WO2001007072A1 - Modulation de l'activation plaquettaire - Google Patents

Modulation de l'activation plaquettaire Download PDF

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Publication number
WO2001007072A1
WO2001007072A1 PCT/US1999/019158 US9919158W WO0107072A1 WO 2001007072 A1 WO2001007072 A1 WO 2001007072A1 US 9919158 W US9919158 W US 9919158W WO 0107072 A1 WO0107072 A1 WO 0107072A1
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par4
parl
activity
compound
thrombin
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PCT/US1999/019158
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English (en)
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Shaun R. Coughlin
Mark Kahn
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The Regents Of The University Of California
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Priority to CA002378473A priority Critical patent/CA2378473A1/fr
Priority to JP2001511955A priority patent/JP2003526625A/ja
Publication of WO2001007072A1 publication Critical patent/WO2001007072A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • This invention relates to inhibition of platelet activation, and particularly to such inhibition mediated through thrombin receptors.
  • Thrombin a coagulation protease generated at sites of vascular injury, activates platelets, leukocytes, and mesenchymal cells (T.-K.H. Vu et al, Cell 64:1057-1068 (1991)). Activation of platelets by thrombin is thought to be critical for hemostasis and thrombosis. In animal models, thrombin inhibitors block platelet-dependent thrombosis, which is the cause of most heart attacks and strokes in humans. Available data in humans suggests that thrombosis in arteries can be blocked by inhibitors of platelet function and by thrombin inhibitors.
  • thrombin's actions on platelets contribute to the formation of clots that cause heart attack and stroke.
  • thrombin's other actions on vascular endothelial cells and smooth muscle cells, leukocytes, and f ⁇ broblasts may mediate inflammatory and proliferative responses to injury, as occur in normal wound healing and a variety of diseases (atherosclerosis, restenosis, pulmonary inflammation (ARDS), glomerulosclerosis, etc.).
  • ARDS pulmonary inflammation
  • glomerulosclerosis glomerulosclerosis
  • Thrombin signaling is mediated at least in part by a family of G protein-coupled protease-activated receptors (PARs) for which PARl is the prototype.
  • PARs G protein-coupled protease-activated receptors
  • the synthetic peptide SFLLRN which mimics the first six amino acids of the new amino terminus unmasked by receptor cleavage, functions as a PARl agonist and activates the receptor independent of thrombin and proteolysis.
  • thrombin-mediated platelet activation by inhibition or enhancement of protease-activated receptor 4 (PAR4) and protease-activated receptor 1 (PARl) activity are disclosed.
  • This method provides a way of substantially blocking all thrombin-mediated activation of platelets by 1) inhibiting signaling through PARl and 2) inhibiting signaling through PAR4.
  • Signaling through each PAR receptor may be inhibited at various molecular levels, including: the level of ligand binding (e.g. by administration of an antagonist), the level of the receptor activity (e.g. blocking expression of the receptor in the relevant cells) and/or intracellularly, (e.g. blocking expression or activity of a molecule required for activity of the receptor).
  • this method provides a method for enhancing thrombin-mediated platelet activation by specifically activating PARl and PAR4.
  • These methods may take place in vivo, through administration of the appropriate compounds, or in vitro, e.g. the ex vivo treatment of a sample.
  • compositions that are effective for prevention of thrombin-associated platelet activation.
  • These compositions are comprised of agents that inhibit the activity of PARl and PAR4, e.g. antagonists of PARl and PAR4, and are effective in the treatment of disorders.
  • useful antagonists are small molecules, modeled proteins, and antibodies.
  • compositions may comprise dominant negative PAR receptors, which may be employed to substantially decrease or eliminate the expression of either PARl or PAR4.
  • These compositions may also contain nucleic acid sequences that encode for antagonists of PARl and PAR4, which may be administered for expression in vivo.
  • compositions that are effective for enhancing thrombin-associated platelet activation, either in vitro and/or in vivo.
  • These compositions are comprised of agents that inhibit the activity of both PARl and PAR4, e.g. antagonists of PARl and PAR4, and are effective in the treatment of disorders.
  • Examples of useful antagonists are small molecules, modeled proteins, and antibodies.
  • These compositions may also contain nucleic acid sequences that encode for antagonists of PARl and PAR4, which may be administered for expression in vivo.
  • compositions are comprised of agents that enhance the activity of both PARl and PAR4, e.g. antagonists of PARl and PAR4, and are effective in the treatment of disorders wherein there is insufficient activation of platelets, e.g. hemophilia.
  • compositions may also contain nucleic acid sequences that encode for antagonists of PARl and PAR4, which may be administered for expression in vivo.
  • the invention provides therapeutic uses of the inhibiting compositions of the invention in the treatment of disorders such as such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses.
  • One method of treatment comprises the administration of protonated acidified nucleic acids to the animal in an amount sufficient to inhibit or prevent tissue occlusion.
  • a sample may be taken from an animal (e.g. a blood sample), treated ex vivo with the inhibiting composition of the invention, and returned to the animal.
  • the invention provides therapeutic uses of the activating compositions of the invention in disorders involving insufficient clotting.
  • the dual activation of PARl and PAR4 may increase the activation of platelets, since thrombin has the ability to activate both receptors.
  • the invention also provides specific PAR4 antibodies for use in the methods of the invention. Such antibodies effectively block signaling through PAR4 and thus effectively block PAR4's contribution to thrombin-mediated platelet activation.
  • compositions of the invention are effective at treating a variety of ailments.
  • Figure 1 is the summary of studies of a competitive RT-PCR assay of RNA prepared from platelets, neutrophils and Dami cells.
  • Figure 2 is a graph illustrating antibody binding to the surface of receptor-expressing COS cells.
  • Figures 3 A - 3D is a series of graphs illustrating the results of flow cytometric analysis of human platelets .
  • Figures 4A - 4D is a series of graphs illustrating the results of flow cytometric analysis of Dami cells.
  • Figure 5 illustrates the ability of the PARl and PAR4 tethered ligand peptides to activate PARl and PAR4 heterologously expressed in Xenopus oocytes.
  • Figures 6 A - 6C illustrate the ability of the PARl and PAR4 tethered ligand peptides to activate PARl and PAR4 human platelets.
  • Figure 7 is a graph illustrating the ability of the PARl and PAR4 antibodies to block thrombin cleavage of PARl and PAR4in rat I fibroblasts expressing FLAG epitope-tagged PARl and PAR4.
  • Figures 8A and 8B illustrate the increases in cytoplasmic calcium in response to thrombin in cells expressing PAR 1 ( Figure 8 A) or PAR 4 (Figure 8B).
  • Figures 9A - 9E illustrate the contribution of PARl and PAR4 signaling to thrombin activation of human platelets with and without treatment with PAR activation peptides or antibodies. DESCRIPTION OF THE PREFERRED EMBODIMENTS Before the present protease-activated receptor assays and methods of using such are described, it is to be understood that this invention is not limited to the particular DNA sequences, materials, methods, or processes described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.
  • protease-activated receptor 1 By “protease-activated receptor 1", “PARl”, “PARl receptor” and the like, is meant all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist thereby activating PARl -mediated signaling events.
  • the polypeptide is characterized as having the properties (including the agonist activating and antagonist inhibiting properties) described herein and in Vu et al. (1991) Cell, 64: 1057-1068, which is incorporated herein by reference.
  • PARl may refer to a naturally occurring form of the receptor and/or a recombinantly produced form of the receptor. In addition, the term may include variants of the PARl protein that retain the same activity and properties.
  • protease-activated receptor 4" By “protease-activated receptor 4", "PAR4", “PAR4 receptor” and the like, is meant all or part of a vertebrate cell surface protein which is specifically activated by thrombin or a thrombin agonist thereby activating PAR4-mediated signaling events.
  • the polypeptide is characterized as having the properties (including the agonist activating and antagonist inhibiting properties) described herein and in U.S. Application No. 09/032,397, which is incorporated herein by reference.
  • PAR4 may refer to a naturally occurring form of the receptor and/or a recombinantly produced form of the receptor. In addition, the term may include variants of the PAR4 protein that retain the same activity and properties.
  • polypeptide any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation).
  • substantially pure is meant that the protease-activated receptor 4 polypeptide provided by the invention is at least 60%, by weight, free from the proteins and naturally - occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, PAR polypeptide.
  • a substantially pure PAR polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a PAR polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The protein is substantially pure if it can be isolated to a band in a gel.
  • substantially identical amino acid sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for leucine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the biological activity of the receptor.
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for leucine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the biological activity of the receptor.
  • Such equivalent receptors can be isolated by extraction from the tissues or cells of any animal which naturally produces such a receptor or which can be induced to do so, using the methods described below, or their equivalent; or can be isolated by chemical synthesis; or can be isolated by standard techniques of recombinant DNA technology, e.g., by isolation of cDNA or genomic DNA encoding such a receptor.
  • Substantially identical receptors have the same biological function, e.g. are activated by the same compound.
  • derived from is meant encoded by the genome of that organism and present on the surface of a subset of that organism's cells.
  • isolated DNA DNA that is not in its native environment in terms of not being immediately contiguous with (i.e., covalently linked to) the complete coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the DNA of the invention is derived.
  • the term therefore includes, for example, recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g.
  • a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion independent of other sequences. It also includes any recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • transformed cell By “transfected cell” “transfected cell”, “genetically engineered cell”, and the like, is meant a cell into which (or into an ancestor of which) has been introduced, by means of genetic engineering, a DNA molecule encoding PARl , PAR4 or a biologically active fragment of either.
  • a DNA molecule is "positioned for expression” meaning that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of a PARl or PAR4 protein, or fragment or analog thereof).
  • antibody is meant an immunoglobulin protein which is capable of binding an antigen.
  • Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g. F(ab') 2 , Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • Antibodies of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to either PARl or PAR4 protein.
  • Antibodies for PARl or PAR4 are preferably immunospecific — i.e., not substantially cross-reactive with related materials, e.g., with each other.
  • the term "antibody” encompasses all types of antibodies (e.g., monoclonal) the antibodies of the invention are preferably produced using the phage display methodology described herein.
  • the preferred antibody of the invention is a purified antibody. By purified antibody is meant one which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody "preferentially binds" to a specific PAR protein (or an antigenic fragment thereof), i.e., does not substantially recognize and bind to other antigenically-unrelated molecules.
  • thrombin a thrombin analog
  • PAR agonist or other chemical agent including polypeptides such as an antibody, which activates protease-activated receptor, receptor polypeptide or a fragment or analog thereof to initiate PAR-mediated biological events as described herein, but which does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally includes a protease-activated receptor polypeptide.
  • an agent such as a thrombin analog, a PAR4 antagonist or other chemical agent including polypeptides such as an antibody, which inhibits activation of protease-activated receptor 4, receptor polypeptide or a fragment or analog thereof, such as by inhibiting thrombin or by blocking activation of PAR4 by thrombin or other PAR4 activator.
  • the agent activates or inhibits the biological activity in vivo or in vitro of the protein to which it binds.
  • biological activity is meant the ability of the PAR to bind thrombin or an agonist and signal the appropriate cascade of biological events (e.g., phosphoinositide hydrolysis, Ca 2+ efflux, and platelet aggregation, and the like).
  • substantially increase is meant an increase in activity or other measurable phenotypic characteristic that is at least approximately a 2-fold increase over control level (where control assays are performed in the absence of activator), preferably at least approximately a 5 -fold increase, more preferably at least approximately a 10-fold increase in activity over a control assay.
  • substantially decrease or “substantial reduction” is meant a decrease or reduction in activity or other measurable phenotypic characteristic that is approximately 80% or the control level, preferably reduced to approximately 50% of the control level, or more preferably reduced to approximately 10% or less of the control level.
  • screening method and “assay method” are used to describe a method of screening a candidate compound for its ability to act as an agonist or antagonist of a PAR4 ligand.
  • the method involves: a) contacting a candidate agonist compound with a recombinant protease-activated receptor 4 (or PAR4 agonist-binding fragment or analog); b) measuring activation of the receptor, the receptor polypeptide or the receptor fragment or analog; and c) identifying agonist compounds as those which interact with the recombinant receptor and trigger or block PAR4 activation. Interaction may be cleavage of the receptor to unmask an intramolecular receptor activating peptide or by mimicking the intramolecular receptor-activating peptide.
  • a tethered ligand may be more difficult to block than a free agonist.
  • blocking thrombin is the acid test for an antagonist which will block responses by other thrombin substrates.
  • an agonist is meant a molecule which mimics a particular activity, in this case, interacting with a PAR or PAR ligand in a manner which activates thereby triggering the biological events which normally result from the interaction (e.g. , phosphoinositide hydrolysis, Ca 2+ efflux, and platelet aggregation).
  • an agonist initiates a substantial increase in receptor activity relative to control assays in the absence of activator or candidate agonist.
  • An agonist may possess the same, less, or greater activity than a naturally-occurring PAR ligand.
  • an “antagonist” is meant a molecule which blocks activation of a PAR receptor.
  • a particular activity such as the ability of thrombin, for example
  • An antagonist may bind to and thereby block activation of a PAR receptor, either PARl , PAR4 or both.
  • antagonist assay refers to a method of screening a candidate compound for its ability to antagonize interaction between a naturally-occurring activating ligand or an agonist and the PAR, either PARl, PAR 4 or both.
  • the method involves: a) contacting a candidate antagonist compound with a first compound which includes a recombinant PAR (or agonist-binding fragment or analog) on the one hand and with a second compound which includes thrombin or a PAR agonist on the other hand; b) determining whether the first and second compounds interact or are prevented from interaction by the candidate compound; and c) identifying antagonistic compounds as those which interfere with the interaction of the first compound (PAR receptor) to the second compound (PAR agonist) and which thereby substantially reduce thrombin or PAR agonist- activated biological events (e.g., phosphoinositide hydrolysis, Ca 2+ efflux, and platelet aggregation).
  • a contacting a candidate antagonist compound with a first compound which includes a recombinant PAR (or agonist-binding fragment or analog) on the one hand and with a second compound which includes thrombin or a PAR agonist on the other hand; b) determining whether the first and second compounds interact or are prevented from interaction by
  • treatment means obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particular a human, and includes:
  • extracorporeal blood includes blood removed in line from a patient, subjected to extracorporeal treatment, and returned to the patient in processes such as dialysis procedures or blood filtration or blood bypass during surgery.
  • the term also includes blood products which are stored extracorporeally for eventual administration to a patient. Such products include whole blood, platelet concentrates and any other blood fraction in which inhibition or enhancement of platelet aggregation and platelet release is desired.
  • the present invention is based upon the discovery that inhibition of PARl and PAR4 virtually abolishes the ability of platelets to respond to thrombin. Functional studies with PARl and PAR4 activating peptides confirmed that activation of either receptor was sufficient to trigger platelet secretion and aggregation. Given thrombin's remarkable potency as a platelet activator, blocking thrombin signaling in platelets is useful in any number of biological phenomena, such as prevention of thrombosis.
  • the invention thus provides a method of treatment for reducing the level of thrombin response in a mammalian host by administering a composition which inhibits both PARl and PAR4 activity. In general, such compounds will reduce the activity of the PARl and PAR4 receptors.
  • PARl -specific antagonists permit direct blockade of PARl signaling, while leaving the signaling through the other PAR molecules generally unaffected.
  • Inhibition of PARl function by antibody, antagonist, or desensitization markedly inhibits platelet responses at low (InM) concentrations of thrombin.
  • PAR4 may be inhibited using similar mechanisms, including but not limited to peptide agonists, antibodies, and PAR4-specific antagonists. PAR4 inhibition is preferably accomplished using an antibody to PAR4's thrombin cleavage site. This antibody preparation blocks PAR4 activation by thrombin without interfering with other PAR activity. Moreover, an antibody to this region of PAR4 does not inhibit activation of either PAR4 or platelets by the PAR4-activating peptide GYPGKF. In contrast to PARl inhibition, inhibition of PAR4 alone had no significant effect on platelet aggregation.
  • the treatment of the present invention may take place in vivo, e.g. through the administration of PARl and PAR4 antagonists or introduction and expression of PARl and PAR4 antagonists. Alternatively, the treatment may take place ex vivo, e.g. a patient's extracorporeal blood may be treated with compositions of the invention and the blood replaced.
  • Novel methods employ compounds that are effective in decreasing the level of PARl and PAR4 in mammalian cells.
  • Candidate compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological compounds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The agonists or antagonists of the present invention may relate to synthetic polypeptides that bind to of PARl and/or PAR4. Such synthetic antiplatelet polypeptides may be prepared by conventional chemical synthesis techniques, for example, synthesis on a solid support.
  • the present invention also relates to recombinant and synthetic DNA molecules which encode molecules that either inhibit or enhance PAR activity.
  • the synthesis of these DNA molecules may be achieved by methods well known in the art.
  • the recombinant DNA molecules may be isolated from a human hematopoetic cDNA library.
  • the synthesis of cDNA libraries and the choice of vector into which the cDNA molecules may be cloned are conventional techniques, see e.g. T. Maniatis et al., "Molecular Cloning ⁇ A Laboratory Manual", Cold Spring Harbor (1982).
  • a wide variety of methods may be used in locating and identifying cDNA sequences corresponding to a the compositions of the present invention.
  • oligonucleotide probe based on the amino acid sequence of the of PARl or PAR4 ligands
  • immunoscreening which utilizes antibodies against the extracellular domains of PARl and PAR4 to detect clones which express cDNA sequences corresponding to potential agonist or antagonist activity. It will be obvious to those of skill in the art that the choice of oligonucleotides probes will be based upon those strengths of amino acids which are encoded by the least redundant DNA sequences.
  • the immunoscreening technique requires that the cDNA library be contained in a vector capable of expression.
  • vectors include lambda gtl 1, lambda gtlO and other expression vectors known in the art.
  • Antibodies employed in the immunoscreening technique include antibodies against intact PARl and PAR4, antibodies against denatured PARl and PAR4 and antibodies against peptide portions of PARl and PAR4.
  • Partial cDNAs may themselves be used to reprobe the cDNA library and to locate full-length cDNAs.
  • the DNA molecules of this invention may be synthesized from nucleotides by chemical means using an synthesizer.
  • Such nucleic acids may be designed based on identified amino acid sequence of the PAR agonists or antagonists. Standard methods may be applied to synthesize a gene encoding such a peptide. For example, the complete amino acid sequence may be used to construct a back-translated gene.
  • a DNA oligomer containing a nucleotide sequence capable of coding for the desired polypeptide may be synthesized in a single step.
  • oligonucleotides coding for portions of the PARl and/or PAR4 agonist or antagonist may be synthesized and subsequently ligated together.
  • the antiplatelet polypeptide gene is synthesized as 10-20 separate oligonucleotides which are subsequently linked together.
  • the individual oligonucleotides contain 5' or 3' overhangs for complementary assembly.
  • assembly of the antiplatelet polypeptide gene may be achieved in one or more steps by techniques well known in the art. Once assembled, the gene will be characterized by sequences which are recognized by restriction endonucleases, including unique restriction sites for direct assembly into a cloning or an expression vector; preferential codons based upon the host expression system to be used: and a sequence which, when transcribed, produces an mRNA with minimal secondary structure. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active antagonist or agonist in a platelet aggregation assay.
  • the present invention relates to compositions for decreasing or preventing platelet aggregation and release and methods which employ them.
  • These compositions may also contain a variety of other conventional antiplatelet or anti-thrombin compounds in addition to a naturally purified, recombinant or synthetic polypeptide inhibitor of platelet activation of this invention.
  • the most widely used antiplatelet agent is aspirin, a cyclooxygenase inhibitor. Although aspirin blocks ADP- and collagen-induced platelet aggregation, it fails to prevent cyclooxygenase-independent platelet aggregation initiated by agonists, such as thrombin.
  • Alternative anti-thrombin compounds are hirudin derivatives.
  • composition of the invention containing additional anti-platelet activation compounds may be a single dosage form, wherein a polypeptide inhibitor of platelet activation of this invention may be chemically conjugated to a conventional polypeptide platelet inhibitor or to a conventional anti-thrombin polypeptide.
  • a single dosage form which contains the polypeptide inhibitor of platelet activation and the other polypeptide in the same composition, but as separate compounds.
  • the composition may also contain multiple dosage forms, wherein the PARl and PAR4 inhibitors and the other polypeptide that inhibits of platelet activation are administered separately, but concurrently, or wherein the two forms are administered sequentially.
  • a polypeptide inhibitor of PARl and/or PAR4 may also be cross-linked to a conventional carrier polypeptide, which may be carried out by chemical cross-linking methods well known in the art. Most preferably, such combinations are formed by cross-linking a natural or recombinant polypeptide PAR antagonists to carriers that have been synthesized with a cross-linking moiety, such as dinitrofluorobenzene, at its NH 2 terminus.
  • the carrier peptide may be conjugated to a natural or recombinant PAR 1 and/or PAR4 antagonist by the use of agents such as glutaraldehyde, dimethyladipimidate, or any other bifunctional cross-linkers known in the art.
  • the conjugated antagonist preferably involves a 1 : 1 stoichiometry with the carrier peptide.
  • the agonists or antagonists of the present invention may be present invention relates to synthetic polypeptides of platelet activation.
  • Such synthetic antiplatelet polypeptides may be prepared by conventional chemical synthesis techniques, for example, synthesis on a solid support.
  • antibodies detecting the extracellular domain of either PARl or PAR4 may be generated by immunizing rabbits or mice with a portion of the extracellular domain of each molecule, or a peptide fragment derived therefrom. Only antibodies with affinity at least 4 fold higher for PARl or PAR4, respectively as compared to their affinity for any other PAR molecules should be selected.
  • the method of antibody generation, purification, labeling and detection may vary.
  • the IgG or Fab's may be purified from different sources by affinity HPLC using protein A column and Size exclusion HPLC.
  • the purified antibodies may be labeled with Europium and detected by time resolved fluorescence.
  • the antibody binding to different PAR proteins may be measured by time-resolved, dissociation-enhanced fluorescence.
  • the system of detection of PAR-bound IG on solid support in situ or in solution may vary.
  • direct or indirect immunological methods including direct radiolabels, fluorescence, luminescence, avidin-biotin amplification, or enzyme-linked assays with color or luminescent substrates.
  • the preferred PARl antibody which can be used in the invention is disclosed in Hung et al. (1992) J. Clin. Invest., 89:1350-1353.
  • an indication that no binding occurs means that the equilibrium or affinity constant Kg is 10 6 1/mole or less.
  • binding will be recognized as existing when the K a is at 10 7 1/mole or greater, preferably 10 8 1/mole or greater.
  • the binding affinity of 10 7 1/mole or more may be due to (1) a single monoclonal antibody (i.e., large numbers of one kind of antibodies) or (2) a plurality of different monoclonal antibodies (e.g. , large numbers of each of five different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations of (1) - (3). Selected preferred antibodies will bind at least 4-fold more avidly to one PAR (e.g. PARl) than to a different PAR (e.g. PAR4).
  • the four fold differential in binding affinity may be accomplished by using several different antibodies as per (1) - (3) above and as such some of the antibodies in a mixture could have less than a four fold difference.
  • the methods of the present invention may be practiced using one or more different antibodies to PARl and/or PAR4.
  • antibodies may be labeled with known labels and used with currently available robotics, sandwich assays, electronic detectors, flow cytometry, and the like.
  • the compound may be administered to the host using any convenient means capable of resulting in the desired target protein activity modulation.
  • the compound can be incorporated into a variety of formulations for therapeutic administration.
  • the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, transdermal patches, suppositories, injections, inhalants and aerosols.
  • administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc. , administration.
  • the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules.
  • additives are conventional additives, such as lactose, mannitol, corn starch or potato starch; binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants, such as talc or magnesium stearate; and if desired, diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • diluents buffering agents, moistening agents, preservatives and flavoring agents.
  • the compounds of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol. If desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives may also be added. The concentration of therapeutically active compound in the formulation may vary from about 0.5-100 wt. % .
  • the compounds can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbo waxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit (e.g. , a teaspoonful, tablespoonful, tablet or suppository) contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • Compounds for use in the method of the invention may also be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate compounds often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate compounds are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • the compounds are added to a host in a physiologically acceptable carrier, at a dosage from 5 mg to 1400 mg, more usually from 100 mg to 1000 mg, preferably 500 to 700 for a dose of 0.5 to 20 mg/kg weight.
  • the dosage for compounds suppressing thrombin response is elected so that the PARl and PAR4 activity is reduced by 10 to 80%, more preferably 20 to 70% and even more preferably 25-50% .
  • the dosage for compounds inhibiting the activity of PARl and PAR4 is elected so that the ability of platelets to respond to thrombin is reduced by about 20 to 80% , preferably 40 to 50% .
  • Platelet activation may be induced by a number of biological phenomenon, including injury, response to certain compounds, etc.
  • the subject compositions will generally be administered daily, in an amount to provide at least about a 50 to 100% , more preferably 75-95%, even more preferably 80-90% decrease in platelet activation.
  • the total daily dosage will be at least about 10 mg, usually at least about 400 mg to 500 mg, preferably about 700 mg, and not more than about 1500 mg, usually not more than about 1000 mg.
  • the amount may vary with the general health of the patient, the response of the patient to the drug, whether the composition is used by itself or in combination with other drugs, and the like.
  • Daily administrations may be one or more times, usually not more than about four times, particularly depending upon the level of drug which is administered.
  • Administration of the compounds of the invention is particularly useful in the treatment of diseases such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood thromboses. Inhibition of platelet activation in such disorders may allow localized treatment at the site of the clotting, thus eliminating some of the more unpleasant side effects of systemic treatment, e.g. hemorrhage.
  • the synthetic peptides GGDDSTPSILPAPRGYPGQVC (PAR4 amino acids 34-55, SEQ ID NO:l), AKPTLPIKTFRGAPPNSFEEFPFSALEGC (PAR3 amino acids 31-58 plus carboxyl glycine-cysteine, SEQ ID NO:2) and NATLDPRSFLLRNPNDKYEPFWEDEEGC (PARl amino acids 35-61 plus carboxyl glycine-cysteine, SEQ ID NO:3) were used to generate polyclonal antisera in rabbits.
  • Ig was purified by Protein-A affinity chromatography to generate the PAR4 IgG, PAR3 IgG and PARl IgG preparations used in this study.
  • a competitive RT-PCR assay was developed to assess expression of mRNAs encoding the known PARs.
  • a competitor RNA cRNA
  • Each cRNA was identical to the sequence of native mRNA to be reverse transcribed and amplified by PCR except for mutation of a single restriction endonuclease site; digestion with the cognate restriction endonuclease thus allowed differentiation of PCR products generated from cRNA vs. mRNA.
  • Varying amounts of competitor cRNAs were added to 200ng of total cellular RNA and the mixtures were reverse transcribed and amplified by PCR using PAR-specific primers. In the absence of reverse transcription, no products were amplified.
  • Dami cells were grown in suspension in RPMI with 10% fetal bovine serum. Platelets were separated from human blood as previously described. Platelet preparations contained less than 0.1% leukocytes as assessed by light microscopic analysis. A discontinuous Percoll gradient was used to separate monocytes plus lymphocytes from neutrophils according to the manufacturer's instructions (Pharmacia). The monocyte/ lymphocyte preparations contained less than 0.1% neutrophils and the neutrophil preparations contained less than 0.1% monocytes or lymphocytes. Total RNA was prepared from all cells using Trizol (GIBCO), treated with DNAse I (Boehringer-Mannheim), and quantified by OD 260 .
  • Trizol Trizol
  • DNAse I Boehringer-Mannheim
  • Each receptor cDNA was mutated so as to ablate an endogenous restriction endonuclease site. Sites were mutated by digesting cDNA encoding the receptor with the selected endonuclease, exposing the digested plasmid to T4 DNA polymerase then religating. Each of these mutant cDNAs was subcloned into Bluescript (KS- or SK-, Stratagene) such that sense cRNA could be transcribed in vitro using T7 RNA polymerase. Competitor cRNAs so generated were added to total cell RNA prior to reverse transcription (RT) reactions.
  • Bluescript KS- or SK-, Stratagene
  • RT reactions were performed at 42 °C using 200 ng of total RNA with varying amounts of competitor cRNA in a 10 ⁇ l reaction volume using a commercial kit (GIBCO) and receptor specific primers (see below). 2 ⁇ l of the RT product was then subjected to PCR amplification in a 50 ⁇ l volume containing a final concentration of 2 ⁇ M primers and 5U of Taq polymerase (GIBCO). Reaction conditions were as follows: 94 °C for 4 min, 72 °C for 1 min with addition of Taq; then 94°C for 45 sec, 55 °C for 1 min, 72°C for 1 min for 30-36 cycles (see below); then 72 °C for 8 min.
  • RNA from the various sources was:
  • Anti-sense primer for PCR AAA CTG TTG CCC ACA CCA GTC CAC (SEQ ID NO: 12).
  • Primer for RT TGA GTA GCT GGG ATT ACA G (1519-1501; SEQ ID NO: 13).
  • Sense strand primer for PCR AAC CTC TAT GGT GCC TAC GTG C (SEQ ID NO: 14).
  • Anti-sense primer for PCR CCA AGC CCA GCT AAT TTT TG (SEQ ID NO: 15). Resulting PCR product: 949-1490.
  • reaction product was digested overnight with the appropriate restriction endonuclease (Agel, 2U at 25 °C; Sfil, 20U at 50°C; Ncol, 10U at 37°C; or BamHI, 20U at 37°C).
  • the products were then separated by 1.5%, agarose gel electrophoresis (Separide gel matrix, GIBCO) and visualized by ethidium bromide staining.
  • GIBCO agarose gel electrophoresis
  • the cRNA concentration at which the intensity of the cRNA-derived product (uncleaved band) matched that of the endogenous mRNA-derived product (cleaved band) was used to estimate the quantity of each PAR mRNA in the original sample.
  • RNA prepared from platelets, neutrophils and Dami cells is presented in Fig. 1.
  • Dami cells a human cell line that express some megakaryocyte markers were analyzed first.
  • Competitive RT PCR of Dami cell RNA yielded results that were concordant with Northern and protein analysis.
  • 2 ⁇ g of poly(A)+ RNA isolated from Dami cells was electrophoresed on a denaturing formaldehyde agarose gel and transferred onto a supported nitrocellulose membrane (Schleicher & Schuell).
  • PARl mRNA was detected with a 400 bp Pstl/PvuII cDNA probe; PAR2 mRNA was detected with a 260 bp Sfil/BstEII cDNA probe; PAR3 mRNA was detected with a 610 bp Kpnl/Nsil cDNA probe; PAR4 mRNA was detected using a 450 bp Sacl/Pstl cDNA probe using high stringency conditions. In all such assays, PARl, PAR3, and PAR4 were detected in Dami cells while PAR2 was not (Fig. 1). Competitive RT-PCR of platelet RNA revealed PARl mRNA to be present at approximately one attomole/200 ng total RNA.
  • PARl mRNA represents one in three thousand platelet mRNAs.
  • PAR4 mRNA was also readily detected in platelet RNA at 10-30% of PARl mRNA levels.
  • PAR3 mRNA was undetectable in human platelet RNA.
  • PAR3 competitor cRNA added to platelet RNA was detectable at 0.001 attomole/200ng total RNA, suggesting that PAR3 mRNA was at least 1000 fold less abundant than PARl mRNA in these samples.
  • PAR2 mRNA was not detected in platelet RNA from one individual and only 0.001 attomole/200ng was detected in the other.
  • the latter may be due to trace contamination of the platelet preparation by neutrophils, which do express PAR2.
  • Inability to detect significant PAR2 mRNA in platelets is consistent with the observation that the specific PAR2 agonist peptide SLIGKV is unable to activate human platelets.
  • IgG was purified from rabbit anti-peptide antisera directed against the amino terminal exodomains of PARl, PAR3, or PAR4. To characterize the ability of each IgG preparation to recognize native PARs and to assess cross-reactivity, antibody binding to the surface of receptor-expressing COS cells was measured (Fig. 2). Each IgG preparation bound to the surface of cells expressing the appropriate receptor without significant cross-reactivity.
  • the IgG preparations were then used for flow cytometric analysis of human platelets (Figs. 3 A - 3D). Washed platelets were fixed in paraformaldehyde for 20 minutes at 40 °C, washed three times with platelet buffer (20mM Tris-HCl pH 7.4, 140mM NaCl, 2.5mM KC, lmM MgCl 2 , lmg/ml glucose, 0.5% BSA), then incubated with primary IgG in platelet buffer at 40 °C for 1 hour. PARl and PAR3 IgG were used at 10 ⁇ glml and PAR4 IgG at 100 ⁇ g/ml.
  • Platelets were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Molecular Probes) at 4 ⁇ glml for 0.5 hour. Platelets were then washed three times and analyzed by flow cytometry. Some fixed platelet samples were exposed to 30 nM thrombin at 37°C prior to incubation with primary antibody. Dami cells were treated like platelets for flow cytometry.
  • FITC fluorescein isothiocyanate
  • IgG Molecular Probes
  • PAR3 immune IgG showed no specific binding to human platelets.
  • this experiment was repeated with Dami cells (Figs. 4A - 4D), which had been shown by Northern blot to express PAR3 mRNA.
  • Figs. 4A - 4D As was observed for platelets, a significant increase in fluorescence was seen using PARl and PAR4 antibodies (Figs. 4A, 4C and 4D). In contrast to platelets, a significant increase in fluorescence was also observed with PAR3 antibody. This result is consistent with the presence of PAR3 mRNA in Dami cells by RT-PCR and Northern blot analysis. It further suggests that the absence of detectable PAR3 protein on the surface of human platelets is not due to insensitivity of the assay. These results are consistent with the analysis of platelet RNA and confirm the presence of PARl and PAR4 but not PAR3 on the surface of human platelets.
  • Synthetic peptides that mimic the tethered ligands of PARl and PAR2 function as agonists for their respective receptors and have been used as pharmacological tools to probe the function of these receptors in various cell types.
  • the cognate peptide for PAR3 appears to be insufficiently avid to function as a free ligand.
  • Peptides mimicking the tethered ligand for PAR4 can function as an agonist for that receptor, albeit at a concentration higher than that seen for the PARl and PAR2 peptides and their cognate receptors.
  • the PAR4 peptide GYPGKF did show minimal activity at PARl.
  • PARl is overexpressed in the oocytes and the sensitivity for detection of PARl activation in the oocyte assay is 10-100 fold greater than in platelets; it is likely that PARl activation at 500 ⁇ M GYPGKF is unimportant in the platelet studies described below.
  • a Par 1 ⁇ ' ⁇ mouse lung fibroblast cell line that showed no thrombin signaling was used to generate stable cell lines expressing FLAG epitope-tagged PARl and PAR4.
  • PARl IgG markedly attenuated such signaling and non-immune antibody was without effect.
  • the PARl antagonist BMS20026121 attenuated PARl signaling even at high thrombin concentrations (Figs. 8A and 8B).
  • the peptide-based PARl antagonist BNIS200261 was synthesized as previously described in N. I. Bernatowicz, et al., J. Med. Chem. 39:4879-87 (1996). Responsiveness to lysophosphaticlic acid was unaffected by the antagonist (Figs. 8A and 8B), suggesting that its inhibitory effect was specific.
  • PAR4 IgG had no effect on platelet aggregation even at low (InM) thrombin responses (Figs. 9A - 9E).
  • PARl IgG or PARl antagonist markedly inhibited platelet aggregation in response to 1 nM thrombin, as did prior desensitization of platelets with the PARl agonist SFLLRN. None of these maneuvers inhibited platelet aggregation in response to GYPGKF or submaximal concentrations of ADP.

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Abstract

L'invention concerne des méthodes de modulation de l'activation plaquettaire induite par la thrombine par inhibition ou stimulation de l'activité des récepteurs PAR4 et PAR1 activés par des protéases. Cette méthode permet de bloquer sensiblement toute l'activation plaquettaire induite par la thrombine grâce à (1) l'inhibition de la signalisation par PAR1 et (2) l'inhibition de la signalisation par PAR4. La signalisation par chaque récepteur PAR peut être inhibée à différents niveaux moléculaires, tels que le niveau de liaison des ligands (par exemple lors de l'administration d'un antagoniste), le niveau d'activité d'un récepteur (par exemple lors du blocage de l'expression de ce récepteur dans les cellules appropriées) et/ou le niveau intracellulaire (par exemple lors du blocage de l'expression ou de l'activité d'une molécule nécessaire à l'activité dudit récepteur). Dans un autre mode de réalisation, cette méthode permet de stimuler l'activation plaquettaire induite par la thrombine par activation spécifique de PAR1 et PAR4. Ces méthodes peuvent s'appliquer in vivo, par administration des composés appropriés, ou in vitro, par exemple lors du traitement ex vivo d'un échantillon.
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WO2001060401A1 (fr) * 2000-02-17 2001-08-23 The Regents Of The University Of California Methodes de modulation de l'activation du recepteur 4 active par des proteases (par4) avec de la cathepsine g
EP1982997A1 (fr) 2004-09-01 2008-10-22 Allergan, Inc. Toxines clostridiennes dégradables
WO2009012401A1 (fr) * 2007-07-17 2009-01-22 Irm Llc Anticorps d'antagoniste de récepteur 1 activé par protéase (par 1)
EP2043686A2 (fr) * 2006-07-18 2009-04-08 Irm Llc Antagonistes de recepteur 1 active par protease (par1)
AU2006232644B2 (en) * 2005-04-08 2011-11-10 AKL Research and Development Ltd. Anti-inflammatory formulation
WO2013088164A1 (fr) * 2011-12-14 2013-06-20 Cambridge Enterprise Limited Molécules d'anticorps se liant à la thrombine et leurs utilisations
WO2015061733A1 (fr) * 2013-10-25 2015-04-30 Bristol-Meyers Squibb Company Méthodes de diagnostic pour thérapie par antagonistes de par4
CN105392495A (zh) * 2013-06-28 2016-03-09 比奥根Ma公司 具有xten的凝血酶可裂解连接子和其用途
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires
WO2019046912A1 (fr) * 2017-09-11 2019-03-14 Monash University Protéines de liaison au récepteur de la thrombine humaine, par4
US11192936B2 (en) 2014-01-10 2021-12-07 Bioverativ Therapeutics Inc. Factor VIII chimeric proteins and uses thereof

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WO2001060401A1 (fr) * 2000-02-17 2001-08-23 The Regents Of The University Of California Methodes de modulation de l'activation du recepteur 4 active par des proteases (par4) avec de la cathepsine g
EP1982997A1 (fr) 2004-09-01 2008-10-22 Allergan, Inc. Toxines clostridiennes dégradables
EP1982996A1 (fr) 2004-09-01 2008-10-22 Allergan, Inc. Toxines clostridiennes dégradables
US7892565B2 (en) 2004-09-01 2011-02-22 Allergan, Inc. Degradable clostridial toxins
US7998489B2 (en) 2004-09-01 2011-08-16 Allergan, Inc. Degradable clostridial toxins
AU2006232644B2 (en) * 2005-04-08 2011-11-10 AKL Research and Development Ltd. Anti-inflammatory formulation
EP2043686A2 (fr) * 2006-07-18 2009-04-08 Irm Llc Antagonistes de recepteur 1 active par protease (par1)
EP2043686A4 (fr) * 2006-07-18 2010-03-24 Irm Llc Antagonistes de recepteur 1 active par protease (par1)
US7888483B2 (en) 2006-07-18 2011-02-15 Irm Llc Antagonists of protease activated receptor-1 (PAR1)
WO2009012401A1 (fr) * 2007-07-17 2009-01-22 Irm Llc Anticorps d'antagoniste de récepteur 1 activé par protéase (par 1)
CN101977936A (zh) * 2007-07-17 2011-02-16 Irm责任有限公司 蛋白酶激活受体1(par1)的拮抗剂抗体
US9605082B2 (en) 2011-12-14 2017-03-28 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
EP3275903A1 (fr) * 2011-12-14 2018-01-31 Janssen Pharmaceuticals, Inc. Molécules d'anticorps de liaison à la thrombine et leurs utilisations
US10370454B2 (en) 2011-12-14 2019-08-06 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
WO2013088164A1 (fr) * 2011-12-14 2013-06-20 Cambridge Enterprise Limited Molécules d'anticorps se liant à la thrombine et leurs utilisations
RU2642276C2 (ru) * 2011-12-14 2018-01-24 Янссен Фармасьютикалз, Инк. Тромбин-связывающие молекулы антител и их применение
US11155637B2 (en) 2011-12-14 2021-10-26 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988463B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988461B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10287363B2 (en) 2011-12-14 2019-05-14 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
CN105392495A (zh) * 2013-06-28 2016-03-09 比奥根Ma公司 具有xten的凝血酶可裂解连接子和其用途
WO2015061733A1 (fr) * 2013-10-25 2015-04-30 Bristol-Meyers Squibb Company Méthodes de diagnostic pour thérapie par antagonistes de par4
US11192936B2 (en) 2014-01-10 2021-12-07 Bioverativ Therapeutics Inc. Factor VIII chimeric proteins and uses thereof
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires
US20210179707A1 (en) * 2017-09-11 2021-06-17 Monash University Binding proteins to the human thrombin receptor, par4
CN111670198A (zh) * 2017-09-11 2020-09-15 莫纳什大学 人凝血酶受体par4的结合蛋白
WO2019046912A1 (fr) * 2017-09-11 2019-03-14 Monash University Protéines de liaison au récepteur de la thrombine humaine, par4
US11780915B2 (en) 2017-09-11 2023-10-10 Monash University Binding proteins to the human thrombin receptor, PAR4
CN111670198B (zh) * 2017-09-11 2023-12-15 莫纳什大学 人凝血酶受体par4的结合蛋白
IL273202B1 (en) * 2017-09-11 2024-04-01 Univ Monash Human thrombin receptor binding proteins, PAR4

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