US20150259665A1 - Factor vii conjugates - Google Patents

Factor vii conjugates Download PDF

Info

Publication number
US20150259665A1
US20150259665A1 US14/433,151 US201314433151A US2015259665A1 US 20150259665 A1 US20150259665 A1 US 20150259665A1 US 201314433151 A US201314433151 A US 201314433151A US 2015259665 A1 US2015259665 A1 US 2015259665A1
Authority
US
United States
Prior art keywords
kda
factor vii
conjugate
heparosan
polypeptide
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US14/433,151
Other languages
English (en)
Inventor
Carsten Behrens
Henrik Oestergaard
Henning Ralf Stennicke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk Health Care AG
Original Assignee
Novo Nordisk Health Care AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk Health Care AG filed Critical Novo Nordisk Health Care AG
Priority to US14/433,151 priority Critical patent/US20150259665A1/en
Publication of US20150259665A1 publication Critical patent/US20150259665A1/en
Assigned to NOVO NORDISK HEALTHCARE AG reassignment NOVO NORDISK HEALTHCARE AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEHRENS, CARSTEN, STENNICKE, HENNING RALF, OESTERGAARD, HENRIK
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A61K47/4823—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)

Definitions

  • the present invention relates to the conjugation of Factor VII polypeptides with heparosan polymers.
  • haemostasis An important part of haemostasis is coagulation of the blood and the formation of a clot at the site of the injury.
  • the coagulation process is highly dependent on the function of several protein molecules. These are known as coagulation factors.
  • Some of the coagulation factors are proteases which can exist in an inactive zymogen or an enzymatically active form. The zymogen form can be converted to its enzymatically active form by specific cleavage of the polypeptide chain catalyzed by another proteolytically active coagulation factor.
  • Factor VII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein.
  • the Factor VII zymogen is converted into an activated form (Factor VIIa) by specific proteolytic cleavage at a single site, i.e. between R152 and 1153 of the Factor VII sequence (wild type human coagulation Factor VII) resulting in a two chain molecule linked by a single disulfide bond.
  • the two polypeptide chains in Factor VIIa are referred to as light and heavy chain, corresponding to residues 1-152 and 153-406, respectively, of the Factor VII sequence.
  • Factor VII circulates predominantly as zymogen, but a minor fraction is on the activated form (Factor VIIa).
  • the blood coagulation process can be divided into three phases: initiation, amplification and propagation.
  • the initiation and propagation phases contribute to the formation of thrombin, a coagulation factor with many important functions in haemostasis.
  • the coagulation cascade starts if the single-layered barrier of endothelial cells that line the inner surface of blood vessels becomes damaged. This exposes subendothelial cells and extravascular matrix proteins to which platelets in the blood will stick to. If this happens, Tissue Factor (TF) which is present on the surface of sub-endothelial cells becomes exposed to Factor VIIa circulating in the blood.
  • TF is a membrane-bound protein and serves as the receptor for Factor VIIa.
  • Factor VIIa is an enzyme, a serine protease, with intrinsically low activity. However, when Factor VIIa is bound to TF, its activity increases greatly. Factor VIIa interaction with TF also localizes Factor VIIa on the phospholipid surface of the TF bearing cell and positions it optimally for activation of Factor X to Xa. When this happens, Factor Xa can combine with Factor Va to form the so-called “prothombinase” complex on the surface of the TF bearing cell. The prothrombinase complex then generates thrombin by cleavage of prothrombin. The pathway activated by exposing TF to circulating Factor VIIa and leading to the initial generation of thrombin is known as the TF pathway.
  • the TF:Factor VIIa complex also catalyzes the activation of Factor IX to Factor IXa. Then activated Factor IXa can diffuse to the surface of platelets which are sticking to the site of the injury and have been activated. This allows Factor IXa to combine with FVIIIa to form the “tenase” complex on the surface of the activated platelet.
  • This complex plays a key role in the propagation phase due to its remarkable efficiency in activating Factor X to Xa.
  • the efficient tenase catalyzed generation of Factor Xa activity in turn leads to efficient cleavage of prothrombin to thrombin catalyzed by the prothrombinase complex.
  • Thrombin formed initially by the TF pathway serves as a pro-coagulant signal that encourages recruitment, activation and aggregation of platelets at the injury site. This results in the formation of a loose primary plug of platelets.
  • this primary plug of platelets is unstable and needs reinforcement to sustain haemostasis. Stabilization of the plug involves anchoring and entangling the platelets in a web of fibrin fibres.
  • Replacement therapy is the traditional treatment for hemophilia A and B, and involves intravenous administration of Factor VIII or Factor IX. In many cases, however, patients develop antibodies (also known as inhibitors) against the infused proteins, which reduce or negate the efficacy of the treatment.
  • Recombinant Factor VIIa (Novoseven®) has been approved for the treatment of hemophilia A or B patients that have inhibitors, and also is used to stop bleeding episodes or prevent bleeding associated with trauma and/or surgery.
  • Recombinant Factor VIIa also has been approved for the treatment of patients with congenital Factor VII deficiency. It has been proposed that recombinant FVIIa operates through a TF-independent mechanism.
  • recombinant FVIIa is directed to the surface of the activated blood platelets by virtue of its Gla-domain where it then proteolytically activates Factor X to Xa thus by-passing the need for a functional tenase complex.
  • the low enzymatic activity of FVIIa in the absence of TF as well as the low affinity of the Gla-domain for membranes could explain the need for supra-physiological levels of circulating FVIIa needed to achieve haemostasis.
  • Recombinant Factor VIIa has a pharmacological half-life of 2-3 hours which may necessitate frequent administration to resolve bleedings in patients. Further, patients often only receive Factor VIIa therapy after a bleed has commenced, rather than as a precautionary measure, which often impinges upon their general quality of life. A recombinant Factor VIIa variant with a longer circulation half-life would decrease the number of necessary administrations and support less frequent dosing thus hold the promise of significantly improving Factor VIIa therapy to the benefit of patients and care-holders.
  • WO03031464 disclose an enzymatic approach where PEG groups can be attached to glycans present on the polypeptide.
  • the present invention derives from the finding that the polymer heparosan can be bound to Factor VII in order to extend its half-life.
  • One advantage with heparosan is that heparosan polymers are biodegradable, avoiding any potential accumulation problems related to non-biodegradable polymers.
  • the use of heparosan polymers in this way can lead to improved properties of Factor VII polypeptide conjugates such as increased FIXa and FXa generation potential and improved clot activity.
  • the present invention provides a conjugate between a Factor VII polypeptide and a heparosan polymer.
  • the polymer may have a polydispersity index (Mw/Mn) of less than 1.10 or less than 1.05. In another interesting embodiment, the polymer may have a size between 13 kDa and 65 kDa.
  • the heparosan Factor VII polypeptide conjugate of the invention may have increased circulating half-life compared to an un-conjugated Factor VII polypeptide; or increased functional half-life compared to an un-conjugated Factor VII polypeptide.
  • the heparosan Factor VII polypeptide conjugate of the invention may have increased mean residence time compared to an un-conjugated Factor VII polypeptide; or increased functional mean residence time compared to an un-conjugated Factor VII polypeptide.
  • the Factor VII polypeptide may be a variant of a Factor VII polypeptide carrying a free cysteine, such as FVIIa-407C, in which the heparosan polymer may be attached to the cysteine at position 407 of said Factor VII polypeptide.
  • the polymer may be attached to the polypeptide via N- or O-glycans.
  • compositions comprising the conjugates described herein, such as a pharmaceutical composition comprising a conjugate of the invention and a pharmaceutically acceptable carrier or diluent.
  • a conjugate or composition of the invention may be provided for use in a method of treating or preventing a bleeding disorder. That is, the invention relates to methods of treating or preventing a bleeding disorder, wherein said methods comprise administering a suitable dose of a conjugate of the invention to a patient in need thereof, such as an individual in need of Factor VII, such as an individual having haemophilia A or haemophilia B.
  • FIG. 1 Structure of (A) heparosan and (B) a heparosan polymer with maleimide functionality at its reducing end.
  • FIG. 2 Assessment of conjugate purity by SDS-PAGE.
  • A SDS-PAGE analysis of final FVIIa conjugates. Gel was loaded with HiMark HMW standard (lane 1); FVIIa (lane 2); 13k-HEP-[C]-FVIIa (lane 3); 27k-HEP-[C]-FVIIa (lane 4); 40k-HEP-[C]-FVIIa (lane 5); 52k-HEP-[C]-FVIIa (lane 6); 60k-HEP-[C]-FVIIa (lane 7); 65k-HEP-[C]-FVIIa (lane 8); 108k-HEP-[C]-FVIIa (lane 9) and 157k-HEP-[C]-FVIIa407C (lane 10).
  • FIG. 3 Analysis of FVIIa clotting activity levels of heparosan conjugates and glycoPEGylated FVIIa references.
  • FIG. 4 Proteolytic activity of heparosan conjugates and glycoPEGylated FVIIa references.
  • FIG. 6 PK results (Clot Activity) in Sprague Dawley rats. Comparison of unmodified FVIIa (2 studies), 13k-HEP-[C]-FVIIa407C, 27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C, 65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]-FVIIa407C and 157k-HEP-[C]-FVIIa407C, glycoconjugated 52k-HEP-[N]-FVIIa and reference molecules (40 kDa-PEG-[N]-FVIIa (2 studies) and 40 kDa-PEG-[C]-FVIIa407C). Data are shown in a semilogarithmic plot.
  • FIG. 7 Relationship between HEP-size and mean residence time (MRT) for a number of HEP-[C]-FVIIa407C conjugates. MRT values from PK studies are plotted against heparosan polymer size of conjugates.
  • the plot represent values for non-conjugated FVIIa, 13k-HEP-[C]-FVIIa407C, 27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C, 65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]-FVIIa407C and 157k-HEP-[C]-FVIIa407C.
  • MRT LOCI
  • the invention relates to conjugates between Factor VII (FVII) polypeptides and heparosan (HEP) polymers, as well as to methods for preparing such conjugates and uses for such conjugates.
  • FVII Factor VII
  • HEP heparosan
  • Factor VII or “FVII” denote Factor VII polypeptides. Suitable polypeptides may be produced by methods including natural source extraction and purification, and by recombinant cell culture systems. The sequence and characteristics of wild-type human Factor VII are set forth, for example, in U.S. Pat. No. 4,784,950.
  • Factor VII polypeptide biologically active factor VII equivalents and modified forms of Factor VII, e.g., differing in one or more amino acid(s) in the overall sequence.
  • the terms used in this application are intended to cover substitution, deletion and insertion amino acid variants of Factor VII or posttranslational modifications.
  • Factor VII polypeptide encompasses, without limitation, Factor VII, as well as Factor VII-related polypeptides.
  • Factor VII-related polypeptides include, without limitation, Factor VII polypeptides that have either been chemically modified relative to human Factor VII and/or contain one or more amino acid sequence alterations relative to human Factor VII (i.e., Factor VII variants), and/or contain truncated amino acid sequences relative to human Factor VII (i.e., Factor VII fragments).
  • Such factor VII-related polypeptides may exhibit different properties relative to human Factor VII, including stability, phospholipid binding, altered specific activity, and the like.
  • Factor VII is intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor VIIa. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor VIIa.
  • Factor VII is also intended to encompass, without limitation, polypeptides having the amino acid sequence 1-406 of wild-type human Factor VII (as disclosed in U.S. Pat. No. 4,784,950), as well as wild-type Factor VII derived from other species, such as, e.g., bovine, porcine, canine, murine, and salmon Factor VII. It further encompasses natural allelic variations of Factor VII that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
  • Vector VII-related polypeptides encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild-type human Factor VII. These polypeptides include, without limitation, Factor VII or Factor VIIa that has been chemically modified and Factor VII variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
  • polypeptides with a modified amino acid sequence for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human Factor VIIa.
  • polypeptanides with a modified amino acid sequence for instance, polypeptides having a modified C-terminal end including C-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human Factor VIIa.
  • Factor VII-related polypeptides including variants of Factor VII, exhibiting substantially the same or better bioactivity than wild-type Factor VII, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild-type Factor VII by insertion, deletion, or substitution of one or more amino acids.
  • Factor VII-related polypeptides having substantially the same or improved biological activity relative to wild-type Factor VIIa encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific activity of wild-type Factor VIIa that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay.
  • the Factor VII polypeptide may be a Factor VII-related polypeptide, in particular a variant, wherein the ratio between the activity of said Factor VII polypeptide and the activity of native human Factor VIIa (wild-type FVIIa) is at least about 1.25 when tested in an in vitro hydrolysis assay; in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
  • the Factor VII polypeptide may be a Factor VII analogue, in particular a variant, wherein the ratio between the activity of said Factor VII polypeptide and the activity of native human Factor VIIa (wild-type FVIIa) is at least about 1.25 when tested in an in vitro proteolysis assay; the ratio may be at least about 2.0; the ratio may be at least about 4.0; the ratio may be at least about 8.0.
  • the Factor VII polypeptide may be human Factor VII, as disclosed, e.g., in U.S. Pat. No. 4,784,950 (wild-type Factor VII).
  • the Factor VII polypeptide may be human Factor VIIa.
  • Factor VII polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140%, and most preferably at least about 160%, of the specific biological activity of human Factor VIIa.
  • the Factor VII polypeptide may be a variant Factor VII polypeptide having a reduced interaction with antithrombin III when compared to that of human Factor VIIa.
  • the Factor VII polypeptide may have less than 100%, less than 95%, less than 90%, less than 80%, less than 70% or less than 50% of the interaction with antithrombin III of wild type human Factor VIIa.
  • a reduced interaction with antithrombin III may be present in combination with another improved biological activity as described herein, such as an improved proteolytic activity.
  • the Factor VII polypeptide may have an amino acid sequence that differs from the sequence of wild-type Factor VII by insertion, deletion, or substitution of one or more amino acids.
  • the Factor VII polypeptide may be a polypeptide that exhibits at least about 70%, preferably at least about 80%, more preferably at least about 90%, and most preferable at least about 95%, of amino acid sequence identity with the sequence of wild-type Factor VII as disclosed in U.S. Pat. No. 4,784,950.
  • Amino acid sequence homology/identity is conveniently determined from aligned sequences, using a suitable computer program for sequence alignment, such as, e.g., the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22: 4673-4680).
  • Non-limiting examples of Factor VII variants having substantially the same or improved biological activity as wild-type Factor VII include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/
  • Factor VII variants falling within the scope of Factor VII polypeptides herein are those described in WO 2007/031559 and WO 2009/126307.
  • Preferred Factor VII polypeptides for use in accordance with the present invention are those in which an additional cysteine residue has been added compared to an existing FVII sequence, such as a wild type FVII sequence.
  • the cysteine may be appended to a Factor VII polypeptide at the C-terminal.
  • the cysteine may be appended to a Factor VIIa polypeptide at the C-terminal residue 406 of the amino acid sequence of wild-type human Factor VII, leading to FVIIa 407C.
  • the cysteine may be positioned in the amino acid sequence of a Factor VII molecule at a surface exposed position that will not seriously impede tissue factor binding, Factor X binding or binding to phospholipids.
  • the structure of Factor VIIa is known and a suitable position meeting these requirements may therefore be identified by the skilled person.
  • the numbering of amino acids in the Factor VII polypeptide set out herein is based on the amino acid sequence for wild type human Factor VII as disclosed in U.S. Pat. No. 4,784,950. It will be apparent that equivalent positions in other Factor VII polypeptides may be readily identified by the skilled person by carrying out an alignment of the relevant sequences.
  • the biological activity of Factor VIIa in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
  • Factor VII polypeptide The biological activity of a Factor VII polypeptide may be measured by a number of ways as described below:
  • the peptidolytic activity of a FVII polypeptide or a FVII conjugate can be estimated using a chromogenic peptide (S-2288; Chromogenix) as substrate.
  • a chromogenic peptide S-2288; Chromogenix
  • the proteolytic activity of a FVII polypeptide or a FVII conjugate can be estimated using plasma-derived factor X (FX) as substrate.
  • FX plasma-derived factor X
  • a way of performing the assay is as follows: All proteins are initially diluted in 50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM CaCl 2 , 1 mg/mL BSA, and 0.1% (w/v) PEG8000.
  • biological activity of Factor VII polypeptides may also be quantified by measuring the ability of a preparation to promote blood clotting using Factor VII-deficient plasma and thromboplastin, as described, e.g., in U.S. Pat. No. 5,997,864 or WO 92/15686.
  • biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to “Factor VII units” by comparison with a pooled human serum standard containing 1 unit/ml Factor VII activity.
  • Factor VIIa biological activity may be quantified by measuring the physical binding of Factor VIIa or a Factor VII-related polypeptide to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413:359-363, 1997).
  • Potencies can be estimated using a commercial FVIIa specific clotting assay; STACLOT® VIIa-rTF from Diagnostica Stago.
  • the assay is based on the method published by J. H. Morrissey et al, Blood. 81:734-744 (1993). It measures sTF initiated FVIIa activity-dependent time to fibrin clot formation in FVII deficient plasma in the presence of phospholipids.
  • Test compounds are diluted in Pipes+1% BSA assay dilution buffer and tested in 4 dilutions in 4 separate assay runs. Clotting times can be measured on an ACL9000 (ILS) coagulation instrument and results calculated using linear regression on a bilogarithmic scale based on a FVIIa calibration curve.
  • IFS ACL9000
  • the pharmacokinetic properties of a FVII polypeptide or a FVII conjugate can be estimated in sprauge Dawley rats.
  • a suitable buffer such as 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween80 80, pH 6.0 and FVII polypeptide or FVII conjugate concentration in formulation buffer is determined by light chain quantification on HPLC.
  • Male Sprague Dawley rats are obtained for the study. The animals are allowed at least one week acclimatisation period, and are allowed free access to feed and water before start of the experiment.
  • the FVII polypeptide or FVII conjugate formulations are then given as a single iv bolus injection in the tail vein.
  • Blood is then samples according to a predetermined schedual. Blood can be sampled the following way: 45 ⁇ l of blood is transferred to an Eppendorf tube containing 5 ⁇ l Stabilyte; 200 ⁇ l PIPES buffer (0.050 M Pipes, 0.10 M sodium chloride, 0.002 M EDTA, 1% (w/v) BSA, pH 7.2) is added and inverted gently 5 times. The diluted citrate-stabilised blood is kept at room temperature until centrifugation at 4000 G for 10 minutes at room temperature.
  • the supernatant is divided to three Micronic tubes; 70 ul for clot activity, 70 ul for antigen analysis and the rest as extra sample.
  • the samples are immediately frozen on dry ice and storage at ⁇ 80° C. until plasma analysis for example as described below can be carried out.
  • FVIIa clotting activity levels of FVII polypeptide or a FVII conjugate in rat plasma can be estimated using a commercial FVIIa specific clotting assay; such as STACLOT® VIIa-rTF from Diagnostica Stago.
  • the assay is based on the method published by J. H. Morrissey et al, Blood. 81:734-744 (1993). It measures soluble tissue factor (sTF) initiated FVIIa activity-dependent time to fibrin clot formation in FVII deficient plasma in the presence of phospholipids. Samples can be measured on an ACL9000 coagulation instrument against FVIIa calibration curves with the same matrix as the diluted samples (like versus like).
  • FVII polypeptide or FVII conjugate antigen concentrations in plasma can be determined using LOCI technology.
  • two monoclonal antibodies against human FVII are used for detection. The principle is described in Thromb Haemost 100(5):920-8 (2008). Samples are measured against drug substance calibration curves.
  • Pharmacokinetic analysis can be carried out by non-compartmental methods (NCA) using for example WinNonlin (Pharsight Corporation St. Louis, Mo.) software. From the data the following parameters can be estimated: C max (maximum concentration), T max (time of maximum concentration), AUC (area under the curve from zero to infinity), AUC extrap (percentage of AUC that are extrapolated from the last concentration to infinity), T 1/2 (half-life), Cl (clearance) Vz (volume of distribution), and MRT (mean residence time).
  • factor VIIa or Factor VII polypeptides to generate thrombin can also be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations (minus factor VIII when mimicking hemophilia A conditions) and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, which is hereby incorporated as reference)
  • the activity of the Factor VII polypeptides may also be measured using a one-stage clot assay (assay 4) essentially as described in WO 92/15686 or U.S. Pat. No. 5,997,864. Briefly, the sample to be tested is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 ⁇ l is incubated with 100 ⁇ l of Factor VII deficient plasma and 200 ⁇ l of thromboplastin C containing 10 mM Ca 2+ . Clotting times are measured and compared to a standard curve using a reference standard or a pool of citrated normal human plasma in serial dilution.
  • Human purified Factor VIIa suitable for use in the present invention may be made by DNA recombinant technology, e.g. as described by Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or as described in European Patent No. 200.421 (ZymoGenetics, Inc.). Factor VII may also be produced by the methods described by Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983. These methods yield Factor VII without detectable amounts of other blood coagulation factors.
  • Factor VII preparation may be obtained by including an additional gel filtration as the final purification step.
  • Factor VII is then converted into activated factor VIIa by known means, e.g. by several different plasma proteins, such as factor XIIa, IX a or Xa Alternatively, as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like, or by autoactivation in solution.
  • ion-exchange chromatography column such as Mono Q® (Pharmacia fine Chemicals) or the like
  • Factor VII-related polypeptides may be produced by modification of wild-type Factor VII or by recombinant technology.
  • Factor VII-related polypeptides with altered amino acid sequence when compared to wild-type Factor VII may be produced by modifying the nucleic acid sequence encoding wild-type factor VII either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural factor VII by known means, e.g. by site-specific mutagenesis.
  • the introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure that utilizes a super coiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation.
  • the oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated.
  • Dpnl is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
  • Other procedures known in the art for creating, identifying and isolating variants may also be used, such as, for example, gene shuffling or phage display techniques.
  • Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like.
  • Factor VII polypeptides may be further purified. Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-Factor VII antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem. 261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988); hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like.
  • affinity chromatography such as, e.g., on an anti-Factor VII antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem. 261:11097, 1986; and Thim et al., Biochem.
  • the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and most preferably less than about 1%, of non-Factor VII polypeptides derived from the host cell.
  • Factor VII polypeptides may be activated by proteolytic cleavage, using Factor XIIa or other proteases having trypsin-like specificity, such as, e.g., Factor IXa, kallikrein, Factor Xa, and thrombin.
  • Factor IXa Factor IXa
  • kallikrein Factor Xa
  • thrombin e.g., thrombin.
  • Factor VII polypeptides may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia) or the like, or by autoactivation in solution.
  • the resulting activated Factor VII polypeptide may then be conjugated with a heparosan polymer, formulated and administered as described in the present application.
  • Heparosan is a natural sugar polymer comprising (-GlcUA-beta1,4-GlcNAc-alpha1,4-) repeats (see FIG. 1A ). It belongs to the glycosaminoglycan polysaccharide family and is a negatively charged polymer at physiological pH. It can be found in the capsule of certain bacteria but it is also found in higher vertebrate where it serves as precursor for the natural polymers heparin and heparan sulphate. Although not proven in detail, heparosan is believed to be degraded in the lysosomes. An injection of a 100 kDa heparosan polymer labelled with Bolton-Hunter reagents has shown that heparosan is secreted as smaller fragments in body fluids/waste (US 2010/0036001).
  • heparosan polymers and methods of making such polymers are described in US 2010/0036001, the content of which is incorporated herein by reference.
  • the heparosan polymer may be any heparosan polymer described or disclosed in US 2010/0036001.
  • heparosan polymers can be produced by any suitable method, such as any of the methods described in US 2010/0036001 or US 2008/0109236.
  • Heparosan can be produced using bacterial-derived enzymes.
  • the Escherichia coli K5 enzymes KfiA (alpha GlcNAc transferase) and KfiC (beta GlcUA transferase) can together also form the disaccharide repeat of heparosan.
  • a heparosan polymer for use in the present invention is typically a polymer of the formula (-GlcUA-beta1,4-GlcNAc-alpha1,4-)n.
  • the size of the heparosan polymer may be defined by the number of repeats n in this formula.
  • the number of said repeats n may be, for example, from 2 to about 5000.
  • the number of repeats may be, for example 50 to 2000 units, 100 to 1000 units or 200 to 700 units.
  • the number of repeats may be 200 to 250 units, 500 to 550 units or 350 to 400 units. Any of the lower limits of these ranges may be combined with any higher upper limit of these ranges to form a suitable range of numbers of units in the heparosan polymer.
  • the size of the heparosan polymer may be defined by its molecular weight.
  • the molecular weight may be the average molecular weight for a population of heparosan polymer molecules, such as the weight average molecular mass.
  • heparosan polymer size of 40 kDa denotes 40 kD+/ ⁇ 5%, e.g. 40 kDa could for example in practise mean 38.8 kDa or 41.5 kDa.
  • the heparosan polymer may have a molecular weight of, for example, 500 Da to 1,000 kDa.
  • the molecular weight of the polymer may be 500 Da to 650 kDa, 5 kDa to 750 kDa, 10 kDa to 500 kDa, 15 kDa to 550 kDa or 25 kDa to 250 kDa.
  • the molecular weight may be selected at particular levels within these ranges in order to achieve a suitable balance between activity of the Factor VII polypeptide and half-life or mean residence time of the conjugate.
  • the molecular weight of the polymer may be in a range selected from 15-25 kDa, 25-35 kDa, 35-45 kDa, 45-55 kDa, 55-65 kDa or 65-75 kDa.
  • the molecular weight may be 20 kDa to 35 kDa, such as 22 kDa to 32 kDa such as 25 kDa to 30 kDa, such as about 27 kDa.
  • the molecular weight may be 35 to 65 kDa, such as 40 kDa to 60 kDa, such as 47 kDa to 57 kDa, such as 50 kDa to 55 kDa such as about 52 kDa.
  • the molecular weight may be 50 to 75 kDa such as 60 to 70 kDa, such as 63 to 67 kDa such as about 65 kDa.
  • the heparosan polymer of the Factor VII conjugate, of the invention has a size in a range selected from 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa and 38-42 kDa.
  • the heparosan polymer may have a narrow size distribution (i.e. monodisperse) or a broad size distribution (i.e. polydisperse).
  • the polydispersity value using this equation for an ideal monodisperse polymer is 1.
  • a heparosan polymer for use in the present invention is monodisperse.
  • the polymer may therefore have a polydispersity that is about 1, the polydispersity may be less than 1.25, preferably less than 1.20, preferably less than 1.15, preferably less than 1.10, preferably less than 1.09, preferably less than 1.08, preferably less than 1.07, preferably less than 1.06, preferably less than 1.05.
  • the molecular weight size distribution of the heparosan may be measured by comparison with monodisperse size standards (HA Lo-Ladder, Hyalose LLC) which may be run on agarose gels.
  • the size distribution of heparosan polymers may be determined by high performance size exclusion chromatography-multi angle laser light scattering (SEC-MALLS). Such a method can be used to assess the molecular weight and polydispersity of a heparosan polymer.
  • Polymer size may be regulated in enzymatic methods of production. By controlling the molar ratio of heparosan acceptor chains to UDP sugar, it is possible to select a final heparosan polymer size that is desired
  • the heparosan polymer may further comprise a reactive group to allow its attachment to a Factor VII polypeptide.
  • a suitable reactive group may be, for example, an aldehyde, alkyne, ketone, maleimide, thiol, azide, amino, hydrazide, hydroxylamine, carbonate, ester, chelator or a combination of any thereof.
  • FIG. 1B illustrates a heparosan polymer comprising a maleimide group.
  • Hydroxylamine group added at the reducing or non-reducing terminus, react with aldehydes and ketones.
  • maleimide functionalized heparosan polymers of defined size may be prepared by an enzymatic (PmHS1) polymerization reaction using the two sugar nucleotides UDP-GlcNAc and UDP-GlcUA in equimolar amount.
  • a priming trisaccharide (GlcUA-GlcNAc-GlcUA)NH 2 may be used for initiating the reaction, and polymerization run until depletion of sugar nucleotide building blocks.
  • Terminal amine (originating from the primer) may then be functionalized with suitable reactive groups such as a reactive group as described above, such as a maleimide functionality designed for conjugation to free cysteines.
  • the size of the heparosan polymers can be pre-determined by variation in sugar nucleotide: primer stoichiometry. The technique is described in detail in US 2010/0036001.
  • the reactive group may be present at the reducing or non-reducing termini or throughout the sugar chain. The presence of only one such reactive group is preferred when conjugating the heparosan polymer to the polypeptide.
  • a Factor VII polypeptide as described herein is conjugated to a heparosan polymer as described herein. Any Factor VII polypeptide as described herein may be combined with any heparosan polymer as described herein.
  • the heparosan polymer may be attached at a single position on the polypeptide, or heparosan polymers may be attached at multiple positions on the polypeptide.
  • the location of attachment of the polymer to the polypeptide may depend on the particular polypeptide molecule being used.
  • the location of attachment of the polymer to the polypeptide may depend on the type of reactive group, if any, that is present on the polymer. As explained above, different reactive groups will react with different groups on the polypeptide molecule.
  • Heparosan polymers may be attached to the glycans of a Factor VII polypeptide using attachment technology described in any of US 2010/0036001, WO03/031464, WO2005/014035 or WO2008/025856, the content of each of which is included herein by reference.
  • WO 03/031464 describes methods for remodelling the glycan structure of a polypeptide, such as a Factor VII or Factor VIIa polypeptide and methods for the addition of a modifying group such as a water soluble polymer to such a polypeptide. Such methods may be used to attach a heparosan polymer to a Factor VII polypeptide in accordance with the present invention.
  • a Factor VII polypeptide may be conjugated to its glycan moieties using sialyltransferase.
  • a HEP polymer first need to be linked to a sialic acid cytidine monophosphate.
  • 5′-glycylamidoneuraminic acid cytidine monophosphate is a suitable starting point for such chemistry, but other sialic acid cytidine monophosphate or fragments of such can be used.
  • Examples set out methods for covalent linking HEP polymers to GSC molecules. By covalent attachment, a HEP-GSC (HEP conjugated glycylamidoneuraminic acid cytidine monophosphate) molecule is created that can be transferred to glycan moieties of FVIIa.
  • HEP-GSC HEP conjugated glycylamidoneuraminic acid cytidine monophosphate
  • WO 2005/014035 describes chemical conjugation that utilises galactose oxidase in combination with terminal galactose-containing glycoproteins such as sialidase treated glycoproteins or asialo glycoproteins.
  • Such method may utilise the reaction of sialidases and galactose oxidase to produce reactive aldehyde groups that can be chemically conjugated to nucleophilic reactive groups to attach a polymer to a glycoprotein.
  • Such methods may be used to attach a heparosan polymer to a Factor VII glycoprotein.
  • a suitable Factor VII polypeptide for use in such methods may be any Factor VII glycopeptide that comprises terminal galactose.
  • Such a glycoprotein may be produced by treatment of a Factor VII polypeptide with sialidase to remove terminal sialic acid.
  • WO2011012850 describes the attachment of polymeric groups to a glycosyl group in a glycoprotein. Such methods may be used in accordance with the present invention to attach a heparosan polymer to a Factor VII polypeptide.
  • Heparosan may be attached to the polypeptide via an engineered extra cysteine in the polypeptide or an exposed sulfhydryl group.
  • the sulfhydryl the cysteine group may be coupled to a functionalised heparosan polymer, such as a maleimide-heparosan polymer to obtain a heparosan-polypeptide conjugate.
  • the heparosan polymer is attached to a FVII polypeptide by conjugation to a cysteine on the FVII molecule.
  • the cysteine may be engineered into a Factor VII polypeptide, such as added to the amino acid sequence of a wild-type Factor VII polypeptide.
  • the cysteine may be positioned at the C-terminal of the Factor VII polypeptide, such as at position 407, or in chain at a surface exposed position that will not seriously impede tissue factor binding, FX binding or binding to phospholipids.
  • the Cys407 can act as site of attachment of a heparosan polymer (e.g. a 13 kDa, 27 kDa, 40 kDa, 52 kDa, 60 kDa, 65 kDa, 108 kDa or 157 kDa heparosan polymer that has been functionalised with maleimide).
  • a heparosan polymer e.g. a 13 kDa, 27 kDa, 40 kDa, 52 kDa, 60 kDa, 65 kDa, 108 kDa or 157 kDa heparosan polymer that has been functionalised with maleimide.
  • a Factor VII polypeptide with unblocked cysteine such as FVIIa-407C
  • a suitable buffer such as HEPES and at near neutral pH.
  • the reaction may be allowed to stand at room temperature for, for example, 3-4 hours.
  • Such a reaction can achieve the conjugation of the heparosan polymer to the Factor VII polypeptide.
  • Factor VII-heparosan conjugates may be purified once they have been produced.
  • purification may comprise by affinity chromatography using immobilised mAb directed towards the Factor VII polypeptide, such as mAb directed against the calcified gla-domain on FVIIa.
  • unconjugated HEP-maleimide may be removed by extensive washing of the column.
  • FVII may be released from the column by releasing the FVII from the antibody.
  • release from the column may be achieved by washing with a buffer comprising EDTA.
  • Size exclusion chromatography may be used to separate Factor VII-heparosan conjugates from unconjugated Factor VII.
  • Pure conjugate may be concentrated by ultrafiltration.
  • Final concentrations of Factor VII-heparosan conjugate resulting from a process of production may be determined by, for example, HPLC quantification, such as HPLC quantification of the FVII light chain.
  • a conjugate of the invention may show various advantages.
  • the conjugate may show one of more of the following advantages when compared to a suitable control Factor VII molecule.
  • the conjugate may show an improvement in any biological activity of Factor VII as described herein and this may be measured using any assay or method as described herein, such as the methods described above in relation to the activity of Factor VII.
  • a conjugate of the invention i.e. a conjugate of interest
  • a suitable control Factor VII molecule may be, for example, an unconjugated Factor VII polypeptide or a conjugated Factor VII polypeptide.
  • the conjugated control may be a FVIIa polypeptide conjugated to a water soluble polymer, or a FVIIa polypeptide chemically linked to a protein.
  • a conjugated Factor VII control may be a Factor VII polypeptide that is conjugated to a chemical moiety (being protein or water soluble polymer) of a similar size as the heparosan molecule in the conjugate of interest.
  • the water-soluble polymer can for example be polyethylene glycol (PEG), branched PEG, dextran, poly(l-hydroxymethylethylene hydroxymethylformal), 2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC).
  • the Factor VII polypeptide in the control Factor VII molecule is preferably the same Factor VII polypeptide that is present in the conjugate of interest.
  • the control Factor VII molecule may have the same amino acid sequence as the Factor VII polypeptide in the conjugate of interest.
  • the control Factor VII may be the same glycosylation pattern as the Factor VII polypeptide in the conjugate of interest.
  • the control Factor VII molecule is preferably the same Factor VII molecule having an additional cysteine at position 407, but having no heparosan attached.
  • the control being used for comparison may be a suitable Factor VII conjugated molecule as described above.
  • the conjugate of the invention preferably shows an improvement in circulating half-life, or in mean residence time when compared to a suitable control.
  • the control is preferably a suitable Factor VII polypeptide conjugated to a water soluble polymer of comparable size to the heparosan conjugate of the current invention.
  • the conjugate may not retain the level of biological activity seen in Factor VII that is not modified by the addition of heparosan.
  • the conjugate of the invention retains as much of the biological activity of unconjugated Factor VII as possible.
  • the conjugate may retain at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 60% of the biological activity of an unconjugated Factor VII control.
  • the control may be a Factor VII molecule having the same amino acid sequence as the Factor VII polypeptide in the conjugate, but lacking heparosan.
  • the conjugate may, however, show an improvement in biological activity when compared to a suitable control.
  • the biological activity here may be any biological activity of Factor VII as described herein such as clotting activity or proteolysis activity.
  • An improved biological activity when compared to a suitable control as described herein may be any measurable or statistically significant increase in a biological activity.
  • the biological activity may be any biological activity of Factor VII as described herein, such as clotting activity, proteolysis activity.
  • the increase may be, for example, an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70% or more in the relevant biological activity when compared to the same activity in a suitable control.
  • conjugates of the invention is that heparosan polymers are enzymatically biodegradable.
  • a conjugate of the invention is therefore preferably enzymatically degradable in vivo and/or in vitro.
  • An advantage of the conjugates of the invention may be that a heparosan polymer linked to Factor VII may reduce or not create inter-assay variability in aPTT-based assays.
  • the present invention provides compositions and formulations comprising conjugates of the invention.
  • the invention provides a pharmaceutical composition comprising one or more conjugates of the invention, formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents.
  • suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline.
  • suitable aqueous carriers include water, buffered water and saline.
  • other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • compositions are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly, or it may be administered by continuous or pulsatile infusion.
  • the compositions for parenteral administration comprise the Factor VII conjugate of the invention in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • the Factor VII conjugate of the invention can also be formulated into liposome preparations for delivery or targeting to the sites of injury.
  • compositions are generally described in, e.g., U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,501,728, and U.S. Pat. No. 4,975,282.
  • the compositions may be sterilised by conventional, well-known sterilisation techniques.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilised, the lyophilised preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the concentration of Factor VII conjugate in these formulations can vary widely, i.e., from less than about 0.5% by weight, usually at or at least about 1% by weight to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution and 10 mg of the Factor VII conjugate.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa. (1990).
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active agent (e.g. conjugate) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the conjugate may be used in conjunction with a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid poly[ethylene glycol], and the like), suitable mixtures thereof, vegetable oils, and combinations thereof.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid poly[ethylene glycol], and the like), suitable mixtures thereof, vegetable oils, and combinations thereof.
  • the proper fluidity of the conjugate may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions may be prepared by incorporating the conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the heparosan conjugate into a sterile carrier that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation may include vacuum drying, spray drying, spray freezing and freeze-drying that yields a powder of the active ingredient (i.e., the heparosan conjugate) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of conjugate calculated to produce the desired therapeutic effect.
  • the specification for the dosage unit forms of the presently claimed and disclosed invention(s) are dictated by and directly dependent on (a) the unique characteristics of the heparosan conjugate and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a subject.
  • compositions of the invention may comprise additional active ingredients as well as a conjugate of the invention.
  • a pharmaceutical composition may comprise additional therapeutic or prophylactic agents.
  • a pharmaceutical composition of the invention may additionally comprise one or more agents intended to reduce the symptoms of the bleeding disorder.
  • the composition may comprise one or more additional clotting factors.
  • the composition may comprise one or more other components intended to improve the condition of the patient.
  • the composition may comprise one or more analgesic, anaesthetic, immunosuppressant or anti-inflammatory agents.
  • composition may be formulated for use in a particular method or for administration by a particular route.
  • a conjugate or composition of the invention may be administered parenterally, intraperitoneally, intraspinally, intravenously, intramuscularly, intravaginally, subcutaneously, intranasally, rectally, or intracerebrally.
  • an advantageous property of the Factor VII polypeptide and heparosan polymer conjugate, of the invention is where the polymer has a polymer size around in the range of 13-65 kDa (e.g. 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa or 38-42 kDa) this may allow for an in vivo useful half-life or mean residence time while also having a suitable viscosity in liquid solution.
  • 13-65 kDa e.g. 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa or 38-42 kDa
  • a conjugate of the invention may be administered to an individual in need thereof in order to deliver Factor VII to that individual.
  • the individual may be any individual in need of Factor VII.
  • the Factor VII conjugates according to the present invention may be used to control bleeding disorders which may be caused by, for example, clotting factor deficiencies (e.g. haemophilia A and B or deficiency of coagulation factors XI or VII) or clotting factor inhibitors, or they may be used to control excessive bleeding occurring in subjects with a normally functioning blood clotting cascade (no clotting factor deficiencies or inhibitors against any of the coagulation factors).
  • the bleeding may be caused by a defective platelet function, thrombocytopenia or von Willebrand's disease. They may also be seen in subjects in whom an increased fibrinolytic activity has been induced by various stimuli.
  • the Factor VII conjugates of the invention will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter.
  • Administration as a coagulant can be by a variety of routes as described herein.
  • the dose of the Factor VII conjugates delivers from about 0.05 mg to 500 mg of the Factor VII polypeptide/day, preferably from about 1 mg to 200 mg/day, and more preferably from about 10 mg to about 175 mg/day for a 70 kg subject as loading and maintenance doses, depending on the weight of the subject and the severity of the condition.
  • a suitable dose may also be adjusted for a particular conjugate of the invention based on the properties of that conjugate, including its in vivo half-life or mean residence time and its biological activity. For example, conjugates having a longer half-life may be administered in reduced dosages and/or compositions having reduced activity compared to wild-type Factor VII may be administered in increased dosages.
  • compositions containing the Factor VII conjugates of the present invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject already suffering from a disease, such as any bleeding disorder as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as “therapeutically effective amount”.
  • amounts effective for this purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. In general, however, the effective delivery amount will range from about 0.05 mg up to about 500 mg of the Factor VII polypeptide per day for a 70 kg subject, with dosages of from about 1.0 mg to about 200 mg of the Factor VII being delivered per day being more commonly used.
  • the conjugates of the present invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threatening situations. In such cases, in view of the minimisation of extraneous substances and general lack of immunogenicity of human Factor VII polypeptide variants in humans, it may be felt desirable by the treating physician to administer a substantial excess of these Factor VII conjugate compositions.
  • compositions containing the Factor VII conjugate of the invention are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability.
  • Such an amount is defined to be a “prophylactically effective dose.”
  • the precise amounts of Factor VII polypeptide being delivered once again depend on the subject's state of health and weight, but the dose generally ranges from about 0.05 mg to about 500 mg per day for a 70-kilogram subject, more commonly from about 1.0 mg to about 200 mg per day for a 70-kilogram subject.
  • the compositions can be carried out with dose levels and patterns being selected by the treating physician.
  • the Factor VII polypeptide conjugates may be administered by continuous infusion using e.g. a portable pump system.
  • a Factor VII conjugate of the present invention such as, for example, topical application may be carried out, for example, by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods.
  • the pharmaceutical compositions should provide a quantity of Factor VII conjugate sufficient to effectively treat the subject.
  • the conjugates of the invention were analysed for purity by HPLC.
  • HPLC was also used to quantify amount of isolated conjugate based on a FVIIa reference molecule.
  • Samples were analysed either in non-reduced or reduced form.
  • a Zorbax 300SB-C3 column (4.6 ⁇ 50 mm; 3.5 um Agilent, Cat. No.: 865973-909) was used. Column was operated at 30° C. 5 ug sample was injected, and column eluted with a water (A)-acetonitrile (B) solvent system containing 0.1% trifluoroacetic acid.
  • the gradient program was as follows: 0 min (25% B); 4 min (25% B); 14 min (46% B); 35 min (52% B); 40 min (90% B); 40.1 min (25% B).
  • Reduced samples were prepared by adding 10 ul TCEP/formic acid solution (70 mM tris(2-carboxyethyl)phosphine and 10% formic acid in water) to 25 ul/30 ug FVIIa (or conjugate). Reactions were left for 10 minutes at 70° C., before analysis on HPLC (5 ul injection).
  • Maleimide functionalized heparosan polymers of defined size are prepared by an enzymatic (PmHS1) polymerization reaction using the two sugar nucleotides UDP-GlcNAc and UDP-GlcUA.
  • a priming trisaccharide (GlcUA-GlcNAc-GlcUA)NH 2 is used for initiating the reaction, and polymerization is run until depletion of sugar nucleotide building blocks.
  • the terminal amine (originating from the primer) is then functionalized with suitable reactive group, in this case a maleimide functionality designed for conjugation to free cysteines.
  • Size of heparosan polymers can be pre-determined by variation in sugar nucleotide: primer stoichiometry. The technique is described in detail in US 2010/0036001.
  • FVIIa407C was reduced as described in US 20090041744 using a glutathione based redox buffer system.
  • Non-reduced FVIIa 407C (15.5 mg) was incubated for 17 h at room temperature in a total volume of 41 ml 50 mM Hepes, 100 mM NaCl, 10 mM CaCl2, pH 7.0 containing 0.5 mM GSH, 15 uM GSSG, 25 mM p-aminobenzamidine and 3 ⁇ M Grx2.
  • the reaction mixture was then cooled on ice, and added 8.3 ml 100 mM EDTA solution while keeping pH at 7.0.
  • Single cysteine reduced FVIIa 407C was reacted with 40K HEP-maleimide (26.8 mg) in 50 mM Hepes, 100 mM NaCl, 10 mM CaCl2, pH 7.0 buffer (8.5 ml) for 22 hours at 5° C.
  • the method essentially follows the principle described by Thim, L et al. Biochemistry (1988) 27, 7785-779.
  • FVIIa 407C (8 mg) was reacted with 65k HEP-maleimide (42 mg 1:4 ratio) in 50 mM Hepes, 100 mM NaCl, 10 mM CaCl 2 , pH 7.0 buffer (8 ml) for 3 hours at room temperature.
  • the method essentially follows the principle described by Thim, L et al. Biochemistry (1988) 27, 7785-779.
  • the products with unfolded Gla-domain was collected and directly applied to a HiTrap Q FF ion-exchange column (Amersham Biosciences, GE Healthcare) equilibrated with 10 mM His, 100 mM NaCl, pH 7.5.
  • the 65k-HEP-[C]-FVIIa 407C was eluted in approximately 10 mM histidine, ⁇ 300 mM NaCl, 10 mM CaCl 2 , 0.01% Tween80, pH 6.0.
  • Yield and concentration was determined by quantifying the FVIIa light chain content against a FVIIa standard after tris(2-carboxyethyl)-phosphine reduction using reverse phase HPLC.
  • a total of 3.10 mg (38%) 65k-HEP-[C]-FVIIa 407C conjugate was obtained in a concentration of 0.57 mg/ml in 10 mM His, ⁇ 300 mM NaCl, 10 mM CaCl 2 , 0.01% Tween80, pH 6.0.
  • the pure conjugate was finally diluted to 0.4 mg/ml (8 uM) by ultrafiltration, and buffer exchange into 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0 by dialysis.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (17 mg) and 13k-HEP-maleimide (8.5 mg). 7.1 mg (41%) 13k-HEP-[C]-FVIIa407C was obtained as a 0.4 mg/ml (8 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (15.7 mg) and 27k-HEP-maleimide (11.2 mg). 6.9 mg (44%) 27k-HEP-[C]-FVIIa407C was obtained as a 0.4 mg/ml (8 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (8.3 mg) and 52k-HEP-maleimide (27 mg). 6.15 mg (71%) 52k-HEP-[C]-FVIIa407C was obtained as a 0.4 mg/ml (8 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (14.3 mg) and 60k-HEP-maleimide (68 mg). 8.60 mg (60%) 60k-HEP-[C]-FVIIa407C was obtained as a 0.4 mg/ml (8 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (20.0 mg) and 108k-HEP-maleimide (174 mg). 3.75 mg (19%) 108k-HEP-[C]-FVIIa407C was obtained as a 0.4 mg/ml (8 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • This conjugate was prepared as described in example 2, using FVIIa 407C (14.5 mg) and 157k-HEP-maleimide (180 mg). 4.93 mg (34%) 157k-HEP-[C]-FVIIa407C was obtained as a 0.3 mg/ml (6 uM) solution in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween 80, pH 6.0.
  • Step 1 Preparation of 5[(4-mercaptobutanoyl)glycylamido]neuraminic acid cytidine monophosphate
  • N-glycyl neuraminic acid cytidine monophosphate 200 mg; 0.318 mmol was dissolved in water (2 ml), and thiobutyrolactone (325 mg; 3.18 mmol) was added. The two phase solution was gently mixed for 21 h at room temperature. The reaction mixture was then diluted with water (10 ml) and applied to a reverse phase HPLC column (C18, 50 mm ⁇ 200 mm).
  • the heparosan GSC reagent was prepared by coupling GSC-SH (5′-[(4-mercaptobutanoyl)glycylamido]neuraminic acid cytidine monophosphate) with 52k-HEP-maleimide in a 1:1 molar ratio as follows: GSC-SH (0.50 mg) dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (50 ul) was added 15.80 mg of the 52k-HEP-maleimide dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (1350 ul). The clear solution was left for 2 hours at 25° C.
  • GSC-SH 5′-[(4-mercaptobutanoyl)glycylamido]neuraminic acid cytidine monophosphate
  • 52k-HEP-maleimide in a 1:1 molar ratio as follows: GSC-SH (0.50 mg) dissolved in 50 mM He
  • the excess of GSC-SH was removed by dialysis, using a Slide-A-Lyzer cassette (Thermo Scientific) with a cut-off of 10 kD.
  • the reaction mixture was dialyzed twice for 2.5 hours.
  • the recovered material was used as such in step 4 below, assuming a quantitative reaction between GSC-SH and HEP-maleimide.
  • FVIIa (28 mg) was added sialidase ( Arthrobacter ureafaciens, 200 ul, 0.3 mg/ml, 200 U/ml) in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0 (18 ml), and left for 1 hour at room temperature. The reaction mixture was then diluted with 50 mM Hepes, 150 mM NaCl, pH 7.0 (30 ml), and cooled on ice. 100 mM EDTA solution (6 ml) was added in small portions. After each addition pH was measured. pH was maintained within 5.5-9.0.
  • the EDTA treated sample was then applied to a 2 ⁇ 5 ml HiTrap Q FF ion-exchange columns (Amersham Biosciences, GE Healthcare) equilibrated with 50 mM Hepes, 150 mM NaCl, pH 7.0.
  • Sialidase was eluted with 50 mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0 (4 CV), before eluting asialo FVIIa with 50 mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0 (10 CV).
  • AsialoFVIIa was isolated in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0. Yield (24 mg) and concentration (3.0 mg/ml) was determined by quantifying the FVIIa light chain content against a FVIIa standard after tris(2-carboxyethyl)phosphine reduction using reverse phase HP
  • Step 4 Enzymatic Heparosan Conjugation Using 52k-HEP-GSC Reagent
  • Step 5 Isolation of 52k-HEP-[N]-FVIIa
  • buffer A 50 mM Hepes, 100 mM NaCl, 10 mM CaCl 2 , pH 7.4
  • buffer B 50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4
  • the products with unfolded Gla-domain was collected and directly applied to a 5 ml HiTrap Q FF ion-exchange column (Amersham Biosciences, GE Healthcare) equilibrated with a buffer containing 10 mM His, 100 mM NaCl, pH 7.5.
  • FVIIa clotting activity levels of 65K HEP-FVIIa 407C conjugates in rat plasma were estimated using a commercial FVIIa specific clotting assay; STACLOT®VIIa-rTF from Diagnostica Stago. The assay is based on the method published by J. H. Morrissey et al, Blood. 81:734-744 (1993). It measures sTF initiated FVIIa activity-dependent time to fibrin clot formation in FVII deficient plasma in the presence of phospholipids. Samples were measured on an ACL9000 coagulation instrument against FVIIa calibration curves with the same matrix as the diluted samples (like versus like). The lower limit of quantification (LLOQ) was estimated to 0.25 U/ml.
  • LLOQ lower limit of quantification
  • HEP-FVIIa conjugates were formulated in 10 mM Histidine, 100 mM NaCl, 10 mM CaCl 2 , 0.01% Tween80 80, pH 6.0.
  • Sprague Dawley rats (three to six per group) were dosed intravenously with 20 nmol/kg test compound.
  • StabyliteTM TriniLize Stabylite Tubes; Tcoag Ireland Ltd, Ireland
  • Plasma samples were analysed for FVIIa clot activity level using a commercial FVIIa specific clotting assay; STACLOT®VIIa-rTF from Diagnostica Stago and antigen concentrations in plasma were determined using LOCI technology.
  • PK-profiles (LOCI and FVIIa:clot) are shown in FIGS. 5 and 6 .
  • FIG. 7 A plot of all LOCI based mean-residence times, as obtained from the non-compartmental analysis methods is shown in FIG. 7 .
  • the conjugate is composed of a FVII polypeptide and a heparosan polymer.
  • the heparosan polymer has a mass of between 5k and 200k. In one embodiment the heparosan polymer has a polydispersity index (Mw/Mn) of less than 1.10
  • the heparosan polymer has a polydispersity index (Mw/Mn) of less than 1.07
  • the heparosan polymer has a polydispersity index (Mw/Mn) of less than 1.05
  • the FVII polypeptide is conjugated to a heparosan polymer having a size of 10 kDa ⁇ 5 kDa.
  • the FVII polypeptide is conjugated to a heparosan polymer having a size of 20 kDa ⁇ 5 kDa
  • the FVII polypeptide is conjugated to a heparosan polymer having a size of 30 kDa ⁇ 5 kDa.
  • the FVII polypeptide is conjugated to a heparosan polymer having a size of 40 kDa ⁇ 5 kDa.
  • the FVII polypeptide is conjugated to a heparosan polymer having a size of 50 kDa ⁇ 5 kDa.
  • the heparosan polymer is branched via a chemical linker.
  • said heparosan polymers each have a size equal to 20 kDa ⁇ 3 kDa.
  • said heparosan polymers each have a size equal to 30 kDa ⁇ 5 kDa.
  • the heparosan polymer is conjugated to FVII polypeptide via an N-glycan.
  • one of the two N-glycans at position 145 and 322 are removed by PNGase F treatment, and Heparosan is coupled to the remaining N-glycan.
  • the heparosan polymer is conjugated via a sialic acid moiety on FVIIa.
  • heparosan is coupled to a FVII polypeptide mutant via a single surface exposed cysteine residue.
  • a conjugate between a Factor VII polypeptide and a heparosan polymer is A conjugate between a Factor VII polypeptide and a heparosan polymer.
  • a conjugate according to embodiments 1-6 wherein said conjugate has (a) increased circulating half-life compared to the same Factor VII polypeptide which is not conjugated to a heparosan polymer; or (b) increased functional half-life compared to the same Factor VII polypeptide which is not conjugated to a heparosan polymer.
  • a conjugate according to embodiments 1-6 wherein said conjugate has (a) increased mean residence time compared to the same Factor VII polypeptide which is not conjugated to a heparosan polymer; or (b) increased functional mean residence time compared to the same Factor VII polypeptide which is not conjugated to a heparosan polymer.
  • a pharmaceutical composition comprising a conjugate according to any one of the preceding embodiments and a pharmaceutically acceptable carrier or diluent.
  • a conjugate comprising a Factor VII polypeptide and a heparosan polymer.
  • Mw/Mn polydispersity index
  • a pharmaceutical composition comprising a conjugate according to any one of the preceding embodiments and a pharmaceutically acceptable carrier or diluent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
US14/433,151 2012-10-15 2013-10-15 Factor vii conjugates Abandoned US20150259665A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/433,151 US20150259665A1 (en) 2012-10-15 2013-10-15 Factor vii conjugates

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP12188472 2012-10-15
EP12188472.0 2012-10-15
US201261715929P 2012-10-19 2012-10-19
US14/433,151 US20150259665A1 (en) 2012-10-15 2013-10-15 Factor vii conjugates
PCT/EP2013/071499 WO2014060397A1 (en) 2012-10-15 2013-10-15 Factor vii conjugates

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/071499 A-371-Of-International WO2014060397A1 (en) 2012-10-15 2013-10-15 Factor vii conjugates

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/453,188 Continuation US10179905B2 (en) 2012-10-15 2017-03-08 Factor VII conjugates

Publications (1)

Publication Number Publication Date
US20150259665A1 true US20150259665A1 (en) 2015-09-17

Family

ID=47022565

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/433,151 Abandoned US20150259665A1 (en) 2012-10-15 2013-10-15 Factor vii conjugates
US15/453,188 Expired - Fee Related US10179905B2 (en) 2012-10-15 2017-03-08 Factor VII conjugates

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/453,188 Expired - Fee Related US10179905B2 (en) 2012-10-15 2017-03-08 Factor VII conjugates

Country Status (5)

Country Link
US (2) US20150259665A1 (ja)
EP (1) EP2906247A1 (ja)
JP (1) JP2015533152A (ja)
CN (1) CN104661685A (ja)
WO (1) WO2014060397A1 (ja)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9370583B2 (en) 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
US10179905B2 (en) * 2012-10-15 2019-01-15 Novo Nordisk Health Care Ag Factor VII conjugates

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
AR099328A1 (es) * 2014-02-12 2016-07-13 Novo Nordisk As Conjugados de factor vii

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9370583B2 (en) * 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides

Family Cites Families (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4456591A (en) 1981-06-25 1984-06-26 Baxter Travenol Laboratories, Inc. Therapeutic method for activating factor VII
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
GR860984B (en) 1985-04-17 1986-08-18 Zymogenetics Inc Expression of factor vii and ix activities in mammalian cells
US4975282A (en) 1985-06-26 1990-12-04 The Liposome Company, Inc. Multilamellar liposomes having improved trapping efficiencies
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5580560A (en) 1989-11-13 1996-12-03 Novo Nordisk A/S Modified factor VII/VIIa
EP0575464B1 (en) 1991-02-28 2004-08-11 Zymogenetics, Inc. Modified factor vii
US5997864A (en) 1995-06-07 1999-12-07 Novo Nordisk A/S Modified factor VII
US8088604B2 (en) 1998-04-02 2012-01-03 The Board Of Regents Of The University Of Oklahoma Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
ES2325877T3 (es) 2000-02-11 2009-09-23 Bayer Healthcare Llc Moleculas de tipo factor vii o viia.
AU2001254624A1 (en) 2000-05-03 2001-11-12 Novo-Nordisk A/S Human coagulation factor vii variants
JP4361728B2 (ja) 2000-09-13 2009-11-11 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト ヒト凝固因子ｖｉｉ変異型
US7235638B2 (en) * 2001-03-22 2007-06-26 Novo Nordisk Healthcare A/G Coagulation factor VII derivatives
CA2441580A1 (en) * 2001-03-22 2002-10-03 Novo Nordisk Health Care Ag Coagulation factor vii derivatives
ES2516041T3 (es) 2001-10-10 2014-10-30 Ratiopharm Gmbh Remodelación y glicoconjugación de la hormona del crecimiento humano (hGH)
JP2007501811A (ja) 2003-08-08 2007-02-01 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト 治療的な関心対象のタンパク質に対する遅延分子の選択的な化学物質接合のためのガラクトースオキシダーゼの使用。
US20060040856A1 (en) 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
WO2005075635A2 (en) 2004-02-03 2005-08-18 Novo Nordisk Health Care Ag Coagulation factor vii/viia variants lacking a functional lipid membrane binding domain
JP5216580B2 (ja) 2005-05-25 2013-06-19 ノヴォ ノルディスク アー／エス グリコペグ化第ｉｘ因子
WO2006134174A2 (en) * 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Dimeric and multimeric fviia compound
JP2009515508A (ja) 2005-08-19 2009-04-16 ネオス テクノロジーズ インコーポレイテッド グリコｐｅｇ化因子ｖｉｉおよび因子ｖｉｉａ
JP5690047B2 (ja) 2005-09-14 2015-03-25 ノボ ノルディスク ヘルス ケア アーゲー ヒト凝固第ｖｉｉ因子ポリペプチド
US20100056428A1 (en) 2006-09-01 2010-03-04 Novo Nordisk Health Care Ag Modified proteins
ES2521490T3 (es) * 2006-12-15 2014-11-12 Baxter International Inc. Conjugado de factor VIIa - ácido (poli)siálico con una vida media in vivo prolongada.
WO2008127702A2 (en) 2007-04-13 2008-10-23 Catalyst Biosciences, Inc. Modified factor vii polypetides and uses thereof
EP2235042A1 (en) * 2008-01-23 2010-10-06 Novo Nordisk Health Care AG New blood coagulation factor inhibitors
US9687559B2 (en) 2008-03-19 2017-06-27 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
TWI538916B (zh) 2008-04-11 2016-06-21 介控生化科技公司 經修飾的因子vii多肽和其用途
JP5642065B2 (ja) * 2008-05-23 2014-12-17 ノボ ノルディスク ヘルス ケア アーゲー ペグ化されたｇｌａ−ドメイン含有タンパク質を含む低粘度組成物
WO2010030342A2 (en) * 2008-09-09 2010-03-18 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US20120093840A1 (en) 2009-04-06 2012-04-19 Novo Nordisk A/S Targeted delivery of factor viii proteins to platelets
ES2590679T3 (es) 2009-07-27 2016-11-23 Lipoxen Technologies Limited Glicopolisialilación de proteínas diferentes a proteínas de coagulación de la sangre
JP2013517782A (ja) 2010-01-28 2013-05-20 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー 因子ｖｉｉ融合ポリペプチド
WO2011101277A1 (en) 2010-02-16 2011-08-25 Novo Nordisk A/S Conjugated proteins
WO2011101242A1 (en) 2010-02-16 2011-08-25 Novo Nordisk A/S Factor viii molecules with reduced vwf binding
EP2593130A2 (en) 2010-07-15 2013-05-22 Novo Nordisk A/S Stabilized factor viii variants
EP2616486B1 (en) 2010-09-15 2019-01-02 Stichting Sanquin Bloedvoorziening Factor viii variants having a decreased cellular uptake
JP2015533152A (ja) * 2012-10-15 2015-11-19 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー 第ｖｉｉ因子コンジュゲート
JP2015532307A (ja) 2012-10-15 2015-11-09 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー 凝固因子ｖｉｉポリペプチド
WO2014140103A2 (en) 2013-03-12 2014-09-18 Novo Nordisk A/S Thrombin sensitive coagulation factor x molecules
AR099328A1 (es) * 2014-02-12 2016-07-13 Novo Nordisk As Conjugados de factor vii

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9370583B2 (en) * 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
US9371370B2 (en) * 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10179905B2 (en) * 2012-10-15 2019-01-15 Novo Nordisk Health Care Ag Factor VII conjugates
US9370583B2 (en) 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
US9371370B2 (en) 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides

Also Published As

Publication number Publication date
CN104661685A (zh) 2015-05-27
US20170355974A1 (en) 2017-12-14
US10179905B2 (en) 2019-01-15
JP2015533152A (ja) 2015-11-19
EP2906247A1 (en) 2015-08-19
WO2014060397A1 (en) 2014-04-24

Similar Documents

Publication Publication Date Title
US11203749B2 (en) Factor VII polypeptides that are modified and uses thereof
EP1517710B1 (en) Pegylated factor vii glycoforms
US8053410B2 (en) Pegylated factor VII glycoforms
ES2344887T3 (es) Glicoformas de factor vii.
JP5558106B2 (ja) 凝固第ｖｉｉ因子関連ポリペプチドの皮下投与
US10179905B2 (en) Factor VII conjugates
ES2338425T3 (es) Variantes de dominio de gla del factor vii o viia.
BRPI0720282B1 (pt) Construção proteica, composição farmacêutica, e, kit
JP2005530683A (ja) 第ｖｉｉ因子ポリペプチドの液体組成物
JP2011520999A (ja) 高濃度の芳香族保存料を有するペグ官能化セリンプロテアーゼの製剤
US20150225711A1 (en) Factor VII Conjugates
JP2013121967A (ja) 第ｖｉｉ因子ポリペプチドの液体組成物
EP2014299A1 (en) Subcutaneous administration of coagulation factor VII
WO2008023052A1 (en) Combination of a fvii and a thrombin-activable factor x

Legal Events

Date Code Title Description
AS Assignment

Owner name: NOVO NORDISK HEALTHCARE AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BEHRENS, CARSTEN;OESTERGAARD, HENRIK;STENNICKE, HENNING RALF;SIGNING DATES FROM 20150709 TO 20151020;REEL/FRAME:036846/0068

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION