WO2005042011A1

WO2005042011A1 - Pharmaceutical composition comprising a protein c polypeptide and a blood glucose regulator

Info

Publication number: WO2005042011A1
Application number: PCT/DK2004/000758
Authority: WO
Inventors: Jakob Michael Back
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2003-11-04
Filing date: 2004-11-03
Publication date: 2005-05-12
Anticipated expiration: 2006-05-04

Abstract

The present invention relates to the combinatorial use of a protein C polypeptide and a blood glucose regulator for treating thrombotic or coagulopathic related diseases, respi-ratory diseases and inflammatory diseases.

Description

PHARMACEUTICAL COMPOSITION COMPRISING A PROTEIN C POLYPEPTIDE AND A BLOOD GLUCOSE REGULATOR

FIELD OF THIS INVENTION The present invention relates to a pharmaceutical composition comprising a protein C polypeptide and a blood glucose regulator. The invention also relates to the use of a combination of a protein C polypeptide and a blood glucose regulator for the manufacture of a medicament for treatment of thrombotic or coagulopathic related diseases, respiratory diseases and inflammatory diseases.

BACKGROUND OF THE INVENTION Activated Protein C (APC) is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of vascular homeostasis by inactivating Factors Va and Villa in the coagulation cascade. Human Protein C (PC) is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. In concert with other proteins, Protein C functions' as an important down-regulator of blood coagulation factors that promote thrombosis. In other words, the Protein C enzyme system represents a major physiological mechanism of anticoagulation. The critical role of protein C in controlling hemostasis is exemplified by the in- creased rate of thrombosis in heterozygous deficiency, protein C resistance (e.g., due to the common Factor V Leiden mutation) and the fatal outcome of untreated homozygous protein C deficiency. Human activated protein C, both plasma-derived and recombinant, have been shown to be effective and safe antithrombotic agents in a variety of animal models for both venous and arterial thrombosis. Activated protein C in recent clinical studies has been shown to be effective in human thrombotic diseases including the treatment of protein C deficiencies and microvascular thrombosis, such as disseminated intravascular coagulation associated with sepsis. Today, Protein C is commercially available as Xigris® from Eli Lilly, Inc. (recom- binantly produced, activated human protein C), or Ceprotin® from Baxter (plasma- derived human protein C). Critical illness polyneuropathy (OP) is a syndrome that was first extensively described in the early 1980s, mainly in subjects with failure to wean from mechanical ventilation. The syndrome is further characterized by limb muscle weakness, usually more pronounced distally than proximally, and is often accompanied by atrophy. The facial musculature is often strikingly spared, Reduced or absent deep-tendon reflexes and loss of peripheral sensation to light touch and pin prick often accompany the syndrome. Involvement of the phrenic nerve has been shown to further contribute to delayed weaning from the ventilator in many subjects. The ectrophysiologic studies are consistent with a predominantly motor and, often to a lesser extent, sensory axonal polyneuropathy. The incidence of CIP is high, with often more than 50% of subjects in major medical and surgical critical care units suffering from the syndrome. The systemic inflammatory response syndrome (SIRS) is strongly associated with CIP and, among the multiorgan failure often seen in SIRS, CIP is thought to represent a neurologic manifestation of SIRS. CIP occurs in about 70% of subjects who have the systemic inflammatory response syndrome (SIRS). 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 electro- physiologic studies. Morbidity and mortality rates are high. If the underlying problem causing sepsis and/or SIRS can be treated successfully, full recovery from CIP can occur. This recovery often occurs in a matter of weeks in milder cases and in months in more severe cases. Knowledge of CIP is essential for intensivists and other specialists who care for critically ill subjects. However, clinical signs are often absent and it remains an occult problem in many ICUs worldwide. Nonetheless, it is an important clinical entity as it is a frequent cause of difficulty to wean subjects from the ventilator and it leads to problems with rehabilitation after the acute illness has been treated and cured. If the underlying condition (e.g., sepsis or SIRS) can be successfully treated, recovery from and/or prevention of CIP can be expected. This will occur in a matter of weeks in mild cases and in months in more severe cases. The pathophysiology of this type of neuropathy remains unknown. It has been speculated to be directly related to inflammatory diseases or disorders such as, e.g., sep- sis and its mediators. Indeed, cytokines released in sepsis have histamine-like properties which may increase microvascular permeability and induce disturbed microcirculation. International Publication No. WO 01185256 relates to a medicament for critically ill subjects and a method of treatment; the medicament is a blood glucose regulator. There is still a need in the art for improved compositions and treatments having anticoagulant and anti-inflammatory activity which can be administered at relatively low doses and do not produce the undesirable side effects associated with traditional anticoagulant compositions. The present invention is the first to describe the combination of protein C and a blood glucose regulator in the treatment of thrombotic or coagulopathic related diseases, respiratory diseases and inflammatory diseases. The combination of protein C and a blood glucose regulator results in a treatment that will allow for improved properties. The improved properties of the combinatorial treatment may include, without limitation, reduction of dosages, e.g., of protein C, of blood glucose regulator, or of both; clinical treatment options, outcome of the subject being treated, and reduced side effects that may occur with either agent.

SUMMARY OF THE INVENTION 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. Other objects of the present invention will become apparent upon reading the present description. In a first aspect the invention provides a pharmaceutical composition comprising a protein C polypeptide and a blood glucose regulator. In a second aspect, the invention provides the use of a protein C polypeptide 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. In one embodiment thereof, 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 Syndrome (ARDS), pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular restenosis, 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 disease, endotoxin induced lung damage, non cell lung cancer; inflammatory bowel disease, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, rheumatoid arthritis, cystic fibrosis, stroke, acute bronchitis, chronic bron- chitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic 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, Luegenec's granulomatosis, polymyalgia rheumatica, joint cell arteritis, calcium crystal deposition arthropathies, pseudo gout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (typing), miscellaneous forms of arthritis, neuropathic joint disease (e.g., charco and joint), hemarthrosis, Henoch Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, scoliosis, hemochromatosis, sickle cell disease and other hemoglobi- nopathies, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing and critical illness polyneuropathy (CIP), and/or multiple organ failure resulting from any of the preceding pathologic processes. In one embodiment, the diseases or disorders are respiratory disease and in- flammatory disease. In one embodiment, respiratory disease and inflammatory disease include 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, en- dotoxin induced lung damage, non cell lung cancer; 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, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy or juvenile ankylosing spondylitis, re- active 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, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathris, pseudo gout, non-articular rheumatism, bursitis, tenosynomitis. epi- condylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (e.g., from typing), miscellaneous forms of arthritis, neuropathic joint disease (e.g., charco and joint), hemarthrosis, Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis. arthritis associated with certain diseases, scoliosis, hemochromatosis, sickle cell disease and other hemoglobinopathies, hyperlipoproteineimia, hypogam- maglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing and critical illness polyneuropathy (CIP), and/or multiple organ failure resulting from any of the preceding pathologic processes In another embodiment, the diseases or disorders are thrombotic or coagulopatic related diseases or disorders. In one embodiment, thrombotic or coagulopatic related disease include vascular diseases and inflammatory responses such as deep venous thrombosis, arterial thrombo- sis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous trans- dermal 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, sys- temic 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. In a preferred embodiment, 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. In another embodiment, the medicament is formulated for intravenous administration, preferably injection or infusion, in particular injection. The composition can be administered in any suitable form. For example, in one embodiment, 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 protein C polypeptide and a second unit dosage form comprising a preparation of a blood glucose regulator. In a further aspect, 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 protein C polypeptide 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 protein C polypeptide and the blood glucose regulator can be administered in any amounts that together suffice to cure, alleviate or partially arrest a disease and its complications. For example, in one embodiment the protein C polypeptide and the blood glucose regulator are present in a ratio by mass of between about 100: 1 and about 1: 100 (w/w protein C:blood glucose regulator). In another embodiment, the pharmaceutical composition is formulated for intravenous administration, preferably injection or infusion, in particular injection. In one embodiment of the invention, the protein C polypeptide and the blood glucose regulator are administered simultaneously. In another embodiment, the Protein C polypeptide and the blood glucose regulator are administered sequentially. In one embodiment of the present invention, the pharmaceutical composition is in single-dosage form and consists essentially of a preparation of a protein C polypeptide 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, deter- gents, neutral salts, antioxidants, preservatives, and protease inhibitors. In another embodiment, 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 protein C polypeptide and one or more of the components selected from the list of pharmaceutical acceptable excipients or carriers, stabi- lizers, 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. In one embodiment, the blood glucose regulator are selected from the list of insulin, active insulin derivatives, insulin analogues, compounds that stimulate signal transduction mediated by an insulin receptor type tyrosine kinase in a cell, certain pro- tein-tyrosine phosphatases (PTP's), other type II antidiabetica, and other biologically active substances having insulin releasing action. In one embodiment, the blood glucose regulator is porcine insulin, human insulin, or a zinc or protamine salt of any thereof. In one embodiment, the blood glucose regulator is insulin aspart (i.e., Asp628 human insulin), insulin lispro (i.e., LysB28, ProB29 human insulin), insulin glagin (i.e., GlyA21, ArgB31, ArgB32 human insulin), or insulin detemir (i.e., des-ThrB30 human insulin gamma-LysB29 tetradecanoyl). In one embodiment, the subject is a non-diabetic sub- ject.

DETAILED DESCRIPTION OF THIS INVENTION

Protein C polypeptides: In practicing the present invention, any protein C polypeptide may be used that is effective in preventing or treating bleeding. This includes protein C polypeptides derived from blood or plasma, or produced by recombinant means. The present invention encompasses protein C polypeptides, such as, e.g., those having the amino acid sequence disclosed by Beckmann et al., (Nucleic Acids Research

13:5233 (1985) (wild-type human protein C); by Foster et al. (PNAS.USA 1986; 82, 4673-4677); in European patent No. EP 191606 and US patents Nos. US 4775624, US

5151268, and US 5270040 (Eli Lilly); in European patent No. EP 215548 and US patents Nos. US 5073609, US 5302529, and US 5516650 (ZymoGenetics); and in US 5009889 (Oklahoma Medical Research Foundation). In some embodiments, the protein C polypeptide is human activated protein C, as disclosed, e.g., in US 4,981,952. In various embodiments, the protein C polypeptide is protein C, human protein C, a protein C-related polypeptide, a protein C sequence variant, zymogen protein C, activated protein C, and human activated protein C. In one embodiment, the protein C polypeptide is recombinantly made. In preferred embodiments, the protein C polypeptide is recombinant human protein C or recombinant activated human protein C. In one series of embodiments, protein C polypeptides include polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70%, of the specific biological activity of human activated protein C when tested in one or more assays for Protein C activity. Protein C activity can, for example, be tested by means of the "Protein C assay" disclosed herein. In one series of embodiments, protein C polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140%, and most preferably at least about 160%, of the specific biological activity of human activated protein C. In one series of embodiments, protein C 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 sequence of wild-type protein C as disclosed in Beckmann et al., (Nucleic Acids Research 13:5233 (1985)). As used herein, "protein C polypeptide" encompasses, without limitation, protein C, as well as protein C-related polypeptides. The term "protein C" is intended to encompass, without limitation, polypeptides having the amino acid sequence of wild-type human protein C (as disclosed supra), as well as wild-type protein C derived from other species, such as, e.g., bovine, porcine, canine, murine, rat and salmon protein C, said protein C derived from blood or plasma, or produced by recombinant means. It further encompasses natural allelic variations of protein C that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. The term "protein C" is also intended to encompass protein C 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 aPC. "Protein C-related polypeptides" include, without limitation, protein C polypeptides that have either been chemically modified relative to human protein C and/or contain one or more amino acid sequence alterations relative to human protein C (i.e., protein C variants), and/or contain truncated amino acid sequences relative to human protein C (i.e., protein C fragments). Such protein C-related polypeptides may exhibit different properties relative to human protein C, including stability, phospholipid binding, altered specific activity, and the like. The term "protein C-related polypeptides" are intended to encompass such 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 "aPC-related polypeptides" or "activated protein C-related polypeptides" As used herein, "protein C-related polypeptides" encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild-type human protein C, as well as polypeptides in which the protein C biological activity has been substantially modified or reduced relative to the activity of wild-type human protein C. These polypeptides include, without limitation, protein C or activated protein C that has been chemically modified and protein C variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide. It further encompasses 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 protein C. Protein C-related polypeptides, including variants, encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific activity of wild-type activated protein C that has been produced in the same cell type, when tested in the "Protein C assay" as described herein. In some embodiments the protein C polypeptides are protein C-related polypeptides, in particular variants, wherein the ratio between the activity of said protein C polypeptide and the activity of native human activated protein C (wild-type aPC) is at least about 1.25 when tested in the "protein C Assay" as described herein; in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0, in further embodiments, the ratio is at least about 8.0. Protein C is commercially available as Xigris® from Eli Lilly, Inc. (recombinantly produced, activated human protein C), or Ceprotin® from Baxter (plasma-derived human protein C). Examples of protein C polypeptides that can be used in accordance with the present invention include, but are not limited to Xigris® and Ceprotin®. Blood glucose regulators: In practicing. the present invention, any compound may be used, which is able to regulate the blood glucose level. Blood glucose levels may be controlled by insulin treat- ment. However, it will be clear for the man skilled in the art that also active insulin derivatives and its physiologically tolerated salts and other blood glucose regulators can be used to obtain the same result. Furthermore, it will be clear for the man skilled in the art, that compounds of the group of biologically active substances having insulin releasing action can be used in practising the present invention. Such compounds, capable of pro- moting 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). Furthermore, it will be clear for the man skilled in the art, that compounds of the group of compounds that stimulate signal transduction mediated by an insulin receptor type tyrosine kinase in a cell can be used when practising the present invention. It is known that insulin binding to the insulin receptor triggering a variety of metabolic and growth promoting effects. Metabolic effects include glucose transport, biosynthesis of glycogen and fats, inhibition of triglyceride breakdown, and growth promoting effects include DNA synthesis, cell division and differentiation. It is known that some of these bio- logical effects of insulin can be mimicked by vanadium salts such as vanadates and per- vanadates. However, this class of compounds appears to inhibit phosphotyrosine phosphatases generally, and are potentially toxic because they contain heavy metal (US 5,155,031; Fantus etal., 1989, Biochem. 28:8864-71; Swarup et al., 1982, Biochem. Biophys. Res. Commun. 107: 1104-9). Furthermore, it has been demonstrated that cer- tain protein-tyrosine phosphatases (PTPs), in particular, RPTP. alpha and RPTP.epsilon., specifically regulate the insulin receptor signalling pathway (Lammers Reiner et al., 19 Jan 1999, US 5861266 and WO 9523217). 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 antidiabetica, and other biologically active substances having insulin releasing action. The term "insulin", as used herein, refers to insulin from any species such as porcine insulin, bovine insulin, and human insulin and salts thereof such as zinc salts, and protamine salts. The term "active derivatives of insulin", as used herein, are what a skilled art worker generally considers derivatives, including, for example, insulin having a substitu- ent not present in the parent insulin molecule. The term "insulin analogues", as used herein 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. Using results from the so-called free fat cell assay, 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 6,011,007. Examples of specific insulin analogues are insulin aspart (i.e., AspB28 human insulin), insulin lispro (i.e., LysB28, ProB29 human insulin), and insulin glagin (i.e., GIyA21, ArgB31, ArgB32 human insulin). Also compounds which can be considered being both an insulin derivative and an insulin analogue can be used to practice the present invention. Examples of such com- pounds are described in the following patents and equivalents thereto: US 5,750,497, and US 6,011,007. An example of a specific insulin analogues and derivatives is insulin detemir (i.e., des-ThrB30 human insulin gamma-LysB29 tetradecanoyl).

The terms "aPC", "APC", "Activated Protein C", "raPC", "rAPC", and "recombinant Activated Protein C" are synonymous for the purpose and practice of this invention and can be used interchangeably. Protein C Activity: any property of activated human Protein C or its derivatives responsible for proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro- fibrinolytic) activities. Methods for testing for Protein C anticoagulant and amidolytic ac- tivity are well known in the art, i.e., see Grinnell et.al., 1987, Bio/Technology 5: 1189- 1192. RhaPC: Recombinant activated human protein C, produced by activating r-HPC in vitro or by direct secretion of the activated form of Protein C from prokaryotic cells, eu- karyotic cells, or from transgenic animals. Zymogen: an enzymatically inactive precursor of a proteolytic enzyme. Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chain, of protein C. The term "systemic inflammatory response syndrome (SIRS)", as used herein refers to the uncontrolled disease process which ensures an initial insult and which gives raise to a multisystem disturbance secondary to inflammatory mediators released during shock. The term "sepsis", as used herein refers to "SIRS", as described above, which is particularly caused by an infectious insult leading to the initial shock phase. The term "mediators of sepsis", as used herein refers to factors released by inflammatory cells, such as TNFs, interleukins, bradykinins etc. The term "insulin receptor type tyrosine kinase", as used herein refers to a post- receptor signal transduction pathway involved in the insulin signaling. The term "endoneural edema", as used herein refers to swelling of the neuronal cells. The term "phrenic nerves", as used herein refers to the left and right nervus phrenicus, The term "non-diabetic subject", as used herein refers to a subject who has not been diagnosed as having diabetes. In its broadest sense, the term a "critically ill subject" (herein designated CIP), as used herein refers to a subject who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury, a subject who is being operated and where complications supervene, and a subject who has been operated in a vital organ within the last week or has been subject to major surgery within the last week. In a more restricted sense, the term a "critically ill subject", as used herein refers to a subject 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 subject who is being operated and where complications supervene. In an even more restricted sense, the term "critically ill subject", as used herein, refers to a subject who has sustained or are at risk of sustaining acutely life-threatening single or multiple organ system failure due to disease or injury. Similarly, these definitions apply to similar expressions such as "critical illness in a subject" and a "subject is critically ill". The term "Intensive Care Unit" (herein designated ICU), as used herein refers to the part of a hospital where critically ill subjects 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. The term "active site" and the like when used herein with reference to protein C, refers to the catalytic and zymogen substrate binding site. The general mechanism of blood clot formation is reviewed by Ganong in "Review of Medical Physiology, 13th ed" (Lange, Los Altos Calif., pp 411-414 (1987)). Co- agulation requires the confluence of two processes, the production of thrombin which induces platelet aggregation and the formation of fibrin which renders the platelet plug stable. The process comprises several stages each requiring the presence of discrete pro- enzymes and profactors. The process ends in fibrin crosslinking and thrombus formation. Fibrinogen is converted to fibrin by the action of thrombin. Thrombin, in turn, is formed by the proteolytic cleavage of prothrombin. This proteolysis is effected by factor Xa which binds to the surface of activated platelets and in the presence of FVa and calcium, cleaves prothrombin. TF/FVIIa is required for the proteolytic activation of factor X by the extrinsic pathway of coagulation. Therefore, a process mediated by or associated with TF/FVIIa, or a TF-mediated coagulation activity includes any step in the coagulation cas- cade from the formation of the TF/FVIIa complex to the formation of a fibrin platelet clot and which initially requires the presence of TF/FVIIa. For example, the TF/FVIIa complex initiates the extrinsic pathway by activation of factor X to factor Xa, FIX to FIXa, and additional FVII to FVIIa. A TF/FVIIa 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/FVIIa and other necessary reagents. It should be noted that 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, citrul- line, 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. In addition, the 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; and Nva, norvaline. The three-letter indication "GUV as used herein means 4-carboxyglutamic acid (gamma-carboxyglutamate). The terms "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. Thrombotic or coagulopathic related diseases or disorders: The term 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. 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. 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, spondyarthropathris, 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, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, cal- cium crystal deposition arthropathris, pseudo gout, non-articular rheumatism, bursitis, tenosynomitis, 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, arthritis associated with certain dis- eases, surcoilosis, hemochromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, fa- milial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing, and multiple organ failure resulting from any of the preceding pathologic processes. The phrase "therapeutically effective interval" is a period of time beginning when one of either (a) the Protein C polypeptide or (b) The blood glucose regulator is adminis- tered 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, as used herein, refers to situations in which the Protein C polypeptide 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 subject 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 coagu- lation 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 subject's haemostatic system. Blood samples are drawn from the subject 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 2001; 12:627-637; Vig et al.. Hematology, Vol. 6 (3) pp. 205- 213 (2001); Vig et al., Blood coagulation & Fibrinolysis, Vol. 12 (7) pp. 555-561 (2001) Oct; Glidden et al., Clinical and applied thrombosis/hemostasis, Vol. 6 (4) pp. 226-233 (2000) Oct; McKenzie et al., Cardiology, Vol. 92 (4) pp. 240-247 (1999) Apr; or Davis et al., Journal of the American Society of Nephrology, Vol. 6 (4) pp. 1250-1255 (1995). The term "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 Protein C polypeptide and a blood glucose regulator is thus included in the term "treatment". The term "subject" is intended to include any animal, in particular mammals, such as humans. The protein C polypeptide 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 coagula- tion factors, or in the form of a kit-of-parts comprising a preparation of a protein C polypeptide as a first unit dosage form and a preparation of a blood glucose regulator as a second unit dosage form. Whenever a first or second or third, etc., unit dose is men- tioned throughout this specification this does not indicate the preferred order of administration, but is merely done for convenience purposes. By "simultaneous" dosing of a preparation of a protein C polypeptide and a preparation of a blood glucose regulator is meant administration of the two coagulation factor proteins in single-dosage form, or administration of the first coagulation factor protein followed by administration of the second coagulation factor protein with a time separation of no more than about 15 minutes, preferably about 10, or about 5, or about 2. Either factor may be administered first. By "sequential" dosing of a preparation of a protein C polypeptide and a prepara- tion of a blood glucose regulator is meant administration of the first coagulation factor protein followed by administration of the 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. Preferably, both products are injected through the same intravenous access. By "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 protein C polypeptide 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 protein C polypeptide-containing preparation with an anti-PC antibody immobilised onto the Elisa plate, subsequently contacting the immobilised antibody-PC complex with a second anti- PC 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 appropri- ate antibodies. The total amount of protein present in a preparation is determined by adding the amounts of the individual proteins. In one embodiment, the preparation comprises isolated coagulation factors. In another embodiment, the preparation is free of coagulation factor II and coagulation factor Ila (prothrombin and thrombin) and/or factor X or Xa. As used herein, the term "isolated" refers to polypeptides, e.g., insulin or protein

C, 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). The term "isolated" also in- eludes polypeptides that have been synthesized using peptide synthesis- and purification methods known in the art. 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; centrifuga- tion 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- protein C antibody column; hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like. Within the present invention an "effective amount" of a protein C polypeptide and a blood glucose regulator is defined as the amount of a protein C polypeptide, e.g., APC, and a blood glucose regulator that together suffices to cure, alleviate or partially arrest a disease and its complications. The phrase "therapeutically effective combination", used in the practice of this invention, means administration of both (a) a protein C polypeptide and (b) a blood glucose regulator, either simultaneously or separately, wherein the two agents, in the administered amounts, together suffices to cure, alleviate or partially arrest a disease and its complications. The term, "Active Ingredient" as used herein refers to a combination of (a) a protein C polypeptide and (b) a blood glucose regulator co-present in a pharmaceutical formulation for the delivery of a treatment regimen that applies this invention. The term, "injectable liquid carrier" refers to a liquid medium containing either or both of (a) a protein C polypeptide, or (b) a blood glucose regulator; wherein (a) and (b) are independently dissolved, suspended, dispersed, or emulsified in the liquid medium.

Abbreviations

TF tissue factor FVII factor VII in its single-chain, unactivated form

FVIIa factor VII in its activated form

APC Activated human Protein C, also called, Activated Protein C.

HPC human Protein C zymogen. rhPC recombinant human Protein C zymogen. APTT activated partial thromboplastin time.

Preparation of compounds: Methods for preparing recombinant proteins including conventional molecular biology, microbiology, and recombinant DNA techniques are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Labo- ratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989") DNA Cloning : A Practical Approach, Volumes I and II /D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984). Briefly, DNA sequences encoding a specific protein (e.g., protein C) 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). For the present pur- pose, 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 Beau- cage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method de- scribed by Matthes et al., EMBO Journal 3 (1984), 801 - 805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors. The 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 polypeptides (for example, capable of gammacarboxylation and glycosylation) and includes yeast, fungi and higher eukaryotic 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. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841 - 845.

Preparation of the Activated Protein C Ingredient. The cloning of human protein C has been described by Beckmann et al., (Nucleic Acids Research 13:5233 (1985) (wild-type human protein C). The expression of recombinant human Protein C in human kidney 293 cells has been described by Grinnell et al. (BioTechnology 5: 1189-1192 (1987)). Recombinant human Protein C (r-hPC) may be produced by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent No. 4,981,952. The gene encoding human Protein C is disclosed and claimed in Bang et al., U.S. Patent No. 4,775,624. A plasmid useful to express human Protein C in 293 cells (pLPC) is disclosed in Bang et al., U.S. Patent No. 4,992,373; the construction of plasmid pLPC is also described in European Patent Publication No. 0 445 939 and in Grinnell et al., 1987, Bio/Technology 5: 1189-1192. Briefly, the plasmid was trans- fected into 293 cells; stable transformants were identified, subcultured and grown in serum-free media. After fermentation, cell-free medium was obtained by microfiltration. Production of recombinant protein C is also described in European patent No. EP 191606 and US patents Nos. US 4775624, US 5151268, and US 5270040 (Eli Lilly); in European patent No. EP 215548 and US patents Nos. US 5073609, US 5302529, and US 5516650 (ZymoGenetics); and in US 5009889 (Oklahoma Medical Research Foundation). Production and isolation of Protein C is also described by Haley et al., J. Biol. Chem., 264; 16303, 1989, and Turkay et al., Thromb. Haemost. 81; 727 1999. Isolation of protein C from plasma is described, e.g., in Develop. Biol. Standard 67 : 51-57 (1987).

Preparation of the glucose regulating agent The preparation of insulin, e.g., pig insulin and human insulin, whether isolated from glands or recombinantly produced, is well known to the skilled worker. Reference to publications describing different types of insulin analogues and derivatives, as well as alternative types of glucose regulating agents are given supra.

Pharmaceutical Compositions and Methods of Use 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 disorders including, without limitation, deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), per- cutaneous 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 prophylactic treatment of mammals with atherosclerotic vessels at risk for thrombosis; asthma, bronchitis, idio- pathic pulmonary fibrosis, pneumonia, pulmonary edema, pulmonary obstructive disease, endotoxin induced lung damage, non cell lung cancer; inflammatory bowel disease, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, rheumatoid arthritis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy or juvenile ankylosing spondylitis, reactive arthropathy, infectious or post- infectious arthritis, gonoccocal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthri- tis, Lyme disease, arthritis associated with "vasculitic syndromes" polyarteritis nodosa, hypersensitivity vasculitis, Luegenec's granulomatosis, polymyalgin rheumatica. joint cell arteritis, calcium crystal deposition arthropathies, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (typing), miscellaneous forms of arthritis, neuropathic joint disease (e.g., charco and joint), hemarthrosis, Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, scoliosis, hemochromatosis, sickle cell disease and other hemoglobinopathries, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing and critical ill- ness polyneuropathy (CIP), and/or multiple organ failure resulting from any of the preceding pathologic processes. The combination therapy of protein C and a blood glucose regulator may be administered in any sequence or combination according to the best combination for a particular subject or disease need. The present invention is intended to encompass all dos- ing regimens employing protein C and a blood glucose regulator for treating a condition disclosed herein. The needs of a particular subject and the preferences of a treating physician may result in protein C and a blood glucose regulator being used in a variety of dosing schedules. For example, protein C may be administered prior to a blood glucose regulator. A blood glucose regulator may be administered prior to protein C. Protein C and a blood glucose regulator may also be administered simultaneous at different combination proportions. Protein C and a blood glucose regulator may also be administered with varying doses and alternating back and forth between the two agents. The below examples of possible dosing agents are not intended to limit the scope of the invention in any way. The essential ingredients (a) a protein C polypeptide and (b) a blood glucose regulator are administered to the subject in such proportion such as to provide an amount of each ingredient that together is a pharmaceutically effective amount to the subject 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. Typically, the weight ratio of protein C polypeptide and the amount of blood glucose regulator (e.g., insulin or a bio- logically active fragment or variant thereof) may vary from a ratio of between about

1: 100 to about 100: 1 (w/w). The ratio of protein C polypeptide to blood glucose regulator (e.g., insulin or biologically active fragment or variant) 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, or between about 70: 1 to about 5:1, or between about 60: 1 to about 10: 1, or between about 50: 1 to about 25: 1, or between about 40: 1 to about 30: 1; or between about 10: 1 to about 1:10, or between about 5: 1 to about 1:5. Whenever a first or second 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.

The dose of the protein C polypeptide 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. Preferably, the blood glucose regulator, e.g., insulin, is capable of maintaining blood glucose at or below 130 mg per deciliter subject plasma, preferably at or below 110 mg/dl. It is however preferred that 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 skilled art worker, for example, the physician, will be able to decide exactly which upper and lower limits to use. Alternatively, 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. It must be kept in mind that the materials of the present invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of protein C polypeptide 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). In making compositions of the invention the essential ingredients; protein C polypeptide 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 protein C polypeptide 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. When the carrier 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. When the carrier 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. For the pharmaceutical formulations containing both (a) protein C polypeptide and (b) a blood glucose regulator 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 intravenous, subcutaneous, intramuscular, or intrame- dullary injection. Intravenous injection or infusion are most preferred as 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/or suspension agents or a mixture of both. For example, for intravenous injection the compounds of the invention may be dissolved at a concentration of about 2 mg/ml in a 4% dextrose/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. Besides inert diluents such 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^). Besides inert diluents such 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. Examples of aqueous solvents or suspending media are distilled water for injection and physiological salt solution. Examples of non-aqueous solvents or suspending media are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, Polysorbate 80 (registered Trade Mark). These compositions may also include adjuvants such as preserving, wetting, emulsifying and dispersing agents stabilizing agents (e.g. lactose) and solubilizers (e.g. glutamic acid and aspar- aginic acid) hey 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 protein C polypeptide 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, tra- gacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes, and cocoa butter. Preferably, the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly; intravenously being most preferred. They may also be administered by continuous or pulsatile infusion. Local delivery of the preparations of the present invention, such as, for example, topical application, 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 cathe- ters, 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 prac- tices, 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 protein C polypeptide and b) a blood glucose regulator; 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 subject may be either continuous or intermittent. The blood glucose regulator and the protein C polypeptide can be delivered si- multaneously. 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 subject separately but simulta- neously. Dosage of a protein C polypeptide can begin simultaneously with the blood glucose regulator administration. The duration of the protein C polypeptide administration can extend past the blood glucose regulator administration, or vice versa. It is however preferred that 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 subject plasma, preferably at or below 110 mg/dl, the protein C polypeptide is administered to the subject. It is preferred that 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 skilled art worker, for example, the physician, will be able to decide exactly which upper and lower limits to use. Alternatively, 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 protein C polypeptide and (b) blood glucose regulator have a therapeutically effective interval, namely, the interval of time in which each agent provides benefit for the subject 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 subject in any order with a therapeutically effective amount of (a) a Protein C polypeptide 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 protein C polypeptide are preferably administered parenterally to a subject to insure their delivery into the bloodstream in an effec- tive form as fast as possible. The amount and relative ratio of blood glucose regulator and protein C polypeptide to be used in the practice of the method of invention is set out in the previous section. It may be appreciated that it may be necessary to make routine variations to the dosage of either agent depending on the age and condition of the subject. 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 respiratory disease.

Assays:

Test for protein C activity: A suitable assay for testing for Protein C anticoagulant and amidolytic activity and thereby selecting suitable protein C variants can be performed as described, for example, in Grinnell et al., 1987, Bio/Technology 5: 1189-1192 ("the Protein C assay") .

The present invention is further illustrated by the following examples, which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

EXAMPLES a. Preparation of Human Protein C Recombinant human Protein C (r-hPC) was produced in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent No. 4,981,952., the entire disclosure of which is herein incorporated by reference. The gene encoding human Protein C is disclosed and claimed in Bang et al., U.S. Patent No. 4,775,624, the entire disclosure of which is incorporated herein by reference. The plasmid used to express human Protein C in 293 cells was plasmid pLPC which is dis- closed in Bang et al., U.S. Patent No. 4,992,373, the entire disclosure of which is incorporated herein by reference. The construction of plasmid pLPC is also described in European Patent Publication No. 0 445 939, the teachings of which are also incorporated herein by reference and in Grinnell et al., 1987, Bio/Technology 5: 1189-1192. Briefly, the plasmid was transfected into 293 cells, then stable transformants were identified, subcultured and grown in serum-free media. After fermentation, cell-free medium was obtained by microfiltration. The human Protein C was separated from the culture fluid by an adaptation of the techniques of Yan, U.S. Patent No. 4,981,952, the entire disclosure of which is herein incorporated by reference. The clarified medium was made 4 mM in EDTA before it was absorbed to an anion exchange resin (Fast-Flow Q, Pharmacia). After washing with 4 column volumes of 20 mm Tris, 200 mM NaCI, pH 7.4 and 2 column volumes of 20 mM Tris, 150 mM NaCI, pH 7.4, the bound recombinant human Protein C zymogen was eluted with 20 mM Tris, 150 mM NaCI, 10 mM CaC12, pH 7.4. The eluted protein was greater than 95% pure after elution as judged by SDS-polyacrylamide gel electrophoresis. Further purification of the protein was accomplished by making the protein 3 M in NaCI followed by adsorption to a hydrophobic interaction resin (Toyopearl Phenyl 650M, Toso- Haas) equilibrated in 20 mM Tris, 3 M NaCI, 10 mM CaC12, pH 7.4. After washing with 2 column volumes of equilibration buffer without CaC12, the recombinant human Protein C was eluted with 20 mM Tris, pH 7.4. The eluted protein was prepared for activation by removal of residual calcium. The recombinant human Protein C was passed over a metal affinity column (Chelex-100, Bio-Rad) to remove calcium and again bound to an anion exchanger (Fast Flow Q, Pharmacia). Both of these columns were arranged in series and equilibrated in 20 mM Tris, 1'50 mM NaCI, 5 mM EDTA, pH 7.4. Following loading of the protein, the Chelex-100 column was washed with one column volume of the same buffer before disconnecting it from the series. The anion exchange column was washed with 3 column volumes of equilibration buffer before eluting the protein with 0.4 M NaCI, 20 mM Tris-acetate, pH 6.5. Protein concentrations of recombinant human protein C and recombinant activated Protein C solutions were measured by UV 280 nm extinction EO.I%=1.85 or 1.95, respectively, b. Activation of recombinant human Protein C Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4 OC. The coupling reaction was done on resin already packed into a column using approximately 5000 units thrombin/ml resin. The thrombin solution was circulated through the column for approximately 3 hours before adding MEA to a concentration of 0.6 ml/1 of circulating solution. The MEA-containing solution was circulated for an additional 10-12 hours to assure complete blockage of the unreacted amines on the resin. Following blocking, the thrombin-coupled resin was washed with 10 column volumes of 1 M NaCI, 20 mM Tris, pH 6.5 to remove all non- specifically bound protein, and was used in activation reactions after equilibrating in acti- vation buffer. Purified rHPC was made 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg/ml with 20 mM Tris, pH 7.4 or 20 mM Tris- acetate, pH 6.5. This material was passed through a thrombin column equilibrated at 37(C with 50 mM NaCI and either 20 irM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allow for approximately 20 min. of contact time between the rHPC and thrombin resin. The effluent was collected and immediately assayed for amidolytic activity. If the material did not have a specific activity (amidolytic) comparable to an established standard of aPC, it was recycled over the thrombin column to activate the rHPC to completion. This was followed by 1: 1 dilution of the material with 20 mm buffer as above, with a pH of either 7.4 or 6.5 to keep the aPC at lower concentrations while it awaited the next processing step. Removal of leached thrombin from the aPC material was accomplished by binding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCI. Thrombin does not interact with the anion exchange resin under these conditions, but passes through the column into the sample application effluent. Once the aPC is loaded onto the column, a 2-6 column volume wash with 20 mM equilibration buffer is done before eluting the bound aPC with a step elution using 0.4 M NaCI in either 5 MM Tris-acetate, pH 6.5 or mM Tris, pH 7.4. Higher volume washes of the column facilitated more complete removal of the dodecapeptide. The material eluted from this column was stored either in a frozen solution (-20 OC) or as a lyophilised powder. The anticoagulant activity of activated Protein C was determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time (APTT) clotting assay. A standard curve was prepared in dilution buffer (1 mg/ml radioimmuno- assay grade BSA, 20 mM Tris, pH 7.4, 150 mM NaCI, 0.02% NaN3) ranging in Protein C concentration from 125 -1000 ng/ml. Samples were prepared at several dilutions in this concentration range. To each sample cuvette, 50 gl of cold horse plasma and 50 gl of reconstituted activated partial thromboplastin time reagent (APTT Reagent, Sigma) were added and incubated at 37 OC for 5 min. After incubation, 50 gl of the appropriate sam- pies or standards were added to each cuvette. Dilution buffer was used in place of sample or standard to determine basal clotting time. The timer of the fibrometer (CoA Screener Hemostasis Analyzer, American Laboratory) was started immediately after the addition of 50 gl 37 (C 30 mM CaC12 to each sample or standard. Activated Protein C concentration in samples are calculated from the linear regression equation of the stan- dard curve. Clotting times reported here are the average of a minimum of three replicates, including standard curve samples.

Claims

I. A pharmaceutical composition comprising a protein C polypeptide and a blood glucose regulator.

2. A composition according to claim 1, wherein the protein C polypeptide is human protein C.

3. A composition according to claim 2, wherein said protein C is activated human protein C.

4. A composition according to claim 2 or claim 3, wherein said protein C is recombinantly made.

5. A composition according to any one of claims 1 to 4, wherein the protein C polypeptide is a protein C amino acid sequence variant.

6 A composition according to claim 5, wherein the ratio between the activity of said protein C polypeptide and the activity of native human plasma protein C (wild-type PC) is at least about 1.25 when tested in the "protein C activity assay" as described in the present description.

7. A composition according to any one of claims 1 to 6, wherein the blood glucose regulator is insulin, or a salt thereof.

8. A composition according to claim 7, wherein said insulin is porcine or human insulin, or zinc salts thereof, or protamine salts thereof.

9. A composition according to claim 7or claim 8, wherein the insulin is recombinantly made.

10. A composition according to any one of claims 1 to 9, wherein the blood glucose regulator is an insulin analogue or an insulin derivative.

II. A composition according to claim 10, wherein the blood glucose regulator is selected from the list of: insulin aspart (i.e., AspB28 human insulin), insulin lispro (i.e.,

LysB28,ProB29 human insulin), insulin glagin (i.e., GlyA21,ArgB31,ArgB32 human insulin), and insulin detemir (i.e., des-ThrB30 human insulin gamma-LysB29 tetradecanoyl).

12. A composition according to any one of claims 1 to 9, wherein said blood glucose regulator is selected from the list of compounds that stimulate signal transduction mediated by an insulin receptor type tyrosine kinase in a cell, protein-tyrosine phosphatases (PTP's), other type II antidiabetica, and other biologically active substances having insulin-releasing action.

13. A composition according to any one of claims 1 to 12, wherein the protein C polypeptide and the blood glucose regulator are present in a ratio by mass of between about 100: 1 and about 1 : 100 (w/w protein C:blood glucose regulator).

14. A composition according to any one of claims 1 to 13, wherein the composition further comprises pharmaceutically acceptable excipients suitable for injection or infusion, in particular injection.

15. Use of a protein C polypeptide in combination with a blood glucose regulator for the manufacture of a medicament for treating thrombotic and coagulopathic related diseases, respiratory diseases and inflammatory diseases.

16. Use according to claim 15, wherein the medicament is for treatment of a disease selected from the list of deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angio- plastry (PTCA), stroke, tumour metastasis, inflammation, septic chock, hypotension, acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), sepsis, pulmonary embolism, disseminated intravascular coagulation (DIC), sepsis, systemic inflammatory response syndrome (SIRS), vascular restenosis, 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 disease, endotoxin induced lung damage, non cell lung cancer; inflammatory bowel disease, pancreatitis, trauma induced shock, bronchial asthma, allergic rhinitis, rheumatoid arthritis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic 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, Luege- nec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathies, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epi- condylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (e.g., from typing), miscellaneous forms of arthritis, neuropathic joint disease (e.g., charco and joint), hemarthrosis, Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytosis, arthritis associated with certain diseases, scoliosis, hemochromatosis, sickle cell disease and other hemoglobinopathies, hyperlipoproteineimia, hypogam- maglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behat's Disease, systemic lupus erythrematosis, relapsing and CIP, and/or multiple organ failure resulting from any of the preceding pathologic processes.

17. Use according to claim 15, wherein the disease is selected from the list of 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; 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, spondylarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic 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, Luegenec's granulomatosis, polymyalgin rheumatica, joint cell arteritis, calcium crystal deposition arthropathies, pseudo gout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), carpal tunnel syndrome, repetitive use injury (e.g., from typing), miscellaneous forms of arthritis, neuropathic joint disease (e.g., charco and joint), hemarthrosis, Henoch-Schonlein Purpura, hypertrophic osteoarthropathy, multicentric reticulo-histiocytosis, arthritis associated with certain dis- eases, scoliosis, hemochromatosis, sickle cell disease and other hemoglobinopathies, hyperlipoproteineimia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Eehat's Disease, systemic lupus erythrematosis, relapsing and CIP, and/or multiple organ failure resulting from any of the preceding pathologic processes.

18. Use according to claim 15, wherein the disease is selected from the list of: 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, myoca rdial infarction, angiogenesis, or the treatment of mammals with atherosclerotic vessels at risk for thrombosis, and CIP and/or multiple organ failure resulting from any of the preceding pathologic processes.

19. Use according to any one of claims 15 to 18, wherein the disease is one or more of systemic inflammatory response syndrome, acute lung injury, acute respiratory distress syndrome, disseminated intravascular coagulation, sepsis, or CIP, and/or multiple organ failure resulting from any of the preceding pathologic processes.

20. Use according to any one of claims 15 to 19, wherein the medicament is formulated for injection or infusion, in particular injection.

21. Use according to any one of claims 15 to 20, wherein the medicament is in single- dosage form.

22. Use according to any one of claims 15 to 20, wherein the medicament is prepared in the form of a first unit dosage form comprising a preparation of a protein C polypeptide and a second unit dosage form comprising a preparation of a blood glucose regulator.

23. A method for treating thrombotic or coagulopatic diseases, respiratory diseases and inflammatory diseases in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a protein C polypeptide 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 diseases, respiratory diseases and inflammatory diseases.

24. Method according to claim 23, wherein the protein C polypeptide and the blood glucose regulator are administered in single-dosage form.

25. Method according to claim 23, wherein the protein C polypeptide and the blood glucose regulator are administered in the form of a first dosage form comprising a prepara- tion of a protein C polypeptide and a second dosage form comprising a preparation of a blood glucose regulator.