WO2020214033A1 - Médicaments à base de cystéine peptidase de type papaïne et leurs utilisations - Google Patents

Médicaments à base de cystéine peptidase de type papaïne et leurs utilisations Download PDF

Info

Publication number
WO2020214033A1
WO2020214033A1 PCT/NL2020/050255 NL2020050255W WO2020214033A1 WO 2020214033 A1 WO2020214033 A1 WO 2020214033A1 NL 2020050255 W NL2020050255 W NL 2020050255W WO 2020214033 A1 WO2020214033 A1 WO 2020214033A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptidase
seq
amino acid
vwf
acid sequence
Prior art date
Application number
PCT/NL2020/050255
Other languages
English (en)
Inventor
Mirjam Maaike MEBIUS
Aloysius Gerard Marie TIELENS
Jaap Jan VAN HELLEMOND
Philip Gerrit De Groot
Rolf Theodoor URBANUS
Original Assignee
Erasmus University Medical Center Rotterdam
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Erasmus University Medical Center Rotterdam filed Critical Erasmus University Medical Center Rotterdam
Publication of WO2020214033A1 publication Critical patent/WO2020214033A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • 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

Definitions

  • This invention is in the field of medicine. More specifically, the invention is in the field of medicaments against disorders in which von Willebrand Factor (VWF) plays an important role.
  • VWF von Willebrand Factor
  • the invention relates to medical methods employing a new class of proteins exhibiting VWF -cleaving peptidase activity, to the proteins themselves and uses thereof and to pharmaceutical compositions comprising such proteins.
  • the proteins identified herein can be used as an antithrombotic.
  • Von Willebrand Factor is a plasma glycoprotein that mediates adhesion and aggregation of platelets.
  • the protein is synthesized by endothelial cells and megakaryocytes as long multimers with a molecular weight of up to more than 20,000 kDa (Kannicht et al., Biologicals, 43: 117— 22 (2015)).
  • the majority of circulating VWF is synthesized by endothelial cells (Lenting et al., Thromb Haemost 104: 449-55 (2010)).
  • Endothelial cells secrete part of the produced VWF via the constitutive pathway, but a substantial part is stored in Weibel-Palade bodies and is released upon stimulation of the endothelium with, for example, the inflammatory cytokines IL-8 and TNF-a (Hulstein et al., J Thromb Haemost., 4: 2569-75 (2006); Bernardo et al., Blood, 104: 100-6 (2004)).
  • the inflammatory cytokines IL-8 and TNF-a Hulstein et al., J Thromb Haemost., 4: 2569-75 (2006); Bernardo et al., Blood, 104: 100-6 (2004).
  • VWF ultra-large VWF
  • AD AMTS 13 A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13
  • ULVWF multimers will adopt a globular conformation in plasma (Crawley et al., Blood, 118: 3212-21 (2011)).
  • a deficiency in the metalloproteinase AD AMTS 13 is associated with thrombotic thrombocytopenic purpura (TTP), a rare but severe form of a group of hematologic disorders referred to as thrombotic micro angiopathy (TMA).
  • TTP thrombotic thrombocytopenic purpura
  • TMA thrombotic micro angiopathy
  • clumping of platelets by ULVWF results in unwanted thrombosis and organ damage (Crawley et al., Blood, 118: 3212-21 (2011); Clark et al., Thromb Haemost., 118: 471-9 (2018)).
  • acquired TTP patients produce autoantibodies against AD AMTS 13.
  • the current first-line treatment for patients with acquired TTP is a plasma exchange (PEX) procedure, in which the patient’s plasma is removed by apheresis and replaced with donor plasma.
  • PEX plasma exchange
  • the aim of PEX is to replace AD AMTS 13 and remove the autoantibodies that inhibit AD AMTS 13 activity from the blood circulation, as well as any residual ULVWF multimers therefrom. This way of correcting AD AMTS 13 deficiency restores proper cleavage of ULVWF multimers, prevents microvascular thrombosis, and reverses symptoms of organ damage.
  • the beneficial effect of PEX is only temporary.
  • HUS haemolytic uremic syndrome
  • HE LLP syndrome HE LLP syndrome
  • catastrophic antiphospholipid syndrome ischaemic stroke which benefits from thrombolytic therapy.
  • the present invention solves these problems by providing papain-like cysteine peptidases that exhibit peptidase activity towards von Willebrand Factor (VWF).
  • VWF-cleaving peptidases can be used in methods of treatment, for instance in treating a variety of hematologic disorders as described herein.
  • the peptidase of this invention is for use as a medicament.
  • SmCB2 tegumental cathepsin B
  • papain-like cysteine peptidases The class of papain-like cysteine peptidases is well-documented (see for instance the MEROPS database, wherein papain-like cysteine peptidases are identified by clan CA - family C1 - subfamily C1A).
  • SmCB2 acts proteolytically on VWF.
  • the present inventors characterized SmCB2 as a VWF-cleaving peptidase.
  • SmCB2 occurs both in the form of a proenzyme, whose amino acid sequence is provided in SEQ ID NO: 1, and in a mature and catalytically active form, whose amino acid sequence is provided in SEQ ID NO:2.
  • the inventors discovered inter alia that SmCB2 cleaves VWF in the same amino acid region as AD AMTS 13 (i.e. the A2 domain).
  • SmCB2 is not the only peptidase from the class of papain-like cysteine peptidases capable of cleaving VWF.
  • the class of papain -like cysteine peptidases with VWF-cleaving activity differentiate themself from the known peptidases that cleave VWF, which are either metalloproteinases (such as AD AMTS 13, MMP-8 and MMP-9), serine peptidases (such as plasmin, leukocyte-derived proteinase 3, neutrophil elastase and cathepsin G), or peptidases that require divalent cations for their catalytic activity (such as calpain).
  • metalloproteinases such as AD AMTS 13, MMP-8 and MMP-9
  • serine peptidases such as plasmin, leukocyte-derived proteinase 3, neutrophil elastase and cathepsin G
  • peptidases that require divalent cations for their catalytic activity such as calpai
  • the present invention provides a papain-like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor (VWF) for use as a medicament, preferably for use in treatments including thrombolytic therapy.
  • VWF von Willebrand Factor
  • the papain-like cysteine peptidase that exhibits peptidase activity towards von Willebrand Factor is for use in treating hematologic disorders, preferably disorders selected from (i) thrombotic micro angiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.
  • the peptidase is a divalent metal ion-independent cysteine peptidase.
  • the peptidase is a cathepsin B.
  • the peptidase acts proteolytically on VWF.
  • the peptidase has:
  • cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity; or
  • a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity, or combinations thereof.
  • the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity.
  • the peptidase is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 50% sequence identity, preferably at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting VWF cleaving activity.
  • the peptidase can be used in the form of its mature protein or a VWF-cleaving part or sequence identity analog thereof as indicated above.
  • the peptidase can be used in the form of its pro-enzyme or pro-peptide or parts thereof, which parts preferably comprise the cysteine active site or histidine active site, or combinations thereof as indicated herein.
  • the pro-enzyme or pro-peptide or part thereof may then be activated in vivo following its administration to a subject in need thereof. Such activation may include activation of the active sites or removal of inhibitory factors.
  • the peptidase is from Schistosoma mansoni.
  • the peptidase is SmCB2.
  • the peptidase is a recombinant protein.
  • the present invention provides a nucleic acid encoding the peptidase of the present invention as described above, for use as a medicament, preferably for use in treatments including thrombolytic therapy.
  • the invention provides a peptidase or nucleic acid as described herein (i) for use in the treatment of a disorder associated with, or mediated by, VWF, (ii) for use in the treatment of a disorder associated with, or mediated by, the presence of VWF (in a subject), (iii) for use in the treatment of a disorder associated with, or mediated by,
  • the nucleic acid is for use in treating hematologic disorders, preferably disorders selected from (i) thrombotic microangiopathy (TMA), preferably thrombotic
  • TTP thrombocytopenic purpura
  • HUS haemolytic uremic syndrome
  • HELLP syndrome catastrophic antiphospholipid syndrome, more preferably acquired TTP;
  • myocardial infarction or
  • stroke preferably ischemic stroke.
  • the peptidase or the nucleic acid are used as an antithrombotic.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a peptidase and/or nucleic acid as defined herein above, and one or more pharmaceutically-acceptable carriers, adjuvants, excipients and/or diluents.
  • the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 and exhibiting peptidase activity towards von Willebrand Factor (VWF).
  • VWF von Willebrand Factor
  • the present invention provides a method of treating a hematologic disorder in a subject, comprising administering to a subject in need thereof a therapeuticahy effective amount of a peptidase or nucleic acid according to the invention as described above.
  • the disorder is selected from (i) thrombotic micro angiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HELLP syndrome or catastrophic antiphospholipid syndrome, more preferably acquired TTP; (h) myocardial infarction; or (iii) stroke, preferably ischemic stroke.
  • cyste peptidase refers to peptidases which are distinguished by the presence of a cysteine residue which plays a critical role in the catalytic process.
  • the terms“peptidase” and“protease” can be used interchangeably.
  • the term“cysteine peptidase” also refers to peptidases which are distinguished by the presence of a cysteine residue which plays a critical role in the catalytic process.
  • cysteine (C) proteases are classified in different clans (CA,CD, CE, CF, CL, CM, CN, CO, CP, CQ, and CR), different families (C1-C118) and different
  • cathepsin B peptidases belong to clan CA, family C1, subfamily CIA (papain -like peptidases) and are identified by MEROPS identifier CO 1.060, preferably according to MEROPS database release 12.0.
  • calpain peptidases belong to clan CA, family C2 and subfamily C2A. Therefore, calpain is not a papain-like cysteine peptidase.
  • cysteine peptidase refers to cysteine peptidases that include clan CA, family C1, subfamily CIA according to the MEROPS database, preferably the MEROPS database release 12.0.
  • papain-like refers to a grouping or family of peptidases that is characterized by a type of enzyme employed, which in the present case is the papain enzyme type ( Carica papaya), which is a family-type peptidase.
  • the cysteine peptidase as described herein is a cysteine peptidase of the papain family- type of cysteine peptidases.
  • the term includes naturally occurring
  • cysteine peptidase as described herein is an endopeptidase.
  • cysteine protease is a papain-like cysteine protease, such as for instance described by Caffrey et al., Mol Biochem Parasitol. 121:49-61 (2002), who first described the peptidase referred to as SmCB2 and classified it as a cathepsin B that belongs to the family of papain-like cysteine peptidases.
  • a papain-like cysteine peptidase as described herein comprises the conserved catalytic active site residues of papain-like cysteine peptidases of, or corresponding to, Cys122 and His291 of SEQ ID NO: 1 or SEQ ID NO:2 (see also Turk et al., Biochim Biophys Acta. 1824:68-88 (2012); Novinec & Lenarcic, BioMol Concepts 4:287-308 (2013)).
  • a papain-like cysteine peptidase as described herein contains one or more amino acid residues of, or corresponding to, Asn311, Gin 116,
  • corresponding to refers to the fact that a conserved amino acid residue in one peptidase may have a different residue number compared to the same conserved amino acid residue in a different peptidase.
  • One of skill in the art would readily identify corresponding amino acid positions of conserved residues, for instance when performing alignment of the amino acid sequence of multiple peptidases, for instance through Protein BLAST®. As an example, an alignment of cathepsin B peptidases of different species is provided in Figure 5.
  • the peptidase as described herein is a metal ion- independent, more preferably a divalent metal ion-independent, cysteine peptidase.
  • the invention provides a metal ion-independent cysteine peptidase, preferably divalent metal ion-independent cysteine peptidase, that exhibits peptidase activity towards von Willebrand F actor (VWF); wherein said peptidase is for use as a medicament.
  • VWF von Willebrand F actor
  • metal ion-independent refers to any cysteine peptidase whose catalytic mechanism or active site does not involve or require a metal ion for peptidase activity. This is in contrast to
  • cysteine peptidase as described herein is a Ca 2+ -independent cysteine peptidase.
  • the cysteine peptidases described herein exhibit peptidase activity towards von Willebrand Factor (VWF).
  • VWF von Willebrand Factor
  • the peptidase activity is an endopeptidase activity. More preferably, the cysteine peptidase exhibits peptidase activity towards, or is a VWF -cleaving peptidase that cleaves in, the A2 domain of VWF.
  • Peptidase activity towards the A2 domain of VWF can for instance be measured using the generally available FRETS-VWF73 substrate (AnaSpec, Fremont, Ca, USA), preferably according to the FRETS-VWF73 substrate assay protocol as described in Example 1.
  • the FRETS-VWF73 substrate is a synthetic 73 amino acid peptide based on the amino acid sequence of the A2 domain of VWF. This fluorogenic substrate is currently used in a diagnostic assay (FRETS-VWF73 assay) to determine activity of AD AMTS 13, the peptidase that normally controls VWF multimer size through limited cleavage.
  • VWF von Willebrand Factor
  • VWF refers to a plasma glycoprotein that mediates adhesion and aggregation of platelets.
  • VWF is synthesized by endothelial cells and megakaryocytes as long multimers with a molecular weight of up to more than 20,000 kDa. The majority of circulating VWF is synthesized by endothehal cells. Most of the secreted VWF consists of ultra-large VWF (ULVWF) multimers (also referred to as“VWF multimers” or“ multimeric VWF” herein) that are prothrombotic.
  • UUVWF ultra-large VWF
  • the VWF is a mammalian VWF, more preferably a human VWF.
  • VWF as described herein is preferably a VWF multimer (multimeric VWF), which is also referred to as ULVWF multimer or multimeric ULVWF.
  • cysteine peptidase described herein is a cathepsin B.
  • cathepsin B refers to a group of peptidases that include clan CA, family C1, subfamily C1A (papain -like peptidases) and are identified by MEROPS identifier CO 1.060, preferably according to the MEROPS database release 12.0.
  • cathepsin B comprises an occluding loop exhibiting dipeptidyl peptidase activity.
  • cathepsin B comprises an occluding loop that exhibits dipeptidyl peptidase activity comprising an amino acid sequence indicated by the amino acid region of Cys200 - Cys211 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence corresponding to the amino acid sequence indicated by amino acid region Cys200 - Cys211 of SEQ ID NO: 1 or SEQ ID NO:2.
  • the term includes reference to naturally occurring cathepsin B peptidases, cathepsin B peptidases that comprise one or more mutations such as substitution, deletion and/or insertion mutations, and cathepsin B
  • FIG. 1 shows sequence homology between cathepsin B peptidases of different species, which all belong to the family of papain-like cysteine peptidases. The sequence alignment shows inter alia conserved active sites around cysteine and histidine residues.
  • a peptidase as described herein preferably has - a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2; and/or - a histidine active site comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
  • the active site when reference is made to an active site having an amino acid sequence having a certain percentage of sequence identity to SEQ ID NO: 1 or SEQ ID NO:2, the active site is to exhibit an activity corresponding to the reference active site in SEQ ID NO: 1 or SEQ ID NO:2.
  • a peptidase as described herein preferably has - a cysteine active site comprising the amino acid sequence of amino acid region 113-137 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 113- 137 of SEQ ID NO: 1 or SEQ ID NO:2; and - a histidine active site
  • amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2 comprising the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of amino acid region 289-314 of SEQ ID NO: 1 or SEQ ID NO:2.
  • % sequence identity is defined herein as the percentage of amino acids in an amino acid sequence that is identical with the amino acids in a reference amino acid sequence or an amino acid sequence of interest, after ahgning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art. Sequence identity is calculated over substantially the whole length, preferably the whole (full) length, of a reference amino acid sequence or amino acid sequence interest. The skilled person understands that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence.
  • the reference amino acid sequence or amino acid sequence of interest is SEQ ID NO: 1 or SEQ ID NO:2, preferably SEQ ID NO:2, or regions thereof when defined as reference amino acid sequence or amino acid sequence interest.
  • a peptidase as described herein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 50%, 60%, 70%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • a peptidase as described herein preferably comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2.
  • a peptidase as described herein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, preferably SEQ ID NO:2.
  • a peptidase as described herein preferably is from Schistosoma mansoni, more preferably is SmCB2.
  • SmCB2 is a cathepsin B cysteine peptidase (UniProtKB - Q95PM1; last modified: March 1, 2002 - v2), the amino acid sequence of which is provided in SEQ ID NO: 1 (proenzyme) and SEQ ID NO:2 (mature protein).
  • SmCB2 includes naturally occurring variants and other variants that have for instance a substitution, deletion or insertion of an amino acid residue in SEQ ID NO: 1 or SEQ ID NO:2, preferably wherein the substitution, deletion or insertion is 1-80, more preferably 1-50, even more preferably 1-20, most preferably 1- 10 amino acid residues, and the naturally occurring variants and other variants exhibit peptidase activity towards von Willebrand Factor (VWF).
  • VWF von Willebrand Factor
  • the peptidase as described herein may comprise the pro-domain of SmCB2, or is in the form of the proenzyme of SmCB2, preferably wherein the peptidase has the amino acid sequence of amino acid region 1-93 or 24-93 of SEQ ID NO: 1.
  • SmCB2 as a therapeutic protein as foreseen herein may entail improving the pharmacokinetic properties of the protein, in particular hmiting its metabolic turnover by peptidases or glomerular filtration by the kidney to thereby increase its in vivo half-life.
  • One typical modification to improve the pharmacokinetics of the protein is through conjugation to either hnear or branched-chain monomethoxy poly-ethylene glycol (PEG) or derivatives thereof (m-PEG), resulting in increases in the molecular mass through a process referred to as PEGylation.
  • the molecular weight of the PEG molecule may vary, and is typically in the range of from about 100 Da to about 160,000 Da, such as e.g. from about 5,000 Da to about 100,000 Da.
  • Methods for PEGylation are well known in the art and involve chemical conjugation of a PEG molecule of appropriate mass to the protein, followed by repurification of the conjugate. Another approach to improve
  • pharmacokinetics of the protein is by modification of glycosylation pattern, resulting in reduced clearance and extension of half-life.
  • improving the pharmacokinetic properties of the protein may occur by providing a fusion protein, for instance by fusion of the protein to human serum albumin (HSA), to the constant fragment (Fc) domain of a human immunoglobulin (Ig) G, or to non -structured
  • Fusion proteins of SmCB2 are therefore also foreseen in aspects of this invention, and may include a linker or spacer sequence to separate protease and albumin roles in the fusion protein.
  • SmCB2 protein according to the present invention may thus have a modified circulatory half life compared to the wild type SmCB2 protein, preferably a circulatory half life that is increased at least 10-100%, more preferably at least 200-500%.
  • the invention also provides a peptidase as defined herein, preferably a peptidase that is pegylated or a peptidase that is fused to HSA, to the constant fragment (Fc) domain of a human immunoglobulin (Ig) G, or to a non-structured polypeptides such as XTEN.
  • the peptidase can be another peptidase than SmCB2.
  • the invention also provides a nucleic acid encoding the peptidase as described herein, wherein said nucleic acid is for use a medicament.
  • nucleic acid refers to DNA and RNA including mRNA or cDNA, as well as synthetic congeners thereof.
  • the nucleic acid can be a recombinant or synthetic nucleic acid. The skilled person is aware of appropriate methods and means for administration of nucleic acids for therapeutic purposes.
  • the invention further provides a peptidase or nucleic acid as described herein for use as an antithrombotic. Since the peptidase as described herein exhibits peptidase activity towards von Willebrand Factor (VWF), antithrombotic effects are provided for upon administration to a subject.
  • VWF von Willebrand Factor
  • antithrombotic refers to a medicament that prevents the formation of blood clots or counteracts existing blood clots.
  • the antithrombotic is an anticoagulant or a thrombolytic agent.
  • An anticoagulant limits the ability of the blood to clot, whereas a
  • thrombolytic agent provides for breakdown (lysis) of blood clots formed in blood vessels.
  • the peptidase or nucleic acid as described herein is for use in the treatment of thrombosis, preferably for use in thrombolysis (thrombolytic therapy).
  • the invention also provides a peptidase or nucleic acid as described herein for use in the treatment of a disorder associated with VWF. It is estabhshed in the present invention that peptidases as described herein exhibit peptidase activity towards von Willebrand Factor (VWF). This allows beneficial use of such peptidases (including nucleic acids encoding such peptidases) in the treatment of disorders that are associated with VWF. Such disorders are preferably hematologic disorders including coagulopathies and hemostatic disorders, more preferably thrombotic disorders. Such disorders can be acquired or inherited.
  • VWF von Willebrand Factor
  • a disorder associated with VWF is preferably (i) a disorder associated with, or mediated by, the presence of VWF (in a subject), (ii) a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject) and/or (iii) for counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject).
  • the peptidase or nucleic acid as described herein may also be for use in correcting AD AMTS 13 deficiency in a subject.
  • AD AMTS 13 deficiency refers to AD AMTS 13 not exhibiting its normal role in hemostasis (controlling VWF multimer size through limited cleavage), regardless of the cause, which could be ADAMTS13 protein levels that are too low or autoantibodies against AD AMTS 13 resulting in functional neutralization of AD AMTS 13.
  • the disorder associated with VWF is (i) a thrombotic
  • TMA microangiopathy
  • TTP thrombotic thrombocytopenic purpura
  • HUS haemolytic uremic syndrome
  • HELLP syndrome or
  • catastrophic antiphospholipid syndrome more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.
  • TMA thrombotic micro angiopathy
  • the present inventors established that the peptidase as described herein is not sensitive to plasma components specific for acquired TTP patients, which is an indication that the peptidase is not inhibited by auto antibodies against AD AMTS 13. This renders the peptidase as described herein an advantageous peptidase in the treatment of a disorder associated with VWF, especially acquired TTP, where auto-antibodies against
  • AD AMTS 13 dysregulate hemostasis.
  • the invention also provides for a use of a peptidase or nucleic acid as described herein for the manufacture of a medicament, preferably an antithrombotic.
  • the invention also provides for use of a peptidase or nucleic acid as described herein for the manufacture of a medicament for (i) the treatment of a disorder associated with, or mediated by, VWF, (ii) the treatment of a disorder associated with, or mediated by, the presence of VWF (in a subject), (iii) the treatment of a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject) or (iv) counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject); preferably including (i) a thrombotic microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HE UUP syndrome or
  • TMA thro
  • catastrophic antiphospholipid syndrome more preferably acquired TTP; (ii) myocardial infarction; or (iii) stroke, preferably ischemic stroke.
  • the invention also provides a method of treating a subject suffering from, or suspected of suffering from, a disorder associated with, or mediated by, VWF, comprising the step of: - administering to said subject a therapeutically effective amount of a peptidase or nucleic acid as described herein.
  • the disorder associated with VWF is (i) a thrombotic microangiopathy (TMA), preferably thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), HE UUP syndrome or
  • the invention provides a method of treating a subject suffering from, or suspected of suffering from, (i) a disorder associated with, or mediated by, the presence of VWF (in a subject) or (ii) a disorder associated with, or mediated by, (pathogenic) accumulation of VWF (in a subject).
  • the invention also provides a method of counteracting (unwanted or pathogenic) prothrombotic effects mediated by accumulation of VWF (in a subject).
  • subject refers to the recipient of a peptidase or nucleic acid as described herein and is a mammal, preferably a human.
  • patient refers to the recipient of a peptidase or nucleic acid as described herein and is a mammal, preferably a human.
  • patient refers to the recipient of a peptidase or nucleic acid as described herein and is a mammal, preferably a human.
  • patient and“subject” may be used interchangeable.
  • terapéuticaally effective amount means that the amount of the active ingredient is of sufficient quantity to achieve the intended purpose, such as, inter alia in this case, to cleave appropriate levels of multimeric VWF.
  • the amount of active ingredient i.e. the amount of peptidase as described herein, preferably is in the range of about 5 mg to 10 gram in a pharmaceutical composition as described herein.
  • the invention also provides a cysteine peptidase that exhibits peptidase activity towards von Willebrand F actor (VWF); wherein said peptidase is for use as a medicament.
  • VWF von Willebrand F actor
  • a peptidase as described herein can be produced by methods and means generally available in the art. For instance, the person skilled in the art will understand how to generate a DNA sequence that encodes an amino acid sequence of a peptidase as described herein and how to manufacture and isolate a nucleic acid molecule with said DNA sequence using generally known recombinant DNA techniques.
  • the sequence of the nucleic acid molecule is preferably codon-optimized for expression in a host cell. In this way codons are used that are favored for high level expression in a specific host cell.
  • Nucleic acid molecules are preferably inserted in an expression vector using recombinant DNA techniques known by the person skilled in the art.
  • Expression vectors in the context of the invention direct the expression of a peptidase as described herein in a host cell. These expression vectors are preferably replicable in a host cell, either as episomes or as part of the chromosomal DNA.
  • the expression vector preferably comprises (i) a strong promoter/enhancer, such as the CMV or SV40 promoter, (ii) an optimal translation initiation sequence, such as a
  • ribosomal binding site and start codon preferably a KOZAK consensus sequence and (iii) a transcription termination sequence, including a poly(A) signal when the protein is expressed in eukaryotic cells.
  • Suitable expression vectors include plasmids and viral vectors such as adenoviruses, adeno- associated viruses and retroviruses. The person skilled in the art will understand that the expression vector to be used is dependent on the host cell that is used for expression of a recombinant protein.
  • An expression vector is preferably suited for expression of a nucleic acid molecule of the invention in a prokaryotic cell including a bacterial cell, or, more preferred, in a eukaryotic host cell, such as a yeast cell and a mammalian cell.
  • a suitable example is mammalian expression vector pCMV4.
  • a nucleic acid molecule may be inserted in the genome of a host cell. Said insertion preferably is at a locus or within a region that ensures expression of a nucleic acid molecule of the invention in the host cell.
  • Suitable host cells include prokaryotic and eukaryotic cells, such as bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells.
  • suitable eukaryotic host cells include, but are not limited to P. pastoris cells, the hamster cell line CHO and BHK-21; the murine host cells NIH3T3, NSO and C127; the simian host cells COS and Vero; and the human host cells HeLa, PER.C6, U-937, HEK 293 and Hep G2. Suitable cells are available from public sources such as ATCC and Life Technologies.
  • transfection techniques are known in the art, see, e.g., Green & Sambrook., 2012.“Molecular Cloning: A Laboratory Manual”, 4 th Edition, CSHL Press; Cold Spring Harbor Protocols, www.cshprotocols.cshlp.org).
  • the person skilled in the art preferably employs techniques as described in these references to introduce one or more exogenous nucleic acid molecules into suitable host cells.
  • An example of a host cell for the production of a peptidase as described herein is a P. pastoris cell or a HEK 293 cell.
  • the peptidase or nucleic acid as described herein are for administration in the form of a pharmaceutical composition.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a peptidase or nucleic acid as described herein, and one or more pharmaceutically-acceptable carriers, adjuvants, excipients and/or diluents.
  • the peptidase comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 and that exhibits peptidase activity towards von Willebrand Factor (VWF).
  • VWF von Willebrand Factor
  • a pharmaceutical composition as described herein preferably comprises one or more of diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art.
  • the characteristics of the carrier will depend on the route of administration, as is known to the skilled person.
  • compositions as described herein refers to a nontoxic material that is compatible with the physical and chemical characteristics of a peptidase as described herein and does not interfere with the effectiveness of the biological activity of said peptidase.
  • a pharmaceutical composition as described herein is preferably adapted for parenteral administration, wherein the composition is for intravenous, intra-arterial, subcutaneous, and/or intramuscular
  • Parenteral administration involves the injection or infusion of a pharmaceutical composition as described herein into a body tissue or body fluid, whereby preferably a syringe, needle, or catheter is used.
  • a syringe, needle, or catheter is used as an alternative.
  • needle-less high-pressure administration may be used as means for parenteral administration.
  • the carrier may be an aqueous or oily solution, dispersions, emulsions and/or suspension.
  • the carrier is an aqueous solution, preferably distilled sterile water, saline, buffered saline, or another pharmaceutically acceptable excipient for injection.
  • composition as described herein preferably is for use as medicament, more preferably for use as an
  • antithrombotic even more preferably for use in the treatment of a disorder associated with VWF, including (i) thrombosis, (ii) a thrombotic
  • TMA microangiopathy
  • TTP thrombotic thrombocytopenic purpura
  • HUS haemolytic uremic syndrome
  • HELLP syndrome or
  • catastrophic antiphospholipid syndrome more preferably acquired TTP; (iii) myocardial infarction; or (iv) stroke, preferably ischemic stroke.
  • a composition of the invention can be administration in a single dosage form once, preferably by parenteral administration, more preferably by intravenous administration.
  • a suitable dose can be selected from the dose range of 5 mg to 10 gram.
  • a composition of the invention can be administered in a single dosage form once or twice a day, weekly, biweekly (such as every other week or every 14 days) or monthly.
  • Such administration can be a parenteral administration, such as an intravenous administration, for instance in a dose of 5 mg to 10 gram.
  • NEM 10 mM various peptidase inhibitors
  • Leu leupeptin
  • PepA pepstatin A
  • PMSF phenylmethylsulfonyl fluoride
  • EDTA L-cysteine
  • L-cys L-cysteine
  • SWF mansoni soluble worm fraction
  • PNP pooled normal platelet-poor plasma
  • EDTA EDTA
  • Closed circles ( ⁇ ) represent PNP
  • open circles (o) represent PNP with addition of EDTA
  • closed squares ( ⁇ ) represent S. mansoni soluble worm fraction (SWF) plus PNP
  • open squares ( ⁇ ) represent S. mansoni soluble worm fraction (SWF) plus PNP in the presence of EDTA.
  • Initial rates of increase in fluorescence are represented as mean ⁇ SD. All values represent three replicates of duplo measurements.
  • rSmCB2 Purified recombinant SmCB2 (rSmCB2) was examined for proteolytic activity towards two fluorescent substrates Z-Phe-Arg-N AMC (panel A) and FRETS-VWF73 with (upper line) and without (lower line) 5 mM EDTA (panel B). Shown are the results of triplicate experiment in which a serial dilution of rSmCB2 was examined. The dashed line in panel B represents the VWF -cleavage activity of normal pooled plasma at a 2% (v/v)
  • SEQ ID NO: 1 Amino acid sequence of SmCB2 (Uniprot Acc. No. Q95PM1-1, last modified March 1, 2002 - v2)
  • SEQ ID NO:2 Mature SmCB2 (a.a. 94-347 of SEQ ID NO: 1)
  • Soluble worm fraction was prepared by subsequent centrifugation of the WH at 14000x g for 30 minutes at 4°C. Protein concentration in the schistosomal samples were determined according to the method of Lowry et al., J Biol Chem., 193: 265-75 (1951). Anion exchange chromatography fractionation Anion exchange chromatography fractionation was performed on Q
  • Sepharose fast flow resin (GE Healthcare Life Sciences, Boston, MA, USA) in a Tricorn 10/50 column (bed volume 4 ml, GE Healthcare Life Sciences, Boston, MA, USA).
  • the column was run on an ⁇ KTA pure system (GE Healthcare Life Sciences, Boston, MA, USA) at 1 ml/min with running buffer (20 mM triethanolamine, pH 8.0).
  • S. mansoni soluble protein extract was prepared in running buffer as described above and 9 ml was loaded onto the column using a 10 ml Superloop (GE Healthcare Life Sciences, Boston, MA, USA).
  • Fractions (500 pi per fraction) were step-wise eluted with 250 mM, 500 mM, and 1 M NaCl in running buffer and kept at 4 °C. Peptidase activity was tested in 40 ml fraction with the FRETS-VWF73 substrate assay. The three fractions with the highest activity in the FRETS-VWF73 substrate assay were pooled. Anion exchange chromatography was performed in triplicate.
  • the lanes of the SDS-PAGE were subsequently divided into 4 bands approximately equal in size (only including proteins with molecular weights smaller than ⁇ 80 kDa) and proteins were subjected to in-gel tryptic digestion as described by Shevchenko et al., Nat Protoc. 1: 2856-60 (2006). After digestion, samples were analyzed on an Orbitrap Q-Exactive (Thermo Fisher Scientific, Waltham, MA, USA) connected to a UHPLC Proxeon Easy-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Peptides were trapped on a double-fritted trap column (Dr. Maisch Reprosil C18, 3 pm, 2 cm x 100 pm (Dr.
  • Solvent A consisted of 0.1 M acetic acid, solvent B of 0.1 M acetic acid in 80%
  • Peptidases were identified based upon the presence of a peptidase KEGG domain and cysteine peptidases were further identified by the presence of“G0:0004197” or the term“Cathepsin” or present on AmiGO 2 as“cysteine-type
  • FRETS-VWF73 substrate assay Cleavage of the FRETS-VWF73 substrate (AnaSpec, Fremont, Ca, USA) was measured as published with some modifications (Kokame et al., Br J
  • FRETS-VWF73 substrate was dissolved at a concentration of 100 mM in 25% (v/v) dimethyl sulfoxide (DMSO) and diluted to 4 mM in assay buffer (5 mM Bis-Tris, 25 mM CaC12, 0.005% Tween-20, pH 6.0). Plasma samples or schistosomal fractions, where indicated supplemented with peptidase inhibitors, were diluted in assay buffer and FRETS-VWF73 substrate (2 pM) was added to initiate the assay.
  • DMSO dimethyl sulfoxide
  • Peptidase inhibitors used were N-ethylmaleimide (NEM; 10 mM), leupeptin (Leu; 100 pM), pepstatin A (PepA; 5 mM), phenylmethylsulfonyl fluoride (PMSF; 1 mM), ethylenediaminetetraacetic acid (EDTA; 5 mM), and L-cysteine (L-cys; 50 mM). Fluorescence was monitored for 1 hour at 30°C on a Spectromax M2 fluorescence microplate reader (MTX Lab Systems, Bradenton, FL,
  • Schistosoma mansoni preparations contain proteolytic activity towards FRETS-VWF73 substrate and multimeric VWF
  • FRETS-VWF73 substrate is a synthetic 73 amino acid peptide based on the amino acid sequence of the A2 domain of von Willebrand Factor (VWF). This fluorogenic substrate is used in a diagnostic assay (FRETS-VWF73 assay) to determine activity of AD AMTS 13 (Kokame et al., Br J Haematol, 129: 93- 100(2005), the peptidase that normally controls VWF multimer size through limited cleavage.
  • the FRETS-VWF73 peptide forms the minimal substrate for AD AMTS 13, containing the AD AMTS 13 cleavage site and the minimal sequence required for binding of AD AMTS 13.
  • SWF mansoni soluble worm fraction
  • EDTA ethylenediaminetetraacetic acid
  • AD AMTS 13 as the peptidase responsible for the observed cleavage of FRETS-VWF73.
  • VWF needs to be (partially) unraveled, either through sheer stress or through the addition of a denaturing agent, which is done in this experimental setup by the addition of urea (Tsai et al., Blood, 87: 4235-44 (1996)).
  • Purified VWF was incubated with S. mansoni WH or pooled normal platelet -poor plasma (PNP) for 8 hours. Incubations were performed both in the presence and absence of EDTA, to exclude effects of possible contamination of the S. mansoni fractions with host AD AMTS 13. Separation of the VWF multimers by gel electrophoresis under non-reducing conditions reveals the multimeric characteristic of VWF ( Figure 1C).
  • VWF triplets Cleavage of VWF results in the appearance of the slower and faster migrating satellite bands flanking the major VWF-multimer bands to form the so-called VWF triplets (Fischer et al., Biochem J., 331: 483-8 (1998). These satellite bands result from variation in the number of N-terminal fragments that the VWF -multimers contain after cleavage. Uncleaved, purified VWF shows two bands after gel electrophoresis, corresponding to the slower migrating and intermediate triplet band. Incubation of VWF with PNP results in cleavage of VWF by AD AMTS 13, which can be observed as the appearance of the faster migrating triplet band (black arrow).
  • cleavage of the FRETS-VWF73 substrate by S. mansoni fractions indicates that (at least one of) the cleavage site(s) of the peptidase is located close to the AD AMTS 13 cleavage site in the A2-domain of VWF.
  • the VWF-cleaving peptidase belongs to the class of cysteine peptidases
  • N- ethylmaleimide NEM
  • Leupeptin Leu
  • Leupeptin is a reversible small-molecule inhibitor of a broad range of cysteine, serine, and threonine peptidase classes.
  • Pepstatin A is a reversible small-molecule inhibitor of aspartic peptidases.
  • Phenylmethane sulfonylfluoride (PMSF) is an irreversible small- molecule inhibitor of serine peptidases and ethylenediaminetetraacetic acid (EDTA) is a chelating agent that inhibits metallopeptidases through chelation of the metal ion required for proteolytic activity.
  • Proteolytic cleavage of FRETS-VWF73 by S. mansoni soluble worm fraction (SWF) was strongly inhibited by both NEM and leupeptin, but was unaffected by the addition of pepstatin A or PMSF (Figure 2A).
  • VWF-cleaving peptidase belongs to the class of cysteine peptidases is indeed further strengthened by the observation that both EDTA and L-cysteine enhance proteolytic activity, which is characteristic behaviour for (papain-like) cysteine peptidases.
  • the VWF cleaving peptidase retains activity in TTP patient plasma
  • S. mansoni soluble worm fraction contains a VWF- cleaving peptidase
  • Cleavage of FRETS-VWF73 substrate by S. mansoni soluble worm fraction in the presence of EDTA was therefore examined with addition of 2% PNP or platelet-poor plasma obtained from 4 different TTP patients that had 0%
  • AD AMTS 13 activity as previously determined by FRETS-VWF73 assay (patient data not shown) ( Figure 3A).
  • AD AMTS 13 activity in PNP was strongly inhibited after addition of plasma from TTP patient 1. This would indicate the presence of autoantibodies against ADAMTS13 in plasma from this TTP patient, which interferes with the AD AMTS 13 activity from PNP.
  • no significant differences in VWF cleavage of FRETS-VWF73 by S. mansoni soluble worm fraction were observed upon addition of TTP patient plasma, indicating that the VWF -cleaving S. mansoni peptidase is not sensitive to components specific for TTP patient plasma at low plasma concentrations.
  • Mansoni peptidase as cleavage of multimeric VWF in the presence of EDTA occurs more efficiently in the absence of TTP patient plasma, which is visible by the increased disappearance of high multimer bands in the absence of TTP patient plasma.
  • Anion exchange chromatography fractionation combined with mass spectrometry identifies SmCB2 as a candidate gene for the S. mansoni VWF- cleaving cysteine peptidase
  • Proteins present in the active fraction were compared to the protein content of the flow-through fraction (control fraction) containing low VWF -cleaving activity at equal protein concentrations.
  • Five cysteine peptidases were identified in the active fraction (Table 1, see below)), however, only one of these cysteine peptidases was consistently enriched in both mascot ion score and peptide spectrum matches (PSM) in the active fraction compared to the control fraction throughout three independent experiments.
  • This cysteine peptidase, Schistosoma mansoni cathepsin B (Smp_141610) showed 17.2 ⁇ 2.3 fold enrichment in score and 12.4 ⁇ 3.1 fold enrichment in PSM compared to the control fraction. This identified the S. mansoni cathepsin B gene as the prime candidate to encode the S.
  • S. mansoni cathepsin B (Smp_141610) was first isolated and characterized by Caffrey et al., Mol Biochem Parasitol. 121:49-61 (2002). This cathepsin B peptidase was named S. mansoni cathepsin B2 (SmCB2, Uniprot Q95PM1) to distinguish it from the first recorded S. mansoni cathepsin, SmCBl
  • Table 1 Cysteine peptidases present in the schistosomal fraction with proteolytic activity towards FRETS-VWF73 substrate obtained after anion exchange chromatography of S. mansoni soluble worm fraction. Score: mascot ion score; PSM: peptide spectrum matches (total number of identified peptide spectra matched for the protein); A, B, and C indicate three independent experiments.
  • Purified recombinant expressed SmCB2 has proteolytic activity towards FRETS-VWF73
  • rSmCB2 Recombinant expressed SmCB2 (rSmCB2) in Pichia pastoris was purified and examined for its proteolytic activity towards its known fluorogenic substrate Z-FR-AMC as described before (Caffrey et al., Mol Biochem
  • Recombinant SmCB2 was produced according to the protocol described in Caffrey et al., 2002. As shown in Figure 4, purified rSmCB2 is an active peptidase that cleaves the FRETS-VWF73 substrate in a dose dependent manner, which demonstrates that SmCB2 is a

Landscapes

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

Abstract

L'invention concerne une peptidase cystéine de type papaïne qui présente une activité peptidase envers le facteur Willebrand; ladite peptidase étant destinée à être utilisée en tant que médicament.
PCT/NL2020/050255 2019-04-17 2020-04-17 Médicaments à base de cystéine peptidase de type papaïne et leurs utilisations WO2020214033A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP19169812 2019-04-17
EP19169812.5 2019-04-17

Publications (1)

Publication Number Publication Date
WO2020214033A1 true WO2020214033A1 (fr) 2020-10-22

Family

ID=66290200

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL2020/050255 WO2020214033A1 (fr) 2019-04-17 2020-04-17 Médicaments à base de cystéine peptidase de type papaïne et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2020214033A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997041206A2 (fr) * 1996-04-29 1997-11-06 Immuno Aktiengesellschaft Multimerase purifiee
EP1347059A1 (fr) * 2002-03-20 2003-09-24 Biofrontera Pharmaceuticals AG Cathepsin Y, une cible pour le développment de médicaments pour le traitement d'accidents vasculaires cérébraux
EP1391516A1 (fr) * 2001-04-25 2004-02-25 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Enzyme de clivage du facteur de von willebrand (vwf)
US20150297685A1 (en) * 2012-11-23 2015-10-22 Velleja Research S.R.L Fibrinolytic compositions for the prevention and treatment of phlebothrombotic states
WO2017075540A1 (fr) * 2015-10-30 2017-05-04 Ultragenyx Pharmaceutical Inc. Méthodes et compositions pour le traitement de l'amyloïdose
US20170224785A1 (en) * 2013-03-15 2017-08-10 Baxalta Incorporated Subcutaneous administration of adamts13

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997041206A2 (fr) * 1996-04-29 1997-11-06 Immuno Aktiengesellschaft Multimerase purifiee
EP1391516A1 (fr) * 2001-04-25 2004-02-25 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Enzyme de clivage du facteur de von willebrand (vwf)
EP1347059A1 (fr) * 2002-03-20 2003-09-24 Biofrontera Pharmaceuticals AG Cathepsin Y, une cible pour le développment de médicaments pour le traitement d'accidents vasculaires cérébraux
US20150297685A1 (en) * 2012-11-23 2015-10-22 Velleja Research S.R.L Fibrinolytic compositions for the prevention and treatment of phlebothrombotic states
US20170224785A1 (en) * 2013-03-15 2017-08-10 Baxalta Incorporated Subcutaneous administration of adamts13
WO2017075540A1 (fr) * 2015-10-30 2017-05-04 Ultragenyx Pharmaceutical Inc. Méthodes et compositions pour le traitement de l'amyloïdose

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
"UniProtKB", Database accession no. Q95PM1
BERNARDO ET AL., BLOOD, vol. 104, 2004, pages 100 - 6
CAFFREY CONOR R ET AL: "SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.", MOLECULAR AND BIOCHEMICAL PARASITOLOGY 30 APR 2002, vol. 121, no. 1, 30 April 2002 (2002-04-30), pages 49 - 61, XP002794433, ISSN: 0166-6851 *
CAFFREY ET AL., MOL BIOCHEM PARASITOL, vol. 121, 2002, pages 49 - 61
CLARK ET AL., THROMB HAEMOST., vol. 118, 2018, pages 471 - 9
CRAWLEY ET AL., BLOOD, vol. 118, 2011, pages 3212 - 21
EL RIDI RASHIKA ET AL: "Cysteine peptidases as schistosomiasis vaccines with inbuilt adjuvanticity.", PLOS ONE 2014, vol. 9, no. 1, 2014, pages e85401, XP002794432, ISSN: 1932-6203 *
FISCHER ET AL., BIOCHEM J., vol. 331, 1998, pages 483 - 8
GREENSAMBROOK: "Molecular Cloning: A Laboratory Manual", 2012, CSHL PRESS
HULSTEIN ET AL., J THROMB HAEMOST., vol. 4, 2006, pages 2569 - 75
KAIL ET AL., JPROTEOME RES, vol. 7, 2008, pages 29 - 34
KANNICHT ET AL., BIOLOGICALS, vol. 43, 2015, pages 117 - 22
KOKAME ET AL., BR J HAEMATOL, vol. 129, 2005, pages 93 - 100
KOKAME ET AL., BR J HAEMATOL., vol. 129, 2005, pages 93 - 100
LENTING ET AL., THROMB HAEMOST, vol. 104, 2010, pages 449 - 55
LOWRY ET AL., J BIOL CHEM., vol. 193, 1951, pages 265 - 75
MERLYN KEZIAH S ET AL: "Focalization of thrombosis and therapeutic perspectives: a memoir", ORIENTAL PHARMACY AND EXPERIMENTAL MEDICINE, KYUNG HEE ORIENTAL MEDICINE RESEARCH CENTER, KYUNG HEE UNIVERSITY, KR, vol. 18, no. 4, 18 August 2018 (2018-08-18), pages 281 - 298, XP036617549, ISSN: 1598-2386, [retrieved on 20180818], DOI: 10.1007/S13596-018-0331-0 *
NOVINECLENARCIC, BIOMOL CONCEPTS, vol. 4, 2013, pages 287 - 308
SHEVCHENKO ET AL., NAT PROTOC, vol. 1, 2006, pages 2856 - 60
SPIVAK ET AL., J PROTEOME RES., vol. 8, 2009, pages 3737 - 45
TSAI ET AL., BLOOD, vol. 87, 1996, pages 4235 - 44
TURK ET AL., BIOCHIM BIOPHYS ACTA, vol. 1824, 2012, pages 68 - 88

Similar Documents

Publication Publication Date Title
Nagai et al. Recombinant human microplasmin: production and potential therapeutic properties
JP2023133564A (ja) アンジオテンシン変換酵素２（ａｃｅ２）の活性な低分子量変異体
JP2020090541A (ja) 急性及び慢性血栓を予防または治療することに用いる方法
CA2658582C (fr) Procollagene c-proteinase liee a la bmp-1, destinee au diagnostic et au traitement des defauts et des affections des os et des tissus mous
US20050181978A1 (en) Therapeutic use of factor XI
JP2020511537A (ja) 組換え成熟補体因子ｉ
JP2022134139A (ja) 出血治療のための止血促進タンパク質
US8940297B2 (en) Expression of thrombin variants
JPH06505494A (ja) Ｉｘ因子の調製
WO2020214033A1 (fr) Médicaments à base de cystéine peptidase de type papaïne et leurs utilisations
AU2004290869A1 (en) Therapeutic use of factor XI
JP6629744B2 (ja) 第Ｘａ因子の半減期を延ばす組成物および方法
US20230242896A1 (en) Adamts13 protein variants and uses thereof
Mebius et al. Cleavage of von Willebrand Factor by a surface protease (SmCB2) of the flatworm pathogen, Schistosoma mansoni
US20180340163A1 (en) Recombinant serine proteases
DK2813567T3 (en) C-terminally bound amino acids and their therapeutic fibrinolytic applications
KR20050043733A (ko) 인자 ⅶ 폴리펩티드와 ｐａｉ-1 폴리펩티드를 포함하는약학적 조성물
US9145452B2 (en) Application of fibrinogen-420 and its active domain
KR20230147138A (ko) Adamts13 변이체
JP2005510516A (ja) Ｖｉｉ因子ポリペプチドおよびプロテインｓ阻害剤を含む薬学的組成物
JPH09176041A (ja) α２プラスミンインヒビターを含有するＤＩＣ治療用薬剤
EA045357B1 (ru) Прогемостатические белки для лечения кровотечений
WO2012031240A2 (fr) Produits thérapeutiques pour coagulopathie de consommation de facteur v induite par traumatisme
JPH08143470A (ja) 汎発性血管内凝固症候群治療剤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20720537

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20720537

Country of ref document: EP

Kind code of ref document: A1