including acute lung injury, acute respiratory distress syndrome, and systemic inflammatory re- sponse syndrome.
  • DIC disseminated intravascular coagulation
  • CIP Critical illness polyneuropathy
  • CIP systemic inflammatory response syndrome
  • SIRS systemic inflammatory response syndrome
  • CIP occurs in about 70% of patients who have the systemic inflammatory response syndrome (SIRS).
  • SIRS systemic inflammatory response syndrome
  • the neurologic effects of SIRS are thought to be mediated by released mediators like cytokines and free radicals, affecting the microcirculation of the central and peripheral nervous system. Examination of the peripheral nervous system is often unreliable, and the only way to establish a definitive diagnosis is by performing electrophysiologic studies. Morbidity and mortality rates are high.
  • compositions which act to inhibit the cellular functions of TF, which is implicated in Respiratory and Inflammatory conditions like sepsis, inflamma- tion, atherosclerosis, restenosis, or cancer.
  • One object of the present invention is to provide compositions, which can effectively be used in the treatment or prophylaxis of Thrombotic or Coagulopathic related disease, Respiratory diseases and inflammatory diseases.
  • the invention provides a pharmaceutical composition comprising a TF antagonist and a blood glucose regulator.
  • the invention provides the use of a TF antagonist in combination with a blood glucose regulator for the manufacture of a medicament for treating Thrombotic and Coagulopathic related diseases or disorders, Respiratory diseases or disorders, and Inflammatory diseases or disorders.
  • the Thrombotic and Coagulopathic related diseases or disorders, Respiratory diseases or disorders, and Inflammatory diseases or disorders include deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplastry (PTCA), stroke, tumour metastasis, inflammation, septic chock, hypotension, acute lung injury (ALI), Acute Respiratory Distress Syn- drome (ARDS), pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular restenosis, platelet deposition, myocar- dial infarction, angiogenesis, or the prophylactic
  • hypogammaglobulinemia hyperparathyroidism, ac- romegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing, and critical illness polyneuropathy (CIP) andlor multiple organ failure resulting from any of the preceding pathologic processes.
  • CIP critical illness polyneuropathy
  • the diseases or disorders are Respiratory disease and Inflammatory disease.
  • Respiratory disease and Inflammatory disease include lower respiratory diseases such as systemic inflammatory response syndrome, asthma, bronchitis, acute lung injury, acute resporatory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, pulmonary edema, pulmonary obstructive disease, endotoxin induced lung damage, non cell lung cancer; inflammatory bowel disease, sepsis, septic shock, acute respiratory distress syndrome, pan- creatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, rheumatoid arthritis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enterapathric spond
  • lower respiratory diseases
  • epicondylitis tennis elbow
  • carpal tunnel syndrome repetitive use injury (e.g., from typing), miscellaneous forms of arthritis, neuropathic joint disease (charco and joint), hemarthrosis (hemarthrosic), Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis.
  • the diseases or disorders are Thrombotic or Coagulopatic related diseases or disorders.
  • Thrombotic or Coagulopatic related disease include vascular diseases and inflammatory responses such as deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary an- gioplastry (PTCA), stroke, tumour metastasis, inflammation, septic chock, hypotension, acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular restenosis, platelet deposition, myocardial infarction, angiogenesis, or the treatment of mammals with atherosclerotic vessels at risk for thrombosis, and critical illness polyneuropathy (CIP) and/or multiple organ failure resulting from any of the preceding pathologic processes
  • CIP critical illness polyneuropathy
  • the disease or disorder is one or more of systemic inflammatory response syndrome, acute lung injury, acute respiratory distress syndrome, disseminated intravascular coagulation, sepsis, or critical illness polyneuropathy (CIP) and/or multiple organ failure resulting from any of the preceding pathologic processes.
  • the medicament is formulated for intravenous administration, preferably injection or infusion, in particular injection.
  • the medicament is formulated in single-unit dosage form; in another it is formulated in the form of a first unit dosage form comprising a preparation of a TF antagonist and a second unit dosage form comprising a preparation of a blood glucose regula- tor.
  • the invention provides a method for treating Thrombotic or Coagulopathic related disease, Respiratory disease and Inflammatory disease in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a TF antagonist and a second amount of a preparation of a blood glucose regulator, wherein the first and second amount together are effective to treat Thrombotic or Coagulopathic related disease, Respiratory disease and Inflammatory disease.
  • the TF antagonist and the blood glucose regulator are present in a ratio by mass of between about 100:1 and about 1 :100 (w/w TF antagonis blood glucose regulator).
  • the pharmaceutical composition is formulated for intravenous administration, preferably injection or infusion, in particular injection.
  • the composition contains at least one pharmaceutical acceptable excipients or carrier.
  • the TF antagonist and the blood glucose regulator are administered simultaneously.
  • the TF antagonist and the blood glucose regulator are administered sequentially.
  • the pharmaceutical composition is in sin- gle-dosage form and consists essentially of a preparation of a TF antagonist and a preparation of a blood glucose regulator, and one or more of the components selected from the list of pharmaceutical acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors.
  • the pharmaceutical composition is in the form of a first-unit dosage form and a second-unit dosage form, where the first-unit dosage form consists essentially of a preparation of a TF antagonist and one or more of the components selected from the list of pharmaceutical acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors; and the second-unit dosage form consists essentially of a preparation of a blood glucose regulator and one or more of the components selected from the list of pharmaceutical acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors.
  • the TF antagonist is factor VII polypeptides chemically inactivated in the active site.
  • the TF antagonist is an antibody against TF.
  • the antibody is a monoclonal antibody.
  • the antibody is a fully human monoclonal antibody.
  • the antibody is a humanized monoclonal antibody such as a mouse/human chimera antibody.
  • the antibody is an antibody against human TF.
  • the TF antagonist is selected from a list of a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 or F(ab')2 fragment; a bivalent fragment comprising two Fab frag- ments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment; an isolated complementarity determining region (CDR); and single chain Fv (scFv).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab)2 or F(ab')2 fragment a bivalent fragment comprising two Fab frag- ments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting of the VH and CH1 domains
  • the TF antagonist is selected from a list consisting of human and bovine Factor VII, wherein the active site residue Ser344 is modified, replaced with Gly, Met, Thr, or more preferably, Ala. Such substitution can be made separately or in combination with substi- tution(s) at other sites in the catalytic triad, which includes His193 and Asp242.
  • the TF antagonist is human factor VII, which has an amino acid substitution of the lysine corresponding to position 341 of SEQ ID NO: 1. In one embodiment of the invention, he TF antagonist is human factor VII, which has an amino acid substitution of the serine corresponding to position 344 of SEQ ID NO: 1.
  • the TF antagonist is human factor VII, which has an amino acid substitution of the aspartic acid corresponding to position 242 of SEQ ID NO: 1. In one embodiment of the invention, the TF antagonist i s human factor VII, which has an amino acid substitution of the histidine corresponding to position 193 of SEQ ID NO: 1.
  • the TF antagonist is a factor VII polypeptide selected from the list consisting of FVII-(K341A), FVII-(S344A), FVII-(D242A) and FVII-(H193A). In one embodiment, the TF antagonist is a fragment of FVII.
  • the TF antagonist is a factor VII polypeptide inactivated in the active site by reaction with a reagent selected from the list of peptide chloromethylketones or pep- tidyl cloromethanes; azapeptides; acylating agents such as various guanidinobenzoate derivatives and 3-alkoxy-4-chloroisocoumarins; sulphonyl fluorides such as 5 phenylmethylsulphon- ylfluoride (PMSF); diisopropylfluorophosphate (DFP); tosylpropylchloromethyl ketone (TPCK); to- sylysylchloromethyl ketone (TLCK); nitrophenylsulphonates; heterocyclic protease inhibitors such as isocoumarines, and coumarins.
  • a reagent selected from the list of peptide chloromethylketones or pep- tidyl cloromethanes; azapeptides; acy
  • the TF antagonist is a factor VII polypeptide inactivated in the active site by reaction with a reagent selected from the list of Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone, D-Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg chloromethylketone Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg chloromethyl ketone, 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-D-Phe
  • the blood glucose regulator are selected from the list of insulin, active insulin derivatives, insulin analogues, compounds that stimulate signal transduction medi- ated by an insulin receptor type tyrosine kinase in a cell, certain protein-tyrosine phosphatases (PTP's), other type II antidiabetica, and other biologically active substances having insulin releasing action.
  • the blood glucose regulator is porcine insulin, human insulin, or zinc salts thereof, or protamine salts thereof.
  • the blood glucose regulator is insulin aspart (i.e., Asp628 human insulin), insulin lispro (i.e., LysB28, ProB29 human insulin), insu- lin glagin (i.e., GlyA21, ArgB31, ArgB32 human insulin), or insulin detemir (i.e., des-ThrB30 human insulin gamma-LysB29 tetradecanoyl).
  • the patient is a non-diabetic patient.
  • TF antagonist or "TF antagonists”, as used herein is intended to mean any compound binding directly to TF and inhibiting the activation, or conversion, of factor X to factor Xa.
  • any such compound binding directly to tissue factor and inhibiting conversion of factor X to factor Xa may be used.
  • Anti-TF antibodies include, without limitation, antibodies preventing or inhibiting binding of FVII to tissue factor and antibodies pre- venting or inhibiting binding of FIX or FX to TF/FVlla complex.
  • the TF antagonists bind to tissue factor with high affinity and specificity but do not initiate blood coagulation.
  • TF antagonists encompass those that exhibit at least about 10%, at least about 20%, at least about 25%. at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%. at least about 80%, at least about 90%, at least about 100%, at least about 1 10%, at least about 120%, or at least about 130% of the specific TF-binding affinity of wild-type factor Vila, when tested in one or more of the TF binding assays as described in the present specification.
  • the TF antagonists exhibit at least about 75% of the binding affinity of wild-type factor Vila.
  • TF binding activity means the ability of a FVIIa polypeptide or TF antagonist to inhibit the binding of recombinant human 1251-FVIIa to cell surface human TF.
  • the TF binding activity may be measured as described in Assay 3 (of the present specification).
  • TF antagonists encompass those that exhibit less than about 50%, preferably less than about 25%, more preferably less than about 10%, or 5%, or 3%, or 2%, most preferably less than about 1 % of the specific activity of wild-type factor Vila, when tested in one or more of a clotting assay, or proteolysis assay as described in the present specification.
  • the TF antagonists for use in the present invention include, without limitation, immunoglobulin molecules and fragments thereof that have the ability to specifically bind to an antigen (i.e., TF) such as (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab')2 fragments; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment; (vi) an isolated complementarity determining region (CDR); (vii) single chain Fv (scFv); and (viii) diabodies.
  • an antigen i.e., TF
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH I domains
  • antibodies having variable and constant regions derived from human germline immunoglobulin sequences include antibodies having variable and constant regions derived from human germline immunoglobulin sequences; human antibodies including amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3; antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, e.g. the so-called humanized antibodies or human/mouse chimera anti- bodies.
  • human antibodies including amino acid residues not encoded by human germline immunoglobulin sequences e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo
  • CDRs and in particular CDR3 antibodies in which CDR sequences derived from the germline of another mammalian species, such as
  • the TF antagonists for use in the present invention also encompass, without limitation, factor VII polypeptides that has substantially reduced ability to catalyse the conversion of factor X to factor Xa ("inactivated" factor VII polypeptides).
  • factor VII polypeptides that has substantially reduced ability to catalyse the conversion of factor X to factor Xa
  • activated factor VII polypeptides include, without limitation, native Factor VII, as well as factor Vll-related polypeptides that have either been chemically modified relative to human factor VII and/or contain one or more amino acid sequence alterations relative to native Factor VII (i.e., Factor VII variants), and/or contain truncated amino acid sequences relative to native Factor VII (i.e., Factor VII fragments).
  • polypeptides with a slightly modified amino acid sequence for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human factor Vila.
  • factor Vll-related polypeptides may exhibit different properties relative to na- tive Factor VII, including stability, phospholipid binding, altered specific proteolytic activity, and the like.
  • Factor Vll-related polypeptides also include proteolytically inactive variants of Factor VII.
  • variant or “variants”, as used herein, is intended to designate human Factor VII having the sequence of SEQ ID NO: 1, wherein one or more amino acids of the parent protein have been substituted by another amino acid and/or wherein one or more amino acids of the parent protein have been deleted and/or wherein one or more amino acids have been inserted in the protein and/or wherein one or more amino acids have been added to the parent protein.
  • Factor VII polypeptides are intended to include Factor VII polypeptides in their uncleaved (zymogen) form as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated "factor Vila polypeptides" (or “activated factor VII polypeptides”). Typically, FVII is cleaved between residues 152 and 153 to yield FVIIa.
  • factor VII polypeptide is also intended to encompass, without limitation, polypeptides having the amino acid sequence 1-406 of wild-type human Factor VII (as disclosed in U.S. Patent No.
  • factor VII polypeptides include polypeptides that exhibit at least about 70%, preferably at least about 80 %, more preferably at least about 90 %, and most preferable at least about 95 %, of identity with the amino acid sequence of wild-type factor VII as disclosed in U.S. Patent No. 4,784,950.
  • Non-limiting examples of factor VII variants having substantially reduced or modified biological activity relative to wild-type factor VII include R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270:66-72, 1995). FFR-FVIIa (Hoist et al., Eur. J. Vase. Endovasc. 5urg. 15:515-520. 1998). and factor Vila lacking the Gla domain, (Nicolaisen et al., FEBS Letts. 317:245-249, 1993).
  • Non-limiting examples also include human FVIIa, which has an amino acid substitution of the lysine corresponding to position 341 of SEQ ID NO: 1; human FVIIa, which has an amino acid substitution of the serine corresponding to position 344 of SEQ ID NO: 1; human FVIIa, which has an amino acid substitution of the aspartic acid corresponding to position 242 of SEQ ID NO: 1; human FVIIa, which has an amino acid substitution of the histidine corresponding to position 193 of SEQ ID NO: 1; FVII-(K341A); FVII-(S344A); FVIL(D242A); and FVII-(H193A).
  • Non-limiting examples of chemically modified factor VII polypeptides and sequence variants are described, e.g., in US. Patent No. 5,997,864.
  • Non-limiting examples of FVII-derived peptides having TF/FVlla antagonist activity are described in International patent applications WO 90/03390, WO 95/00541, WO 96/18653, and European Patent EP 500800.
  • the catalytic activity of Factor Vila can be inhibited by chemical derivatization of the catalytic centre, or triad. Derivatization may be accomplished by reacting Factor VII with an irreversible inhibitor such as an organophosphor compound, a sulfonyl fluoride, a peptide ha- lomethyl ketone or an azapeptide, or by acylation, for example, peptide chloromethylketones or peptidyl cloromethanes; azapeptides; acylating agents such as various guanidinobenzoate derivatives and 3-alkoxy-4-chloroisocoumarins; sulphonyl fluorides such as phenylmethylsulphon- ylfluoride (PMSF); diisopropylfluorophosphate (DFP); tosylpropylchloromethyl ketone (TPCK); to- sylysylchloromethyl ketone (TLCK); nitrophenylsulphon
  • amino acid substitutions are made in the amino acid se- quence of the Factor VII catalytic triad, defined herein as the regions which contain the amino acids which contribute to the Factor Vila catalytic site.
  • the substitutions, insertions or deletions in the catalytic triad are generally at or adjacent to the amino acids which form the catalytic site.
  • the amino acids which form a catalytic "triad" are Ser344, Asp242, and His193 (subscript numbering indicating position in SEQ ID NO:1).
  • the cata- lytic sites in Factor VII from other mammalian species may be determined using presently available techniques including, among others, protein isolation and amino acid sequence analysis.
  • Catalytic sites may also be determined by aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined (Sigler et al., J. Mol. Biol., 35:143-164 (1968). incorporated herein by reference), and therefrom determining from said alignment the analogous active site residues.
  • the amino acid substitutions, insertions or deletions are made so as to prevent or otherwise inhibit activation by the Factor Vila of Factors X and/or IX.
  • the Factor VII so modified should, however, also retain the ability to com- pete with authentic Factor VII and/or Factor Vila for binding to tissue factor in the coagulation cascade.
  • Such competition may readily be determined by means of, e.g., a clotting assay as described herein, or a competition binding assay using, e.g., a cell line having cell-surface tissue factor, such as the human bladder carcinoma cell line J82 (Sakai et al. J. Biol. Chem. 264: 9980-9988 (1989)).
  • a cell line having cell-surface tissue factor such as the human bladder carcinoma cell line J82 (Sakai et al. J. Biol. Chem. 264: 9980-9988 (1989)).
  • the amino acids which form the catalytic site in Factor VII such as Ser344, Asp242, and
  • His193 in human and bovine Factor VII may either be substituted or deleted. It is preferred to change only a single amino acid, thus minimizing the likelihood of increasing the antigenicity of the molecule or inhibiting its ability to bind tissue factor; however two or more amino acid changes (substitutions, additions or deletions) may be made and combinations of substitution(s), addition(s) and deletion(s) may also be made.
  • Ser344 is preferably substituted with Ala, but Gly, Met, Thr or other amino acids can be substituted. It is preferred to replace Asp with Glu and to replace His with Lys or Arg. In general, substitutions are chosen to disrupt the tertiary protein structure as little as possible.
  • One may introduce residue alterations as described above in the catalytic site of appropriate Fac- tor VII sequence of human, bovine or other species and test the resulting protein for a desired level of inhibition of catalytic activity and resulting anticoagulant activity as described herein.
  • the active site residue Ser344 is modified, replaced with Gly, Met, Thr, or more preferably, Ala. Such substitution could be made separately or in combination with substitution(s) at other sites in the catalytic triad, which includes His193 and Asp242.
  • TF antagonists that can be used in accordance with the present invention include, but are not limited to:
  • CNTO-859 Humanised TF-MAb blocking Factor X binding to the TF/FVlla complex, CNTO-859 from Centocor (subsidiary of J&J); CNTO-859 is a humanised version of the murine TF-MAb, 5G8, developed by Scripps investigated for treatment of sepsis.
  • Immunoconjugate proteins constructed as a dimer of two identical chains, each having an effector domain which is the Fc region of an IgGI immunoglobulin conjugated to a targeting domain which is a mutant form of factor VII that binds to tissue factor but does not initiate blood coagulation, as disclosed in WO 01/02439 (Garen).
  • any compound may be used, which is able to regulate the blood glucose level. Blood glucose levels may be controlled by insulin treatment.
  • active insulin derivatives and its physiologically tolerated salts and other blood glucose regulators can be used to obtain the same result.
  • compounds of the group of biologically active substances having insulin releasing action can be used in practising the present invention.
  • Such compounds, capable of promoting the secretion of insulin are known, e.g., the Islets-Activating Proteins (Ui; Michio et al. US 5000953, 19 March 1991) and the glucagon-like peptides (Habener; Joel F. Newton Highlands, MA, US 5614492, 25 March 1997).
  • Compounds that specifically modulate the activity of the controlling RPTP, thereby prolonging or enhancing signal transduction mediated by the insulin receptor can thus be used in accordance with the present invention.
  • Such compounds have low toxicity since they are specific for the PTPs associated with insulin receptor activity, and do not significantly affect the activity of other PTPs that are non-specific.
  • Non-limiting examples of blood glucose regulators are insulin, active insulin derivatives, insulin analogues, compounds that stimulate signal transduction mediated by an insulin receptor type tyrosine kinase in a cell, certain protein-tyrosine phosphatases (PTP's), other type II antidia- betica, and other biologically active substances having insulin releasing action.
  • insulin refers to insulin from any species such as porcine insulin, bovine insulin, and human insulin and salts thereof such as zinc salts, and protamin salts.
  • active derivatives of insulin are what a skilled art worker generally considers derivatives, including, for example, insulin having a substituent not present in the parent insulin molecule.
  • insulin analogues refers to insulin wherein one or more of the amino acid residues have been exchanged with another amino acid residue and/or from which one or more amino acid residue has been deleted and/or from which one or more amino acid residue has been added with the proviso that said insulin analogue has a sufficient insulin activity.
  • any skilled art worker for example, a physician, knows when and which dosages to administer of the insulin analogue. Examples of insulin analogues are described in the following patents and equivalents thereto: US 5,618,913, EP 254,516, EP 280,534, US 5,750,497, and US 6,01 1,007.
  • insulin analogues examples include insulin aspart (i.e., AspB28 human insulin), insulin lispro (i.e., LysB28, ProB29 human insulin), and insulin glagin (i.e., GlyA21, ArgB31, ArgB32 human insulin).
  • insulin aspart i.e., AspB28 human insulin
  • insulin lispro i.e., LysB28, ProB29 human insulin
  • insulin glagin i.e., GlyA21, ArgB31, ArgB32 human insulin.
  • insulin detemir i.e., des-ThrB30 human insulin gamma-LysB29 tetradecanoyl.
  • amino acids mentioned herein are L-amino acids. It is to be understood, that the first letter in, for example, K337 represent the amino acid naturally present at the indicated position wild-type factor VII, and that, for example, [K337A]-FVIIa designates the FVII-variant wherein the amino acid represented by the one-letter code K naturally present in the indicated position is replaced by the amino acid represented by the one-letter code A.
  • Table 1 Abbreviations for amino acids:
  • factor Vila or "FVIIa” may be used interchangeably.
  • systemic inflammatory response syndrome refers to the uncontrolled disease process which ensues an initial insult and which gives raise to a multisystem disturbance secondary to inflammatory mediators released during shock.
  • SIRS infectious insult leading to the initial shock phase
  • inflammatory cells such as TNFs, interleukins, bradykinins etc.
  • insulin receptor type tyrosine kinase refers to a post- receptor signal transduction pathway involved in the insulin signaling.
  • endoneural edema refers to swelling of the neuronal cells.
  • phrenic nerves refers to the left and right nervus phrenicus
  • non-diabetic patient refers to a patient who has not been diagnosed as having diabetes.
  • a “critically ill patient” refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, a patient who is being operated and where complications supervene, and a patient who has been operated in a vital organ within the last week or has been subject to major surgery within the last week.
  • CIP critically ill patient
  • the term a “critically ill patient”, as used herein refers to a patient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, or a patient who is being operated and where complications supervene.
  • critically ill patient refers to a pa- tient who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury.
  • these definitions apply to similar expressions such as "critical illness in a patient” and a "patient is critically ill " .
  • Intensive Care Unit refers to the part of a hospital where critically ill patients are treated. Of course, this might vary from country to country and even from hospital to hospital and said part of the hospital may not necessary, officially, bear the name "Intensive Care Unit” or a translation or derivation thereof. Of course, the term “Intensive Care Unit” also covers a nursing home, a clinic, for example, a private clinic, or the like if the same or similar activities are performed there.
  • active site and the like when used herein with reference to FVIIa, refers to the catalytic and zymogen substrate binding site, including the 'S1 " site of FVIIa as that term is defined by Schecter, I. and Berger, A, (1967) Biochem. Biophys. Res. Commun. 7:157-162.
  • TF-mediated coagulation activity means coagulation initiated by TF through the formation of the TF/FVlla complex and its activation of FIX and Factor X to FIXa and FXa, re- spectively.
  • TF-mediated coagulation activity is measured in a FXa generation assay.
  • FXa generation assay as used herein is intended to mean any assay where activation of FX is measured in a sample comprising TF, FVIIa, FX, calcium and phospholipids. Examples of FXa generation assays are described in assay 1 and assay 2 (of the present specification).
  • a TF/FVlla mediated or associated process or event, or a process or event associated with TF-mediated coagulation activity, is any event, which requires the presence of TF/FVlla.
  • Such processes or events include, but are not limited to, formation of fibrin which leads to thrombus formation; platelet deposition; proliferation of smooth muscle cells (SMCs) in the vessel wall, such as, for example, in intimal hyperplasia or restenosis, which is thought to result from a complex interaction of biological processes including platelet deposition and thrombus formation, release of chemotactic and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the intima of an arterial segment; and deleterious events associated with post-ischemic reperfusion, such as, for example, in patients with acute myocardial infarction undergoing coronary thrombolysis.
  • SMCs smooth muscle cells
  • a process mediated by or associ- ated with TF/FVlla, or a TF-mediated coagulation activity includes any step in the coagulation cascade from the formation of the TF/FVlla complex to the formation of a fibrin platelet clot and which initially requires the presence of TF/FVlla.
  • the TF/FVlla complex initiates the extrinsic pathway by activation of factor X to factor Xa, FIX to FIXa, and additional FVII to FVIIa.
  • a TF/FVlla mediated or associated process, or TF-mediated coagulation activity can be conveniently measured employing standard assays such as, e.g., those described in Roy, S., (1991) J. Biol. Chem. 266:4665-4668, and O'Brien, D. et al., (1988) J. Clin. Invest. 82:206-212 for the conversion of factor X to factor Xa in the presence of TF/FVlla and other necessary reagents.
  • TF related diseases or disorders means any disease or disor- der, where TF is involved. This includes, but are not limited to diseases or disorders related to TF- mediated coagulation activity, thrombotic or coagulopathic related diseases or disorders or diseases or disorders such as inflammatory responses and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular disorders such as deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percu- taneous transdermal coronary angioplastry (PTCA), stroke, tumour metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis following angioplastry, acute and chronic indications such as inflammation, septic chock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or the prophylactic treatment of mammals
  • the TF related diseases or disorders are not limited to in vivo coagulopatic disorders such as those named above, but includes ex vivo TF/FVlla related processes such as coagulation that may result from the extracorporeal circulation of blood, including blood removed in-line from a patient in such processes as dialysis procedures, blood filtration, or blood bypass during surgery.
  • peptides, proteins and amino acids as used herein can comprise or refer to "natural", i.e., naturally occurring amino acids as well as “non-classical" D-amino acids including, but not limited to, the D-isomers of the common amino acids, alpha-isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, designer amino acids such as beta- methyl amino acids, C-alpha-methyl amino acids, N-alpha-methyl amino acids, and amino acid analogues in general.
  • amino acids can include Abu, 2-amino butyric acid; gamma-Abu, 4-aminobutyric acid; epsilon-Ahx, 6-aminohexanoic acid; Aib, 2-amino-isobutyric acid; beta-Ala, 3-aminopropionic acid; Orn, ornithine; Hyp, trans-hydroxyproline; Nle, norleucine; Nva, norvaline.
  • GLA' as used herein means 4-carboxyglutamic acid (gamma- carboxyglutamate).
  • human tissue factor or "human TF” as used herein, refers to the full length polypeptide receptor comprising the amino acid sequence 1-263 of native human tissue factor.
  • the term 'antibody' as used herein, is intended to refer to immunoglobulin molecules and fragments thereof, which have the ability to specifically bind to an antigen (e.g., human TF).
  • Full-length antibodies comprises four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, Each heavy chain is comprised of a heavy chain vari- able region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity deter- mining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity deter- mining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1, CDRI, FR2, CDR2, FR3, CDR3, FR4.
  • an antibody within the definition of an antibody are also one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TF). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • an antigen e.g., human TF
  • binding fragments encompassed within the term "antibody” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab)2 and F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546 ), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab)2 and F(ab')2 fragments a bivalent fragment comprising two
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombi- nant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv
  • single chain antibodies are also intended to be encompassed within the term "antibody”.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak. R. J., et al. (1994) Structure 2:1121-1123).
  • human TF may have one or more antigenic determinants comprising (1) pep- tide antigenic determinants which consist of single peptide chains within human TF, (2) confor- mational antigenic determinants which consist of more than one spatially contiguous peptide chains whose respective amino acid sequences are located disjointedly along the human TF polypeptide sequence; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to human TF after translation, such as carbohydrate groups, or the like.
  • antigenic determinants comprising (1) pep- tide antigenic determinants which consist of single peptide chains within human TF, (2) confor- mational antigenic determinants which consist of more than one spatially contiguous peptide chains whose respective amino acid sequences are located disjointedly along the human TF polypeptide sequence; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to human TF after translation
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • humanized antibody is intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences; the term “humanized antibody” can be used interchangeably with “chimeric antibody”, and “chimera antibodies”, (e.g., human/mouse chimera antibodies”).
  • a FVIIa inhibitor may be identified as a substance, which reduces the amidolytic activity by at least 50% at a concentration of the substance at 400 micromolar ( ⁇ M) in the FVIIa amidolytic assay described by Persson et al. (Persson et al., J. Biol. Chem. 272: 19919-19924 (1997)).
  • Preferred are substances reducing the amidolytic activity by at least 50% at a concentration of the substance at 300 ⁇ M; more preferred are substances reducing the amidolytic activity by at least 50% at a concentration of the substance at 200 ⁇ M.
  • the " FVIIa inhibitor” may be selected from any one of several groups of FVIIa directed inhibitors. Such inhibitors are broadly categorised for the purpose of the present invention into i) inhibitors which reversibly bind to FVIIa and are cleavable by FVIIa, ii) inhibitors which reversi- bly bind to FVIIa but cannot be cleaved, and iii) inhibitors which irreversibly bind to FVIIa .
  • inhibitors of serine proteases see Proteinase Inhibitors (Research Monographs in cell and Tissue Physiology; v. 12) Elsevier Science Publishing Co., Inc., New York (1990).
  • the FVIIa inhibitor moiety may also be an irreversible FVIIa serine protease inhibitor.
  • Such irreversible active site inhibitors generally form covalent bonds with the protease active site.
  • Such irreversible inhibitors include, but are not limited to, general serine protease inhibitors such as peptide chloromethylketones (see, Williams et al., J. Biol. Chem.
  • peptidyl cloromethanes or peptidyl cloromethanes; azapeptides; acylating agents such as various guanidinobenzoate derivatives and the 3-alkoxy-4-chloroisocoumarins; sulphonyl fluorides such as phenylmethylsul- phonyl-fluoride (PMSF); diisopropylfluorophosphate (DFP); tosylpropylchloromethyl ketone (TPCK); tosyllysylchloromethyl ketone (TLCK); nitrophenylsulphonates and related compounds; heterocyclic protease inhibitors such as isocoumarines, and coumarins.
  • PMSF phenylmethylsul- phonyl-fluoride
  • DFP diisopropylfluorophosphate
  • TPCK tosylpropylchloromethyl ketone
  • TLCK tosyllysylchloromethyl ket
  • peptidic irreversible FVIIa inhibitors include, but are not limited to, 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, L- Glu-Gly-Arg chloromethylketone and D-Glu-Gly-Arg chloromethylketone.
  • FVIIa inhibitors also include benzoxazinones or heterocyclic analogues thereof such as described in PCT/DK99/00138.
  • FVIIa inhibitors include, but are not limited to, small peptides such as for example Phe-Phe-Arg, D-Phe-Phe-Arg, Phe-Phe-Arg, D-Phe-Phe-Arg, Phe-Pro-Arg, D-Phe-Pro- Arg.
  • Phe-Pro-Arg, D-Phe-Pro-Arg, L- and D-Glu-Gly-Arg; peptidomimetics; benzamidine systems; heterocyclic structures substituted with one or more amidino groups; aromatic or heteroaromatic systems substituted with one or more C( NH)NHR groups in which R is H, C1-3alkyl. OH or a group which is easily split of in vivo.
  • Thrombotic or coagulopathic related diseases or disorders includes vascular diseases and inflammatory responses including, without limitation, deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplastry (PTCA), stroke, tumour metastasis, inflammation, septic chock, hypotension, acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular restenosis, platelet deposition, myocardial infarction, angiogenesis, or the treatment of mammals with atherosclerotic vessels at risk for thrombosis, and multiple organ failure resulting from any of the preceding pathologic processes.
  • vascular diseases and inflammatory responses including, without limitation, deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass
  • Respiratory Diseases or disorders Exemplified by lower respiratory diseases such as systemic inflammatory response syndrome, asthma, bronchitis, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, pulmonary edema, pulmonary obstructive disease, endotoxin induced lung damage, non cell lung cancer, and multiple organ failure resulting from any of the preceding pathologic processes.
  • lower respiratory diseases such as systemic inflammatory response syndrome, asthma, bronchitis, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, pneumonia, pulmonary edema, pulmonary obstructive disease, endotoxin induced lung damage, non cell lung cancer, and multiple organ failure resulting from any of the preceding pathologic processes.
  • Inflammatory Diseases or disorders refers to diseases such as inflammatory bowel disease, sepsis, septic shock, acute respiratory distress syndrome, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, rheumatoid arthritis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spond- yarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enterapathric spondylitis, juvenile arthropathy or juvenile ankylosing spondylitis, reactive arthropathy, infectious or post-infectious arthritis, gonoccocal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitic syndromes, polyarteritis nodosa, hypersensitivity vasculitis
  • terapéuticaally effective interval is a period of time beginning when one of either (a) the TF antagonist or (b) The blood glucose regulator is administered to a mammal and ending at the limit of the beneficial effect in preventing or ameliorating respiratory or inflammatory disease or associated CIP and/or organ failure of (a) or (b).
  • sole agents or factors refers to situations in which the TF antagonist and the blood glucose regulator, taken together, are the only haemostatic agents, or active haemostatic agents, or coagulation factors contained in the pharmaceutical composition or kit, or are the only haemostatic agents, or active haemostatic agents, or coagulation factors administered to the patient in the course of a particular treatment, such as, e.g., in the course of a particular bleeding episode. It will be understood that these situations encompass those in which other haemostatic agents or coagulation factors, as applicable, are not present in either sufficient quantity or activity so as to significantly influence one or more coagulation parameter.
  • Clot lysis time, clot strength, fibrin clot formation, and clotting time are clinical parameters used for assaying the status of patient's haemostatic system. Blood samples are drawn from the patient at suitable intervals and one or more of the parameters are assayed by means of, e.g., thromboelastograpy as described by, e.g., Meh et al., Blood Coagulation & Fibrinolysis
  • treatment is meant to include both prevention of an expected unwanted clotting, and regulation of an already occurring clotting. Prophylactic administration of a preparation of a TF antagonist and a blood glucose regulator is thus included in the term “treatment”.
  • subject as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term “patient”.
  • the TF antagonist and the blood glucose regulator as defined in the present specification may be administered simultaneously or sequentially.
  • the factors may be supplied in single- dosage form wherein the single-dosage form contains both coagulation factors, or in the form of a kit-of-parts comprising a preparation of a TF antagonist as a first unit dosage form and a preparation of a blood glucose regulator as a second unit dosage form.
  • a first or sec- ond or third, etc., unit dose is mentioned throughout this specification this does not indicate the preferred order of administration, but is merely done for convenience purposes.
  • a preparation of a TF antagonist and a preparation of a blood glucose regulator administration of the coagulation factor proteins in single- dosage form, or administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of no more than 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes. Either factor may be administered first.
  • sequential dosing administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of more than 15 minutes.
  • Either of the two unit dosage form, or coagulation factor proteins, may be administered first.
  • both products are injected through the same intravenous access.
  • APTT or "aPTT” is meant the activated partial thromboplastin time (described by, e.g., Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin; a simple screening test for first-stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212, 1961).
  • Half-life refers to the time required for the plasma concentration of a TF antagonist or a blood glucose regulator to decrease from a particular value to half of that value.
  • the total amount of protein in a preparation may be measured by generally known methods, e.g, by measuring optical density.
  • Amounts of proteins (“antigen ") may be measured by generally known methods such as standard Elisa immuno assays. In general terms, such assay is conducted by contacting, e.g., a solution of the factor Vll-containing preparation with an anti- FVII antibody immobilised onto the Elisa plate, subsequently contacting the immobilised anti- body-FVII complex with a second anti-FVII antibody carrying a marker, the amounts of which, in a third step, are measured. The amounts of each polypeptide present may be measured in a similar way using appropriate antibodies.
  • the total amount of protein present in a preparation is detemined by adding the amounts of the individual proteins.
  • the preparation comprises isolated coagulation factors.
  • the preparation i s free of coagulation factor II and coagulation factor Ma (prothrombin and thrombin) and/or factor X or Xa.
  • isolated refers to polypeptides, e.g., insulin, that have been separated from the cell in which they were synthesized or the medium in which they are found in nature (e.g., plasma or blood). Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like.
  • Separation of polypeptides from the medium in which they naturally occur may be achieved by any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-factor VII antibody column; hydro- phobic interaction chromatography; ion-exchange chromatography; size exclusion chromatogra- phy; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like.
  • affinity chromatography such as, e.g., on an anti-factor VII antibody column
  • hydro- phobic interaction chromatography such as, e.g., on an anti-factor VII antibody column
  • ion-exchange chromatography such as, e.g., on an anti-factor VII antibody column
  • size exclusion chromatogra- phy size exclusion chromatogra- phy
  • electrophoretic procedures e.g., preparative isoelectric
  • an "effective amount" of a TF antagonist and a blood glucose regulator is defined as the amount of a TF antagonist, e.g., inactivated FVIIa or a TF an- tibody, and a blood glucose regulator that together suffices to cure, alleviate or partially arrest the disease and its complications.
  • terapéuticaally effective combination means administration of both (a) a TF antagonist and (b) a blood glucose regulator, either simultaneously or separately.
  • Active Ingredient refers to a combination of (a) a TF antagonist and (b) a blood glucose regulator co-present in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention.
  • injectable liquid carrier refers to a liquid medium containing either or both of (a) a TF antagonist, or (b) a blood glucose regulator; wherein (a) and (b) are independently dissolved, suspended, dispersed, or emulsified in the liquid medium.
  • DNA sequences encoding a specific protein may be isolated by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the protein by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., above).
  • the DNA sequence encoding the protein is preferably of human origin, i.e. derived from a human genomic DNA or cDNA library.
  • Polypeptide variants may be made by amino acid sequence alterations of the polypeptide, which may be accomplished by a variety of techniques. Modification of the DNA sequence may be by site-specific mutagenesis. Techniques for site-specific mutagenesis are well known in the art and are described by, for example, Zoller and Smith (DNA 3:479-488, 1984).
  • the DNA sequences encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • DNA sequences may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202, Saiki et al., Science 239 (1988), 487 - 491, or Sambrook et al., supra.
  • the host cell into which the DNA sequences encoding the polypeptides is introduced may be any cell, which is capable of producing the posttranslational modified human FVII polypeptides and includes yeast, fungi and higher eucaryotic cells. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327- 341; Loyter et al., Proc. Natl. Acad. Sci.
  • the antibodies are monoclonal antibodies.
  • Monoclonal antibodies may be prepared, e.g., according to the methods of E. Harlow and D. Lane, edi- tors, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York.
  • a cloned wild-type FVII DNA sequence is used for the preparation of recombinant human FVII polypeptides. This sequence may be modified to encode a desired FVII variant.
  • the complete nucleotide and amino acid sequences for human FVII are known; see US. Pat. No. 4,784,950, wherein the cloning and expression of recombinant human FVII is described.
  • the bovine FVII sequence is described in Takeya et al., J. Biol. Chem, 263:14868-14872 (1988), which is incorporated by reference herein.
  • DNA sequences for use will typically encode a pre-pro peptide at the amino-terminus of the FVII protein to obtain proper post-translational processing (e.g. gamma-carboxylation of glu- tamic acid residues) and secretion from the host cell.
  • the pre-pro peptide may be that of FVII or another vitamin K-dependent plasma protein, such as factor IX, factor X, prothrombin, protein C or protein S.
  • additional modifications can be made in the amino acid sequence of FVII where those modifications do not significantly have impact on the ability of the protein to act as a coagulation factor.
  • FVII modified in the catalytic triad can also be modified in the activation cleavage site to inhibit the conversion of zymogen FVII into its activated two-chain form, as generally described in US. Pat. No. 5,288,629.
  • Factor VII polypeptides for use in the present invention may be prepared, e.g., as described in International Applications Nos. WO 92/15686. WO 94/27631 and WO 96/12800; Wildgoose et al., Biochem 29:3413-3420, 1990; Kazama et al., J. Biol. Chem. 27056-72, 1995; Hoist et al., Eur. J. Vase. Endovasc. Surg. 15:515-520, 1998; and Nicolaisen et al., FEBS Letts. 317:245- 249, 1993.
  • FVII polypeptides produced as described above may be purified by affinity chromatography on an anti-FVII antibody column.
  • the immunoadsorption column comprises a high- specificity monoclonal antibody, such as, e.g., a calcium-dependent monoclonal antibody as described by Wakabayashi et al., J. Biol. Chem, 261 :11097-11 108, (1986) and Thim et al., Biochem. 27: 7785-7793, (1988).
  • Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography.
  • Conversion of single-chain FVII to active two-chain FVIIa may be achieved using factor Xlla as described by Hedner and Kisiel (1983, J. Clin. Invest. 71 : 1836-1841), or using other proteases having trypsin-like specificity (Kisiel and Fujikawa, Behring Inst. Mitt. 73: 29-42, 1983).
  • Alter- natively FVII may be autoactivated by passing it through an ion-exchange chromatography column such as mono Q.RTM. (Pharmacia Fire Chemicals) or the like (Bjoern et al., 1986, Research Disclosures 269564-565).
  • the preparations of the present invention may be used to treat thrombotic and coagulopathic related diseases or disorders, respiratory diseases or disorders, and inflammatory diseases or dis- orders including, without limitation, deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplastry (PTCA), stroke, tumour metastasis, inflammation, septic chock, hypotension, acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular resteno- sis, platelet deposition, myocardial infarction, angiogenesis, or the prophylactic treatment of mammals with atherosclerotic vessels at risk for thrombosis; asthma, bronchitis, idiopathic pulmonary fibrosis, pneumonia, pulmonary edema, pulmonary obstructive
  • joint cell arteritis calcium crystal deposition arthropathris, pseudogout, non-articular rheumatism, bursitis, tenosynomitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (typing), miscellaneous forms of arthritis, neuropathic joint disease (charco and joint), hemarthrosis (hemarthroic), Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, surcoilosis, hemo- chromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypo- gammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing, and multiple organ failure resulting from any of the preceding pathologic processes.
  • the essential ingredients (a) a TF antagonist and (b) a blood glucose regulator are present in the formulation in such proportion that a dose of the formulation provides an amount of each ingredient that together is a pharmaceutically effective amount to the patient being treated.
  • the dose of composition of the invention to be administered is determined depending upon age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment etc.
  • the weight ratio of TF antagonist and the amount of blood glucose regulator e.g., insulin or a biologically active fragment or variant thereof
  • the ratio of TF antagonist to blood glucose regulator may thus be, e.g., about 1 :100, or 1 :90, or 1 :80 or 1 :70 or 1 :60, or 1 :50 or 1 :40, or 1 :30, or 1 :20, or 1:10, or 1 :5, or 1 :2, or 1 :1, or 2:1, or 5:1, or 10:1, or 20:1, or 30:1, or 40:1, or 50:1, or 60:1, or 70:1, or 80:1, or 90:1, or 100:1; or between about 1 :90 to about 1 :1, or between about 1 :80 to about 1:2, or between about 1 :70 to about 1 :5, or between about 1 :60 to about 1 :10 , or between about 1 :50 to about 1 :25, or between about 1 :40 to about 1 :30, or between about 90:1 to about 1 :1, or between about 80:1 to about 2:1,
  • the dose of the TF antagonist ranges from about 0.05 to about 500 mg/day; e.g., from about 1 to about 200 mg/day, or, e.g., from about 5 to about 175 mg/day for a 70-kg subject as loading and maintenance doses, depending on the weight of the subject, the condition and the severity of the condition.
  • the dose of the blood glucose regulator is regulated in such a way that the blood glucose level is reduced.
  • the blood glucose regulator e.g., insulin
  • the blood glucose regulator is capable of main- taing blood glucose at or below 130 mg per deciliter patient plasma, preferably at or below 110 mg/dl.
  • the blood glucose levels are regulated and maintained within a range where the lower limit can be selected to be about 60, about 70 or about 80 mg/dL and the upper limit can be selected to be about 110, about 120 or about 130 mg/dL, more specifically to the normal range (i.e., from about 80 to about 1 10 mg/dL).
  • the range is from about 60 to about 130, preferably, from about 70 to about 120, more preferred, from about 80 to about 110 mg/dL.
  • compositions of the invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threaten- ing situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of factor Vila and insulin in humans, it is possible and may be felt desirable by the treating physician to administer a substantial excess of these compositions.
  • a dose may be given continuously or intermittently (once or several times a day).
  • the essential ingredients; TF antagonist and blood glucose regulator are co-present and may be mixed in any homogeneous or non-homogeneous manner or adjacently or otherwise proximately placed together in an individual dosage unit suitable for practicing the method of the invention.
  • the dosage unit of the TF antagonist and/or blood glucose regulator will usually be admixed with a carrier or inert ingredients, or diluted by a carrier, or enclosed within a carrier which may be in the form of an ampoule (e.g., for use in a pen device, a pump device, or other injection or infusion device), capsule, time release dosing device, sachet, paper or other container.
  • a carrier or inert ingredients e.g., for use in a pen device, a pump device, or other injection or infusion device
  • a carrier which may be in the form of an ampoule (e.g., for use in a pen device, a pump device, or other injection or infusion device), capsule, time release dosing device, sachet, paper or other container.
  • the carrier when it serves as a diluent, it may be a solid, semi-solid, paste, or liquid material which acts as a vehicle, or can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), or ointment, containing, for example, up to 10% by weight of the active compound.
  • the dosage unit of the active ingredients will usually be admixed with a liquid carrier and/or other inert ingredients or enclosed within a carrier which may be in the form of an ampoule, bottle, time release dosing device or other container.
  • the carrier when it serves as a diluent, it may be a liquid material which acts as a vehicle, or can be in the form of solutions containing, for example, up to 10% by weight of the active compound.
  • the carrier may be an injectable liquid medium such as is well known in the art.
  • the injectable liquid must be such that permits parenteral administration, that is, introduction of substances to a mammal being treated by intervenous, sub- cutaneous, intramuscular, or intramedullary injection. Intravenous injection is most preferred as a means of administration.
  • the Active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile water containing saline and/or sugars and- lor suspension agents or a mixture of both.
  • a pharmaceutically acceptable carrier such as sterile water, sterile water containing saline and/or sugars and- lor suspension agents or a mixture of both.
  • the compounds of the invention may be dissolved at a concentration of about 2 mg/ml in a 4% dex- trose/0.5% sodium citrate aqueous solution.
  • Liquid compositions for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art such as distilled water or ethanol.
  • compositions may also comprise adjuvants such as wetting and suspending agents, and sweetening, flavouring, perfuming and preserving agents.
  • Other compositions for oral administration include spray compositions which may be prepared by known methods and which comprise one or more of the active compound(s).
  • compositions may also comprise stabilizers such as sodium bisulfite and buffer for isotonicity, for example sodium chloride, sodium citrate or citric acid. The manufacturing methods of spray compositions for inhalation therapy is described in detail in the art.
  • Preparations for injection according to the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions.
  • aqueous solvents or suspending media examples are distilled water for injection and physiological salt solution.
  • non-aqueous solvents or suspending media are pro- pylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, Poly- sorbate 80 (registered Trade Mark).
  • These compositions may also include adjuvants such as pre- serving, wetting, emulsifying and dispersing agents stabilizing agents (e.g. lactose) and solubiliz- ers (e.g. glutamic acid and asparaginic acid).They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporation of sterilizing agents in the compositions or by irradiation. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
  • the TF antagonist and/or the blood glucose regulator may be in the form of powder, tablet or capsule.
  • a solid carrier can be one or more substances which may also act as flavouring agents, lubricants, solubilizers, suspending agents, binders, tablet disintegrating agents and encapsulating material. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes, and cocoa butter.
  • the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly; intravenously being most preferred.
  • compositions of the invention may also be administered by continuous or pulsatile infusion.
  • Local delivery of the preparations of the present invention may be carried out, e.g., by means of a spray, perfusion, double balloon catheters, stents, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods.
  • One skilled in this art may formulate the compositions of the invention an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.
  • This invention is a method of treating or preventing thrombotic and coagulopathic diseases, Inflammatory Disease or Respiratory Disease by administering to a mammal in need thereof a therapeutically effective amount of (a) a TF antagonist and b) a blood glucose regula- tor; wherein (a) and (b) are both administered within a therapeutically effective interval.
  • the administration of (a) or (b) to, e.g., a septic or CIP patient may be either continuous or intermittent.
  • the blood glucose regulator and the TF antagonist can be delivered simultaneously.
  • One convenient method of simultaneous delivery is to use the compositions of the invention, wherein the Active Ingredient has the essential ingredients co-present in a unit dosage form. Solutions or suspensions of mixed essential ingredients may, if desired, be delivered from the same liquid holding bag.
  • Another method of simultaneous delivery is to deliver the two compounds to the patient separately but simultaneously.
  • some TF antagonists may be given as an oral formulation at the same time as a blood glucose regulator is given par- enterally. Dosage of a TF antagonist can begin simultaneously with the blood glucose regulator administration. The duration of the TF antagonist administration can extend past the blood glucose regulator administration, or vice versa.
  • the blood glucose regulator is administered first; the level of blood glucose is monitored, and when the level of blood glucose has been reduced to a level at or below 130 mg per deciliter of patient plasma, prefera- bly at or below 110 mg/dl, the TF antagonist is administered to the patient.
  • the blood glucose levels are regulated and maintained within a range where the lower limit can be selected to be about 60, about 70 or about 80 mg/dL and the upper limit can be selected to be about 110, about 120 or about 130 mg/dL, more specifically to the normal range (i.e., from about 80 to about 110 mg/dL).
  • the range is from about 60 to about 130, preferably, from about 70 to about 120, more preferred, from about 80 to about 110 mg/dL.
  • Each of the essential ingredients viz., a therapeutically effective amount of (a) a TF antagonist and (b) Blood glucose regulator have a therapeutically effective interval, namely, the interval of time in which each agent provides benefit for the patient being treated with Thrombotic or Coagulopathic related disease, Inflammatory Disease or Respiratory Disease.
  • the method of the invention may be practiced by separately dosing the patient in any order with a therapeutically effective amount of (a) a TF antagonist and (b) Blood glucose regulator provided that each agent is given within the period of time that that the other agent is therapeutically effective against Thrombotic or Coagulopathic related disease, Inflammatory Disease or Respiratory Disease, or organ failure resulting from these pathologic processes.
  • the Blood glucose regulator and TF antagonist are preferably administered parenterally to a patient to insure their delivery into the bloodstream in an effective form as fast as possible.
  • the amount and relative ratio of blood glucose regulator and TF antagonist to be used in the practice of the method of invention is set out in the previous section. It may be appreci- ated that it may be necessary to make routine variations to the dosage of either agent depending on the age and condition of the patient. The decision to begin the therapy will be based upon the appearance of the clinical manifestations of Thrombotic or Coagulopathic related disease, Inflammatory Disease or Repiratory Disease.
  • factor Vila in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of factor IX or factor X to produce activated factor IX or X (factor IXa or Xa, respectively).
  • Biological activity of factor VII polypeptides (“factor VII biological activity") may be quantified by measuring the ability of a preparation to promote blood clotting using factor VII- deficient plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864.
  • factor Vila biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "factor VII units" by comparison with a pooled human serum standard containing 1 unit/ml factor VII activity.
  • factor Vila biological activity may be quantified by (i) Measuring the ability of factor Vila or a factor Vila -related polypeptide to produce activated factor X (factor Xa) in a system comprising TF embedded in a lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272:19919-19924, 1997);
  • Native (wild-type) factor Vila and factor Vila variant may be assayed for specific activities. They may also be assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • the ab- sorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of variant and wild-type factor Vila:
  • Ratio (A 405 nm factor Vila variant)/(A 405 noir m factor Vila wild-type). Based thereon, factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above 1.0.
  • factor Vila or factor Vila variants may also be measured using a physio- logical substrate such as factor X, suitably at a concentration of 100-1000 nM, where the factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765).
  • a suitable chromogenic substrate eg. S-2765
  • the activity assay may be run at physiological temperature.
  • factor Vila Native (wild-type) factor Vila and factor Vila variant (both hereafter referred to as "factor Vila") are assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark), factor Vila (10 nM) and factor X (0.8 mi- croM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI2 and 1 mg/ml bo- vine serum albumin, are incubated for 15 min.
  • factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/ml bovine serum albumin.
  • the amount of factor Xa generated is measured by addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVIIa, is used to calculate the ratio between the proteolytic activities of variant and wild-type factor Vila:
  • Ratio (A405 nm factor Vila variant)/(A405 nm factor Vila wild-type). Based thereon, factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above 1.0.
  • the amount of FXa generated is determined by adding substrate S2765 (0.6 mM, Chromogenix, and measuring absorbance at 405 nm continuously for 10 min.
  • IC50 values for TF antagonist inhibition of FVIIa/lipidated TF-mediated activation of FX may be calculated.
  • the IC50 value for FFR-rFVIIa is 51 +/- 26 pM in this assay.
  • Monolayers of human lung fibroblasts WI-38 (ATTC No. CCL-75) or human bladder carcinoma cell line J82 (ATTC No. HTB-1) or human keratinocyte cell line CCD 1102KerTr (ATCC no. CRL-2310) constitutively expressing TF are employed as TF source in FVIIa/TF catalyzed activation of FX.
  • Confluent cell monolayers in a 96-well plate are washed one time in buffer A (10 mM Hepes, pH 7.45, 150 mM NaCI, 4 mM KCI, and 11 mM glucose) and one time in buffer B (buffer A supplemented with with 1 mg/ml BSA and 5 mM Ca2+).
  • buffer A 10 mM Hepes, pH 7.45, 150 mM NaCI, 4 mM KCI, and 11 mM glucose
  • buffer B buffer A supplemented with with 1 mg/ml BSA and 5 mM Ca2+
  • FX (135 nM) and varying concentrations of TF antagonist or FFR- rFVIIa in buffer B are simultaneously added to the cells. FXa formation is allowed for 15 min at 37°C.
  • FXa 50- ⁇ l aliquots are removed from each well and added to 50 ⁇ l stopping buffer (Buffer A supplemented with 10 mM EDTA and 1 mg/ml BSA).
  • the amount of FXa generated is determined by transferring 50 ⁇ l of the above mixture to a microtiter plate well and adding 25 ⁇ l Chromozym X (final concentration 0.6 mM) to the wells.
  • the absorbance at 405 nm is measured continuously and the initial rates of colour development are converted to FXa concentrations using a FXa standard curve.
  • the IC50 value for FFR-rFVIIa is 1.5 nM in this assay.
  • Binding studies are employed using the human bladder carcinoma cell line J82 (ATTC No. HTB-1) or the human keratinocyte cell line (CCD1102KerTr ATCC No CRL-2310) or NHEK P166 (Clonetics No. CC-2507) all constitutively expressing TF.
  • Confluent monolayers in 24-well tissue culture plates are washed once with buffer A (10 mM Hepes, pH 7.45, 150 mM NaCI, 4 mM KCI, and 11 mM glucose) supplemented with 5 mM EDTA and then once with buffer A and once with buffer B (buffer A supplemented with with 1 mg/ml BSA and 5 mM Ca2+).
  • the monolayers are preincubated 2 min with 100 ⁇ l cold buffer B. Varying concentrations of Mabs (or FFR-FVIIa) and radiolabelled FVIIa (0.5 nM 1251-FVIIa) are simultaneously added to the cells (final volume 200 ⁇ l). The plates are incubated for 2 hours at 4 °C. At the end of the incubation, the unbound material is removed, the cells are washed 4 times with ice-cold buffer B and lysed with 300 ⁇ l lysis buffer (200 mM NaOH, 1 % SDS and 10 mM EDTA). Radioactivity is measured in a gamma counter (Cobra, Packard Instruments). The binding data are analyzed and curve fitted using GraFit4 (Erithacus Software, Ltd., (U.K.). The IC50 value for FFR-rFVIIa is 4 nM in this assay.
  • Biosensor assay (Assay 4):
  • TF antagonists are tested on the Biacore instrument by passing a standard solution of the TF antagonist over a chip with immobilized TF. This is followed by different concentrations of sTF in 10 mM hepes pH 7.4 containing 150 mM NaCI, 10 mM CaCI2 and 0.0003 % polysorbate 20.
  • Kd's are calculated from the sensorgrams using the integrated Biacore evaluation software.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Cardiology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Pulmonology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention a trait à une composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un régulateur de glycémie, et son utilisation dans le traitement des maladies thrombotiques et de coagulopathie associées, des maladies respiratoires et des maladies inflammatoires.
PCT/DK2003/000751 2002-11-06 2003-11-04 Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie WO2004041302A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003275947A AU2003275947A1 (en) 2002-11-06 2003-11-04 Pharmaceutical composition comprising a tissue factor antagonist and a blood glucose regulator

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200201710 2002-11-06
DKPA200201710 2002-11-06

Publications (1)

Publication Number Publication Date
WO2004041302A1 true WO2004041302A1 (fr) 2004-05-21

Family

ID=32309252

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2003/000751 WO2004041302A1 (fr) 2002-11-06 2003-11-04 Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie

Country Status (2)

Country Link
AU (1) AU2003275947A1 (fr)
WO (1) WO2004041302A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004064870A2 (fr) * 2003-01-22 2004-08-05 Novo Nordisk A/S Agent se liant au facteur tissulaire et son utilisation
EP1676574A2 (fr) 2004-12-30 2006-07-05 Johnson & Johnson Vision Care, Inc. Procédé favorisant la survie des tissus ou cellules griffées
WO2008107362A2 (fr) * 2007-03-07 2008-09-12 Novo Nordisk Health Care Ag Nouveaux inhibiteurs du facteur de coagulation sanguine
US9150658B2 (en) 2008-12-09 2015-10-06 Genmab A/S Human antibodies against tissue factor and methods of use thereof
US9168314B2 (en) 2010-06-15 2015-10-27 Genmab A/S Human antibody drug conjugates against tissue factor

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027631A1 (fr) * 1993-05-21 1994-12-08 Zymogenetics, Inc. Facteur vii modifie
WO2001030333A2 (fr) * 1999-10-27 2001-05-03 Sunol Molecular Corporation Antagonistes de facteur tissulaire et leurs procedes d'utilisation
WO2001070984A2 (fr) * 2000-03-16 2001-09-27 Genentech, Inc. Anticorps de facteur anti-tissulaire a pouvoir anticoagulant ameliore
WO2001085256A2 (fr) * 2000-05-05 2001-11-15 Novo Nordisk A/S Maladie neurophatique grave

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994027631A1 (fr) * 1993-05-21 1994-12-08 Zymogenetics, Inc. Facteur vii modifie
WO2001030333A2 (fr) * 1999-10-27 2001-05-03 Sunol Molecular Corporation Antagonistes de facteur tissulaire et leurs procedes d'utilisation
WO2001070984A2 (fr) * 2000-03-16 2001-09-27 Genentech, Inc. Anticorps de facteur anti-tissulaire a pouvoir anticoagulant ameliore
WO2001085256A2 (fr) * 2000-05-05 2001-11-15 Novo Nordisk A/S Maladie neurophatique grave

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A. P. B. DACKIW ET AL: "The Role of Tyrosine Phosphorylation in Lipopolysaccharide- and Zymosan-Induced Procoagulant Activity and Tissue Factor Expression in Macrophages", INFECTION AND IMMUNITY, vol. 65, no. 6, June 1997 (1997-06-01), pages 2362 - 2370, XP002273421 *
AHMAD ALJADA ET AL: "Insulin Inhibits the Pro-Inflammatory Transcription Factor Early Growth Response Gene-1 (Egr)-1 Expression in Mononuclear Cells (MNC) and Reduces Plasma Tissue Factor (TF) and Plasminogen Activator Inhibitor-1 (PAI-1) Concentrations", THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 87, no. 3, 2002, pages 1419 - 1422, XP002273420 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004064870A2 (fr) * 2003-01-22 2004-08-05 Novo Nordisk A/S Agent se liant au facteur tissulaire et son utilisation
WO2004064870A3 (fr) * 2003-01-22 2005-04-28 Novo Nordisk As Agent se liant au facteur tissulaire et son utilisation
EP1676574A2 (fr) 2004-12-30 2006-07-05 Johnson & Johnson Vision Care, Inc. Procédé favorisant la survie des tissus ou cellules griffées
WO2008107362A2 (fr) * 2007-03-07 2008-09-12 Novo Nordisk Health Care Ag Nouveaux inhibiteurs du facteur de coagulation sanguine
WO2008107362A3 (fr) * 2007-03-07 2008-10-23 Novo Nordisk Healthcare Ag Nouveaux inhibiteurs du facteur de coagulation sanguine
US9150658B2 (en) 2008-12-09 2015-10-06 Genmab A/S Human antibodies against tissue factor and methods of use thereof
US9714297B2 (en) 2008-12-09 2017-07-25 Genmab A/S Human antibodies against tissue factor and methods of use thereof
US9168314B2 (en) 2010-06-15 2015-10-27 Genmab A/S Human antibody drug conjugates against tissue factor
US9492565B2 (en) 2010-06-15 2016-11-15 Genmab A/S Human antibody drug conjugates against tissue factor

Also Published As

Publication number Publication date
AU2003275947A1 (en) 2004-06-07

Similar Documents

Publication Publication Date Title
RU2571931C2 (ru) Полипептиды на основе модифицированного фактора vii и их применение
ES2605801T3 (es) Formulación de dosis unitaria de antídoto para inhibidores del factor Xa para su uso en la prevención de la hemorragia
EP3013366B1 (fr) Thérapie combinée utilisant un inhibiteur du facteur xii et l'inhibiteur c1
KR102186924B1 (ko) 출혈 장애를 치료하기 위한 변형된 세르핀
KR20180132831A (ko) 죽상동맥경화증의 치료 방법
US20220288172A1 (en) Prohemostatic proteins for the treatment of bleeding
US20210355231A1 (en) Treatment and prevention of hemophilic arthropathy with an antibody against endothelial cell protein c receptor (epcr)
JP2017502036A (ja) 血友病の予防的処置のための第ｉｘ因子を含む融合タンパク質およびその方法
WO2004041302A1 (fr) Composition pharmaceutique comprenant un antagoniste de facteur tissulaire et un regulateur de glycemie
US20040198660A1 (en) Tissue factor antagonist and protein C polypeptide compositions
US20050232925A1 (en) Protease activity of thrombin inhibits angiogenesis
Kurasawa et al. Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII
WO2004041296A2 (fr) Composition pharmaceutique comportant un antagoniste de facteur tissulaire et des polypeptides de la proteine c
US20040143099A1 (en) Tissue factor antagonist and blood glucose regulator compositions
KR102446476B1 (ko) 사람에서의 장기-작용성 인자 ix의 피하 투여
WO2023245335A1 (fr) Activateurs du facteur de coagulation x et formulations de ceux-ci pour le traitement de troubles hémorragiques
KR102597725B1 (ko) 혈우병의 예방적 치료를 위해 인자 ix 및 사람 알부민을 포함하는 융합 단백질에 대한 21-일 투여 용법 및 이의 방법
Dagher et al. Toward non-factor therapy in hemophilia: an antithrombin insensitive Gla-domainless factor Xa as tissue factor pathway inhibitor bait
WO2005042011A1 (fr) Composition pharmaceutique contenant un polypeptide de proteine c et un regulateur de glucose sanguin
AU2013204377B2 (en) Modified factor vii polypeptides and uses thereof
ES2787512T3 (es) Anticuerpos que son capaces de unirse específicamente al inhibidor de la vía del factor tisular

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP