WO2006071801A2 - Polymer-von willebrand factor-conjugates - Google Patents
Polymer-von willebrand factor-conjugates Download PDFInfo
- Publication number
- WO2006071801A2 WO2006071801A2 PCT/US2005/046879 US2005046879W WO2006071801A2 WO 2006071801 A2 WO2006071801 A2 WO 2006071801A2 US 2005046879 W US2005046879 W US 2005046879W WO 2006071801 A2 WO2006071801 A2 WO 2006071801A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- vwf
- construct
- molecule
- fviii
- peg
- Prior art date
Links
- XAZKFISIRYLAEE-UHFFFAOYSA-N CC1CC(C)CC1 Chemical compound CC1CC(C)CC1 XAZKFISIRYLAEE-UHFFFAOYSA-N 0.000 description 2
- QRMPKOFEUHIBNM-UHFFFAOYSA-N CC1CCC(C)CC1 Chemical compound CC1CCC(C)CC1 QRMPKOFEUHIBNM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- 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
-
- 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
- A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
Definitions
- the present invention relates to a proteinaceous construct comprising plasmatic and/or recombinant von Willebrand factor (VWF), said VWF being bound to at least one physiologically acceptable polymer molecule, as well as to a complex between said proteinaceous construct and at least one factor VIII (FVIII) protein. Further the present invention relates to methods for prolonging the in vivo half-life of VWF or FVIII in the blood of a mammal having a bleeding disorder associated with functional defects or deficiencies of at least one of FVIII or VWF.
- VWF von Willebrand factor
- FVIII factor VIII
- VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741- residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett.
- VWF Upon secretion into plasma VWF circulates in the form of various species with different molecular sizes. These VWF molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. VWF can be usually found in plasma as one dimer up to multimers consisting of 50 - 100 dimers (Ruggeri et al. Thromb. Haemost. 82: 576-584, 1999). The in vivo half-life of human VWF in the human circulation is approximately 12 to 20 hours.
- VWD von Willebrand's disease
- FVIII FVIII
- VWD von Willebrand's disease
- a method for purifying proteins that bind to VWF by using a rVWF immobilized on a carrier is disclosed in WO 98/25969.
- the pharmaceutical use of plasma derived and recombinant VWF pro-peptides (pro- VWF) for treating blood coagulation disorders is described in EP 0 977 584.
- US 6,037,452 describes the binding of FVIII and factor IX (FIX) to a poly(alkylene oxide) through a linker or a coupling agent.
- FIX factor IX
- EP 0 774 261 it is shown that the use of recombinant VWF having a prolonged biological in vivo half-life stabilizes FVIII in the blood of a mammal and induces the production of endogenous FVIII. Nevertheless, there exists a need for patients having VWF- or FVIII-based bleeding disorders to further increase the in vivo half-life of VWF and FVIII.
- VWF is known to stabilize FVIII in vivo and, thus, plays a crucial role to regulate plasma levels of FVIII and as a consequence is a central factor to control primary and secondary hemostasis. It is also known that after application of therapeutic products containing VWF an increase in endogenous FVMI:C to 1 to 3 units per ml in 24 hours can be observed demonstrating the in vivo stabilizing effect of VWF on FVIII.
- the present invention provides a novel system for prolonging the in vivo half-life of VWF and/or of FVIII in the blood of a mammal. It is a further object of the present invention to provide methods for the improved treatment of bleeding disorders associated with functional defects of or deficiencies of one or both of FVIII and VWF.
- the present invention relates to a proteinaceous construct comprising plasmatic and/or recombinant von Willebrand factor (VWF) or biologically active derivatives thereof, said VWF or said biologically active derivatives thereof being bound to one or more physiologically acceptable polymer molecules, wherein the in vivo half-life of the proteinaceous construct is prolonged in the blood of a mammal, particularly a human. Further, the present invention relates to a complex between said proteinaceous construct and at least one factor FVIII (FVIII) protein or a biologically active derivative thereof, wherein the in vivo half-life of said FVIII protein or said biologically active derivative thereof is also prolonged in the blood of a mammal.
- VWF von Willebrand factor
- compositions containing said proteinaceous construct or said complex as well as methods for prolonging the in vivo half-life of VWF or FVIII in the blood of a mammal having a bleeding disorder associated with functional defects of or deficiencies of at least one of FVIII and VWF using said proteinaceous construct or said complex are provided according to the present invention.
- Methods for making the proteinaceous construct are also provided.
- One aspect of the present invention relates to a proteinaceous construct (in the following also designated as "polymer-VWF-conjugate”) comprising plasmatic and/or recombinant VWF or biologically active derivatives thereof, said VWF or said biologically active derivatives thereof being bound to one or more types of physiologically acceptable polymer molecules, wherein the in vivo half-life of said VWF or said biologically active derivatives thereof is prolonged in the blood of a mammal.
- polymer-VWF-conjugate comprising plasmatic and/or recombinant VWF or biologically active derivatives thereof, said VWF or said biologically active derivatives thereof being bound to one or more types of physiologically acceptable polymer molecules, wherein the in vivo half-life of said VWF or said biologically active derivatives thereof is prolonged in the blood of a mammal.
- One form stably carries the polymer conjugated to VWF and will be eliminated as an integral molecule over time after application to a mammal.
- the other form is characterized by reversibility of the polymer conjugated to VWF.
- the polymer molecules bound to VWF will be gradually released from VWF and non- conjugated VWF will become available as the pharmacologically functional agent. The release characteristics will depend on the conjugation chemistry and on the composition and the structure of the polymer molecules bound to VWF.
- the polymer-VWF conjugates are useful either alone for treatment of VWD, or combined with FVIII to stabilize FVIII for increased half-life or both.
- the conjugate may take one of two forms.
- the first form is where the polymer is releasably bound to the VWF. In this manner the VWF becomes active as the polymer is released or degrades.
- the second form is where the polymer concentration bound to the VWF is such so as to not interfere with the VWF activity.
- the conjugate is prepared to bind with and stabilize FVIII, the degree or level of polymer bound to the VWF is provided so as to not interfere with the binding region of the VWF.
- the degree of polymer conjugation can also be controlled or modified to maintain VWF activity while also maintaining the ability of the VWF to bind FVIII.
- the polymer-VWF conjugates provide a therapeutically active VWF, while also stabilizing FVIII for increased half-life.
- the VWF and FVIII molecules useful for the present invention include the full length protein, precursors of the protein, subunits or fragments of the protein, and functional derivatives thereof. Reference to VWF and FVIII or FVIII is meant to include all potential forms of such proteins.
- biologically active derivative includes any derivative of a molecule having substantially the same functional and/or biological properties of said molecule, such as binding properties, and/or the same structural basis, such as a peptidic backbone or a basic polymeric unit.
- the VWF useful for the present invention includes all potential forms, including the monomeric and multimeric forms.
- One particularly useful form of VWF are homo-multimers of at least two VWFs.
- the VWF proteins may be either a biologically active derivative, or when to be used solely as a stabilizer for FVIII the VWF may be of a form not biologically active.
- the present invention encompasses different forms of VWF to be used in combination.
- a composition useful for the present invention may include different multimers, different derivatives and both biologically active derivatives and derivatives not biologically active.
- VWF serves as a bridge between platelets and specific components of the extracellular matrix, such as collagen.
- the biological activity of VWF in this process can be measured by two different in vitro assays (Turecek et al., Semin. Thromb. Hemost. 28: 149-160, 2002).
- the ristocetin cofactor assay is based on the agglutination of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF.
- the degree of platelet agglutination depends on the VWF concentration and can be measured by the turbidimetric method, e.g. by use of an aggregometer (Weiss et al., J. Clin. Invest. 52: 2708-2716, 1973; Macfarlane et al., Thromb. Diath. Haemorrh.
- the second method is the collagen binding assay, which is based on ELISA technology (Brown et Bosak, Thromb. Res. 43: 303-311, 1986; Favaloro, Thromb. Haemost. 83: 127-135, 2000).
- a microtiter plate is coated with type I or III collagen.
- the VWF is bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody.
- the last step is the substrate reaction, which can be photometrically monitored with an ELISA reader.
- plasma-derived VWF includes all forms of the protein found in blood including the mature VWF obtained from a mammal having the property of in wVo-stabilizing, e.g. binding, of at least one FVIII molecule.
- the invention is not limited to the mature VWF.
- One, biologically active derivative of said pVWF is pro-VWF which contains the pro-peptide.
- pre-pro-VWF the precursor VWF molecule synthesized by endothelial cells and megakaryocytes
- pro-VWF the VWF propeptide
- mature pdVWF obtained upon cleavage of the signal peptide and pro-peptide, respectively of the precursor molecule.
- biologically active derivatives of pdVWF include pro-drugs which are processed or converted into the biologically active form, or is biologically active as such, truncated forms, forms having deletions, forms having substitutions, forms having additions other than pro- forms, fragments of the mature form, chimeric forms, and forms having post- translational modifications as compared to the natural form.
- PdVWF useful for the present invention also includes those forms not biologically active. This may be accomplished by modification of the mature VWF or other naturally occurring forms found in blood.
- the source for VWF useful for the invention is mammalian, including porcine and human versions. [016]
- recombinant VWF (rVWF) includes VWF obtained via recombinant DNA technology.
- One form of useful rVWF has at least the property of in wVo-stabilizing, e.g. binding, of at least one FVIII molecule and having optionally a glycosylation pattern which is pharmacologically acceptable.
- wVo-stabilizing e.g. binding
- FVIII molecule e.g. binding
- GVF fragment from VaI 449 to Asn 730 including the glycoprotein Ib-binding domain and binding sites for collagen and heparin (Pietu et al., Biochem. Biophys. Res. Commun. 164: 1339-1347, 1989).
- the determination of stabilizing at least one FVIII molecule can be carried out in VWF- deficient mammals according to methods known in the state in the art. For example, as described in Example 8 below, VWF-deficient mice are treated intravenously via the tail vein with VWF, and the level of FVIII activity in their plasma is followed over time. The level of FVIII activity can be measured by, for instance, a chromogenic assay such as published in the European Pharmacopoeia (Ph. Eur., 3 rd Ed. 1997:2.7.4).
- FVIII FVIII
- FIXa activated factor IX
- FX factor X
- FIXa activated factor IX
- FX factor X
- a buffer containing calcium FVIII
- FXa activated factor IX
- FXa phospholipids
- FXa factor X
- FXa phospholipids
- FXa factor X
- This complex activates FX to FXa, which in turn cleaves a chromogenic substrate (e.g. (AcOH*CH 3 OCO-D-CHA-Gly-Arg-pNA).
- pNA para-nitroaniline
- the rVWF of the present invention may be produced by any method known in the art.
- One specific example is disclosed in WO86/06096 published on October 23, 1986 and U.S. Patent Application No. 07/559,509, filed on July 23, 1990, in the name of Ginsburg et al., which is incorporated herein by reference with respect to the methods of producing recombinant VWF.
- This may include any method known in the art for (i) the production of recombinant DNA by genetic engineering, e.g. via reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into procaryotic or eucaryotic cells by transfection, e.g.
- the rVWF can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable VWF molecule.
- eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2.
- reagents or conditions used for producing or isolating VWF according to the present invention and any system known in the art or commercially available can be employed.
- rVWF is obtained by methods as described in the state of the art.
- vectors can be used for the preparation of the rVWF and can be selected from eukaryotic and prokaryotic expression vectors.
- vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.
- vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1 , GAP, GAL1, AUG1, etc; (ii) for expression in insect cells, vectors such as pMT, pAc ⁇ , plB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OplE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1 , UbC, RSV, ADV,
- the FVIII useful with the present invention includes those forms, which are biologically active including the full length FVIII and any derivative capability of acting as a cofactor in the activation of coagulation FIX and the capability of forming a complex with VWF.
- the FVIII used according to the present invention may be a plasma-derived FVIII (pdFVIII) or a recombinant FVIII (rFVIII) or biologically active derivatives thereof.
- the pdFVIII and the rFVIII may be produced by any method known in the art.
- PdFVIII may be purified by any suitable means.
- One useful method is described in U.S. Patent No. 5,470,954, which is incorporated herein by reference.
- RFVIII proteins may be prepared by any suitable means.
- examples of such rFVIII include RECOMBINATE and ADVATE both manufactured and sold by Baxter Healthcare Corporation; REFACTO, a B-domain deleted form of FVIII manufactured and sold by Wyeth Corporation; and KOGENATE, manufactured and sold by Bayer Corporation. Methods and examples of rFVIII are described in U.S. Patent Nos. 4,757,006; 4,965,199; and 5,618,788, all of which are incorporated herein by reference.
- physiologically acceptable polymer includes polymers which are soluble in an aqueous solution or suspension and have no negative impact, such as side effects, to mammals upon administration of the polymer-VWF- conjugate in a pharmaceutically effective amount.
- physiologically acceptable polymer used according to the present invention.
- the polymers are typically characterized as having preferably from 2 to about 300 repeating units. Examples of such polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (PEG), poly (propylene glycol)
- PPG copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), polyvinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and combinations of any of the foregoing.
- the physiologically acceptable polymer is not limited to a particular structure and can be linear (e.g. alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g. forked PEG or PEG attached to a polyol core), dendritic, or with degradable linkages.
- the internal structure of the polymer can be organized in any number of different patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
- These polymers also include poly(alkylene oxide) polymers, poly(maleic acid), poly(DL-alanine), such as carboxymethylcellulose, dextran, hyaluronic acid and chitin, and poly(meth)acrylates.
- the physiologically acceptable polymer is PEG and derivatives thereof.
- the PEG side chain can be linear, branched, forked or can consist of multiple arms.
- a particularly useful PEG has a molecular weight in the range of 3,000 - 20,000.
- Other useful PEG molecules are disclosed in WO 03/040211; US 6,566,506; US 6,864,350; and US 6,455,639, for example.
- the physiologically acceptable polymer is polysialic acid (PSA) and derivatives thereof.
- PSA can be bound to VWF by the method described in US 4,356,170, which is herein incorporated by reference.
- the polysaccharide compound may be naturally occurring polysaccharide, a derivative of a naturally occurring polysaccharide, or a naturally occurring polysaccharide derivative.
- the polysaccharide portion of the compound has more than 5, typically at least 10, and in another embodiment at least 20 to 50 sialic acid residues in the polymer chain.
- Readily available polysaccharide compounds may have up to 500 saccharide residues in total, but usually have fewer than 300 residues in the polymer chain. Generally, all of the saccharide residues in the compound are sialic acid residues.
- the saccharide unit may contain other functional groups, such as, amine, hydroxyl or sulphate groups, or combinations thereof. These groups may be present on naturally occurring saccharide compounds, or introduced into derivative polysaccharide compounds.
- Polysaccharide compounds of particular use for the invention are those produced by bacteria. Some of these naturally occurring polysaccharides are known as glycolipids. It is particularly advantageous if the polysaccharide compounds are substantially free of terminal galactose units, which tend to be recognized by galactose receptors of hepatocytes and Kupffer cells.
- the VWF multimers may be covalently linked to the polysaccharide compounds by any of various techniques known to those of skill in the art. Examples include linkage through the peptide bond between a carboxyl group on one of either the vWF or polysaccharide and an amine group of the other, or an ester linkage be-
- the polymer can also be coupled to the
- the polymer molecule can be coupled to the VWF by affinity interaction.
- the VWF can be biotinylated and avidin or strepavidin conjugated polymers can be bound to the VWF.
- polyclonal or monoclonal anti-VWF antibodies as well as fragments thereof can be bound to a polymer and then this complex can be bound to
- Polymers can be bound to the VWF also by enzymatical methods such as, for example, the transfer of saccharides with polyglycosyltransferase as taught in US 6,379,933 or glycopegylation as taught in US 2004 0132640 A1, all of which teachings are incorporated herein by reference.
- Another approach is the binding of polymers to the VWF on the basis of their biological function like the binding of PEGylated collagens or collagen fragments to the A1 and A3 domains of the VWF.
- collagens from type I and III e.g. from human placenta, showing a strong interaction with the VWF can be used.
- the binding of the polymers may be stable or reversible after in vivo application of the proteinaceous construct.
- PEGylated VWF includes VWF which is bound to one or more PEGs
- PEGylation includes the process of binding one or more PEGs to VWF. Suitable methods of PEGylation are disclosed in U.S. Patent Nos. 5,122,614 and 5,539,063, all of which PEGylation methods are incorporated by reference.
- the physiologically acceptable polymer is PEG or a PEG derivative, which is covalently linked to VWF by any strategy and method known in the art.
- the most common modification strategies are the binding of at least one polymer molecule via amino groups of lysine residues, the binding of at least one polymer molecule via carbohydrate side chains, the binding of at least one polymer molecule via sulfhydryl groups, the binding of at least one polymer molecule via carboxyl groups of aspartic acids and glutamic acids as well as the binding of at least one polymer molecule of hydroxyl groups and the binding of at least one polymer molecule of the N-terminus.
- the binding of at least one polymer molecule to VWF can be performed by covalently coupling said polymer molecule to the amino groups of the lysine side chains of VWF.
- Human VWF contains free 108 lysine residues with NH2 groups in the side chains, which are susceptible to binding of at least one polymer molecule. Examples of lysine residues of VWF to which the polymer molecule may be covalently linked according to the present invention are shown in Fig. 1A.
- Suitable PEG derivatives which may be covalently linked to the lysine residues of VWF are, for example, polyethylene glycols with an active N-hydroxysuccinimide ester (NHS) such as succinimidyl succinate, succinimidyl glutarate or succinimidyl propionate, which react with the lysine residues under mild conditions by forming an amide bond.
- NHS N-hydroxysuccinimide ester
- activated PEG are those with an active carbonate such as succinimidyl carbonate (SC-PEG) and benzotriazole carbonate (BTC-PEG) (see page 463 of Roberts et al., Advanced Drug Delivery Reviews 54: 459-476, 2002).
- SC-PEG and BTC-PEG react preferentially with lysine residues to form a carbamate linkage, but are also known to react with histidine and tyrosine residues.
- Alternative methods are the binding of at least one polymer molecule with PEG carbonates forming urethane bonds or with aldehydes or ketones forming secondary amines, e.g. after reduction with sodium cyano borohydride.
- PEG acylating reagents which produce urethane linked proteins include p-nitrophenyl carbonate, trichlorophenyl carbonate, and carbonylimidazole.
- reagents are prepared by reacting chloroformates or carbonylimidazole with the terminal hydroxyl group on monomethoxyPEG (mPEG) (see p. 464 of Roberts et al., above).
- mPEG monomethoxyPEG
- Another example relates to so-called "second generation" PEGylation chemistry whereby mPEG-propionaldehyde, under acidic conditions, is selective for the N-terminal a-amine (see page 464 of Roberts et al., above).
- Releasable PEG reagents such as PEG maleic anhydride, mPEG phenyl ether succinimidyl carbonates, and mPEG benzamide succinimidyl carbonates, may be used to produce a conjugate which, under physiological conditions, will release the therapeutic protein with "no tag", as described on page 469 of Roberts et al., above.
- VWF can be also bound to at least one polymer molecule via its carbohydrate residues.
- This can be carried out by e.g. mild oxidation of the carbohydrate chains, such as with NaIO 4 , forming an aldehyde function and subsequent coupling to a PEG, such as PEG-hydrazide.
- a PEG such as PEG-hydrazide.
- This figure shows an example of the asparagine N-linked linked GIcNAc and the threonine- or serine O-linked GaINAc residues, which can be oxidized and subsequently bound by at least one polymer molecule. Due to the fact that the carbohydrates of the VWF are clustered in certain domains, the VWF might be site directed bound by at least one polymer molecule by use of this modification procedure. The binding of polymer to the carbohydrate residue is particularly advantageous when the therapeutically active form of the VWF is desired. This can be enhanced by selective reaction of the polymer to either the N-linked or O-linked residues through known methods.
- glucose oxidase can be used to oxidate carbohydrate residues on VWF to generate multiple reactive aldehyde groups, which can be reacted with PEG-hydrazide to produce a hydrazone linkage or with PEG-amine to produce a reversible Schiff base (see page 467 of Roberts et al., above).
- an N-terminal serine or threonine of VWF can be used for site-specific conjugation by converting it to a glyoxylyl derivative by periodate oxidation (see page 467 of Roberts et al., above).
- Another embodiment of the present invention is the binding of at least one polymer molecule to VWF via sulfhydryl groups.
- Human VWF has 177 free SH - groups, which can be modified, for example, by PEG maleimide forming a stable sulfide.
- PEGylation of cysteine residues may also be carried out using, for instance, PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide (see page 466 of Roberts et al., above).
- the VWF molecule has several binding sites to specific receptors or ligands (Girma et al., Thromb Haemost. 74:156-60, 1995).
- One important binding site is the FVIII binding domain, which is located at the N-terminus of the mature subunit (amino acids 1 - 272). This epitope can be protected by incubation with free FVIII and formation of a FVIII/VWF complex. Subsequently the complex is chemically modified (e.g. PEGylated or polysialylated) and the polymer-conjugated i VWF with free FVIII binding site is separated from the FVIII (e.g.
- the VWF can be bound to an affinity resin with immobilized FVIII. Subsequently the VWF is chemically conjugated with a polymer (e.g. a polyethylene glycol or polysialic acid derivative) and eluted from this matrix (e.g. under high salt conditions such as 0.3 M CaCI2 or 2 M NaCI) in a batch mode or by use of a chromatography column.
- a polymer e.g. a polyethylene glycol or polysialic acid derivative
- eluted from this matrix e.g. under high salt conditions such as 0.3 M CaCI2 or 2 M NaCI
- the FVIII binding epitope of VWF is nearly identical with the heparin-binding site.
- the FVIII binding site can be blocked and protected by binding of VWF to heparin or to an affinity resin with immobilized heparin during the chemical modification procedure.
- Factor Vlll-binding-site-protecting agent refers to any agent which binds to the FVIII binding domain or epitope on the VWF molecule.
- a Factor Vlll-binding-site-protecting agent may be selected from Factor VIII, derivatives of FVIII, heparin, and derivatives of heparin.
- the present invention is directed to increasing in vivo half-life of VWF or biologically active derivatives thereof as compared to the in vivo half-life of VWF not linked to at least one physiologically acceptable polymer molecule.
- the in vivo half-life of VWF is prolonged for at least a factor of two, while in another embodiment the in vivo half-life is increased for at least a factor of three.
- the in vivo half-life is increased by a factor of five by binding of at least one physiologically acceptable polymer molecule.
- the increase or prolonging of VWF half-life can be assessed by measuring the pharmacokinetics of VWF in FVIII deficient mice, as described in
- VWF premixed with FVIII via the tail vein, and VWF antigen levels are measured in plasma samples at various time points.
- VWF antigen, as well as FVIII antigen, can be measured via ELISA assay.
- a further aspect of the present invention relates to a complex formed between at least one polymer-VWF-conjugate and at least one FVIII molecule, wherein the in vivo half-life of the FVIII is prolonged in the blood of a mammal by the polymer-VWF- conjugate.
- the binding of the polymer-VWF-conjugate with FVIII prolongs or increases the in vivo half-life of said FVIII as compared to the in vivo half-life of a FVIII forming a complex with VWF not linked to at least one physiologically acceptable polymer molecule.
- the in vivo half-life of FVIII is prolonged for at least a factor of 1.5, in another embodiment for at least a factor of two, in another embodiment for at least a factor of three, and in a further embodiment for at least a factor of five.
- the polymer-VWF-conjugates of the present invention can be used for the treatment of hemophilia A and/or VWD or subtypes of these two diseases.
- VWD the use of polymer-VWF-conjugates will be similar to current substitution therapy with VWF containing concentrates by either regular or irregular treatment, also referred to as prophylaxis protocols or on demand treatment respectively.
- Polymer-VWF-conjugates can also be used as an adjuvant treatment of hemophilia A prophylaxis.
- polymer-VWF- conjugates will be administered in time intervals and independently FVIII concentrates, either plasma-derived or recombinant, the same which are currently used for regular treatment of hemophilia A, will be given as usual, however, with prolonged treatment intervals due to the half-life prolonging capabilities of polymer- VWF-conjugates.
- a polymer- VWF-conjugate will be administered together with FVIII or in the form of a complex with FVIII to patients with hemophilia A or VWD.
- arrest of internal or external bleedings needs to be achieved immediately by raising the otherwise low plasma levels of either VWF and/or FVIII to therapeutically effective levels.
- the polymer-VWF-conjugates can also be used for immunotolerance therapy to eradicate inhibitory antibodies, which have developed against FVIII, a clinical situation also known as inhibitor hemophilia, or against VWF.
- FIX cofactor activity For the determination of FIX cofactor activity a FVIII or FVIIIa sample (FVIII completely activated with thrombin) is added to a prepared mixture of FIXa, FX, phospholipid and CaCk. This reaction mix is incubated at 37 0 C to allow complex formation and subsequent FXa generation. Subsamples are drawn at intervals up to 20 minutes and added to a chromogenic substrate, which is selectively split by FXa. After 15 minutes of incubation, the reaction is terminated by the addition of acetic acid. The absorbance (A405) values, which are proportional to the FXa concentrations, are measured in an ELISA reader and plotted against the incubation time in the reaction mix.
- a further aspect of the present invention is the provision of a method for prolonging the in vivo half-life of FVIII in the blood of a mammal having a bleeding disorder associated with functional defects of or deficiencies of at least one of FVIII and VWF, comprising the steps of: a) providing at least one proteinaceous construct as defined above; b) providing at least one FVIII as defined above; and c) forming a complex between said proteinaceous construct and said
- the complex of step (c) is formed "extracorporeal" (i.e. outside of the body of a mammal) by e.g. mixing the proteinaceous construct and said FVIII, and then administering the thus formed complex in an effective amount to the mammal having said bleeding disorder.
- the complex of step (c) is formed intracorporeal (i.e. inside of the body of a mammal) between the proteinaceous construct and endogenous FVIII present in the blood of a mammal having said bleeding disorder upon administering the proteinaceous construct in an effective amount to said mammal.
- step (c) is formed "intracorporeal" between the proteinaceous construct and exogenous FVIII present in the blood of a mammal having said bleeding disorder upon administering the proteinaceous construct in an effective amount to said mammal.
- Exogenous FVlII may be administered simultaneously in an effective amount with said proteinaceous construct or sequentially, i.e. before or after administering said proteinaceous construct.
- endogenous FVIII includes FVIII which originates from said mammal. It also includes FVIII transcribed from a transgene or any other foreign DNA present in said mammal.
- exogenous FVIII includes FVIII which does not originate from said mammal including pdFVIII and rFVIII as outlined above, as well as pdFVIII which is re-administered to the mammal from which it was isolated after forming the above-defined complex, and rFVIII which is administered to the mammal whose DNA has been used in the production of said rFVIII.
- an effective amount includes a dose suitable for treating a mammal having a bleeding disorder as outlined above; for example, for humans preferably in a range from 5 to 1 ,000 IU per infusion and more preferably in a range from 10 to 250 IU per infusion.
- the route of administration does not exhibit particular limitations, and in one embodiment the proteinaceous construct or the complex of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.
- the present invention is also directed at treating bleeding disorders associated with functional defects of or deficiencies of at least one of FVIII and VWF as used herein, including bleeding disorders wherein the cause of the bleeding disorder may be selected from the group consisting of a shortened in vivo half-life of
- FVIII and/or VWF altered binding properties of FVIII and/or VWF, genetic defects of
- the bleeding disorder is selected from the group consisting of hemophilia A, VWD, or other diseases associated with an impaired function of VWF or impaired interaction of VWF with other molecules.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising an effective amount of a proteinaceous construct as defined above or an effective amount of a complex as defined above.
- the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient.
- the pharmaceutical composition can be used for treating the above- defined bleeding disorders.
- the pharmaceutical composition of the invention may be a solution or a lyophilized product.
- Figure 1A and 1B show the schematic structure of VWF /with examples for target sites for conjugation.
- the grey dots in Fig. 1A indicate the lysine residues of VWF which can be bound by at least one polymer molecule
- Fig. 1B shows the carbohydrate residues in VWF which can be bound by at least one polymer molecule.
- Figure 2 shows the pharmacokinetics of a polymer-VWF-conjugate compared to non-conjugated VWF in mice with VWD.
- Figure 3 shows the pharmacokinetics of a polymer-VWF-conjugate compared to non-conjugated VWF in hemophilic mice.
- Figure 4 shows the pharmacokinetics of FVIII in VWD mice treated with rVWF or
- Figure 5 shows the recovery of rFVIII in hemophilic mice after application of rFVIII complexed with polymer-VWF-conjugate and rFVIII complexed with VWF.
- Figure 6 shows the recovery of VWF in hemophilic mice after application of rFVIII complexed with polymer-VWF-conjugate and rFVIII complexed with VWF.
- Figure 7 shows the pharmacokinetics of rFVIII and PEG-rVWF in FVIII x VWF double knockout mice.
- Figure 8 shows the pharmacokinetics of rFVIII and PEG-rVWF in crossbreed-mice.
- Figure 9 shows VWF:Ag increase in mouse plasma (lysine and carbohydrate coupling).
- Figure 10 shows a biomolecular interaction study (left diagram) and comparison of binding capacity of FVIII (right diagram) of PEG-conjugated rVWF compared to non- conjugated rVWF.
- Figure 11 shows the mass increase of VWF after polymer conjugation measured by
- Figure 12 shows the mass increase of VWF after polymer conjugation measured by agarose electrophoresis to analyze VWF multimers.
- Figure 13 shows the pharmacokinetics of rFVIII in FVIII-K.O.-mice after application of rFVIII complexed with polymer-VWF-conjugate (rVWF with PEGylated lysine residues) and rFVIII complexed with rVWF.
- Figure 14 shows the pharmacokinetics of rVWF in FVIII-K.O.-mice after application of rFVIII complexed with polymer-VWF-conjugate (rVWF with PEGylated lysine residues) and rFVIII complexed with rVWF.
- Figure 15 shows the determination of the FVIII binding capacity of PEG-conjugated rVWF compared to non-conjugated rVWF using a combined ELISA and chromogenic assay (ECA).
- Figure 16 shows the determination of the FVIII binding capacity of PEG-conjugated rVWF using the surface plasmon resonance technology.
- Figure 17 shows the mass increase of VWF after polymer conjugation measured by SDS-PAGE as described in Example 13.
- Figure 18 shows the determination of the FVIII binding capacity of furin-maturated rVWF compared to a pdVWF and non-treated rVWF using a combined ELISA and chromogenic assay (ECA).
- Figure 19 shows the determination of the FVIII binding capacity of furin-maturated rVWF compared to a pdVWF and non-treated rVWF using the surface plasmon resonance technology.
- Figure 20 shows the isoelectric focusing patterns of rVWF-polysialic acid conjugate and rVWF under reducing conditions.
- Figure 21 shows the pharmacokinetics of rVWF-polysialic acid conjugate and rVWF in VWF-deficient mice.
- Figure 22 shows the time course of mouse FVIII activity after application of rVWF- polysialic acid conjugate or rVWF in VWF-deficient mice.
- Figure 23 shows the pharmacokinetics of PEG-rVWF (branched PEG 2OK SG) and rVWF in FVIII deficient mice.
- Figure 24 shows the pharmacokinetics of rFVIII, co-infused with PEG-rVWF
- Figure 25 shows the pharmacokinetics of rFVIII, co-infused with various amounts of
- PEG-rVWF branched PEG 2OK SG
- Figure 26 shows the pharmacokinetics of PEG-rVWF #A (5 mg PEG/mg protein), PEG-rVWF #B (20 mg PEG/mg protein) and native rVWF in FVIII deficient mice.
- Figure 27 shows the pharmacokinetics of rFVIII, co-infused with PEG-rVWF #A (5 mg PEG/mg protein), PEG-rVWF #B (20 mg PEG/mg protein) or native rVWF.
- PEG-rVWF #A 5 mg PEG/mg protein
- PEG-rVWF #B 20 mg PEG/mg protein
- native rVWF native rVWF
- Example 1 Preparation of polymer - VWF - conjugate by modification of carbohydrate residues
- a solution of rVWF (final concentration: 500 ⁇ g/ml) was prepared in 20 mM sodium acetate buffer, pH 6.0 and NaIO 4 was added (final concentration 5 mM) for the oxidation of carbohydrate residues. The oxidation was carried out for 20 min at 4 0 C, then sodium bisulfite (final concentration 5 mM) was added to stop the reaction. Subsequently mPEG-hydrazide (chain length: 3 kD) was added (final concentration 10 mM) and the PEGylation of the VWF was performed for 1 h at room temperature.
- the PEGylated VWF was purified by size-exclusion chromatography.
- the reaction mixture was applied onto a chromatographic column (size: 26 mm x 840 mm) filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWF was separated from the reagents by using 20 mM HEPES - buffer, 150 mM NaCI, pH 7.4 containing 5% trehalose.
- the modified VWF was eluted in the void volume as indicated by measurements of VWF-antigen levels and OD 280 nm.
- the VWF containing fractions were directly applied to an anion-exchange column (size: 10 mm x 108 mm) filled with EMD TMAE 650 M (Merck) for further purification. Then the PEGylated VWF was eluted with 20 mM HEPES buffer, containing 5% trehalose and 100O mM NaCI.
- rVWF final concentration: 500 ⁇ g/ml
- mPEG succinimidyl succinate chain length: 5 kD
- the VWF was PEGylated for 1 h at room temperature. Subsequently the PEGylated VWF was purified by size-exclusion chromatography.
- the reaction mixture was applied onto a chromatographic column filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWF was separated by the same buffer system used for the PEGylation reaction.
- the VWF was eluted in the void volume as indicated by measurements of VWF-antigen levels and OD 280 nm.
- the VWF containing fractions were directly applied to an anion-exchange column (size: 26 mm x 840 mm) filled with EMD TMAE 650 M (Merck) for further purification. Then the PEGylated VWF was eluted with 20 mM HEPES buffer, containing 5% sucrose and 1000 mM NaCI.
- the reaction mixture was applied onto a chromatographic column filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWF was separated by the same buffer system used for the PEGylation reaction.
- the VWF was eluted in the void volume as indicated by measurements of VWF-antigen levels and OD 280 nm.
- the reaction mixture was applied onto a chromatographic column filled with Sephacryl S-300 HR (Amersham) and the PEGylated VWF was separated by the same buffer system used for the PEGylation reaction.
- the modified VWF was eluted in the void volume as indicated by measurements of VWF-antigen levels and OD 280 nm.
- a dextran (MW 40 kD) solution of 6 mg/ml was prepared in 20 mM sodium acetate buffer, pH 6.0 and NaIO 4 was added (final concentration 10 mM) to generate free aldehyde groups. The oxidation was carried out for 1 h at 4°C in the dark, then sodium bisulfite (final concentration 5 mM) was added to stop the reaction. The activated dextran was dialyzed against 0.1 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaCI (PBS - buffer). Then 2.4 ml of this solution of activated dextran were added to 10 ml of a solution of rVWF (concentration: 0.6 mg/ml in PBS buffer).
- the dextran coupled rVWF derivative was further purified by size-exclusion chromatography by applying the mixture onto a chromatography column (size: 50 mm x 860 mm) filled with Sephacryl S-300 HR (buffer: 20 mM HEPES, 5% sucrose, pH 7.4).
- the rVWF derivative was eluted in the void volume as indicated by measurements of VWF- antigen levels and OD 280 nm. These fractions were collected and concentrated by ultrafiltration using a 100 kD regenerated cellulose membrane (Millipore).
- VWF-deficient mice described in detail by Denis et al. (PNAS 95: 9524-9529, 1998) were used as a model of human VWD resembling severe type III VWD.
- Groups of 5 mice received a bolus injection via the tail vein either with PEG-rVWF (chain length 3 kD, PEGylation of rVWF according to Example 1) or native rVWF as control in a dose of 40 U VWF:Ag/kg bodyweight, based on detectable VWF (ELISA) after PEGylation.
- PEG-rVWF chain length 3 kD, PEGylation of rVWF according to Example 1
- native rVWF native rVWF
- the PEG-rVWF groups were anesthetized 5 min, 30 min, 1 h, 2 h, 6 h, 10 h and 24 h after injection (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h and 24 h for the control groups) and citrate plasma was prepared from heart puncture. VWF antigen levels were followed in plasma.
- Fig. 2 Native rVWF is eliminated from the circulation in a typical biphasic manner, as described in the literature (Lenting et al., J. Biol. Chem. 279: 12102-12109, 2004) and falls below the limit of detection between 600 min and 1440 min, equivalent to 10 and 24 h.
- FVIII deficient mice described in detail by Bi et al. were used as a model of severe human hemophilia A.
- Groups of 5 mice received a bolus injection (13 ml/kg) via the tail vein with either PEG-rVWF (chain length 3 kD, PEGylation of rVWF was performed according to example 1), or native rVWF, each premixed with rFVIII to achieve 3 U FVIII/ml and 3 U VWF:Ag/ml.
- citrate plasma was prepared by heart puncture from the respective groups, 5 min and 6 h after product injection.
- mice received a volume dose of 10 ml/kg.
- PEG-rVWF 5 min, 1 h, 3 h, 6 h, 10 h, 24 h and 48 h
- native rVWF 5 min, 15 min, 30 min 1 h, 2 h, 6 h, 24 h and 32 h
- groups of 4-5 mice were anesthetized, citrate plasma was prepared from heart puncture and the level of FVIII activity (chromogenic in house assay) was followed in plasma.
- the control-group was bled 15 minutes after injection. The results of this experiment are summarized in Fig. 4.
- mice were used as a model of severe human hemophilia A.
- Groups of 5 mice received a bolus injection (13 ml/kg) via the tail vein with either PEG-rVWF (HZ-
- VWF antigen recovery levels were measured in plasma samples. The results of this experiment are summarized in Fig. 5 and 6.
- FVIII x VWF double knockout mice were obtained by cross-breeding of FVIII deficient and VWF-deficient mice. Those mice suffer from FVIII deficiency as well as from VWF deficiency, thus providing an ideal model for studying FVIII - VWF interactions in an animal model.
- the VWF-antigen values were measured by use of an ELISA method as published (Ingerslev, Scand. J. Clin. Invest. 47: 143-149, 1987).
- the functional VWF:RCo activity reflecting the platelet binding properties of the VWF in the process of primary hemostasis was measured according to Macfarlane et al. (Thromb. Diath. Haemorrh. 34: 306-308, 1975). Five min, 3 h, 6 h, 10 h and 24 h after injection, citrate plasma by heart puncture after anesthesia was prepared from the respective groups. FVIII activity and VWF antigen levels were measured in plasma samples.
- Half-life of FVIII and VWF was calculated using the MicroMath Engineer program (Micromath Research, Saint Luis, MO, US) employing one compartment model from the pharmacokinetic library.
- Half-life for FVIII, co-infused with either rVWF or PEGylated rVWF increased from 1.88 h to 2.58 h, the area under the curve
- rVWF was PEGylated according to Example 2 using mPEG succinimidyl succinate (chain length: 5 kD) in various concentrations (1 mM, 2.5 mM, 5 mM, 7.5 mM and 10 mM).
- the PEGylated VWF species were analyzed with two different methods: SDS Polyacrylamide gel electrophoresis and VWF multimer analysis.
- SDS gel electrophoresis was performed under reducing conditions using a 3 - 8% gradient gel (Tris Acetat Gel/ Bio-Rad).
- VWF multimer analysis was carried out according to Ruggeri et Zimmerman (Blood 57: 1140-43, 1981) using a 1.6 % agarose gel.
- the visualization of VWF-multimers was carried out according to Aihara et al. (Thromb. Haemost. 55: 263-67, 1986).
- the SDS gel electrophoresis (Fig. 11) shows a rVWF preparation consisting of the mature VWF (lower band) and pro-VWF (upper band) and the increase of the molecular weight after PEGylation by use of different reagent concentrations.
- HSA human serum albumin
- PEGylation was carried out according to Example 2 is shown as a reference preparation. It is demonstrated that the molecular weight of HSA is shifting from 66,000 Da to 190,000 Da showing the efficacy of the PEGylation procedure.
- Fig. 12 shows the multimeric pattern of rVWF before and after PEGylation with different reagent concentrations. A broadening and a shift to higher molecular weight of the different multimers with increasing reagent concentration is clearly demonstrated.
- Example 14 PEGylation of lysine residues in VWF with mPEG succinimidyl qlutarate
- a solution of rVWF (final concentration: 500 ⁇ g/ml) was prepared in 20 mM HEPES - buffer, 150 mM NaCI, pH 7.4, 5% sucrose) and mPEG succinimidyl glutarate (chain length: 5 kD) was added (final concentration: 200 mg PEG succinimidyl glutarate / mg protein). Then the pH value was adjusted to 7.4 with 0.1 M NaOH. The VWF was PEGylated for 1 h at room temperature and purified as described in Example 2.
- FVIII deficient mice described in detail by Bi et al. (Nat. Genet. 10: 119-121 , 1995) were used as a model of severe human hemophilia A.
- Groups of 5 mice received a bolus injection (10 ml/kg) via the tail vein with either PEG-rVWF (SS- PEG, 5K) prepared according to Example 2 or native rVWF, each premixed with recombinant FVIII to achieve 10 U FVIII/ml and 10 U VWF/ml.
- Citrate plasma by heart puncture after anesthesia was prepared from the respective groups, 5 min, 3, 9 and 24 h after injection.
- FVIII activity and VWF antigen recovery levels were measured in plasma samples. The results of this experiment are summarized in Fig. 13 and 14.
- Example 16 Determination of FVIII binding capacity of SS-PEGylated VWF preparations by different methods
- Plates were subsequently washed after each step with PBS-Tween buffer (100 mM Na 2 HPO 4 ZKH 2 PO 4 , 150 mM NaCI, pH 7.6 and 0.05% Tween 20). Plates were blocked for 1 h at 37°C in 0.1% dry-milk/ 2mM benzamidine in PBS-Tween. Increasing amounts of VWF were preincubated for 25 min at 37°C with 0.2 U/ml rFVIII (ADVATE, Baxter AG, Vienna, Austria), and 100 ⁇ l of these mixtures were added to the plates. After incubation, the amount of FVIII bound to the captured VWF was measured by FVIII chromogenic assay (Technoclone, Vienna, Austria).
- PBS-Tween buffer 100 mM Na 2 HPO 4 ZKH 2 PO 4 , 150 mM NaCI, pH 7.6 and 0.05% Tween 20. Plates were blocked for 1 h at 37°C in 0.1% dry-milk/ 2m
- the FVIII binding capacity has been expressed as changes in the absorbance measured at 405 nm (dA405) in 1 min.
- Fig. 15 shows the VWF dose-dependent FVIII binding of two PEGylated VWF preparations, both modified with mPEG- succinimidyl succinate (SS).
- the FVIII binding capacity of the modified VWF preparations was calculated as % of the unmodified starting VWF preparations and found to be 20 % for PEG-SS-rVWF-1 and 50 % of PEG-SS-rVWF- 2.
- Fig. 16 shows the FVIII binding capacity of the two PEG-SS-rVWF preparations, as measured by the surface plasmon resonance method as described in Example 12. The calculated binding capacity was 25 and 45 %, respectively.
- PEGylated rVWF is separated from the reagents by ultrafiltration/diafiltration (UF/DF) using a 100 kD membrane consisting of regenerated cellulose (Millipore).
- UF/DF ultrafiltration/diafiltration
- Example 18 PEGylation of carboxyl groups in VWF with mPEG hvdrazide/EDC
- a furin maturated rVWF is prepared and purified according to Example 23.
- the preparation is dialyzed against 50 mM phosphate buffer, pH 6.2 and diluted to a concentration of 400 ⁇ g/ml.
- mPEG hydrazide mPEG Hz
- mPEG Hz mPEG hydrazide
- concentration 60 mg mPEG Hz / mg VWF.
- EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
- the reagents are separated from the PEGylated rVWF by UF/DF against 20 mM HEPES - buffer (150 mM NaCI, pH 7.4) using a 100 kD membrane (regenerated cellulose/Millipore).
- Example 19 Modification of lysine residues in VWF with polvsialic acid
- the solution was dialyzed overnight against 0.05 M sodium phosphate buffer, pH 7.2 in the dark. Subsequently an aliquot of this solution was added to a rVWF solution (400 ⁇ g/ml) in 0.05 M sodium phosphate to give a final concentration of 50 mg activated CA per mg VWF. This mixture was stirred for 30 minutes at room temperature in the dark. NaCNBH3 was added (1 mg/mg rVWF) and the mixture was incubated for 18 h at room temperature in the dark under gentle shaking. An aqueous 1 M TRIS-solution, pH 7.2 was added (50 ⁇ l per mg NaCNBHa) and stirred for 1 h to terminate the reaction. The free reagents were separated from the rVWF-polysialic acid conjugate by UF/DF using a 100 kD membrane (regenerated cellulose/ Millipore).
- rVWF conjugation of rVWF with polysialic acid was demonstrated by the shift of the isoelectric point (Pl) detected by isoelectric focusing (IEF) under reducing conditions.
- Fig. 20 shows a comparison of a VWF preparation before and after conjugation with polysialic acid according to this example.
- the Ampholine PAGplate system (pH 3.5 - 9.5) was used according to the instruction of the manufacturer
- a FVIII binding capacity of 54% as described in Example 16 was determined by use of the ECA test.
- a FVIII binding capacity of 70% was determined using the surface plasmon resonance method (Example 12).
- a perfusion chamber was manufactured as described by Sakariassen et al. (J. Lab. Clin. Med. 102: 522-535, 1983) and used for the PEGylation of rVWF under shear stress conditions.
- a solution of rVWF (500 ⁇ g/ml) in 20 mM HEPES, pH 7.4 containing 3% trehalose was prepared.
- mPEG succinimidyl succinate final concentration: 5 mM
- pH value adjusted with 0.1 M NaOH to 7.4 and immediately filled in the perfusion system.
- pdVWF The preparation of pdVWF was carried out according to Thorell et Blomback (Thromb. Res. 35: 431-450, 1984) with modifications.
- pdVWF 1.5 kg of cryoprecipitate was dissolved at 20-30 0 C in 6 I of water. After stirring for 1h, the fibronectin precipitate was removed by centrifugation. 8 g NaCI per liter supernatant were added, the solution was warmed to room temperature and SD-stock reagent (1% Tween 80 + 0.18% acetyltriethylcitrate final concentration) was added for virus inactivation.
- the solution was further purified on a EMD-TMAE Fractogel 650 M column (XK50/180) preequilibrated with 0.2M NaCI, 0.02M sodium acetate, pH 6.5 (washing buffer). After washing with 20 column volumes of washing buffer, VWF was eluted with 0.5M NaCI, 0.02M Na-citrate, pH 6.9. For precipitation of VWF glycine (1M final concentration) and NaCI (3M final concentration) were added. The precipitate was dissolved in buffer and applied onto a Sephacryl S-400 HR (Amersham) column equilibrated in 0.3M CaCI 2 , 0.15M NaCI, 2OmM HEPES, pH 7.4 for final purification.
- Example 22 PEGylation of lysine residues in pdVWF with PEG SS
- PdVWF was prepared according to Example 21 and diluted with 20 mM HEPES - buffer pH 7.4 (containing 150 mM NaCI and 3% sucrose) to a final concentration of 400 ⁇ g/ml. Then mPEG succinimidyl succinate (chain length: 5 kD) was added (concentration: 10 mg PEG SS 5000 / mg VWF) and the pdVWF was PEGylated for 1 h at room temperature. Then the reagent was separated from the PEGylated VWF by UF/DF using a 100 kD membrane consisting of regenerated cellulose (Millipore).
- Furin-maturated VWF was prepared according to Example 23 and dialyzed against 20 mM HEPES - buffer pH 7.4 (containing 150 mM NaCI and 3% sucrose). Then the solution was diluted to a final concentration of 300 ⁇ g/ml with 20 mM
- Example 26 Conjugation of VWF with polvsialic acid by cross-linking with glutaraldehvde
- VWF-deficient mice described by Denis et al. were used as an animal model of human VWD.
- Groups of 5 mice received a bolus injection (10 ml/kg) via the tail vein with either rVWF-polysialic acid conjugate prepared according to Example 19 or native rVWF to achieve 100 U VWF:Ag/kg.
- Citrate plasma by heart puncture after anesthesia was prepared from the respective groups, 5 min, 1 , 3, 6, 9 and 21 h after injection.
- VWF antigen recovery and endogenous mouse FVIII activity levels were measured in the plasma samples. The results of this experiment are summarized in Fig. 21 and 22.
- Half-iife of VWF increased from 1.3 h (native rVWF) to 2.4 h (rVWF-polysialic acid conjugate), AUC for FVIII increased from 3.3 U*hr/ml to 5.3 U*hr/ml respectively.
- Example 28 Coupling of mPEG propionaldehvde to lysine groups by reductive ami- nation
- a rVWF solution (400 ⁇ g/ml) is prepared in 0.05 M sodium phosphate buffer, pH 7.2 and mPEG propionaldehyde (chain length 5kDa) is added to give a final concentration of 10 mg mPEG propionaldehyde per mg VWF. This mixture is stirred for 30 minutes. Then NaCNBH3 is added (1 mg/mg rVWF) and the mixture is incubated for 15 h at room temperature under gentle shaking. An aqueous 1 M TRIS-solution, pH 7.2 is added (50 ⁇ l per mg NaCNBH 3 ) and stirred for 1 h to terminate the reaction. Subsequently, the PEGylated rVWF is separated from the reagents by ultrafiltration/diafiltration using a 100 kD membrane (regenerated cellulose/ Millipore).
- N-terminal PEGylation of rVWF is carried out as described by Lee et al. (Pharm. Res. 20: 818-825, 2003).
- a solution of rVWF (final concentration: 500 ⁇ g/ml) is prepared in 50 mM sodium acetate buffer, pH 5.5 and mPEG propionaldehyde (chain length: 5kD) is added (concentration: 10 mg mPEG propionaldehyde/mg VWF).
- the PEGylation is carried out for 24 h at room temperature in the presence of 2 mM NaCNBH3 as reducing agent.
- the PEGylated rVWF is separated from the reagents by ultrafiltration/diafiltration using a 100 kD membrane (regenerated cellulose/ Millipore).
- RVWF is PEGylated via lysine residues with mPEG succinimidyl succinate (chain length: 5 kD) according to Example 2.
- the PEGylation is performed at room temperature for 1h and the free reagents are separated from the rVWF PEG conjugate by UF/DF against 20 mM HEPES-buffer, pH 7.4, containing 5% saccharose using a 100 kD membrane (regenerated cellulose/ Millipore). Then the pH value of the solution is adjusted to 7.6 with 0.1 M NaOH and mPEG maleimide (chain length 5kD / final concentration 10 mM) is added for PEGylation of free SH- groups. The PEGylation is carried out for 2 h at room temperature under gentle shaking. Then the reagents are separated again from the reaction mixture using a 100 kD membrane (regenerated cellulose/ Millipore).
- Example 31 Enzymatic oxidation of carbohydrate residues and subsequent PEGylation with PEG-Hz
- mPEG hydrazide (mPEG Hz) with a chain length of 5 kD (final concentration: 40 mg mPEG-Hz/mg VWF) is added.
- the mixture is incubated at room temperature under gentle shaking for 3 h.
- the reagents are separated from the PEGylated rVWF by UF/DF against 20 mM HEPES - buffer (150 mM NaCI, pH 7.4) containing 5% saccharose using a 100 kD membrane (regenerated cellulose/Millipore).
- the mixture is diluted with an equal volume of HEPES - buffer, pH 7.4 containing 2M NaCI.
- the gel is separated from the supernatant by filtration. Then the gel is washed 3x with 2 ml HEPES- buffer, pH 7.4 (20 mM HEPES, 1 M NaCI) and the supernatant and the washing solutions are combined. Subse- quently the solution containing the PEGylated VWF is concentrated by ultrafiltration and diafiltrated against 20 mM HEPES - buffer, pH 7.4 (150 mM NaCI, 3% saccharose) using a 100 kD membrane consisting of regenerated cellulose (Millipore). The derivative obtained shows full FVIII binding capacity by use of the Biacore technology or the ECA test (the test systems are described in U.S. Provisional Patent Appli- cation Serial No. 60/668,378 filed April 04, 2005).
- RVWF was polysialylated according to Example 19.
- the ratio VWF:RCo/VWF:Ag was calculated.
- the VWF antigen level was determined by use a commercially available assay system (Asserachrom vWF, Roche, Basel, Switzerland).
- the functional activity of the preparation was determined by the Ristocetin Co- factor assay as described by Macfarlane et al. (Thromb. Diath. Haemorrh 34: 306- 308, 1975).
- the ratio of 0.39 calculated for the rVWF starting material decreases to 0.13 after polysialylation.
- Example 36 Conjugation of rVWF with branched PEG via lysine residues
- a solution of a mature rVWF (35 U VWF:Ag/ml) in 20 mM HEPES buffer, 150 mM NaCI, pH 7.4, containing 0.5 % sucrose was prepared according to Example 23. Then branched mPEG succinimidyl glutarate (PE-SG / chain length: 20 kD) supplied by NOF corporation (NOF Europe, Grobbendonk, Belgium) was added to this solu- tion under gentle stirring (5 mg PEG-SG / mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH. Then the PEGylation was carried out under gentle stirring for 1 h at room temperature.
- PE-SG / chain length: 20 kD branched mPEG succinimidyl glutarate supplied by NOF corporation (NOF Europe, Grobbendonk, Belgium) was added to this solu- tion under gentle stirring (5 mg PEG-SG / mg protein) and the pH value was adjusted to 7.4 by drop
- reaction mixture was applied onto an equilibrated ion-exchange chromatography resin (Fractogel EMD TMAE 650M) in 20 mM HEPES buffer, 150 mM NaCI, pH 7.4, containing 0.5 % sucrose. Then the column was washed with 20 CV equilibration buffer to remove excess reagent and the PEGylated rVWF was eluted with elution buffer (20 mM HEPES, 0.5 M NaCI, 0.5% sucrose, pH 7.4).
- equilibrated ion-exchange chromatography resin Frazier EMD TMAE 650M
- the eluate was concentrated by ultrafil- tration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 100 kD using a buffer system consisting of 20 mM HEPES, 150 mM NaCI, 0.5 % sucrose, pH 7.4.
- the PEGylated derivative obtained showed a slightly diminished VWF:RCo/VWF:Ag ratio of 0.79 as compared with the rVWF starting material (VWF:Rco/VWF:Ag ratio: 0.89).
- the PEGylated rVWF starting material had a FVIII binding capacity of 83% as measured by the ECA test (Example 16).
- Example 37 Pharmacokinetics of VWF conjugated with branched PEG's in FVIII- K.O.-mice
- FVIII deficient mice (Bi et al., Nat. Genet.10: 119-121, 1995) were used as a model of severe human hemophilia A. Groups of 5 mice received a bolus injection (10 ml/kg) via the tail vein with either a mixture of PEG-rVWF (branched PEG, SG) and rFVIII or a mixture of native rVWF and rFVIII to achieve 30 U FVIII/ml and 25 L) VWF/ml. Five min, 3, 9, 24 and 32 h after injection citrate plasma was prepared from the respective groups by heart puncture after anesthesia. FVIII activity and VWF antigen recovery levels were measured in plasma samples.
- VWF half-life increased from 1.4 to 9.7 h
- AUC for VWF increased from 11.8 to 49.2 U*h/ml
- Half-life for FVIII increased from 1.2 h (in the presence of native rVWF) to 4.4 h (when applied together with PEG-rVWF), area under curve (AUC) increased from 12.1 to 30.5 U * h/ml.
- Example 38 Comparative pharmacokinetics of FVIII mixed with different amounts of PEG-VWF in FVIII-K.O.-mice
- Example 39 Comparative pharmacokinetics using PEG-VWF preparations with dif- ferent degrees of PEGylation
- FVIII x VWF double knockout mice were obtained by cross-breeding of FVIII deficient and VWF-deficient mice. Those mice suffer from FVIII deficiency as well as from VWF deficiency. Groups of 5 FVIII x VWF double knockout mice were infused via the tail vein with a mixture of native rVWF/ rFVIII (100/150 IU/kg) or with PEGy- lated rVWF #A mixed with rFVIII (100/150 IU/kg) or with PEGylated rVWF #B mixed with rFVIII (150/150 IU/kg).
- PEG-rVWF #A (5 mg PEG-SS 5000/mg protein) and PEG-rVWF #B (20 mg PEG-SS 5000/mg protein) were prepared according to Example 24.
- preparation #A a ratio of 2.5 mole PEG/mole lysine and for prepara- tion #B a ratio of 10 mole PEG/mole lysine was calculated.
- citrate plasma samples were prepared 5 min, 1, 3, 9 and 24 h after sample application. Plasma levels of VWF:Ag and FVIII activity were measured and expressed as percent of the maximum plasma level, generally reached 5 min after injection. The elimination curves for VWF and FVIII are shown in Fig. 26 and Fig. 27, respectively.
- Half-life was 6.3 h and 8.1 h for PEG-rVWF #A and #B respectively.
- For the native rVWF a half-life of 2.0 h was calculated.
- the normalized AUC (% of maximum x h) for both PEGylated rVWFs increased from 360 %*h (native rVWF) to 901 %*h (#A) and 1064 %*h (#B).
- the circulation time for co-infused rFVIII was improved by PEGylated rVWF compared with native VWF.
- the half-life of FVIII was 0.8 h in the presence of native rVWF, and increased to 1.5 and 1.8 h when infused with PEGylated rVWF #A and #B respectively.
- the AUC for FVIII was 214, 370 and 358 %*h.
- a solution of a VWF dimer (58 IU VWF:Ag / ml), which was purified from the conditioned medium of a recombinant CHO cell line (Baxter BioScience), was prepared in 20 mM HEPES buffer, 15OmM NaCI, pH 7.4, containing 0.5 % sucrose was prepared. Then branched mPEG succinimidyl glutarate (PE-SG / chain length: 20 kD) supplied by NOF corporation was added to this solution under gentle stirring (5 mg PEG-SG / mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH. The PEGylation was carried out under gentle stirring for 1 h at room temperature.
- reaction mixture was applied onto an equilibrated ion-exchange chromatography resin (Fractogel EMD TMAE 650M) in 20 mM HEPES buffer, 150 mM NaCI, pH 7.4, containing 0.5 % sucrose. Then the column was washed with 20 CV equilibration buffer to remove excess reagent and the PE- Gylated rVWF dimer was eluted with elution buffer (20 mM HEPES, 0.5 M NaCI, 0.5% sucrose, pH 7.4).
- equilibrated ion-exchange chromatography resin Frazier EMD TMAE 650M
- the eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose (Millipore) and with a molecular weight cut-off of 100 kD using a buffer system consisting of 20 mM HEPES, 15OmM NaCI, 0.5 % sucrose, pH 7.4.
- Example 41 PEGylation and in vitro characterization of low multimer rVWF
- Mature rVWF was purified according to Example 23.
- the purification procedure included ion-exchange chromatography steps as well as a final gelfiltration step on Superose 6, performed in 20 mM HEPES, 15OmM NaCI, pH 7.4, where the high multimer rVWF (17 multimers) was eluted in the void volume.
- the VWF multimer analysis was performed according to Ruggeri and Zimmerman (Blood 57: 1140-43, 1981) using a 1.0 % agarose gel.
- a low multimer rVWF preparation (6 multimers) was obtained from a side fraction, eluting at higher retention times. This fraction was stabilized by addition of 0.5 % sucrose, pH 7.4.
- the low multimer rVWF was PEGylated using mPEG succinimidyl succinate (PEG-SS).
- PEG-SS was added to this solution under gentle stirring (5 mg PEG-SS / mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH.
- the PEGylation was carried out under gentle stirring for 1 h at room temperature.
- the excess reagent was removed by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 100 kD using a buffer system consisting of 20 mM HEPES, 15OmM NaCI, 0.5 % sucrose, pH 7.4.
- the FVIII binding capacity determined by the ECA assay according to Example 16 slightly de- creased from 49% for the starting material to 34% for the PEGylated preparation.
- the VWF:RCo/VWF:Ag ratio of 0.02 measured for the low multimer rVWF was not affected by the PEGylation procedure.
- Example 42 Derivatization of VWF with reversiblv blocked FVIII binding epitopes (blocking with FVIII and heparin)
- a chromatographic column (15 mm X 148 mm) was filled with Heparin Hy- perD (Bio-Sepra) and equilibrated with an equilibration buffer consisting of 20 mM HEPES, 68 mM NaCI, 0.5% sucrose, pH 7.4. Then a solution of mature rVWF (48 IU VWF:Ag / ml) in 20 mM HEPES, 150 mM NaCI, 0.5 sucrose was diluted with H 2 O to give a conductivity of 7-8 mS/cm and applied onto this column using a linear flow rate of 1.5 cm/min.
- an equilibration buffer consisting of 20 mM HEPES, 68 mM NaCI, 0.5% sucrose, pH 7.4. Then a solution of mature rVWF (48 IU VWF:Ag / ml) in 20 mM HEPES, 150 mM NaCI, 0.5 sucrose was diluted with H 2 O to give a conduct
- a mature rVWF is purified according to Example 23. Then a solution of this rVWF (40 U VWF:Ag/ml) in 20 mM HEPES buffer, 150 mM NaCI, pH 7.4, containing 0.5 % sucrose is prepared. Subsequently the PEGylation is carried out by adding mPEG-(carboxymethyl)-3-hydroxy-butanoic acid N-hydroxysuccinimide ester (chain length: 5 kD) to this solution under gentle stirring (5 mg PEG-reagent / mg protein) and the pH value is adjusted to 7.4 by drop wise addition of 0.5 M NaOH. Then the PEGylation reaction is carried out under gentle stirring for 1 h at room temperature.
- the excess reagent is separated from the PEGylated rVWF by ultrafil- tration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 100 kD using a buffer system consisting of 20 mM HEPES 1 150 mM NaCI, 0.5 % sucrose, pH 7.4.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BRPI0519562-4A BRPI0519562A2 (en) | 2004-12-27 | 2005-12-23 | proteinaceous construct, complex, method for prolonging the in vivo half-life of factor viii (fviii) or a biologically active derivative of the same pharmaceutical composition, and method for forming a proteinaceous construct |
CA002591852A CA2591852A1 (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
AU2005322067A AU2005322067B8 (en) | 2004-12-27 | 2005-12-23 | Polymer-von Willebrand factor-conjugates |
JP2007548552A JP5022231B2 (en) | 2004-12-27 | 2005-12-23 | Polymer-von Willebrand factor conjugate |
ES05855442T ES2434035T3 (en) | 2004-12-27 | 2005-12-23 | Polymer-von Willebrand factor conjugates |
MX2007007877A MX2007007877A (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates. |
EP05855442.9A EP1835938B1 (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
SI200531778T SI1835938T1 (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
PL05855442T PL1835938T3 (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
CN2005800445983A CN101163506B (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
DK05855442.9T DK1835938T3 (en) | 2004-12-27 | 2005-12-23 | Polymer-von Willebrand factor conjugates |
IL183634A IL183634A (en) | 2004-12-27 | 2007-06-04 | Polymer-von willebrand factor-conjugates |
HK07111722.5A HK1105276A1 (en) | 2004-12-27 | 2007-10-30 | Polymer-von willebrand factor-conjugates |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US63924404P | 2004-12-27 | 2004-12-27 | |
US60/639,244 | 2004-12-27 | ||
US66837805P | 2005-04-04 | 2005-04-04 | |
US60/668,378 | 2005-04-04 | ||
US67190105P | 2005-04-15 | 2005-04-15 | |
US60/671,901 | 2005-04-15 | ||
US68508605P | 2005-05-26 | 2005-05-26 | |
US60/685,086 | 2005-05-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2006071801A2 true WO2006071801A2 (en) | 2006-07-06 |
WO2006071801A3 WO2006071801A3 (en) | 2007-08-16 |
Family
ID=36540284
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/046879 WO2006071801A2 (en) | 2004-12-27 | 2005-12-23 | Polymer-von willebrand factor-conjugates |
Country Status (16)
Country | Link |
---|---|
US (4) | US7884075B2 (en) |
EP (1) | EP1835938B1 (en) |
JP (2) | JP5022231B2 (en) |
KR (1) | KR20070092754A (en) |
CN (1) | CN101163506B (en) |
AU (1) | AU2005322067B8 (en) |
BR (1) | BRPI0519562A2 (en) |
CA (1) | CA2591852A1 (en) |
DK (1) | DK1835938T3 (en) |
ES (1) | ES2434035T3 (en) |
HK (1) | HK1105276A1 (en) |
IL (1) | IL183634A (en) |
MX (1) | MX2007007877A (en) |
PL (1) | PL1835938T3 (en) |
PT (1) | PT1835938E (en) |
WO (1) | WO2006071801A2 (en) |
Cited By (42)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008025856A2 (en) | 2006-09-01 | 2008-03-06 | Novo Nordisk Health Care Ag | Modified glycoproteins |
WO2008151817A1 (en) | 2007-06-13 | 2008-12-18 | Csl Behring Gmbh | Use of vwf stabilized fviii preparations and of vwf preparations without fviii for extravascular administration in the therapy and prophylactic treatment of bleeding disorders |
WO2009000522A1 (en) * | 2007-06-26 | 2008-12-31 | Baxter International Inc. | Hydrolysable polymeric fmoc-linker |
WO2008082669A3 (en) * | 2006-12-27 | 2009-04-09 | Nektar Therapeutics Al Corp | Von willebrand factor- and factor viii-polymer conjugates having a releasable linkage |
WO2009135888A2 (en) * | 2008-05-06 | 2009-11-12 | Octapharma Ag | Complex |
WO2010014258A2 (en) * | 2008-08-01 | 2010-02-04 | Nektar Therapeutics Al, Corporation | Conjugates having a releasable linkage |
WO2010014708A3 (en) * | 2008-08-01 | 2010-04-22 | Baxter International Inc. | Factor viii polymer conjugates |
JP2010512753A (en) * | 2006-12-15 | 2010-04-30 | バクスター・インターナショナル・インコーポレイテッド | Factor VIIa- (poly) sialic acid conjugate with extended in vivo half-life |
JP2010527588A (en) * | 2007-05-18 | 2010-08-19 | バクスター・インターナショナル・インコーポレイテッド | A method for producing mature VWF from a VWF propeptide. |
EP2222329A1 (en) * | 2007-11-09 | 2010-09-01 | Baxter International Inc. | Modified recombinant factor viii and von willebrand factor and methods of use |
WO2011017055A2 (en) | 2009-07-27 | 2011-02-10 | Baxter International Inc. | Blood coagulation protein conjugates |
US7982010B2 (en) | 2006-03-31 | 2011-07-19 | Baxter International Inc. | Factor VIII polymer conjugates |
US7985839B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US8053561B2 (en) | 2006-03-31 | 2011-11-08 | Baxter International Inc. | Pegylated factor VIII |
WO2012016131A1 (en) | 2010-07-30 | 2012-02-02 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
WO2012087838A1 (en) | 2010-12-22 | 2012-06-28 | Baxter International Inc. | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
WO2012166622A1 (en) | 2011-05-27 | 2012-12-06 | Baxter International Inc. | Therapeutic proteins with increased half-life and methods of preparing same |
US20120316116A1 (en) * | 2011-06-10 | 2012-12-13 | Baxter Healthcare S.A. | Treatment of coagulation disease by administration of recombinant vwf |
WO2013120939A1 (en) | 2012-02-15 | 2013-08-22 | Csl Behring Gmbh | Von willebrand factor variants having improved factor viii binding affinity |
US8575104B2 (en) | 2008-06-24 | 2013-11-05 | Csl Behring Gmbh | Factor VIII, von willebrand factor or complexes thereof with prolonged in vivo half-life |
WO2013173557A1 (en) | 2012-05-16 | 2013-11-21 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage and use of nmr analyses of the same |
US8642737B2 (en) | 2010-07-26 | 2014-02-04 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
US8809501B2 (en) | 2009-07-27 | 2014-08-19 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
EP2796145A1 (en) * | 2013-04-22 | 2014-10-29 | CSL Behring GmbH | Complex |
JP2015155469A (en) * | 2015-06-01 | 2015-08-27 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | Fviii-polymer conjugates |
WO2016000039A1 (en) | 2014-07-02 | 2016-01-07 | Csl Limited | Modified von willebrand factor |
US9274122B2 (en) | 2008-10-21 | 2016-03-01 | Baxalta Incorporated | Methods for determining active ingredients in pro-drug PEG protein conjugates with releasable PEG reagents (in vitro de-pegylation) |
WO2016142288A1 (en) | 2015-03-06 | 2016-09-15 | Csl Behring Recombinant Facility Ag | Modified von willebrand factor having improved half-life |
EP3093029A1 (en) | 2009-07-27 | 2016-11-16 | Baxalta GmbH | Blood coagulation protein conjugates |
WO2016188907A1 (en) | 2015-05-22 | 2016-12-01 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for treating hemophilia |
WO2016188905A1 (en) | 2015-05-22 | 2016-12-01 | Csl Behring Recombinant Facility Ag | Methods for preparing modified von willebrand factor |
WO2017117631A1 (en) | 2016-01-07 | 2017-07-13 | Csl Limited | Mutated truncated von willebrand factor |
US9795683B2 (en) | 2009-07-27 | 2017-10-24 | Lipoxen Technologies Limited | Glycopolysialylation of non-blood coagulation proteins |
WO2018087267A1 (en) | 2016-11-11 | 2018-05-17 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for treating hemophilia |
WO2018087271A1 (en) | 2016-11-11 | 2018-05-17 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder |
JP2018115170A (en) * | 2018-03-02 | 2018-07-26 | バクスアルタ ゲーエムベーハー | Factor fviii-polymer conjugates |
WO2019038350A1 (en) | 2017-08-23 | 2019-02-28 | Csl Behring Gmbh | Method for virus filtration of von willebrand factor |
US10808023B2 (en) | 2016-01-07 | 2020-10-20 | CSL Behring Lengnau AG | Mutated von Willebrand factor |
US10829539B2 (en) | 2014-06-13 | 2020-11-10 | Csl Limited | Production of recombinant von Willebrand factor in a bioreactor |
US11192936B2 (en) | 2014-01-10 | 2021-12-07 | Bioverativ Therapeutics Inc. | Factor VIII chimeric proteins and uses thereof |
US11529395B2 (en) | 2017-07-07 | 2022-12-20 | Takeda Pharmaceutical Company Limited | Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF |
US12030925B2 (en) | 2018-05-18 | 2024-07-09 | Bioverativ Therapeutics Inc. | Methods of treating hemophilia A |
Families Citing this family (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6263355A (en) * | 1985-09-13 | 1987-03-20 | Hitachi Ltd | Checking method for battery back-up ram |
WO2006071801A2 (en) | 2004-12-27 | 2006-07-06 | Baxter International Inc | Polymer-von willebrand factor-conjugates |
NZ585530A (en) * | 2007-12-27 | 2012-06-29 | Baxter Int | Methods for differentiating plasma-derived protein from recombinant protein in a sample |
KR20160012252A (en) | 2007-12-27 | 2016-02-02 | 백스터 인터내셔널 인코포레이티드 | Method and compositions for specifically detecting physiologically acceptable polymer molecules |
US11197916B2 (en) | 2007-12-28 | 2021-12-14 | Takeda Pharmaceutical Company Limited | Lyophilized recombinant VWF formulations |
BRPI0821474B8 (en) | 2007-12-28 | 2021-05-25 | Baxalta GmbH | stable liquid pharmaceutical formulation |
TWI492951B (en) | 2007-12-31 | 2015-07-21 | Baxter Int | Transgenic non-human animals expressing human blood clotting factors and uses thereof |
CA2731546C (en) * | 2008-07-23 | 2013-11-19 | Hanmi Holdings Co., Ltd. | A polypeptide complex comprising non-peptidyl polymer having three functional ends |
HUE039906T2 (en) | 2008-10-17 | 2019-02-28 | Baxalta GmbH | Modified blood factors comprising a low degree of water soluble polymer |
WO2010089756A2 (en) * | 2008-10-20 | 2010-08-12 | Usv Limited | An improved process for pegylation of proteins |
TWI508735B (en) | 2008-10-21 | 2015-11-21 | 巴克斯特國際公司 | Lyophilized recombinant vwf formulations |
PL2356467T3 (en) * | 2008-12-11 | 2013-12-31 | Baxalta Inc | Detection of physiologically acceptable polymer molecules using near infrared spectroscopy |
WO2011017414A2 (en) | 2009-08-04 | 2011-02-10 | Baxter International Inc. | Transgenic mouse lacking endogenous fviii and vwf - a model of hemophilia a |
JP5838159B2 (en) * | 2009-08-20 | 2015-12-24 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | Purification of VWF to enhance removal of non-lipid enveloped virus |
WO2011028229A1 (en) | 2009-08-24 | 2011-03-10 | Amunix Operating Inc. | Coagulation factor ix compositions and methods of making and using same |
EP2499165B1 (en) * | 2009-11-13 | 2016-09-14 | Grifols Therapeutics Inc. | Von willebrand factor (vwf)-containing preparations, and methods, kits, and uses related thereto |
GB201007357D0 (en) * | 2010-04-30 | 2010-06-16 | Leverton Licence Holdings Ltd | Conjugated factor VIII |
KR101309566B1 (en) * | 2010-12-10 | 2013-09-17 | 포항공과대학교 산학협력단 | Hyaluronic acid-protein conjugate, and preparation method thereof |
PT2804623T (en) * | 2012-01-12 | 2019-11-18 | Bioverativ Therapeutics Inc | Chimeric factor viii polypeptides and uses thereof |
CA2864126A1 (en) | 2012-02-15 | 2013-08-22 | Biogen Idec Ma Inc. | Recombinant factor viii proteins |
LT3564260T (en) | 2012-02-15 | 2023-01-10 | Bioverativ Therapeutics Inc. | Factor viii compositions and methods of making and using same |
BR112015000267B1 (en) * | 2012-07-11 | 2023-01-24 | Bioverativ Therapeutics Inc. | CHIMERIC PROTEINS, AND PHARMACEUTICAL COMPOSITION |
EP3875106A1 (en) * | 2013-08-08 | 2021-09-08 | Bioverativ Therapeutics Inc. | Purification of chimeric fviii molecules |
TW202003554A (en) | 2013-08-14 | 2020-01-16 | 美商百歐維拉提夫治療公司 | Factor VIII-XTEN fusions and uses thereof |
CN104491917B (en) * | 2014-12-30 | 2018-01-09 | 广州市拜特凇医药科技有限公司 | A kind of biomaterial containing PEG or derivatives thereof |
EA201890423A1 (en) | 2015-08-03 | 2018-07-31 | Биовератив Терапьютикс Инк. | SLIGHT PROTEINS OF THE FACTOR IX, METHODS OF THEIR RECEPTION AND APPLICATION |
CN111565741A (en) | 2017-07-07 | 2020-08-21 | 百深公司 | Treatment of patients with severe von willebrand disease undergoing elective surgery by administration of recombinant VWF |
US10934340B2 (en) | 2018-03-21 | 2021-03-02 | Baxalta Incorporated | Separation of VWF and VWF propeptide by chromatographic methods |
US20200261546A1 (en) | 2019-02-01 | 2020-08-20 | Baxalta Incorporated | METHODS OF PROPHYLACTIC TREATMENT USING RECOMBINANT VWF (rVWF) |
EP4028046B1 (en) | 2019-09-11 | 2024-02-14 | Takeda Pharmaceutical Company Limited | Methods of treatment related to complexes of von willebrand factor and complement c1q |
EP4100048A1 (en) | 2020-02-04 | 2022-12-14 | Takeda Pharmaceutical Company Limited | Treatment of menorrhagia in patients with severe von willebrand disease by administration of recombinant vwf |
Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4877608A (en) | 1987-11-09 | 1989-10-31 | Rorer Pharmaceutical Corporation | Pharmaceutical plasma protein formulations in low ionic strength media |
US5328694A (en) | 1990-01-19 | 1994-07-12 | Octapharma Ag | Stable injectable solution of factor VIII |
WO1994015625A1 (en) | 1993-01-15 | 1994-07-21 | Enzon, Inc. | Factor viii - polymeric conjugates |
US5565427A (en) | 1991-04-09 | 1996-10-15 | Behringwerke Aktiengesellschaft | Stabilized factor VIII preparations |
US5605884A (en) | 1987-10-29 | 1997-02-25 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Factor VIII formulations in high ionic strength media |
WO1997011957A1 (en) | 1995-09-29 | 1997-04-03 | Pharmacia & Upjohn Ab | Conjugates of a polypeptide and a biocompatible polymer |
EP0774261A2 (en) | 1995-11-10 | 1997-05-21 | Immuno Ag | Uses of von Willebrand Factor and pharmaceutical preparations |
EP0784632A1 (en) | 1994-10-04 | 1997-07-23 | IMMUNO Aktiengesellschaft | Process for extracting high-purity von willebrand factor |
US5733873A (en) | 1992-10-02 | 1998-03-31 | Pharmacia & Upjohn Ab | Composition comprising coagulation factor VIII formulation, process for its preparation and use of a surfactant as stabilizer |
US5763401A (en) | 1996-07-12 | 1998-06-09 | Bayer Corporation | Stabilized albumin-free recombinant factor VIII preparation having a low sugar content |
WO1998025969A1 (en) | 1996-12-13 | 1998-06-18 | Immuno Aktiengesellschaft | Von willebrand factor derivative and method for isolating proteins |
EP0977584A1 (en) | 1997-05-28 | 2000-02-09 | Baxter Aktiengesellschaft | PHARMACEUTICAL PREPARATION COMPRISING vWF PROPEPTIDE |
US6037452A (en) | 1992-04-10 | 2000-03-14 | Alpha Therapeutic Corporation | Poly(alkylene oxide)-Factor VIII or Factor IX conjugate |
WO2000049047A1 (en) | 1999-02-19 | 2000-08-24 | Baxter Aktiengesellschaft | METHOD FOR PRODUCING A vWF PREPARATION |
US6586573B1 (en) | 1999-02-22 | 2003-07-01 | Baxter International Inc. | Albumin-free Factor VIII formulations |
WO2004075923A2 (en) | 2003-02-26 | 2004-09-10 | Nektar Therapeutics Al, Corporation | Polymer-factor viii moiety conjugates |
Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2004A (en) * | 1841-03-12 | Improvement in the manner of constructing and propelling steam-vessels | ||
US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
US4356170A (en) | 1981-05-27 | 1982-10-26 | Canadian Patents & Development Ltd. | Immunogenic polysaccharide-protein conjugates |
JPS59172425A (en) * | 1983-03-18 | 1984-09-29 | Nippon Chemiphar Co Ltd | Novel blood coagulation factor derivative, its preparation and blood coagulation promoting agent containing the same |
US4757006A (en) | 1983-10-28 | 1988-07-12 | Genetics Institute, Inc. | Human factor VIII:C gene and recombinant methods for production |
US4965199A (en) | 1984-04-20 | 1990-10-23 | Genentech, Inc. | Preparation of functional human factor VIII in mammalian cells using methotrexate based selection |
US4970300A (en) | 1985-02-01 | 1990-11-13 | New York University | Modified factor VIII |
AU591671B2 (en) | 1985-04-11 | 1989-12-14 | Children's Medical Center Corporation | Von willebrand factor |
CA1339946C (en) | 1987-03-31 | 1998-07-07 | Michael J. Griffith | Ultrapurification process for polypeptides |
US5122614A (en) * | 1989-04-19 | 1992-06-16 | Enzon, Inc. | Active carbonates of polyalkylene oxides for modification of polypeptides |
US5595732A (en) | 1991-03-25 | 1997-01-21 | Hoffmann-La Roche Inc. | Polyethylene-protein conjugates |
US5846951A (en) | 1991-06-06 | 1998-12-08 | The School Of Pharmacy, University Of London | Pharmaceutical compositions |
AU2297792A (en) | 1991-06-28 | 1993-01-25 | Rhone-Poulenc Rorer International (Holdings) Inc. | Therapeutic polypeptides based on von willebrand factor |
US5298643A (en) * | 1992-12-22 | 1994-03-29 | Enzon, Inc. | Aryl imidate activated polyalkylene oxides |
US5621039A (en) | 1993-06-08 | 1997-04-15 | Hallahan; Terrence W. | Factor IX- polymeric conjugates |
US5919455A (en) | 1993-10-27 | 1999-07-06 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
US6127153A (en) | 1995-06-07 | 2000-10-03 | Neose Technologies, Inc. | Method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and gene encoding a polyglycosyltransferase |
US6562781B1 (en) | 1995-11-30 | 2003-05-13 | Hamilton Civic Hospitals Research Development Inc. | Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates |
US6214966B1 (en) | 1996-09-26 | 2001-04-10 | Shearwater Corporation | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
AT405403B (en) | 1997-02-27 | 1999-08-25 | Immuno Ag | CLEANING OF WILLEBRAND FACTOR BY CATION EXCHANGER CHROMATOGRAPHY |
DE69929311T2 (en) | 1998-03-24 | 2006-09-07 | Nof Corp. | OXIMANDERIVATES AND METHOD FOR THE PRODUCTION THEREOF |
WO2001005434A2 (en) | 1999-07-20 | 2001-01-25 | Amgen Inc. | Hyaluronic acid-protein conjugates |
SE515295C2 (en) | 1999-11-23 | 2001-07-09 | Medicarb Ab | Process for the preparation of conjugates of a biologically active polysaccharide and a solid substrate, and such conjugates |
AUPR610501A0 (en) | 2001-07-04 | 2001-07-26 | Smart Drug Systems Inc | Treatment of parasitic disease |
US7265084B2 (en) | 2001-10-10 | 2007-09-04 | Neose Technologies, Inc. | Glycopegylation methods and proteins/peptides produced by the methods |
MXPA04004336A (en) | 2001-11-07 | 2005-05-16 | Nektar Therapeutics Al Corp | Branched polymers and their conjugates. |
US20050176108A1 (en) | 2003-03-13 | 2005-08-11 | Young-Min Kim | Physiologically active polypeptide conjugate having prolonged in vivo half-life |
US20040247624A1 (en) * | 2003-06-05 | 2004-12-09 | Unger Evan Charles | Methods of making pharmaceutical formulations for the delivery of drugs having low aqueous solubility |
EP1673453A2 (en) * | 2003-10-07 | 2006-06-28 | Novo Nordisk Health Care AG | Hybrid molecules having factor vii/viia activity |
WO2005058366A2 (en) | 2003-12-10 | 2005-06-30 | Nektar Therapeutics Al, Corporation | Compositions comprising two different populations of polymer-active agent conjugates |
WO2006071801A2 (en) * | 2004-12-27 | 2006-07-06 | Baxter International Inc | Polymer-von willebrand factor-conjugates |
-
2005
- 2005-12-23 WO PCT/US2005/046879 patent/WO2006071801A2/en active Application Filing
- 2005-12-23 AU AU2005322067A patent/AU2005322067B8/en active Active
- 2005-12-23 CA CA002591852A patent/CA2591852A1/en not_active Abandoned
- 2005-12-23 ES ES05855442T patent/ES2434035T3/en active Active
- 2005-12-23 BR BRPI0519562-4A patent/BRPI0519562A2/en not_active IP Right Cessation
- 2005-12-23 EP EP05855442.9A patent/EP1835938B1/en active Active
- 2005-12-23 MX MX2007007877A patent/MX2007007877A/en active IP Right Grant
- 2005-12-23 PT PT58554429T patent/PT1835938E/en unknown
- 2005-12-23 KR KR1020077017261A patent/KR20070092754A/en not_active Application Discontinuation
- 2005-12-23 US US11/317,582 patent/US7884075B2/en active Active
- 2005-12-23 DK DK05855442.9T patent/DK1835938T3/en active
- 2005-12-23 JP JP2007548552A patent/JP5022231B2/en active Active
- 2005-12-23 PL PL05855442T patent/PL1835938T3/en unknown
- 2005-12-23 CN CN2005800445983A patent/CN101163506B/en active Active
-
2007
- 2007-06-04 IL IL183634A patent/IL183634A/en active IP Right Grant
- 2007-10-30 HK HK07111722.5A patent/HK1105276A1/en unknown
-
2011
- 2011-01-14 US US13/007,208 patent/US8076463B2/en active Active
- 2011-11-28 US US13/305,334 patent/US8357779B2/en active Active
-
2012
- 2012-03-30 JP JP2012081700A patent/JP5715983B2/en active Active
- 2012-12-21 US US13/724,831 patent/US8835388B2/en active Active
Patent Citations (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5605884A (en) | 1987-10-29 | 1997-02-25 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Factor VIII formulations in high ionic strength media |
US4877608A (en) | 1987-11-09 | 1989-10-31 | Rorer Pharmaceutical Corporation | Pharmaceutical plasma protein formulations in low ionic strength media |
US5328694A (en) | 1990-01-19 | 1994-07-12 | Octapharma Ag | Stable injectable solution of factor VIII |
US5565427C1 (en) | 1991-04-09 | 2002-07-23 | Aventis Behring Gmbh | Stabilized factor viii preparations |
US5565427A (en) | 1991-04-09 | 1996-10-15 | Behringwerke Aktiengesellschaft | Stabilized factor VIII preparations |
US6037452A (en) | 1992-04-10 | 2000-03-14 | Alpha Therapeutic Corporation | Poly(alkylene oxide)-Factor VIII or Factor IX conjugate |
US5733873A (en) | 1992-10-02 | 1998-03-31 | Pharmacia & Upjohn Ab | Composition comprising coagulation factor VIII formulation, process for its preparation and use of a surfactant as stabilizer |
WO1994015625A1 (en) | 1993-01-15 | 1994-07-21 | Enzon, Inc. | Factor viii - polymeric conjugates |
EP0784632A1 (en) | 1994-10-04 | 1997-07-23 | IMMUNO Aktiengesellschaft | Process for extracting high-purity von willebrand factor |
EP1258497A2 (en) | 1995-09-29 | 2002-11-20 | Biovitrum Ab | Conjugates of factor VIII and a biocompatible polymer |
WO1997011957A1 (en) | 1995-09-29 | 1997-04-03 | Pharmacia & Upjohn Ab | Conjugates of a polypeptide and a biocompatible polymer |
EP1260582A1 (en) | 1995-09-29 | 2002-11-27 | Biovitrum Ab | Conjugates of factor IX and a biocompatible polymer |
EP0774261A2 (en) | 1995-11-10 | 1997-05-21 | Immuno Ag | Uses of von Willebrand Factor and pharmaceutical preparations |
US5763401A (en) | 1996-07-12 | 1998-06-09 | Bayer Corporation | Stabilized albumin-free recombinant factor VIII preparation having a low sugar content |
WO1998025969A1 (en) | 1996-12-13 | 1998-06-18 | Immuno Aktiengesellschaft | Von willebrand factor derivative and method for isolating proteins |
EP0977584A1 (en) | 1997-05-28 | 2000-02-09 | Baxter Aktiengesellschaft | PHARMACEUTICAL PREPARATION COMPRISING vWF PROPEPTIDE |
WO2000049047A1 (en) | 1999-02-19 | 2000-08-24 | Baxter Aktiengesellschaft | METHOD FOR PRODUCING A vWF PREPARATION |
US6586573B1 (en) | 1999-02-22 | 2003-07-01 | Baxter International Inc. | Albumin-free Factor VIII formulations |
WO2004075923A2 (en) | 2003-02-26 | 2004-09-10 | Nektar Therapeutics Al, Corporation | Polymer-factor viii moiety conjugates |
Non-Patent Citations (8)
Title |
---|
FISCHER ET AL., CMLS, vol. 53, 1997, pages 943 - 950 |
FISCHER ET AL., FEBS LETT., vol. 375, 1995, pages 259 - 262 |
ROUSSI ET AL., BLOOD COAG. FIBRINOL., vol. 9, 1998, pages 361 - 372 |
SCHLOKAT ET AL., BIOTECHNOL. APPL. BIOCHEM., vol. 24, 1996, pages 257 - 267 |
SCHWARZ ET AL., HAEMOPHILIA, vol. 4, 1998, pages 53 - 62 |
SCHWARZ ET AL., SEMIN. THROMB. HEMOST, vol. 28, 2002, pages 215 - 225 |
TURECEK ET AL., BLOOD, vol. 90, 1997, pages 3555 - 3567 |
TURECEK ET AL., SEMIN. THROMB. HEMOST., vol. 28, 2002, pages 149 - 160 |
Cited By (100)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7985839B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US8067543B2 (en) | 2006-03-31 | 2011-11-29 | Baxter International Inc. | Factor VIII polymer conjugates |
US8053561B2 (en) | 2006-03-31 | 2011-11-08 | Baxter International Inc. | Pegylated factor VIII |
US8003760B2 (en) | 2006-03-31 | 2011-08-23 | Baxter International Inc. | Factor VIII polymer conjugates |
US7985838B2 (en) | 2006-03-31 | 2011-07-26 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071727B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071724B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US7982010B2 (en) | 2006-03-31 | 2011-07-19 | Baxter International Inc. | Factor VIII polymer conjugates |
US11020458B2 (en) | 2006-03-31 | 2021-06-01 | Takeda Pharmaceutical Company Limited | Factor VIII polymer conjugates |
US8071726B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071725B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
US8071728B2 (en) | 2006-03-31 | 2011-12-06 | Baxter International Inc. | Factor VIII polymer conjugates |
WO2008025856A2 (en) | 2006-09-01 | 2008-03-06 | Novo Nordisk Health Care Ag | Modified glycoproteins |
JP2017141305A (en) * | 2006-12-15 | 2017-08-17 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | Factor viia-(poly) sialic acid conjugate having prolonged in vivo half-life |
JP2010512753A (en) * | 2006-12-15 | 2010-04-30 | バクスター・インターナショナル・インコーポレイテッド | Factor VIIa- (poly) sialic acid conjugate with extended in vivo half-life |
KR101503085B1 (en) * | 2006-12-27 | 2015-03-16 | 넥타르 테라퓨틱스 | Von willebrand factor- and factor viii-polymer conjugates having a releasable linkage |
JP2010514773A (en) * | 2006-12-27 | 2010-05-06 | ネクター セラピューティックス エイエル,コーポレイション | Polymer conjugates of von Willebrand factor and factor VIII having dissociable linkages |
US8133865B2 (en) | 2006-12-27 | 2012-03-13 | Nektar Therapeutics | von Willebrand factor- and factor VIII-polymer conjugates having a releasable linkage |
US9493544B2 (en) | 2006-12-27 | 2016-11-15 | Nektar Therapeutics | Von willebrand factor- and factor VIII-polymer conjugates having a releasable linkage |
WO2008082669A3 (en) * | 2006-12-27 | 2009-04-09 | Nektar Therapeutics Al Corp | Von willebrand factor- and factor viii-polymer conjugates having a releasable linkage |
US8349800B2 (en) | 2006-12-27 | 2013-01-08 | Nektar Therapeutics | Von Willebrand factor- and factor VIII-polymer conjugates having a releasable linkage |
AU2007340382B2 (en) * | 2006-12-27 | 2013-06-27 | Nektar Therapeutics | Von Willebrand Factor- and Factor VIII-polymer conjugates having a releasable linkage |
JP2010527588A (en) * | 2007-05-18 | 2010-08-19 | バクスター・インターナショナル・インコーポレイテッド | A method for producing mature VWF from a VWF propeptide. |
US9107902B2 (en) | 2007-06-13 | 2015-08-18 | Csl Behring Gmbh | Use of VWF stabilized FVIII preparations and of VWF preparations without FVIII for extravascular administration in the therapy and prophylactic treatment of bleeding disorders |
JP2010529155A (en) * | 2007-06-13 | 2010-08-26 | ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー | Use of VWF-stabilized FVIII and non-FVIII VWF formulations for extravascular administration in the treatment and prophylactic treatment of bleeding disorders |
WO2008151817A1 (en) | 2007-06-13 | 2008-12-18 | Csl Behring Gmbh | Use of vwf stabilized fviii preparations and of vwf preparations without fviii for extravascular administration in the therapy and prophylactic treatment of bleeding disorders |
EP2486936A1 (en) | 2007-06-13 | 2012-08-15 | CSL Behring GmbH | A composition comprising VWF and FVIII preparations for use in treating of bleeding disorders |
WO2009000522A1 (en) * | 2007-06-26 | 2008-12-31 | Baxter International Inc. | Hydrolysable polymeric fmoc-linker |
US8445544B2 (en) | 2007-06-26 | 2013-05-21 | Baxter International Inc. | Hydrolysable polymeric FMOC-linker |
EP2222329A1 (en) * | 2007-11-09 | 2010-09-01 | Baxter International Inc. | Modified recombinant factor viii and von willebrand factor and methods of use |
JP2011503101A (en) * | 2007-11-09 | 2011-01-27 | バクスター・インターナショナル・インコーポレイテッド | Modified recombinant factor VIII and von Willebrand factor and methods of use thereof |
WO2009135888A3 (en) * | 2008-05-06 | 2010-07-01 | Octapharma Ag | Complex comprising both heparin binding proteins and heparin-hydroxyalkyl starch conjugates |
WO2009135888A2 (en) * | 2008-05-06 | 2009-11-12 | Octapharma Ag | Complex |
US9290561B2 (en) | 2008-06-24 | 2016-03-22 | Csl Behring Gmbh | Factor VIII, von Willebrand factor or complexes thereof with prolonged in vivo half-life |
US8575104B2 (en) | 2008-06-24 | 2013-11-05 | Csl Behring Gmbh | Factor VIII, von willebrand factor or complexes thereof with prolonged in vivo half-life |
EP2865760A1 (en) | 2008-06-24 | 2015-04-29 | CSL Behring GmbH | Factor VIII, von Willebrand factor or complexes thereof with prolonged in vivo half-life |
JP2013500238A (en) * | 2008-08-01 | 2013-01-07 | バクスター・インターナショナル・インコーポレイテッド | Factor FVIII polymer conjugate |
WO2010014258A2 (en) * | 2008-08-01 | 2010-02-04 | Nektar Therapeutics Al, Corporation | Conjugates having a releasable linkage |
WO2010014708A3 (en) * | 2008-08-01 | 2010-04-22 | Baxter International Inc. | Factor viii polymer conjugates |
US8575102B2 (en) | 2008-08-01 | 2013-11-05 | Nektar Therapeutics | Conjugates having a releasable linkage |
WO2010014258A3 (en) * | 2008-08-01 | 2010-12-02 | Nektar Therapeutics Al, Corporation | Conjugates having a releasable linkage |
US9274122B2 (en) | 2008-10-21 | 2016-03-01 | Baxalta Incorporated | Methods for determining active ingredients in pro-drug PEG protein conjugates with releasable PEG reagents (in vitro de-pegylation) |
US10414793B2 (en) | 2009-07-27 | 2019-09-17 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US10772968B2 (en) | 2009-07-27 | 2020-09-15 | Lipoxen Technologies Limited | Glycopolysialylation of non-blood coagulation proteins |
US8809501B2 (en) | 2009-07-27 | 2014-08-19 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
US11564992B2 (en) | 2009-07-27 | 2023-01-31 | Takeda Pharmaceutical Company Limited | Nucleophilic catalysts for oxime linkage |
EP3093029A1 (en) | 2009-07-27 | 2016-11-16 | Baxalta GmbH | Blood coagulation protein conjugates |
US8637640B2 (en) | 2009-07-27 | 2014-01-28 | Baxter International Inc. | Blood coagulation protein conjugates |
US11040109B2 (en) | 2009-07-27 | 2021-06-22 | Takeda Pharmaceutical Company Limited | Blood coagulation protein conjugates |
WO2011017055A2 (en) | 2009-07-27 | 2011-02-10 | Baxter International Inc. | Blood coagulation protein conjugates |
US9795683B2 (en) | 2009-07-27 | 2017-10-24 | Lipoxen Technologies Limited | Glycopolysialylation of non-blood coagulation proteins |
US9731024B2 (en) | 2009-07-27 | 2017-08-15 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US9492555B2 (en) | 2009-07-27 | 2016-11-15 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US10576160B2 (en) | 2009-07-27 | 2020-03-03 | Baxalta Incorporated | Nucleophilic catalysts for oxime linkage |
US10350301B2 (en) | 2009-07-27 | 2019-07-16 | Baxalta Incorporated | Blood coagulation protein conjugates |
US8642737B2 (en) | 2010-07-26 | 2014-02-04 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
WO2012016131A1 (en) | 2010-07-30 | 2012-02-02 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
EP3505186A1 (en) | 2010-07-30 | 2019-07-03 | Baxalta GmbH | Nucleophilic catalysts for oxime linkage |
EP4023247A1 (en) | 2010-07-30 | 2022-07-06 | Takeda Pharmaceutical Company Limited | Nucleophilic catalysts for oxime linkage |
EP3673921A1 (en) | 2010-12-22 | 2020-07-01 | Baxalta GmbH | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
WO2012087838A1 (en) | 2010-12-22 | 2012-06-28 | Baxter International Inc. | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
EP3808378A1 (en) | 2011-05-27 | 2021-04-21 | Baxalta GmbH | Therapeutic proteins with increased half-life and methods of preparing same |
EP3412314A1 (en) | 2011-05-27 | 2018-12-12 | Baxalta GmbH | Therapeutic proteins conjugated to polysialic acid and methods of preparing same |
WO2012166622A1 (en) | 2011-05-27 | 2012-12-06 | Baxter International Inc. | Therapeutic proteins with increased half-life and methods of preparing same |
US20120316116A1 (en) * | 2011-06-10 | 2012-12-13 | Baxter Healthcare S.A. | Treatment of coagulation disease by administration of recombinant vwf |
US9272021B2 (en) * | 2011-06-10 | 2016-03-01 | Baxalta Incorporated | Treatment of coagulation disease by administration of recombinant VWF |
WO2013120939A1 (en) | 2012-02-15 | 2013-08-22 | Csl Behring Gmbh | Von willebrand factor variants having improved factor viii binding affinity |
US9458223B2 (en) | 2012-02-15 | 2016-10-04 | Csl Behring Gmbh | Von willebrand factor variants having improved factor VIII binding affinity |
EP4019049A1 (en) | 2012-05-16 | 2022-06-29 | Takeda Pharmaceutical Company Limited | Methods for preparing therapeutic protein-polymer conjugates |
WO2013173543A1 (en) | 2012-05-16 | 2013-11-21 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage |
WO2013173557A1 (en) | 2012-05-16 | 2013-11-21 | Baxter International Inc. | Nucleophilic catalysts for oxime linkage and use of nmr analyses of the same |
WO2014173873A1 (en) | 2013-04-22 | 2014-10-30 | Csl Behring Gmbh | Complex |
EP2796145A1 (en) * | 2013-04-22 | 2014-10-29 | CSL Behring GmbH | Complex |
US9878017B2 (en) | 2013-04-22 | 2018-01-30 | Csl Ltd. | Covalent complex of von Willebrand Factor and factor VIII, compositions, and uses relating thereto |
AU2014257637B2 (en) * | 2013-04-22 | 2018-07-19 | Csl Limited | Complex |
US11192936B2 (en) | 2014-01-10 | 2021-12-07 | Bioverativ Therapeutics Inc. | Factor VIII chimeric proteins and uses thereof |
US10829539B2 (en) | 2014-06-13 | 2020-11-10 | Csl Limited | Production of recombinant von Willebrand factor in a bioreactor |
US10253088B2 (en) | 2014-07-02 | 2019-04-09 | CSL Behring Lengnau AG | Modified von Willebrand Factor |
WO2016000039A1 (en) | 2014-07-02 | 2016-01-07 | Csl Limited | Modified von willebrand factor |
WO2016142288A1 (en) | 2015-03-06 | 2016-09-15 | Csl Behring Recombinant Facility Ag | Modified von willebrand factor having improved half-life |
US11155601B2 (en) | 2015-03-06 | 2021-10-26 | CSL Behring Lengnau AG | Modified von Willebrand factor having improved half-life |
US11564976B2 (en) | 2015-05-22 | 2023-01-31 | CSL Behring Lengnau AG | Methods for preparing modified von Willebrand Factor |
WO2016188907A1 (en) | 2015-05-22 | 2016-12-01 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for treating hemophilia |
WO2016188905A1 (en) | 2015-05-22 | 2016-12-01 | Csl Behring Recombinant Facility Ag | Methods for preparing modified von willebrand factor |
US10905747B2 (en) | 2015-05-22 | 2021-02-02 | CSL Behring Lengnau AG | Methods for preparing modified von Willebrand factor |
EP4089109A2 (en) | 2015-05-22 | 2022-11-16 | CSL Behring Lengnau AG | Methods for preparing modified von willebrand factor |
US10772936B2 (en) | 2015-05-22 | 2020-09-15 | CSL Behring Lengnau AG | Methods for preparing modified von Willebrand factor |
JP2015155469A (en) * | 2015-06-01 | 2015-08-27 | バクスター・インターナショナル・インコーポレイテッドBaxter International Incorp0Rated | Fviii-polymer conjugates |
WO2017117631A1 (en) | 2016-01-07 | 2017-07-13 | Csl Limited | Mutated truncated von willebrand factor |
US10806774B2 (en) | 2016-01-07 | 2020-10-20 | CSL Behring Lengnau AG | Mutated truncated von Willebrand Factor |
US10808023B2 (en) | 2016-01-07 | 2020-10-20 | CSL Behring Lengnau AG | Mutated von Willebrand factor |
WO2018087271A1 (en) | 2016-11-11 | 2018-05-17 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder |
WO2018087267A1 (en) | 2016-11-11 | 2018-05-17 | Csl Behring Recombinant Facility Ag | Truncated von willebrand factor polypeptides for treating hemophilia |
US11890327B2 (en) | 2016-11-11 | 2024-02-06 | CSL Behring Lengnau AG | Truncated von Willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder |
US11814421B2 (en) | 2016-11-11 | 2023-11-14 | CSL Behring Lengnau AG | Truncated von Willebrand Factor polypeptides for treating hemophilia |
US12016904B2 (en) | 2017-07-07 | 2024-06-25 | Takeda Pharmaceutical Company Limited | Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF |
US11529395B2 (en) | 2017-07-07 | 2022-12-20 | Takeda Pharmaceutical Company Limited | Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF |
WO2019038350A1 (en) | 2017-08-23 | 2019-02-28 | Csl Behring Gmbh | Method for virus filtration of von willebrand factor |
JP2018115170A (en) * | 2018-03-02 | 2018-07-26 | バクスアルタ ゲーエムベーハー | Factor fviii-polymer conjugates |
US12030925B2 (en) | 2018-05-18 | 2024-07-09 | Bioverativ Therapeutics Inc. | Methods of treating hemophilia A |
Also Published As
Publication number | Publication date |
---|---|
CN101163506B (en) | 2012-09-26 |
US8357779B2 (en) | 2013-01-22 |
JP2012126750A (en) | 2012-07-05 |
MX2007007877A (en) | 2007-08-21 |
BRPI0519562A2 (en) | 2009-01-27 |
CN101163506A (en) | 2008-04-16 |
JP2008525491A (en) | 2008-07-17 |
PT1835938E (en) | 2013-11-06 |
AU2005322067B8 (en) | 2012-07-26 |
US7884075B2 (en) | 2011-02-08 |
US20130190242A1 (en) | 2013-07-25 |
US20120135462A1 (en) | 2012-05-31 |
EP1835938B1 (en) | 2013-08-07 |
IL183634A (en) | 2012-02-29 |
AU2005322067B2 (en) | 2012-07-05 |
EP1835938A2 (en) | 2007-09-26 |
ES2434035T3 (en) | 2013-12-13 |
AU2005322067A1 (en) | 2006-07-06 |
US20060160948A1 (en) | 2006-07-20 |
HK1105276A1 (en) | 2008-02-06 |
IL183634A0 (en) | 2007-09-20 |
JP5022231B2 (en) | 2012-09-12 |
DK1835938T3 (en) | 2013-11-04 |
US20110111455A1 (en) | 2011-05-12 |
KR20070092754A (en) | 2007-09-13 |
PL1835938T3 (en) | 2014-01-31 |
US8076463B2 (en) | 2011-12-13 |
WO2006071801A3 (en) | 2007-08-16 |
US8835388B2 (en) | 2014-09-16 |
JP5715983B2 (en) | 2015-05-13 |
CA2591852A1 (en) | 2006-07-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8835388B2 (en) | Polymer von Willebrand factor-conjugates | |
KR101879838B1 (en) | Factor ⅷ polymer conjugates | |
US20130296243A1 (en) | Modified recombinant factor viii and von willebrand factor and methods of use | |
AU2009305612B2 (en) | Modified blood factors comprising a low degree of water soluble polymer | |
WO2007126808A1 (en) | Pegylated factor viii | |
JP2015155469A (en) | Fviii-polymer conjugates | |
JP2018115170A (en) | Factor fviii-polymer conjugates |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200580044598.3 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 183634 Country of ref document: IL |
|
WWE | Wipo information: entry into national phase |
Ref document number: 4610/DELNP/2007 Country of ref document: IN |
|
ENP | Entry into the national phase |
Ref document number: 2591852 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/a/2007/007877 Country of ref document: MX Ref document number: 2007548552 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005322067 Country of ref document: AU |
|
ENP | Entry into the national phase |
Ref document number: 2005322067 Country of ref document: AU Date of ref document: 20051223 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020077017261 Country of ref document: KR Ref document number: 2005855442 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: PI0519562 Country of ref document: BR |