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WO1999036439A1 - Polypeptides, adn les codant, leurs formulations et leur utilisation dans l'inhibition de l'activation du facteur xii - Google Patents

Polypeptides, adn les codant, leurs formulations et leur utilisation dans l'inhibition de l'activation du facteur xii Download PDF

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Publication number
WO1999036439A1
WO1999036439A1 PCT/GB1999/000147 GB9900147W WO9936439A1 WO 1999036439 A1 WO1999036439 A1 WO 1999036439A1 GB 9900147 W GB9900147 W GB 9900147W WO 9936439 A1 WO9936439 A1 WO 9936439A1
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Prior art keywords
polypeptide
acid sequence
nucleic acid
seq
sequence
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PCT/GB1999/000147
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English (en)
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Lisa Seale
Sarah Finney
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Bio-Discovery Limited
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Application filed by Bio-Discovery Limited filed Critical Bio-Discovery Limited
Priority to AU20685/99A priority Critical patent/AU2068599A/en
Priority to CA002318358A priority patent/CA2318358A1/fr
Priority to JP2000540154A priority patent/JP2002508953A/ja
Priority to EP99901059A priority patent/EP1045862A1/fr
Publication of WO1999036439A1 publication Critical patent/WO1999036439A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a novel class of inhibitors of the initiation steps of blood coagulation and complement that can be derived, for example, from leech tissues or secretions.
  • the present invention relates to polypeptides, cDNA encoding therefor, and the use of the polypeptides and formulations thereof in cardiovascular, autoimmune and inflammatory disease.
  • the blood coagulation system is a cascade of enzymes that results in the formation of fibrin, which is an insoluble, fibrous polymeric protein that plugs leaks in the vasculature and prevents blood loss.
  • the individual enzymes of the coagulation system circulate in the normal state as pro-enzymes (so-called 'zymogens'), which are normally inactive.
  • the system can become activated by either:
  • tissue factor which is a membrane protein that is expressed on the outer surface of many cell types, particularly those found in blood vessel walls and that come into contact with blood at sites of leakage. This is called the 'extrinsic system'.
  • the first phase of the intrinsic pathway involves the binding of factors XII, XI, prekallikrein and high molecular weight kininogen in a complex on the negatively- charged surface.
  • factor XII becomes activated to factor aXQa which, in turn, activates factor XI to XIa.
  • Factors ⁇ XHa, ⁇ XIIa, XIa and kallikrein are all serine proteases of the initiation complex.
  • the coagulation cascade can become activated inappropriately and result in the formation of haemostatic plugs inside the blood vessels. Thereby, vessels can become hlocked and the blood supply to distal organs limited. This process is known as thrombosis and is associated with high mortality.
  • prosthetic devices that are in contact with blood is severely limited because of activation of the coagulation cascade and coating of the prosthetic surface, often compromising its function. Examples of such prosthetic devices are haemodialysers, cardiopulmonary by-pass circuits, vascular stents and in-dwelling catheters.
  • anticoagulants such as heparin
  • some patients are intolerant of heparin, which can cause thrombocytopaenia and the resultant risk of serious bleeding. There is therefore a need for new types of anticoagulant that do not cause such problems and that can be used in affected patients.
  • the complement system in mammalian blood is involved in the defence of the body against infection by foreign organisms. Like the coagulation system, it is also a complex enzyme cascade. When activated, it leads to the clearance of invading organisms, either indirectly, by coating them with proteins that are recognised by phagocytic cells (so- called 'immune adherence') or directly, by lysis. It also causes a localised inflammatory response leading to leukocyte activation and migration; increased vascular permeability; contraction of smooth muscle; and release of biogenic amines.
  • Activation of complement can occur through one or both of two pathways: (a) the classical pathway; and (b) the alternative pathway:
  • the first part of the classical complement pathway is analogous to the coagulation cascade. Activation can take place on an antibody-coated surface or other negatively-charged surface and gives rise to a serine protease, Cl esterase. This, in the presence of an activated cofactor (C4b), specifically cleaves the next protease zymogen (C2) in the cascade and leads eventually to an end-product that, in this case, is the "membrane attack complex", which lyses the targeted cell.
  • C4b activated cofactor
  • the stimulus can be bacterial lipopolysaccharides; various polyanions, the FAb portions of immunoglobulins; and negatively charged phospholipids. It leads to direct activation of C3 without involving Cl, C2 or C4.
  • Cl esterase is therefore the activated initiation complex of the classical complement pathway.
  • the first component of the classical pathway, Cl is a heteropolymer comprising one molecule of Clq, and two each of Clr and Cls.
  • Clq binds either to the Fc portion of either IgG or IgM or to negatively-charged substances, such as DNA, carrageenan, heparin, dextran sulphate, chondroitin sulphate, certain bacterial lipopolysaccharides and viral envelopes.
  • the Clq induces the autocatalytic activation of Clr to a serine protease, which in turn activates Cls to another serine protease.
  • the activated Cls in the complex can cleave both C4 and C2, which is essential in the activation of the whole classical pathway.
  • the complement cascade can also be activated inappropriately, and in such circumstances causes marked inflammation. It is thereby involved in the pathology of a large number of inflammatory and auto-immune diseases. It is therefore desirable to provide new inhibitors of the complement pathway for the treatment of such diseases; to reduce tissue rejection of implanted organs; and to inhibit complement activation on foreign surfaces, such as haemodialysers and cardiopulmonary by-pass circuits.
  • Such inhibitors are expected to have an advantage over inhibitors that act on enzymes lower down the cascades, because the initiation complexes occur in much lower concentrations. This enables them to be inhibited by lower concentrations of their inhibitors, which could then be administered in lower doses, avoiding possible toxicity and reducing the cost of treatment, relative to inhibitors of enzymes lower down the cascades.
  • inhibitors of serine proteases in the initiation complexes of coagulation and complement fall into three categories: low molecular weight amidines or guanidines; small polypeptides that can be isolated from parasites; and the naturally-occurring blood serpin (serine protease inhibitor), Cl inhibitor.
  • the low molecular weight compounds described to date lack specificity and inhibit factor Xlla and Cl esterase much less potently than other proteases. Therefore, they are not suitable for treating human complement-mediated disease.
  • the plasma serpin, Cl inhibitor inhibits both the intrinsic activation of coagulation and Cl esterase, although it is rather non-specific, as it inhibits factors Xlla, XIa, kallikrein, Cls and Clr.
  • Cl inhibitor can be purified from human blood and a recombinant version has also been described. Cl inhibitor from blood is used to treat the disease of its own deficiency, angioneurotic oedema. Recent evidence in pilot human clinical trials indicates that it is also effective in treating both septic shock and capillary leak syndrome. Further the administration of Cl inhibitor in a feline model of myocardial reperfusion injury significantly improved the recovery of cardiac contractility and reduced the infarcted area of the myocardium.
  • Cl inhibitor is a large protein of molecular weight 104,000 Da, containing 478 amino acids; the recombinant protein is consequently very expensive to produce.
  • Cl inhibitor in view of the promising effects of Cl inhibitor in the clinic, but its production difficulties, there is a need for new compounds that mimic the effects of Cl inhibitor but which are easier and less expensive to produce.
  • amino acids are abbreviated to their single letter codes
  • X represents any natural amino acid, preferably alanine or N-acyl alanine or N-alkyl alanine, especially alanine
  • X' may be cysteine [SEQ ID No: 9] or glutamic acid [SEQ ID No: 10], especially cysteine.
  • 'acyl' is meant (C,. 5 alkoxy)carbonyl, such as formyl or acetyl; and by 'alkyl' is meant a straight or branched C,. 5 alkyl chain.
  • polypeptides have the following ten amino acids at the N-terminus, ie wherein, in [SEQ ID No: 1], X is alanine:
  • X 1 is, as above, cysteine or glutamic acid, respectively, especially cysteine.
  • polypeptides according to the present invention comprise the sequence of 37 amino acids [SEQ ID No: 13]; more preferably, the sequence of 60 amino acids [SEQ ID No: 2].
  • polypeptide according to the present invention comprises the sequence of 122 amino acids [SEQ ID No: 3], which exhibits a calculated molecular weight of about 14kDa:
  • the present invention therefore provides a polypeptide, in particular an isolated, purified or synthesised polypeptide, derivable from a Haementeria leech, which polypeptide is suitable for inhibiting Cl esterase and/or factor XII activation and which polypeptide comprises a sequence of amino acids as hereinbefore defined.
  • the present invention further provides a polypeptide that is substantially homologous or analogous to any of the sequences specified herein, including derivatives (such as a chimeric derivative) or bioprecursors thereof (including wherein the polypeptide is linked to a so-called 'leader sequence'), or salts of any of these.
  • Haementeria-de ⁇ vdb e polypeptides encompassed within the scope of this invention are hereinafter collectively referred to as 'haemostasins' .
  • Haemostasins are potent inhibitors of the initiation complexes of blood coagulation and/or of complement. As a consequence, they prolong the activated partial thromboplastin clotting time of human plasma (as shown hereinafter in Example 8) and/or inhibit the haemolysis of antibody-coated sheep erythrocytes in a classical CH 50 assay (as shown hereinafter in Example 10) at concentrations in the range of from 0.01 to 5 ⁇ g/ml.
  • haemostasins of the present invention by 'homologue' is meant a polypeptide in which no more than ( ⁇ ) 20% of the amino acids in the polypeptide chain differ from those listed.
  • the figure of 20% is based on the fact that many homologues of another leech protein, hirudin, occur naturally in Hirudo medicinalis and are described in the literature; the most diverse of these differ in 15 of the 65 amino acids in the polypeptide chain.
  • haemostasins are polymorphic and sequences where amino acid number 16 may be threonine (T) instead of serine (S) [SEQ ID No: 7] and where number 60 may be asparagine (N) instead of aspartic acid (D) [SEQ ID No: 8] are encompassed.
  • 'analogue' is meant that one or more additional amino acids may be interposed in the polypeptide chain, provided that they do not interfere with the pharmacological activity of the haemostasin.
  • the haemostasins according to this invention therefore also encompass amino acid sequences including post-translational modifications, such as sulphation of the aromatic ring of tyrosines 119 and/or 121, as has been observed in the hirudins. Furthermore, since the motif N V T occurs at positions 23 - 25 of [SEQ ID No: 2], which is a well-known site for potential glycosylation, the haemostasins further encompass polypeptides of the [SEQ ID No: 2] where aspargine 23 is modified by an N-linked complex carbohydrate containing not more than 10 ( ⁇ 10) sugars or sugar derivatives in a single or branched chain.
  • haemostasins also encompass truncated and therefore lower molecular weight forms of the polypeptides defined above at the N-terminus, for example forms where one or two of the N-terminal amino acids, such as given in [SEQ ID No: 1], are deleted; and forms where the N-terminus is extended by the addition of one or two amino acids to the N- terminal amino group.
  • haemostasins also encompass truncated or extended (lower or higher molecular weight) forms of the polypeptide of [SEQ ID No: 3, 7 or 8] at the C-terminus.
  • haemostasins also encompass the case where the sequence is cleaved, especially after amino acid 37 (R) of [SEQ ID No: 2, 3, 7 or 8], so that it exists as two or more polypeptide chains normally cross-linked by the usual disulphide bonds.
  • haemostasins encompass the case where a so-called 'leader sequence' is present when the polypeptide is expressed in vivo, especially the case where the leader sequence comprises [M S F K I V L L L F L V V C V V A S L A].
  • Haemostasins can advantageously form salts, preferably pharmaceutically acceptable salts, with any suitable non-toxic metal ion, organic or inorganic acid, or base.
  • inorganic acids include hydrochloric, hydrobromic, sulphuric, phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrosulphate.
  • orgamc acids include mono-, di- and tri-carboxylic acids, such as acetic, glycolic, lactic, pyruvic and sulphonic acids, or the like.
  • bases include ammonia, primary, secondary or tertiary amines, or quaternary ammonium ion.
  • Other suitable salts are known to those skilled in the art.
  • Haemostasins may be extracted from Haementeria leech tissue or secretions by, for example, homogenisation of substantially the whole leech, or its salivary glands, its proboscis or the like, in a suitable buffer. Neither inhibitors of factor XII activation nor complement inhibitors have been previously identified in, or extracted from, Haementeria leeches.
  • the present invention therefore further provides an inhibitor of factor XII activation and/or an inhibitor of Cl esterase, derivable from Haementeria leech tissue or secretions.
  • the present invention provides a haemostasin derivable from a Haementeria leech, which haemostasin is suitable for inhibiting factor XII activation and/or Cl esterase activity.
  • the inhibitor or haemostasin inhibits both factor XII activation and Cl esterase activity.
  • 'derivable' encompasses both material that is directly derived, such as by isolation and/or purification, as well as material which is indirectly derived or converted to a chemically-modified derivative, or which is chemically or biologically synthesised, including genetically-engineered.
  • the inhibitors or haemostasins according to this invention are typically extracted or purified from Haementeria leeches using a combination of known techniques, such as ion-exchange, gel filtration and/or reverse phase chromatography.
  • Leeches of the same genus or even the same species often have polypeptides in their saliva that have similar biochemical effects and highly homologous amino acid sequences.
  • isoforms may exist that differ by only a few amino acids.
  • the present invention also comprises such isoforms and analogues derivable from Haementeria leeches, as described hereinabove.
  • an inhibitor of the intrinsic pathway of blood coagulation which inhibitor is derivable from leech tissue or leech secretions from leeches of the order Rhynchobdellida, of the genus Haementeria, and especially from the species ghilianii or officinalis, more especially, H. ghilianii.
  • Such inhibitors have an anticoagulant effect in plasma, since they prolong the activated partial thromboplastin time but not the prothrombin time or thrombin time.
  • This effect is probably caused by the inhibitory effect of haemostasins on the activation of plasma by surfaces, such as glass or dextran sulphate, that can be measured by a reduction in the appearance of enzymically active factor ⁇ XIIa or factor XIa.
  • a further aspect of this invention provides an inhibitor of Cl esterase, which inhibitor is derivable from leech tissue or leech secretions from leeches of the order Rhynchobdellida, of the genus Haementeria, and especially from the species ghilianii or officinalis, more especially, H. ghilianii.
  • Haemostasins have the ability to inhibit: the enzymic cleavage of 4-nitroanilide from d-Val-Ser-Arg-4-nitroanilide by purified Cls; the whole complement- mediated haemolysis of antibody-sensitised erythrocytes; and the formation of the immunologically-detected membrane attack complex in serum in response to bound antibodies.
  • haemostasins can also be prepared by providing a host, transformed with an expression vector comprising a DNA sequence encoding the haemostasin polypeptide under such conditions that said polypeptide is expressed therein and thereafter, if desired, isolating the polypeptide thus obtained.
  • This approach is typically based on obtaining a nucleotide sequence encoding the polypeptide it is wished to express and expressing the polypeptide in recombinant organisms.
  • the cultivation of the genetically-modified organism leads to the production of the desired product displaying full biological activity.
  • the present invention therefore also comprises a recombinant haemostasin, or a synthetic, or genetically- or protein-engineered, equivalent to a haemostasin polypeptide according to the invention.
  • the present invention further provides a nucleic acid sequence, in particular an isolated, purified or recombinant nucleic acid sequence, comprising:
  • the present invention provides a nucleic acid sequence as defined above, wherein the sequence is a DNA or RNA sequence, such as cDNA or mRNA. More particularly, the present invention provides a DNA sequence identified herein by [SEQ ID No: 4], which sequence corresponds with the polypeptide identified herein as [SEQ ID No: 3] including its leader sequence. Given the polymorphisms already described above with reference to [SEQ ID Nos: 7 and 8], the present invention further provides the corresponding DNA sequences identified herein as [SEQ ID Nos: 5 and 6], respectively.
  • the present invention further provides a method for the preparation of a polypeptide according to the present invention, which method comprises:
  • the present invention further provides: a recombinant construct comprising any nucleic acid sequence according to the invention; a vector comprising such a construct; and a host transformed or transfected by such a vector.
  • the present invention therefore further provides a cell, plasmid, virus, live organism or other vehicle that has been genetically or protein-engineered to produce a polypeptide or haemostasin according to the present invention, said cell, plasmid, virus, live organism or other vehicle having incorporated expressably therein a sequence as disclosed herein.
  • Such cells may include animal, such as mammal, eg human or humanised cells, for use in gene therapy to treat or prevent conditions such as those mentioned herein.
  • the present invention therefore provides a method for the treatment or prevention of a condition or disorder mentioned herein, wherein the polypeptide is administered by means of being expressed in the cells of the patient, which cells have incorporated expressably therein a nucleic acid sequence.
  • the haemostasins of the invention may be administered as a pharmaceutical formulation.
  • the present invention provides the use of a polypeptide, particularly a haemostasin, described herein or a nucleic acid sequence coding for the polypeptide in medicine, including gene therapy; and also the use of such a polypeptide in the manufacture of a medicament.
  • a pharmaceutical formulation comprising a haemostasin according to the invention (as described above) and a pharmaceutically acceptable carrier therefor.
  • pharmaceutically acceptable carrier should be taken to mean any inert, non-toxic, solid or liquid filler, diluent or encapsulating material, or other excipient, which does not react adversely with the active ingredient(s) or with a patient.
  • Such formulations and carriers are well known in the art and include pharmaceutical formulations that may be, for example, administered to a patient systemically, such as parenterally, or orally or topically.
  • parenteral' as used here includes subcutaneous, intravenous, intramuscular, intra-arterial and intra-tracheal injection, and infusion techniques.
  • Parenteral formulations are preferably administered intravenously, either in bolus form or as a constant infusion, or subcutaneously, according to known procedures.
  • Preferred liquid carriers which are well known for parenteral use, include sterile water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants and wetting agents, etc.
  • Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs or the like, or may be presented as a dry product for reconstitution with water or other suitable vehicle for use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.
  • Formulations suitable for topical application may be in the form of aqueous or oily suspensions, solutions, emulsions, gels or, preferably, emulsion ointments.
  • Unit doses of the pharmaceutical formulations according to the invention may contain daily required amounts of the haemostasins, or sub-multiples thereof to make a desired dose.
  • the optimum therapeutically-acceptable dosage and dose rate for a given patient (which may be a mammal, such as a human) depend on a variety of factors, such as the potency of the active ingredient(s); the age, body weight, general health, sex and diet of the patient; the time and route of administration; rate of clearance; the object of the treatment (eg treatment or prophylaxis); and the nature of the disease to be treated.
  • a systemic dose in the range of from 0.005 to 50 mg/kg body weight, preferably between 0.05 and 10 mg/kg and more preferably from 0.1 to 1 mg/kg, will be effective.
  • one single dose may contain in the range of from 0.05 to 10 mg/kg body weight, whether applied systemically or topically.
  • Oral formulations are preferably administered in 2 to 6, preferably 3 to 4, sub-doses per day.
  • a further aspect of this invention provides covalent complexes of the haemostasins with the surfaces of prostheses or extracorporeal circulations that are exposed to blood in order to prevent either the initiation of complement and/or intrinsic coagulation and its pathological sequellae.
  • the present invention provides the use of a polypeptide, particularly a haemostasin, described herein or a pharmaceutical formulation thereof or a nucleic acid sequence coding therefor in the treatment or prophylaxis of a condition or disorder related to Cl esterase initiation and/or factor XII activation.
  • the present invention provides a method for the treatment or prophylaxis of a condition or disorder related to Cl esterase initiation and/or factor XII activation, which method comprises the administration to a patient in need thereof of an effective, inhibitory amount of the polypeptide or a pharmaceutical formulation thereof.
  • the use or method is one wherein the medical condition or disorder is selected from one or more of: cardiovascular disease, inflammation and auto-immune diseases.
  • the haemostasins can potentially be used to inhibit the activation of coagulation, which happens to the detriment of patients in, for example, thrombotic disease selected from deep venous thrombosis, pulmonary embolus, and thrombosis associated with angioplasty and endarterectomy.
  • thrombotic disease selected from deep venous thrombosis, pulmonary embolus, and thrombosis associated with angioplasty and endarterectomy.
  • disease may also be alleviated by the ability of the haemostasins to inhibit both complement activation and the intrinsic pathway of blood coagulation, such as in haemodialysis, cardiopulmonary bypass, or rejection of transplanted organs or tissues, or in the syndromes: sepsis; myocardial infarction; stroke; particularly in the injury caused to tissues by reperfusion after an ischaemic period (such as occurs after heart attack or cerebral stroke); atherosclerosis; shock; vasculitis; rheumatoid arthritis; sickle cell anaemia or angioedema.
  • haemostasins may be used in conditions associated with activation of complement as adjudged by the appearance of activated components or their complexes with natural inhibitors in biological fluids and/or their deposition in diseased tissues, such as: various autoimmune diseases (eg lupus arthritis); glomerulonephritis; nephritis; nephropathy; systemic sclerosis; Behcets syndrome; cerebral lupus; Guillan-Barre disease; multiple sclerosis; myasthenia gravis; pemphigus; bullous pemphigoid; phototoxic reactions; thermal burns; anaphylaxis; asthma; skin reactions; infections; inflammatory bowel disease; thyroiditis; infertility; Alzheimer's disease; paroxysmal nocturnal haemoglobinuria; and haemolytic anaemia.
  • various autoimmune diseases eg lupus arthritis
  • glomerulonephritis glomerulonephritis
  • haemostasin of the invention or formulation thereof may advantageously be administered in combination with an additional anticoagulant or a thrombolytic agent to reduce tissue injury following reperfusion or to prevent complement activation on the surface of haemodialyser or cardiopulmonary bypass apparatus.
  • haemostasins may be used in combination with immunosuppressant agents to decrease transplant rejection or with steroidal or non-steroidal anti-inflammatory drugs.
  • combination is meant the simultaneous or sequential administration of the haemostasins with the other, pharmacologically active, ingredient(s).
  • S2314 refers to H-D-Val-Ser-Arg-4-nitroanilide
  • S2266 refers to H-D-Val-Leu-Arg-4-nitroanilide
  • S2302 refers to H-D-Pro-Phe-Arg-4-nitroanilide
  • S2366 refers to pyroGlu-Pro-Arg-4-nitroanilide
  • S2238 refers to H-D-Phe-Pip-Arg-4-nitroanilide.
  • Extracts of different leech species were prepared by homogenisation of either the whole salivary complex or the individual glands in CAE (Diasorin Ltd, Charles House, Toutley Road, Wokingham, Berks, UK) diluent and assayed in the Diasorin CAE test. Samples containing CAE diluent (0.5 ml), extract (0.095 ml) and normal human serum (0.005 ml) were made up and 0.15 ml aliquots placed in the antibody-coated wells.
  • the number of anti-CAE units was calculated on the basis that the control serum contained 100 CAE units/well and 1 inhibitory unit inhibits 1 CAE unit by 100 %.
  • the inhibitory activity is demonstrable in the salivary complexes of both Haementeria officinalis and H ghilianii and is highest in the salivary glands (Table 1).
  • the presence of inhibitors of the Cls component of complement can be demonstrated by the ability of leech extracts to inhibit a chromogenic assay of the enzyme.
  • An extract of a single whole salivary complex of Haementeria officinalis, comprising anterior, posterior glands and proboscis* was prepared by homogenisation in phosphate buffered saline (0.45 ml).
  • An extract of the anterior salivary glands of a single Haementeria ghilianii was prepared by homogenisation in phosphate buffered saline (0.5 ml). Both extracts were centrifuged at 13,000 rpm for 3 min and the supernatant was used for the assay.
  • Cuvettes were made up as follows: 8 mM S 2314 (0.01 ml); extract or phosphate buffered saline (0.04 ml); and 0.1M sodium phosphate buffer pH 7.3 (0.14 ml). The reaction was started by addition of 0.025 mg/ml Cls (Calbiochem)(0.01 ml) and monitored in a Beckman DU 650 spectrophotometer by the increase in absorbance at 405 nm.
  • Table 2 shows the rate of the enzyme-catalysed reaction after subtraction of the rate of a similar cuvette where water replaced the enzyme.
  • the reaction rate where water replaced enzyme was between 0.03 and 0.11 mAbs/min in each experiment.
  • Table 2 demonstrates the ability of the extracts of both Haementeria officinalis and Haementeria ghilianii to inhibit Cls and indicates the presence of an inhibitor of this enzyme.
  • the active fraction was reconstituted in water (2 ml) and applied to a 16 x 600 mm column of Superdex-75 which had been equilibrated in phosphate-buffered saline pH 7.4.
  • Direct assay of the fractions in the CAE method demonstrated that the inhibitory activity eluted in the major peak in 0.52-0.66 column volumes.
  • the active fraction was homogeneous by SDS PAGE under reducing conditions and had an apparent molecular weight of 24.7 kDa.
  • the fraction containing the peak from ProRPC HPLC described in example 3 contained a protein (hereinafter designated "haemostasin 1") which gave a single amino acid sequence from the N-terminus on an Applied Biosystems 473A automatic sequencer. After identifying the first 10 amino acids, the sequence of the first 29 amino acids was found:
  • EXAMPLE 5 mRNA and Partial cDNA Sequence from H. ghilianii, Recombinant DNA Vectors and Plasmids
  • mRNA was extracted from the complete salivary complexes of 20 Haementaria ghilianii by thawing the frozen tissue in guanidinium thiocyanate lysis solution (8 ml) as described in Ambion Micro (A) Pure kit (Ambion Inc., 2130 Woodward Street, Austin, Texas, USA). Following Dounce homogenisation, dilution buffer (16 ml) was added, mixed and supernatant collected following centrifugation (12000 g, 4°C, 15 min). The supernatant was mixed with oligo dT (deoxythymidine) resin (20 mg) at room temperature for 60 min and collected by centrifugation (4000 g, room temp, 3 min).
  • oligo dT deoxythymidine
  • the oligo dT pellet was then treated to three cycles of addition of high salt binding buffer (1 ml), vortexing and centrifugation (4000 g, room temp, 3 min). The final pellet was re-suspended in wash buffer (0.5 mis) added to a spin column and centrifuged (5000 g, room temp, 20 sec). The column was washed three more times in the same manner by addition of wash buffer (5mls) and centrifugation.
  • the bound Poly A+ RNA was finally collected by adding pre- warmed (65°C) elution buffer (100 ⁇ l), centrifugation and precipitation in the presence of ammonium acetate (20 ⁇ l), glycogen (1 ⁇ l) and ethanol (550 ⁇ l) and stored at -20 °C.
  • RNA reverse transcription reaction
  • dNTPs mix Boehringer Mannheim
  • random hexamer primers 2 ⁇ l
  • sterile water 3 ⁇ l
  • RT-PCR buffer (10 x concentrated, 2 ⁇ l), placental RNAase inhibitor (1 ⁇ l), M-MLV reverse transcriptase (1 ⁇ l).
  • the reaction was allowed to proceed at 42 °C for 80 min and inactivated by incubation at 92°C for 10 min. The reaction was then stored at -20 °C.
  • RT-PCR was used to produce double-stranded cDNA for sequencing.
  • the following reaction was set up (Ambion Retroscript kit): reverse transcriptase reaction above (5 ⁇ l), reaction buffer (10 x concentrated, 5 ⁇ l), dNTPs mix (2.5 mM each) (2.5 ⁇ l), 5 ⁇ M primers A (2.5 ⁇ l), 5 ⁇ M primers B (2.5 ⁇ l), Super Taq+, 2 units (Ambion)(0.5 ⁇ l) and sterile water (33 ⁇ l).
  • the reaction mixture was denatured at 94 °C for 2 min and cycled in a Techne Genius DNA Thermal Cycler for 30 cycles each of 94 °C for 30 sec, 45 °C for 60 sec and 72 °C for 90 sec. The final incubation was at 72 °C for 10 min.
  • the resulting PCR sample was subjected to another round of PCR using different primers based on the amino acid sequences described in example 4 to increase specificity.
  • the reaction mixture comprised: PCR Reaction above (5 ⁇ l), Taq Buffer (Life Technologies Ltd, Inchinnan Business Park, Paisley, UK)(10 x concentrated, 5 ⁇ l, dNTPs mix (2.5 mM each,)(2.5 ⁇ l), primers C (5 ⁇ M)(2.5 ⁇ l), primers D (5 ⁇ M)(2.5 ⁇ l), Taq (Life Tec)(lU)(0.25 ⁇ l) and sterile water (34.75 ⁇ l).
  • the reaction mixture was denatured at 94 °C for 2 min and cycled 30 times in a Techne Genius DNA Thermal Cycler, as follows: 94 °C for 30 sec, 50 °C for 30 sec and 72 °C for 90 sec. The final incubation was 72 °C for 10 min.
  • RACE Rapid Amplification of cDNA Ends
  • first strand buffer (10 x concentrated, 2 ⁇ l), dNTPs mix (10 mM each)(l ⁇ l), AMN reverse transcriptase (20 U)(l ⁇ l) and sterile water (l ⁇ l).
  • the reaction was allowed to proceed at 42°C for 60 min.
  • first strand reaction (10 ⁇ l), sterile water (48.4 ⁇ l), second strand buffer (5 x concentrated, 16 ⁇ l), d ⁇ TPs mix (10 mM each)(1.6 ⁇ l) and 20 x second strand enzyme cocktail (4 ⁇ l). The mixture was incubated at 16°C for 90 min.
  • T4 D ⁇ A polymerase (2 ⁇ l) and incubation was continued for a further 45 min at 16°C.
  • the reaction was terminated by the addition of 10 mM EDTA, and pure D ⁇ A extracted by phenol/chloroform and precipitated by ethanol. The pellet was dissolved in sterile water (10 ⁇ l).
  • Marathon adaptor sequence was ligated to the ends of the double stranded cD ⁇ A to facilitate isolation, by incubation of double stranded cD ⁇ A (5 ⁇ l), 5 x ligation buffer (2 ⁇ l), Marathon adaptor sequence (10 ⁇ M)(2 ⁇ l) and T4 ligase (1U)(1 ⁇ l) for 14 hours at 16 °C and terminated at 70 °C for 5 min. This was followed by the addition of Tricine- EDTA buffer (lOmM Tricine-KOH pH 8.5, 0.1 mM EDTA)(240 ⁇ l).
  • RACE RACE was used to isolate the 5' and 3' ends of the whole sequence.
  • the following incubations were designed: 10 x PCR reaction buffer (5 ⁇ l), dNTP mix (10 mM)(l ml), 50 mM MgCl 2 (1.5 ⁇ l), Taq (2U) (0.5 ⁇ l), sterile water (35 ⁇ l) and for the 5' end as follows: cDNA (5 ⁇ l), API Primer (Clontech)(l ⁇ l), 10 ⁇ M primer E [AGTGTTGCAAGTACAGAA] (1 ⁇ l); or for the 3' end, as follows: cDNA (5 ⁇ l), API Primer (1 ⁇ l), gene-specific primer F [AAGAAATGCGAATGCAGG](10 ⁇ M).
  • reaction mixtures were denatured at 94 °C for 2 min and cycled in a Techne Genius DNA Thermal Cycler for 30 cycles of: 94 °C for 30 sec, 45 °C for 60 sec and 72 °C for 90 sec with a final incubation at 72 °C for 10 min.
  • DNA fragments were purified as in example 5, sequenced and sequences corresponding to the amino acid sequence defined in example 4 identified. This allowed elucidation of two further nucleotide sequences coding for haemostasins: the complete DNA sequence [SEQ D No: 4] and another variant DNA sequence [SEQ ID No: 5]. The complete amino acid sequences of three haemostasin variants [SEQ ID Nos: 3, 7 and 8] were deduced from the DNA. The calculated molecular weight based on the amino acid sequence is approximately 14,200 Da, assuming no post-translational modification.
  • nucleotide sequences [SEQ ID Nos: 5 and 6] both contained code at the 5' end for a leader amino acid sequence [M S F K I V L L L F L V V C V V A S L A], which is not attached to the N-terminus of the extracted native protein.
  • EXAMPLE 7 Inhibition of Factor XII Activation by Haemostasin 1
  • haemostasin 1 (cf. Examples 3 and 4) on the activation of factor XII was investigated in a chromogenic assay.
  • the cuvette contained: the supernatant from acetone-treated plasma (0.025 ml) prepared by treatment of human plasma with 0.33 volumes of acetone and incubation for 10 min; 50 mM Tris HC1 containing 3.36 g/1 EDTA pH 7.9 (0.075 ml); 10. ⁇ M soybean trypsin inhibitor (0.04 ml); inhibitor sample or phosphate-buffered saline (0.1 ml); and 40 ⁇ g/ml dextran sulphate (0.1 ml).
  • haemostasin 1 was investigated on plasma clotting by standard methods. Human plasma samples were made up to contain a range of haemostasin 1 concentrations from 0.02 to 1.23 ⁇ M or with the equivalent buffer controls and the time for clotting to occur when activated by tissue thromboplastin (one stage prothrombin time) or thrombin (thrombin time) or thrombosil (Ortho Diagnostics, Amersham, Bucks.) (activated thromboplastin time) was measured on a Sysmex CA5000 automated coagulometer. There was no effect on the one stage prothrombin time, a measure of the extrinsic coagulation pathway, and no effect on the thrombin time.
  • haemostasin 1 prolonged the activated partial thromboplastin time.
  • a concentration of 3.8 ⁇ g/ml prolonged the activated thromboplastin time by 50%.
  • the effect of haemostasin 1 on factor Xlla is therefore reflected in its ability to inhibit the clotting of plasma.
  • Inhibitors of the classical complement pathway can be demonstrated in a haemolytic assay whereby they inhibit the ability of complement to lyse antibody-coated erythrocytes. Lysis is quantified by measuring the released haemoglobin at 405 nm. The IC 50 of haemostasin 1 prepared as in example 3 was investigated in a standard haemolytic (CH 50 ) assay.
  • Sheep erythrocytes were washed 3 times in triethanolamine-buffered saline (TBS-G) (128.3 mM NaCl, 17.7 mM HC1, 20.6 mM triethanolamine, 0.5 mM MgCl 2 , 0.15 mM CaCl 2 , 0.05 % (w/v) gelatin, pH 7.35) by centrifugation at 2000 rpm for 10 min and re-suspension.
  • TSS-G triethanolamine-buffered saline
  • the erythrocytes were coated with antibody by adding haemolysin (Harlan-SeraLab), diluted 1:200 in TBS-G (4 ml) to erythrocytes (4 ml, diluted 1:4 in TBS-G) and incubating at 37°C for 30 min then at 0°C for a further 30 min with periodic mixing.
  • the coated erythrocytes were washed twice in TBS-G and re-suspended in TBS- G supplemented with 2.5 % (w/v) glucose and 0.1 % (w/v) sodium azide, and diluted in TBS-G to give an absorbance at 405 nm of 0.7 when fully lysed.
  • blank microtitre plate wells contained TBS-G (0.2 ml) or water plus coated erythrocytes (0.05 ml) giving absorbance values for 0 % and 100 % haemolysis, respectively.
  • Test wells contained TBS-G (0.1 ml), haemostasin or phosphate-buffered saline (PBS) (0.05 ml), human serum (complement) diluted in TBS-G to give approximately 75 % haemolysis (0.05 ml) and erythrocytes (0.05 ml).
  • PBS haemostasin or phosphate-buffered saline
  • human serum complement
  • complement can also be activated by the alternative pathway by chelation of calcium ions and provision of magnesium ions [Servais G, Walmagh J, Duchateau J. J Immunol Meth 140:93-100 (1991)].
  • Rabbit erythrocytes were washed 4 times in gelatin veronal buffer (Sigma) containing 10 mM EGTA and 7 mM MgCl 2 pH 7.2 (ie VCM-MEG) by centrifugation at 2000 rpm for 10 min and re- suspension.
  • Microtitre plate wells contained VCM-MEG (0.15 ml), test sample (0.075 ml) human plasma diluted 1:4 with VCM-MEG (0.075 ml), 1 % v/v washed erythrocytes (0.075 ml). The plate was covered and incubated at 37 °C for 45 min before addition of 0.2 M EDTA (0.0225 ml), centrifugation at 1000 rpm for 3 min and transfer of the supernatants (0.1 ml) to a fresh flat-bottomed microtitre plate. Absorbance was read at 405 nm.
  • the percent haemolysis was compared with a PBS buffer blank, samples where the plasma was omitted (0 % haemolysis) or where water (0.015 ml) was substituted for the VCM-MEG buffer (100 % haemolysis). There was no significant inhibition of the alternative pathway by haemostasin 1 at concentrations up to 20 ⁇ g/ml.
  • haemostasin 1 for various serine proteases was determined in chromogenic substrate assays. Assays were set up using commercially-available substrates at concentrations at or near to the published Km. Enzyme rate curves were monitored by the release of 4-nitroanilide from the substrate at 405 nm in a spectrophotometer in cuvettes containing various concentrations of haemostasin 1. Tissue kallikrein was Padutin (Bayer AG, Leverkusen, Germany). Plasma kallikrein was obtained from Quadratech, (Epsom, Surrey, UK).
  • Factor ⁇ XIIa was activated from normal human plasma by treatment of the plasma with 0.33 volumes of acetone, followed by centrifugation.
  • the acetone-treated plasma (0.025 ml), 50 mM Tris HC1 containing 3.36 g/1 EDTA pH 7.9 (0.075 ml), phosphate buffered saline (0.1 ml), 10 ⁇ M soy bean trypsin inhibitor (Sigma)(0.04 ml) and 40 ⁇ g/ml dextran sulphate (0.1 ml) were incubated for 10 min at 37 °C prior to addition of 0.87 mM substrate S2302 in 22 mM Tris HC1 pH 7.9 (0.46 ml) in the presence of either haemostasin 1 or its vehicle, phosphate buffered saline (0.1 ml).
  • Factor XIa was activated similarly from normal human plasma and the assays were carried out with S2366 in the presence of 2.5 ⁇ g/ml corn trypsin inhibitor (Rho reagents, Gerrards Cross, Bucks, UK) to inhibit substrate cleavage by factor ⁇ XIIa.
  • Human thrombin was obtained from NIBSC (Blanche Lane, Potters Bar, UK). The enzymes, Clr (Sigma) and Cls (Calbiochem), were incubated at 37 °C for 1 h prior to use to allow their activation.
  • Complement factor D was purchased from Sigma Chemical Company.
  • Table 4 shows that haemostasin 1 only inhibited complement Cls of all of these enzymes, having no effect at concentrations up to 7.4 ⁇ g/ml on the others.
  • factor XI activation is dependent on the generation of factor ⁇ XIIa, it was expected that its activation would also be inhibited by haemostasin 1.
  • Plasma was activated in the presence or absence of different concentrations of haemostasin 1 in a glass cuvette containing acetone-treated plasma (0.025 ml), 50 mM Tris HCl containing 3.36 g/1 EDTA pH 7.9 (0.075 ml), 10 ⁇ M soybean trypsin inhibitor (0.04 ml), inhibitor sample or phosphate buffered saline (0.1 ml) incubated at 20 °C for 10 min.
  • a 0.3 ml sample was assayed for factor XIa activity by mixing in a cuvette containing 50 mM Tris HCl pH 7.9 (0.26 ml), 50 ⁇ g/ml corn trypsin inhibitor (0.04 ml) and 4 mM S2366 (0.2 ml).
  • Factor XI activation by glass was inhibited by haemostasin 1 with an IC 50 of 7.4 ⁇ g/ml.

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Abstract

L'invention porte sur un polypeptide pouvant être dérivé d'une sangsue Haementeria et qui est approprié pour inhiber l'activation d'estérase C1 et/ou du facteur XII, une séquence d'acide nucléique codant le polypeptide. Ce polypeptide peut être utilisé dans une formulation pharmaceutique ou en thérapie génique pour traiter ou prévenir les maladies cardio-vasculaires, inflammatoires ou autoimmunes.
PCT/GB1999/000147 1998-01-16 1999-01-15 Polypeptides, adn les codant, leurs formulations et leur utilisation dans l'inhibition de l'activation du facteur xii WO1999036439A1 (fr)

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AU20685/99A AU2068599A (en) 1998-01-16 1999-01-15 Polypeptides, dna coding therefor, formulations thereof, and their use in inhibiting factor xii activation
CA002318358A CA2318358A1 (fr) 1998-01-16 1999-01-15 Polypeptides, adn les codant, leurs formulations et leur utilisation dans l'inhibition de l'activation du facteur xii
JP2000540154A JP2002508953A (ja) 1998-01-16 1999-01-15 ポリぺプチド、そのｄｎａコード、その製法、及びｘｉｉ因子活性化の抑制におけるそれらの使用法
EP99901059A EP1045862A1 (fr) 1998-01-16 1999-01-15 Polypeptides, adn les codant, leurs formulations et leur utilisation dans l'inhibition de l'activation du facteur xii

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GBGB9800817.0A GB9800817D0 (en) 1998-01-16 1998-01-16 Serine protease inhibitors
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047963A3 (fr) * 1999-12-24 2002-05-10 Bio Discovery Ltd Inhibiteurs d'activation de complement, leur preparation et leur utilisation
WO2002068641A1 (fr) * 2001-02-28 2002-09-06 Fu Wai Hospital Chinese Academy Of Medical Sciences Facteur d'inhibition d'inflammations fwa116
WO2001098365A3 (fr) * 2000-06-21 2003-07-03 Zymogenetics Inc Inhibiteurs peptidiques et polypeptidiques du complement c1s
EP2497489A1 (fr) * 2011-03-09 2012-09-12 CSL Behring GmbH Inhibiteur du facteur XII pour le traitement de la pénombre ischémique cérébrale et l'ischémie d'autres organes
WO2012120128A1 (fr) * 2011-03-09 2012-09-13 Csl Behring Gmbh Inhibiteurs du facteur xii destinés à être administrés avec des procédures médicales comprenant le contact avec des surfaces artificielles
US8715672B2 (en) 2004-12-23 2014-05-06 Csl Behring Gmbh Treatment of diseases linked to pathological kinin formation
US20140378653A1 (en) * 2012-01-31 2014-12-25 Csl Behring Gmbh Factor XII Inhibitors for the Treatment of Neurological Inflammatory Disorders
WO2014207199A1 (fr) * 2013-06-28 2014-12-31 Csl Behring Gmbh Polythérapie utilisant un inhibiteur du facteur xii et un inhibiteur de c1
WO2015054569A1 (fr) * 2013-10-10 2015-04-16 Viropharma Holdings Limited Procédés d'inhibition de la voie alternative d'activation du système immunitaire complémentaire et compositions utilisées dans ces procédés
US10286047B2 (en) 2013-03-08 2019-05-14 Csl Behring Gmbh Treatment and prevention of remote ischemia-reperfusion injury
WO2019113642A1 (fr) * 2017-12-15 2019-06-20 Csl Limited Utilisation d'un inhibiteur de fxiia dans le traitement d'une fibrose rénale et/ou d'une maladie rénale chronique

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0333356A2 (fr) * 1988-03-04 1989-09-20 Biogen, Inc. Peptides de hirudine
WO1995007986A1 (fr) * 1993-09-14 1995-03-23 Genentech, Inc. Compositions pharmaceutiques contenant l'ecotine et des homologues de celle-ci
WO1996034965A2 (fr) * 1995-05-05 1996-11-07 Chiron Corporation Proteines chimeres mcp et daf a domaine de localisation de surface de cellule

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0333356A2 (fr) * 1988-03-04 1989-09-20 Biogen, Inc. Peptides de hirudine
WO1995007986A1 (fr) * 1993-09-14 1995-03-23 Genentech, Inc. Compositions pharmaceutiques contenant l'ecotine et des homologues de celle-ci
WO1996034965A2 (fr) * 1995-05-05 1996-11-07 Chiron Corporation Proteines chimeres mcp et daf a domaine de localisation de surface de cellule

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARIO TOSI ET AL.: "Molecular cloning of human C1 inhibitor: sequence homologies with alpha1-antitrypsin and other members of the serpins superfamily", GENE, vol. 42, 1986, AMSTERDAM NL, pages 265 - 272, XP002102026 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047963A3 (fr) * 1999-12-24 2002-05-10 Bio Discovery Ltd Inhibiteurs d'activation de complement, leur preparation et leur utilisation
WO2001098365A3 (fr) * 2000-06-21 2003-07-03 Zymogenetics Inc Inhibiteurs peptidiques et polypeptidiques du complement c1s
WO2002068641A1 (fr) * 2001-02-28 2002-09-06 Fu Wai Hospital Chinese Academy Of Medical Sciences Facteur d'inhibition d'inflammations fwa116
US8715672B2 (en) 2004-12-23 2014-05-06 Csl Behring Gmbh Treatment of diseases linked to pathological kinin formation
US9987328B2 (en) 2011-03-09 2018-06-05 Csl Behring Gmbh Factor XII inhibitors for the administration with medical procedures comprising contact with artificial surfaces
EP2497489A1 (fr) * 2011-03-09 2012-09-12 CSL Behring GmbH Inhibiteur du facteur XII pour le traitement de la pénombre ischémique cérébrale et l'ischémie d'autres organes
KR20140011372A (ko) * 2011-03-09 2014-01-28 체에스엘 베링 게엠베하 인공 표면과의 접촉을 포함하는 의료 시술에 투여하기 위한 인자 ｘｉｉ 억제제
WO2012120128A1 (fr) * 2011-03-09 2012-09-13 Csl Behring Gmbh Inhibiteurs du facteur xii destinés à être administrés avec des procédures médicales comprenant le contact avec des surfaces artificielles
WO2012120124A1 (fr) * 2011-03-09 2012-09-13 Csl Behring Gmbh Inhibiteurs du facteur xii pour traiter l'ischémie cérébrale silencieuse et l'ischémie d'autres organes
KR101954052B1 (ko) * 2011-03-09 2019-03-06 체에스엘 베링 게엠베하 인공 표면과의 접촉을 포함하는 의료 시술에 투여하기 위한 인자 ｘｉｉ 억제제
AU2012224510B2 (en) * 2011-03-09 2016-04-14 Csl Behring Gmbh Factor XII inhibitors for the treatment of silent brain ischemia and ischemia of other organs
US9352016B2 (en) 2011-03-09 2016-05-31 Csl Behring Gmbh Factor XII inhibitors for the administration with medical procedures comprising contact with artificial surfaces
US9624307B2 (en) 2011-03-09 2017-04-18 The General Hospital Corporation Factor XII inhibitors for the treatment of silent brain ischemia and ischemia of other organs
US20140378653A1 (en) * 2012-01-31 2014-12-25 Csl Behring Gmbh Factor XII Inhibitors for the Treatment of Neurological Inflammatory Disorders
US9957329B2 (en) 2012-01-31 2018-05-01 Csl Behring Gmbh Factor XII inhibitors for the treatment of neurological inflammatory disorders
US10286047B2 (en) 2013-03-08 2019-05-14 Csl Behring Gmbh Treatment and prevention of remote ischemia-reperfusion injury
US10973891B2 (en) 2013-03-08 2021-04-13 Csl Behring Gmbh Treatment and prevention of remote ischemia-reperfusion injury
WO2014207199A1 (fr) * 2013-06-28 2014-12-31 Csl Behring Gmbh Polythérapie utilisant un inhibiteur du facteur xii et un inhibiteur de c1
AU2014301041B2 (en) * 2013-06-28 2019-10-31 Csl Behring Gmbh Combination therapy using a Factor XII inhibitor and a C1-Inhibitor
WO2015054569A1 (fr) * 2013-10-10 2015-04-16 Viropharma Holdings Limited Procédés d'inhibition de la voie alternative d'activation du système immunitaire complémentaire et compositions utilisées dans ces procédés
WO2019113642A1 (fr) * 2017-12-15 2019-06-20 Csl Limited Utilisation d'un inhibiteur de fxiia dans le traitement d'une fibrose rénale et/ou d'une maladie rénale chronique
US11505619B2 (en) 2017-12-15 2022-11-22 Csl Limited Use of a FXIIa-inhibitor in the treatment of renal fibrosis and/or chronic kidney disease

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