WO2012088123A1 - Conjugués polymère-fraction de facteur vii - Google Patents

Conjugués polymère-fraction de facteur vii Download PDF

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WO2012088123A1
WO2012088123A1 PCT/US2011/066176 US2011066176W WO2012088123A1 WO 2012088123 A1 WO2012088123 A1 WO 2012088123A1 US 2011066176 W US2011066176 W US 2011066176W WO 2012088123 A1 WO2012088123 A1 WO 2012088123A1
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composition
factor vii
moiety
water
daltons
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PCT/US2011/066176
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English (en)
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Mary J. Bossard
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Nektar Therapeutics
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)

Definitions

  • the present invention relates generally to conjugates comprising a Factor VII moiety (i.e., a moiety having Factor VII activity) and a polymer.
  • the invention relates to compositions comprising the conjugates, methods for synthesizing the conjugates, methods for delivering the conjugates, and methods for treating patients.
  • Hemostasis is the process of arresting the outflow of blood from an injured blood vessel.
  • the hemostatic process is critically important for continued survival. Defects in the hemostatic process can result in, for example, the inability to effectively form blood clots that serve to stop the loss of blood following vascular injury.
  • individuals who suffer from an inability to form blood clots are called hemophiliacs.
  • hemophiliacs Of particular concern for hemophiliacs is the life-threatening risk that once started, bleeding will never cease.
  • Factor VII The main role of Factor VII (FVII) is to initiate the process of coagulation in conjunction with tissue factor. Tissue factor is found on the outside of blood vessels - normally not exposed to the bloodstream. Upon vessel injury, tissue factor is exposed to the blood and circulating Factor VII. Once bound to tissue factor, Factor VII is activated to Factor Vila by different proteases, among which are thrombin (factor Ila), Factor Xa, Factor IXa, Factor Xlla, and the FVIIa-TF complex itself. The most important substrates for Factor VIIa-TF are Factor X and Factor IX.
  • a commercial form of Factor VII is available as NovoSeven RT® brand of coagulation Factor Vila [recombinant] room temperature stable (Novo Nordisk, Princeton NJ).
  • blood-derived sources since the latter involves the risk of transmitting viruses and/or other diseases.
  • purity is often higher with the recombinant source, thereby avoiding potential problems arising from administering unwanted blood factors and other proteins generally present in blood-derived sources.
  • compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art.
  • a conjugate comprising a Factor VII moiety covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the molecular weight of the water-soluble polymer is greater than 5,000 Dal tons and less than about 150,000 Daltons.
  • a conjugate comprising a Factor VII moiety covalently attached at an amino acid residue, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the amino acid residue is not attached, either directly or though the spacer moiety, via a -CH 2 -C(0)-0-, -N(H)-C(0)CH 2 -0-, -C(0)-N(H)-, -N(H)-C(0)-CH 2 -0-, -C(0)-CH 2 -0-, -C(0)-CH 2 -CH 2 -C(0)-0-, diazo, or triazine linkage.
  • a conjugate comprising a Factor VII moiety covalently attached, either directly or through a spacer moiety comprising of one or more atoms to a non-linear water-soluble polymer.
  • a composition comprising a plurality of conjugates, wherein at least about 80% of all conjugates in the composition are each comprised of a Factor VII moiety covalently attached to one, two, three or four water-soluble polymers, and further wherein for each water-soluble polymer in the conjugate, the Factor VII moiety is attached either directly or through a spacer moiety comprised of one or more atoms.
  • the compositions encompass all types of formulations and in particular those that are suited for injection such as powders that can be reconstituted, as well as liquids (e.g., suspensions and solutions).
  • a method for preparing a conjugate comprising adding a polymeric reagent composition to a Factor VII composition under conditions sufficient to result in a conjugate comprising a Factor VII moiety covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer.
  • a method for delivering a conjugate comprising administering to the patient a composition comprising a conjugate as described herein.
  • the step of administering the conjugate can be effected by injection (e.g., intramuscular injection, intravenous injection, subcutaneous injection, and so forth) or other approach.
  • PEG polyethylene glycol
  • poly( ethylene glycol) as used herein, are interchangeable.
  • PEGs for use in accordance with the invention comprise the following structure "-(OCH 2 CH 2 ) n - M where (n) is 2 to 4000.
  • PEG also includes "-CH 2 CH 2 -0(CH 2 CH 2 0) n -CH 2 CH 2 -” and “-(OCH 2 CH 2 ) tone0-,” depending upon whether or not the terminal oxygens have been displaced.
  • PEG includes structures having various terminal or "end capping" groups and so forth.
  • PEG also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2 CH 2 - repeating subunits.
  • the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below.
  • end-capped and “terminally capped” are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.
  • the end-capping moiety comprises a hydroxy or Ci -20 alkoxy group, more preferably a CMO alkoxy group, and still more preferably a Ci_ 5 alkoxy group.
  • examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like.
  • the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH 3 0(CH 2 CH 2 0) n -] or not [e.g., "CH 3 " in CH3(OCH2CH 2 ) n -]
  • the end-capping group can also be a silane.
  • the end-capping group can also advantageously comprise a detectable label.
  • the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector.
  • suitable detectors include photometers, films, spectrometers, and the like.
  • the end-capping group can also advantageously comprise a phospholipid.
  • Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines.
  • Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin.
  • Non-naturally occurring with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature.
  • a non-naturally occurring polymer may, however, contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.
  • water soluble as in a "water-soluble polymer” is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35%) (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85%o (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water.
  • Molecular weight in the context of a water-soluble polymer can be expressed as either a number-average molecular weight or a weight-average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight-average molecular weight. Both molecular weight determinations, number-average and weight-average, can be measured using gel permeation chromatography or other liquid chromatography techniques.
  • the polymers of the invention are typically polydisperse (i.e., number-average molecular weight and weight- average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.
  • references will at times be made to a single water-soluble polymer having either a weight-average molecular weight or number-average molecular weight; such references will be understood to mean that the single-water soluble polymer was obtained from a composition of water-soluble polymers having the stated molecular weight.
  • active when used in conjunction with a particular functional group, refer to a reactive functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive” or “inert” group).
  • spacer moiety refers to an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a water-soluble polymer and a Factor VII moiety or an electrophile or nucleophile of a Factor VII moiety.
  • the spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage.
  • Alkyl refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl” includes cycloalkyl as well as cycloalkyl ene-containing alkyl.
  • “Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, /-butyl, and t- butyl.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.
  • Cycloalkylene refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.
  • Alkoxy refers to an -O- group, wherein R is alkyl or substituted alkyl, preferably Ci -6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).
  • substituted refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C 3 _s cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.
  • “Substituted aryl” is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).
  • Noninterfering substituents are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.
  • Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms.
  • Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl” includes heteroaryl.
  • Heteroaryl is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
  • Heterocycle or “heterocyclic” means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.
  • Substituted heteroaryl is heteroaryl having one or more noninterfering groups as substituents.
  • Substituted heterocycle is a heterocycle having one or more side chains formed from noninterfering substituents.
  • An "organic radical” as used herein shall include alkyl, substituted alkyl, aryl and substituted aryl.
  • Electrophile and "electrophilic group” refer to an ion or atom or collection of atoms, that may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.
  • Nucleophile and “nucelophilic group” refers to an ion or atom or collection of atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic center or with an electrophile.
  • a "physiologically cleavable” or “hydrolyzable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions.
  • the tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms.
  • Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and
  • An "enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.
  • hydrolytically stable linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time.
  • hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like.
  • a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
  • “Pharmaceutically acceptable excipient” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • Therapeutically effective amount is used herein to mean the amount of a polymer-Factor VII moiety conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated Factor VII moiety) in the bloodstream or in the target tissue.
  • the precise amount will depend upon numerous factors, e.g., the particular Factor VII moiety, the components and physical characteristics of the therapeutic composition, intended patient population, mode of delivery, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
  • Multi-functional means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different.
  • Multi-functional polymeric reagents will typically contain from about 3-100 functional groups, or from 3-50 functional groups, or from 3-25 functional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.
  • Fractor VII moiety refers to a moiety having Factor
  • the Factor VII moiety will also have at least one electrophilic group or nucleophilic group suited for reaction with a polymeric reagent. Typically, although not necessarily, the Factor VII moiety is a protein.
  • the term "Factor VII moiety” encompasses both the Factor VII moiety prior to conjugation as well as the Factor VII moiety residue following conjugation. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has Factor VII activity.
  • a protein comprising an amino acid sequence corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 are both Factor VII moieties, as well as any protein or polypeptide substantially homologous thereto, whose biological properties result in the activity of Factor VII.
  • the term “Factor VII moiety” includes proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations.
  • the term “Factor VII moiety” also includes derivatives having from 1 to 6 additional glycosylation sites, derivatives having at least one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and derivatives having an amino acid sequence which includes at least one glycosylation site.
  • substantially homologous means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more
  • sequences having greater than 95 percent homology, equivalent biological properties (although potentiality different degrees of activity), and equivalent expression characteristics are considered substantially homologous.
  • sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics are considered substantial equivalents.
  • Exemplary Factor VII moieties for use herein include those proteins having a sequence that is substantially homologous to SEQ ID NO: 1.
  • fragment means any protein or polypeptide having the amino acid sequence of a portion of a Factor VII moiety that retains some degree of Factor VII activity. Fragments include proteins or polypeptides produced by proteolytic degradation of the Factor VII protein or produced by chemical synthesis by methods routine in the art. Determining whether a particular fragment has the biological activity of Factor VII can carried out by conventional, well known tests utilized for such purposes on one or more species of mammals. An appropriate test which can be utilized to demonstrate such biological activity is described herein.
  • a "deletion variant" of a Factor VII moiety is peptide or protein in which one amino acid residue of the Factor VII moiety has been deleted and the amino acid residues preceding and following the deleted amino acid residue are connected via an amide bond (except in instances where the deleted amino acid residue was located on a terminus of the peptide or protein).
  • Deletion variants include instances where only a single amino acid residue has been deleted, as well as instances where two amino acids are deleted, three amino acids are deleted, four amino acids are deleted, and so forth. Each deletion variant must, however, retain some degree of Factor VII activity.
  • a "substitution variant" of a Factor VII moiety is peptide or protein in which one amino acid residue of the Factor VII moiety has been deleted and a different amino acid residue has taken its place.
  • Substitution variants include instances where only a single amino acid residue has been substituted, as well as instances where two amino acids are substituted, three amino acids are substituted, four amino acids are substituted, and so forth. Each substitution variant must, however, have some degree of Factor VII activity.
  • An "addition variant" of a Factor VII moiety is peptide or protein in which one amino acid residue of the Factor VII moiety has been added into an amino acid sequence and adjacent amino acid residues are attached to the added amino acid residue by way of amide bonds (except in instances where the added amino acid residue is located on a terminus of the peptide or protein, wherein only a single amide bond attaches the added amino acid residue).
  • Addition variants include instances where only a single amino acid residue has been added, as well as instances where two amino acids are added, three amino acids are added, four amino acids are added, and so forth. Each addition variant must, however, have some degree of Factor VII activity.
  • patient refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of an active agent (e.g., conjugate), and includes both humans and animals.
  • an active agent e.g., conjugate
  • substantially means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater of the condition.
  • Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
  • Tyrosine is Tyr or Y; Histidine is His or H; Glutarnine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • a conjugate comprising a Factor VII moiety covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer.
  • the conjugates of the invention will have one or more of the following features.
  • the term "Factor VII moiety” shall include the Factor VII moiety prior to conjugation as well as to the Factor VII moiety following attachment to a water-soluble polymer. It is understood, however, that when the Factor VII moiety is attached to a nonpeptidic water-soluble polymer, the Factor VII moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer (or spacer moiety that is attached to the polymer). Often, this slightly altered form of the Factor VII moiety attached to another molecule is referred to a "residue" of the Factor VII moiety.
  • the Factor VII moiety can be derived from either non-recombinant methods or from recombinant methods and the invention is not limited in this regard.
  • the Factor VII moiety can be derived from human sources or from animal sources.
  • the Factor VII moiety can be derived non-recombinantly.
  • the Factor VII moiety can be derived non-recombinantly.
  • Factor VII moiety can be obtained from blood-derived sources wherein fractions of human plasma containing enriched levels of Factor VII can be isolated.
  • the Factor VII moiety can be derived from recombinant methods.
  • the Factor VII moiety can be prepared in accordance with the recombinant methods set forth in U.S. Patent No. 4,784,950.
  • native Factor VII is a single chain glycoprotein of about
  • Exemplary recombinant methods used to prepare a Factor VII moiety can be briefly described. Such methods involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria such as E. coli, yeast such as Saccharomyces cerevisiae, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art.
  • nucleic acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or added in-frame with the coding sequence, thereby producing a fusion protein comprised of the desired polypeptide and a polypeptide suited for binding.
  • Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein.
  • binding moieties e.g., antibodies
  • the recombinant polypeptide can also be identified and purified by lysing the host cells, separating the polypeptide, e.g., by size exclusion chromatography, and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. In one or more embodiments of the present invention, however, it is preferred that the Factor VII moiety is not in the form of a fusion protein.
  • the Factor VII moiety can be unglycosylated or glycosylated and either may be used. That is, the Factor VII moiety can be unglycosylated or the Factor VII moiety can be glycosylated. In one or more embodiments of the invention, it is preferred that the Factor VII moiety is glycosylated.
  • the moiety having Factor VII activity can advantageously be modified to include one or more amino acid residues such as, for example, lysine, cysteine and/or arginine, in order to provide facile attachment of a polymer to an atom within an amino acid.
  • Factor VII moiety can be modified to include a non-naturally occurring amino acid residue.
  • Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art. Reference is made to J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1992).
  • Factor VII moiety can advantageously be modified to include attachment of a functional group (other than through addition of a functional
  • the Factor VII moiety can be modified to include a thiol group.
  • the Factor VII moiety can be modified to include an N-terminal alpha carbon.
  • the Factor VII moiety can be modified to include one or more carbohydrate moieties. Factor VII moieties modified to contain an aminoxy, aldehyde or other functional group can also be used.
  • Factor VII moieties include the following: Factor
  • Factor VII Factor Vila
  • truncated versions of Factor VII hybrid proteins, and peptide mimetics having Factor VII activity.
  • Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of Factor VII activity can also serve as a Factor VII moiety.
  • a one-stage clotting assay essentially as described in W092/15686 can be used where a sample to be tested is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 ⁇ is incubated with 100 ⁇ of FVII deficient plasma and 200 ⁇ of thromboplastin C containing 10 mM Ca ++ . Clotting times are measured and compared to a standard curve using a pool of citrated normal human plasma in serial dilution.
  • each conjugate comprises a Factor VII moiety attached to a water-soluble polymer.
  • the water-soluble polymer is nonpeptidic, nontoxic, non-naturally occurring and biocompatible.
  • biocompatibility a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such a Factor VII moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician.
  • non-immunogenicity a substance is considered nonimmunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. It is particularly preferred that the water-soluble polymer is biocompatible and nonimmunogenic.
  • 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(olefmic alcohol), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(ct-hydroxy acid), poly( vinyl alcohol), polyphosphazene,
  • 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(olefmic alcohol), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(ct-hydroxy acid), poly( vinyl alcohol
  • polyoxazoline poly(N-acryloylmorpholine), and combinations of any of the foregoing.
  • the polymer is not limited in a particular structure and can be linear (e.g., alkoxy PEG or bifunctional PEG), or non-linear such as branched, forked, multi-armed (e.g., PEGs attached to a polyol core), and dendritic.
  • 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.
  • activated PEG and other activated water-soluble polymers are activated with a suitable activating group appropriate for coupling to a desired site on the Factor VII moiety.
  • a polymeric reagent will possess a reactive group for reaction with the Factor VII moiety.
  • Representative polymeric reagents and methods for conjugating these polymers to an active moiety are known in the art and further described in Zalipsky, S., et al., "Use of Functionalized Poly (Ethylene Glycols) for
  • the weight-average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons.
  • Exemplary ranges include weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.
  • PEGs having a molecular weight in one or more of these ranges are preferred.
  • Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1 ,000 Daltons, about 1 ,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 1 1,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Dal
  • Branched versions of the water-soluble polymer e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers
  • the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight-average molecular weight of less than about 6,000 Daltons.
  • PEGs When used as the polymer, PEGs will typically comprise a number of
  • (OCH 2 CH2) monomers [or (CH 2 CH 2 0) monomers, depending on how the PEG is defined].
  • the number of repeating units is identified by the subscript in "(OCH 2 CH 2 ) n .”
  • the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1 30, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1 ,200 to about 1 ,900.
  • n the number of repeating units
  • a conjugate comprising a Factor VII moiety covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the molecular weight of the water-soluble polymer is greater than 5,000 Daltons and less than about 150,000 Daltons.
  • One particularly preferred polymer for use in the invention is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower Ci -6 alkoxy group, although a hydro yl group can also be used.
  • a relatively inert group such as a lower Ci -6 alkoxy group
  • mPEG methoxy-PEG
  • mPEG methoxy-PEG
  • mPEG is a linear form of PEG wherein one terminus of the polymer has a methoxy (- OCH 3 ) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.
  • free or unbound PEG is a linear polymer terminated at each end with hydroxyl groups:
  • PEG-OH methoxy-PEG-OH
  • mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group.
  • the structure of mPEG is given below.
  • Multi-armed or branched PEG molecules such as those described in U.S.
  • Patent No. 5,932,462 can also be used as the PEG polymer.
  • PEG can have the structure:
  • poly a and poly b are PEG backbones (either the same or different), such as methoxy poly(ethylene glycol);
  • R" is a non-reactive moiety, such as H, methyl or a PEG backbone
  • the branched PEG polymer is methoxy poly(ethylene glycol) disubstituted lysine.
  • the reactive ester functional group of the disubstituted lysine may be further modified to form a functional group suitable for reaction with the target group within the Factor VII moiety.
  • the PEG can comprise a forked PEG.
  • An example of a forked PEG is an example of a forked
  • PEG is represented by the following structure:
  • X is a spacer moiety of one or more atoms and each Z is an activated terminal group linked to CH by a chain of atoms of defined length.
  • PCT/US99/05333 discloses various forked PEG structures capable of use in one or more embodiments of the present invention.
  • the chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof.
  • the PEG polymer may comprise a pendant PEG molecule having reactive groups, such as carboxyl, covalently attached along the length of the PEG rather than at the end of the PEG chain.
  • the pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.
  • the polymer can also be prepared with one or more weak or degradable linkages (such as a hydrolytically degradable linkage) in the polymer, including any of the above described polymers.
  • PEG can be prepared with ester linkages in the polymer that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight:
  • hydrolytically degradable linkages useful as a degradable linkage within a polymer backbone, include: carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al.
  • phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between a formate and an alcohol; amide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of another PEG chain; urethane linkages formed from reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example
  • Such optional features of the polymer conjugate may provide for additional control over the final desired pharmacological properties of the conjugate upon administration.
  • a large and relatively inert conjugate e.g., having one or more high molecular weight PEG chains attached to a Factor VII moiety, for example, one or more PEG chains having a molecular weight greater than about 10,000, wherein the conjugate possesses essentially no bioactivity
  • a large and relatively inert conjugate e.g., having one or more high molecular weight PEG chains attached to a Factor VII moiety, for example, one or more PEG chains having a molecular weight greater than about 10,000, wherein the conjugate possesses essentially no bioactivity
  • the properties of the conjugate can be more effectively tailored to balance the bioactivity of the conjugate over time.
  • polymeric reagent generally refers to an entire molecule, which can comprise a water-soluble polymer segment and a functional group.
  • a conjugate of the invention comprises a water-soluble polymer covalently attached (either directly or through a spacer moiety) to a Factor VII moiety.
  • a water-soluble polymer covalently attached to a Factor VII moiety.
  • there will be one to four water-soluble polymers covalently attached to a Factor VII moiety wherein for each water-soluble polymer, the water soluble polymer can be attached either directly to the Factor VII moiety or through a spacer moiety).
  • the conjugate may have 1 , 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers individually attached to a Factor VII moiety.
  • the conjugate may include not more than 8 water-soluble polymers individually attached to a Factor VII moiety, not more than 7 water-soluble polymers individually attached to a Factor VII moiety, not more than 6 water-soluble polymers individually attached to a Factor VII moiety, not more than 5 water-soluble polymers individually attached to a Factor VII moiety, not more than 4 water-soluble polymers individually attached to a Factor VII moiety, not more than 3 water-soluble polymers individually attached to a Factor VII moiety, and not more than 2 water-soluble polymers individually attached to a Factor VII moiety.
  • the particular linkage between the Factor VII moiety and the polymer (or the spacer moiety that is attached to the polymer) depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular Factor VII moiety, the available functional groups within the Factor VII moiety (either for attachment to a polymer or conversion to a suitable attachment site), the possible presence of additional reactive functional groups within the Factor VII moiety, and the like. [0095] In one or more embodiments of the invention, the linkage between the Factor
  • the linkage does not result from reaction of the polymeric reagent bearing triazine, acetyl, hydrazine, diazonium, amino, or succinimidyl ester functional group with the Factor VII moiety.
  • the linkage is not a carbamate linkage and not a carbamide linkage, and furthermore, that no linkage is formed based on the reaction of a polymer derivative bearing an isocyanate or isothiocyanate species to a Factor VII moiety,
  • a preferred hydrolytically stable linkage is an amide.
  • An amide can be readily prepared by reaction of a carboxyl group contained within the Factor VII moiety (e.g., the terminal carboxyl of a peptidic moiety having Factor VII activity) with an amino-terminated polymer.
  • the linkage of the water-soluble polymer (and any spacer moiety) is "cleavable.” That is, the water-soluble polymer (and any spacer moiety) cleaves (either through hydrolysis, enzymatic processes, or otherwise), thereby resulting in the native or unconjugated Factor VII moiety.
  • cleavable linkages result in the polymer (and any spacer moiety) detaching from the Factor VII moiety in vivo without leaving any fragment of the water-soluble polymer (and any spacer moiety).
  • Exemplary degradable linkages include carbonate, carboxylate ester, phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, and orthoesters.
  • Such linkages can be readily prepared by appropriate modification of either the Factor VII moiety (e.g., the carboxyl group C terminus of the protein or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein) and/or the polymeric reagent using coupling methods commonly employed in the art.
  • Most preferred, however, are hydrolyzable linkages that are readily formed by reaction of a suitably activated polymer with a non-modified functional group contained within the moiety having Factor VII activity.
  • a conjugate comprising a Factor VII moiety covalently attached at an amino acid residue, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the amino acid residue is not attached, either directly or though the spacer moiety, via a CH 2 -C(0)-0-, -N(H)-C(0)CH 2 -0-, -C(0)-N(H)-,
  • the conjugates may or may not possess a measurable degree of Factor VII activity. That is to say, a conjugate in accordance with the invention will possesses anywhere from about 0% to about 100% or more of the bioactivity of the unmodified parent Factor VII moiety.
  • compounds possessing little or no Factor VII activity typically contain a hydrolyzable linkage connecting the polymer to the moiety, so that regardless of the lack of activity in the conjugate, the active parent molecule (or a derivative thereof having Factor VII activity) is released upon aqueous- induced cleavage of the linkage.
  • Such activity may be determined using a suitable in-vivo or in-vitro model, depending upon the known activity of the particular moiety having Factor VII activity employed.
  • each water-soluble polymer portion is facilitated through the use of physiologically cleavable and/or enzymatically degradable linkages such as urethane, amide, carbonate or ester-containing linkages.
  • physiologically cleavable and/or enzymatically degradable linkages such as urethane, amide, carbonate or ester-containing linkages.
  • clearance of the conjugate via cleavage of individual water-soluble polymer(s)] can be modulated by selecting the polymer molecular size and the type functional group that would provide the desired clearance properties.
  • One of ordinary skill in the art can determine the proper molecular size of the polymer as well as the cleavable functional group.
  • one of ordinary skill in the art using routine experimentation, can determine a proper molecular size and cleavable functional group by first preparing a variety of polymer-Factor VII conjugates with different polymer weights and cleavable functional groups, and then obtaining the clearance profile for each conjugate by administering the conjugate to a patient and taking periodic blood and/or urine sampling. Once a series of clearance profiles have been obtained for each tested conjugate, a conjugate having the desired clearance can be identified.
  • the conjugate will typically possess a measurable degree of Factor VII activity.
  • such conjugates are typically characterized as having a bioactivity satisfying one or more of the following percentages relative to that of the unconjugated Factor VII moiety: at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 100%, and more than 105% (when measured in a suitable model, such as those presented here and/or well known in the art).
  • conjugates having a hydrolytically stable linkage e.g., an amide linkage
  • the Factor VII moiety is expected to share (at least in part) an amino acid sequence similar or related to native Factor VII.
  • native Factor VII an amino acid sequence similar or related to native Factor VII.
  • the description provided herein for native Factor VII is often applicable to Factor Vila, as well as fragments, deletion variants, substitution variants or addition variants of any of the foregoing.
  • Amino groups on Factor VII moieties can provide a point of attachment between the Factor VII moiety and the water-soluble polymer.
  • Native Factor VII comprises 26 lysine residues, each having an ⁇ -amino group that may be available for conjugation, as well as one amino terminus.
  • exemplary attachment points of such Factor VII moieties include attachment at an amino acid (through the amine-containing side chain of lysine) at any one or more of positions 26, 38, 79, 92, 98, 122, 145, 162, 169, 197, 208, 217, 221 , 257, 259, 376, and 449.
  • the N-terminal amine of any protein having Factor VII activity can also serve as a point of attachment.
  • Conjugation of a polymeric reagent to an amine group of a Factor VII moiety can be accomplished by a variety of techniques.
  • a Factor VII moiety can be conjugated to a polymeric reagent functionalized with a succinimidyl derivative (or other activated ester group, wherein approaches similar to those described for a succinimidyl derivative can be used for other activated ester group-containing polymeric reagents).
  • the polymeric reagent bearing a succinimidyl group can be attached to the Factor VII moiety in aqueous media at a pH of 7.0 to 9.0, although different reaction conditions (e.g., a lower pH such as 6 to 7, or different temperatures and/or less than 15 °C) can result in the attachment of a polymer to a different location on the Factor VII moiety.
  • an amide linkage can be formed by reacting an amine-terminated non-peptidic, water-soluble polymer with a Factor VII moiety bearing an aldehyde or an activated carboxylic acid group.
  • An exemplary conjugate comprises the following structure
  • (n) is an integer having a value of from 2 to 3400;
  • X is a spacer moiety, preferably one of methylene (“-CH 2 -”), ethylene (“-CH2CH2-”) and propylene ("-CH 2 CH 2 CH 2 -");
  • R 1 is an organic radical, preferably H or methyl ("-CH 3 ");
  • F7 is a Factor VII moiety.
  • Typical of another approach useful for conjugating the Factor VII moiety to a polymeric reagent is the use of a reductive amination reaction to conjugate a primary amine of a Factor VII moiety with a polymer functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate and aldehyde hydrate).
  • the primary amine from the Factor VII moiety reacts with the carbonyl group of the aldehyde or ketone (or the corresponding hydroxy-containing group of a hydrated aldehyde or ketone), thereby forming a Schiff base.
  • the Schiff base in turn, can then be reductively converted to a stable conjugate through use of a reducing agent such as sodium borohydride.
  • a reducing agent such as sodium borohydride.
  • Selective reactions are possible, particularly with a polymer functionalized with a ketone or an alpha-methyl branched aldehyde and/or under specific reaction conditions (e.g., reduced pH).
  • Carboxyl groups represent another functional group that can serve as a point of attachment on the Factor VII moiety. Structurally, the conjugate will comprise the following:
  • F7-C-X-POLY where F7 and the adjacent carbonyl group corresponds to the carboxyl-containing Factor VII moiety, X is a spacer moiety, preferably a heteroatom selected from O, N(H), and S, and POLY is a water-soluble polymer such as PEG, optionally terminating in an end-capping moiety.
  • the C(0)-X linkage results from the reaction between a polymeric derivative bearing a terminal functional group and a carboxyl-containing Factor VII moiety.
  • the specific linkage will depend on the type of functional group utilized. If the polymer is end-functionalized or "activated" with a hydroxyl group, the resulting linkage will be a carboxylic acid ester and X will be O. If the polymer backbone is functionalized with a thiol group, the resulting linkage will be a thioester and X will be S.
  • the C(0)X moiety, and in particular the X moiety may be relatively more complex and may include a longer linkage structure.
  • Polymeric reagents containing a hydrazide moiety are also useful for conjugation at a carbonyl.
  • a carbonyl moiety can be introduced by reducing any carboxylic acids (e.g., the C-terminal carboxylic acid) and/or by providing glycosylated or glycated (wherein the added sugars have a carbonyl moiety) versions of the Factor VII moiety.
  • carboxylic acids e.g., the C-terminal carboxylic acid
  • glycosylated or glycated wherein the added sugars have a carbonyl moiety
  • any polymeric reagent comprising an activated ester e.g., a succinimidyl group
  • any polymeric reagent comprising an activated ester can be converted to contain a hydrazide moiety by reacting the polymeric reagent comprising the activated ester with hydrazine (NH 2 -NH 2 ) or tert-butyl carbazate [NH 2 NHC0 2 C(CH 3 ) 3 ].
  • the hydrazone linkage can be reduced using a suitable reducing agent.
  • Thiol groups contained within the Factor VII moiety can serve as effective sites of attachment for the water-soluble polymer.
  • cysteine residues provide thiol groups when the Factor VII moiety is a protein.
  • the thiol groups in such cysteine residues can be reacted with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl polymer or other derivative, as described in U.S. Patent No. 5,739,208 and in International Patent Publication No. WO 01/62827.
  • variable (n) represents the number of repeating monomenc units and "-S-F7" represents the Factor VII moiety following conjugation to the water-soluble polymer. While each polymeric portion [e.g., (OCH 2 CH 2 ) n or (CH 2 CH 2 0) n ] presented in Table 3 terminates in a "CI3 ⁇ 4" group, other groups (such as H and benzyl) can be substituted therefor.
  • the corresponding maleamic acid form(s) of the water-soluble polymer can also react with the Factor VII moiety.
  • the maleimide ring will "open” to form the corresponding maleamic acid.
  • the maleamic acid in turn, can react with an amine or thiol group of a Factor VII moiety.
  • Exemplary maleamic acid-based reactions are
  • POLY represents the water-soluble polymer
  • F7 represents the Factor VII moiety
  • a representative conjugate in accordance with the invention can have the following structure:
  • POLY-Lo , i-C(0)Z-Y-S-S-F7 wherein POLY is a water-soluble polymer, L is an optional linker, Z is a heteroatom selected from the group consisting of O, NH, and S, and Y is selected from the group consisting of C 2 -io alkyl, C2-10 substituted alkyl, aryl, and substituted aryl, and F7 is a Factor VII moiety.
  • Polymeric reagents that can be reacted with a Factor VII moiety and result in this type of conjugate are described in U.S. Patent Application Publication No. 2005/0014903.
  • the attachment between the Factor VII moiety and water-soluble polymer can be direct, wherein no intervening atoms are located between the Factor VII moiety can the polymer, or indirect, wherein one or more atoms are located between the Factor VII moiety and polymer.
  • a "spacer moiety" serves as a link between the Factor VII moiety and the water-soluble polymer.
  • the one or more atoms making up the spacer moiety can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the spacer moiety can comprise an amide, secondary amine, carbamate, thioether, and/or disulfide group.
  • Nonlimiting examples of specific spacer moieties include those selected from the group consisting of -0-, -S-, -S-S-, - C(O)-, -C(0)-NH-, -NH-C(0)-NH-, -0-C(0)-NH-, -C(S)-, -CH 2 -, -CH 2 -CH 2 -,
  • R 6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is zero to six, and (j) is zero to 20.
  • R 6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is zero to six, and (j) is zero to 20.
  • Other specific spacer moieties have the following structures:
  • any of the above spacer moieties may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., - (CH 2 CH 2 0)i- 2 o].
  • the ethylene oxide oligomer chain can occur before or after the spacer moiety, and optionally in between any two atoms of a spacer moiety comprised of two or more atoms. Also, the oligomer chain would not be considered part of the spacer moiety if the oligomer is adjacent to a polymer segment and merely represent an extension of the polymer segment.
  • the spacer moiety does not include sugars or carbohydrates and it is preferred that the conjugate lacks substantially any water-soluble polymers attached directly, or through a spacer moiety, to a sugar or carbohydrate that, in turn, is attached to a Factor VII moiety.
  • the conjugate may only have a single water-soluble polymer associated with a single Factor VII moiety.
  • the water-soluble polymer may be desirous to have the water-soluble polymer be a non-linear water-soluble polymer (and prepare the conjugate using a non-linear polymeric reagent).
  • a preferred non-linear water-soluble polymer is a branched water-soluble polymer, although multi-branched water-soluble polymers are included. By incorporating a branched water-soluble polymer, it is possible, for example, to double the effective molecular weight for each attachment site as compared to a single polymer.
  • Exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form include branched forms comprising a lysine-based branched polymer and a branched approach comprising the structure:
  • each (n) is independently an integer having a value of from 2 to 3400.
  • Exemplary conjugates of the invention comprise the following structure:
  • X is spacer moiety
  • (b) is an integer having a value 2 through 6;
  • (c) is an integer having a value 2 through 6;
  • R in each occurrence, is independently H or lower alkyl
  • F7 is a Factor VII moiety.
  • An exemplary conjugate of the invention comprises the following
  • each (n) is independently an integer having a value of from 2 to 3400; and F7 is a Factor VII moiety.
  • Another exemplary conjugate of the invention comprises the following structure:
  • each (n) is independently an integer having a value of from 2 to 3400;
  • X when present, is a spacer moiety comprised of one or more atoms
  • (b 1 ) is zero or an integer having a value of one through ten;
  • (c) is an integer having a value of one through ten
  • R in each occurrence, is independently H or an organic radical
  • R in each occurrence, is independently H or an organic radical; and F7 is a Factor VII moiety.
  • An exemplary conjugates of the invention comprises the following structure:
  • each (n) is independently an integer having a value of from 2 to 3400;
  • F7 is a Factor VII moiety.
  • the conjugates are typically part of a composition.
  • the composition comprises a plurality of conjugates, preferably although not necessarily, each having one, two, three or four water-soluble polymers separately covalently attached (either directly or through a spacer moiety) to one Factor VII moiety.
  • the compositions can also comprise other conjugates having four, five, six, seven, eight or more polymers attached to any given moiety having Factor VII activity.
  • the invention includes instances wherein the composition comprises a plurality of conjugates, each conjugate comprising one water-soluble polymer covalently attached to one Factor VII moiety, as well as compositions comprising two, three, four, five, six, seven, eight, or more water-soluble polymers covalently attached to one Factor VII moiety.
  • a composition comprising a plurality of conjugates, wherein at least about 80% of all conjugates in the composition are each comprised of a Factor VII moiety covalently attached to one, two, three or four water-soluble polymers, and further wherein for each water-soluble polymer in the conjugate, the Factor VII moiety is attached to the water-soluble polymer either directly or through a spacer moiety comprised of one or more atoms.
  • the composition will typically satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have from one to five polymers attached to the Factor VII moiety; at least about 85% of the conjugates in the composition will have from one to four polymers attached to the Factor VII moiety; at least about 85% of the conjugates in the composition will have from one to three polymers attached to the Factor VII moiety; at least about 85% of the conjugates in the composition will have from one to two polymers attached to the Factor VII moiety; at least about 85% of the conjugates in the composition will have one polymer attached to the Factor VII moiety (i.e., be monoPEGylated); at least about 95% of the conjugates in the composition will have from one to five polymers attached to the Factor VII moiety; at least about 95% of the conjugates in the composition will have from one to four polymers attached to the Factor VII moiety; at least about 95% of the conjugates in the composition will have from
  • the conjugate-containing composition is free or substantially free of albumin. It is also preferred that the composition is free or substantially free of proteins that do not have Factor VII activity. Thus, it is preferred that the composition is 85%, more preferably 95%, and most preferably 99% free of albumin. Additionally, it is preferred that the composition is 85%, more preferably 95%, and most preferably 99% free of any protein that does not have Factor VII activity.
  • exemplary compositions of the invention are substantially free of conjugates comprising a poly(ethylene glycol) polymer linking a residue of a Factor VII moiety to albumin.
  • Control of the desired number of polymers for any given moiety can be achieved by selecting the proper polymeric reagent, the ratio of polymeric reagent to the Factor VII moiety, temperature, pH conditions, and other aspects of the conjugation reaction.
  • reduction or elimination of the undesired conjugates e.g., those conjugates having four or more attached polymers
  • the polymer-Factor VII moiety conjugates can be purified to obtain/isolate different conjugated species.
  • the product mixture can be purified to obtain an average of anywhere from one, two, three, four, five or more PEGs per Factor VII moiety, typically one, two or three PEGs per Factor VII moiety.
  • the strategy for purification of the final conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular Factor VII moiety, the desired dosing regimen, and the residual activity and in vivo properties of the individual conjugate(s).
  • conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography. That is to say, gel filtration chromatography is used to fractionate differently numbered polymer-to-Factor VII moiety ratios (e.g., 1 -mer, 2-mer, 3-mer, and so forth, wherein “1 -mer” indicates 1 polymer attached to a Factor VII moiety, "2-mer” indicates two polymers attached to Factor VII moiety, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble polymer portion).
  • polymer-to-Factor VII moiety ratios e.g., 1 -mer, 2-mer, 3-mer, and so forth, wherein “1 -mer” indicates 1 polymer attached to a Factor VII moiety, "2-mer” indicates two polymers attached to Factor VII moiety, and so on
  • the resulting reaction mixture may contain unmodified protein (having a molecular weight of about 55,000 Daltons), monoPEGylated protein (or "1-mer”) (having a molecular weight of about 75,000 Daltons), diPEGylated protein (or 2-mer" (having a molecular weight of about 95,000 Daltons), and so forth.
  • Gel filtration chromatography can be used to separate from each other mixtures of 1 -mers, 2-mers, 3-mers, and so forth, although each of the recovered PEG-mer compositions may contain PEGs attached to different reactive amino groups (e.g., lysine residues) within Factor VII moiety.
  • Gel filtration columns suitable for carrying out this type of separation include
  • RP-HPLC reverse phase-high performance liquid chromatography
  • CI 8 column or C3 column Amersham Biosciences or Vydac
  • ion exchange chromatography using an ion exchange column, e.g., a SepharoseTM ion exchange column available from Amersham Biosciences.
  • ion exchange column e.g., a SepharoseTM ion exchange column available from Amersham Biosciences.
  • Either approach can be used to separate polymer-active agent isomers having the same molecular weight (positional isomers).
  • compositions are preferably substantially free of proteins that do not have
  • compositions preferably are substantially free of all other noncovalently attached water-soluble polymers.
  • the composition can contain a mixture of polymer-Factor VII moiety conjugates and
  • composition of the invention further comprises a
  • the pharmaceutically acceptable excipient can be added to a conjugate to form a composition.
  • Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose,
  • maltodextrins dextrans, starches, and the like
  • alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
  • an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
  • the composition can also include an antimicrobial agent for preventing or deterring microbial growth.
  • antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
  • An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium
  • a surfactant can be present as an excipient.
  • exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA, zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • the amount of the conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective amount when the composition is stored in a unit dose container (e.g., a vial).
  • a unit dose container e.g., a vial
  • the pharmaceutical preparation can be housed in a syringe.
  • a therapeutically effective amount can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine
  • the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • compositions of the present invention are typically, although not necessarily, administered via injection and are therefore generally liquid solutions or suspensions immediately prior to administration.
  • the pharmaceutical preparation can also take other forms such as syrups, creams, ointments, tablets, powders, and the like.
  • Other modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, subcutaneous, intra-arterial, and so forth.
  • the invention also provides a method for delivering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with conjugate.
  • the method comprises delivering, generally via injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition).
  • the conjugates can be delivered by, for example, intravenous injection, intramuscular injection, subcutaneous injection, and so forth.
  • Suitable formulation types for parenteral administration include ready- for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.
  • the method of delivering may be used to treat a patient having a condition that can be remedied or prevented by administration of the conjugate.
  • the conjugates can be used to treat individuals suffering from hemophilia B, either as a replacement therapy or on a prophylaxis basis.
  • Administration of the conjugate for prophylaxis includes situations where a patient suffering from hemophilia B is about to undergo surgery and the conjugate is administered between one to four hours prior to surgery.
  • the conjugates are suited for use as a prophylactic against uncontrolled bleeding, optionally in patients not suffering from hemophilia.
  • the conjugate can be administered to a patient at risk for uncontrolled bleeding prior to surgery.
  • the actual dose to be administered will vary depend upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered.
  • Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature. Generally, on a weight basis, a therapeutically effective amount will range from about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day. On an activity basis, corresponding doses based on international units of activity can be calculated by one of ordinary skill in the art.
  • the unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical composition) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • the specific dosing schedule will be known by those of ordinary skill in the art or can be determined
  • Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • SEC Size exclusion chromatography
  • Agilent 1100 HPLC system Agilent 1100 HPLC system
  • Agilent 1100 HPLC system Agilent 1100 HPLC system
  • each sample is analyzed using a SHODEX protein KW-804 column (Showa Denko KK, Tokyo Japan), at pH 7.2.
  • the flow rate for the column is set at 0.5 mL/minute.
  • Eluted protein and PEG-protein conjugates are detected using an UV-based approach having a wavelength set at 280 nm,
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • XCELL SURELOCK Mini-Cell electrophoresis system Invitrogen Corporation, Carlsbad CA.
  • 4x LDS Sample Buffer Invitrogen Corporation, Carlsbad CA.
  • the prepared samples are then loaded onto a NuPAGE Novex 4-12% Bis-Tris gel and run for
  • C3 reverse column (Hamilton, Zorbax). A 30-80% gradient of acetonitrile is used along with an elevated temperature over thirty minutes at 0.5 mL/minute.
  • mPEG-Succinimidyl butanoate having a molecular weight of 10,000 Daltons is obtained from Nektar Therapeutics, (Huntsville, AL).
  • the basic structure of the polymer reagent is provided below:
  • Factor Vila is dissolved in amine-free buffer such as phosphate to result in a final pH to 7.2-9. To this solution is then added a 1.5 to 10-fold molar excess of mPEG-SBA. The resulting mixture is stirred at room temperature for several hours.
  • reaction mixture is analyzed by SDS-PAGE to determine the degree of
  • mPEG-2-NHS, 40kDa, stored at -20 °C under argon, is warmed to ambient temperature.
  • a five- fold excess (relative to the amount of Factor VII in a measured aliquot of the stock Factor VII solution) of the warmed mPEG-2-NHS is dissolved in 2 milimolar HCL solution to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of stock Factor VII solution at ph 7.5 and is mixed well.
  • the reaction solution was stirred for five hours at room temperature in the dark, thereby resulting in a conjugate solution.
  • the reaction was quenched with glycine.
  • the conjugate solution is characterized.
  • mPEG-ButyrALD Linear mPEG-Butyraldehyde Derivative, 30kDa
  • mPEG-ButyrALD 30kDa, stored at -20 °C under argon, is warmed to ambient temperature.
  • An eight- fold excess (relative to the amount of Factor VII in a measured aliquot of the stock Factor VII, which has been buffered to a pH of 5.5 using conventional techniques) of the warmed mPEG-ButryALD is dissolved in water to form a 10% reagent solution.
  • the 10% reagent solution is quickly added to the aliquot of stock Factor VII solution and mixed well.
  • the pH of the reaction mixture is determined and is adjusted to 5.5 using conventional techniques, followed by mixing for thirty minutes.
  • a reducing agent sodium cyanoborohydride (NaCNBH 3 )
  • NaCNBH 3 sodium cyanoborohydride
  • the reaction solution is thereafter stirred for five hours at room temperature in the dark. Then, the reaction solution is stirred overnight at 3-8 °C in a cold room in the dark to ensure coupling via a secondary amine linkage to thereby form a conjugate solution.
  • the reaction is quenched with glycine.
  • the conjugate solution is characterized.
  • mPEG-Butyraldehyde Derivative, 40kDa (mPEG2-ButyrALD) [0188] mPEG-ButyrALD, 40kDa, stored at -20 °C under argon, is warmed to ambient temperature. A ten-fold excess (relative to the amount of Factor VII in a measured aliquot of the stock Factor VII solution, which has been buffered to a pH of 5.5 using conventional techniques) of the warmed mPEG-ButryALD is dissolved in water to form a 10% reagent solution. The 10% reagent solution is quickly added to the stock Factor VII solution and mixed well.
  • the pH of the reaction mixture is determined and is adjusted to 5.5 using conventional techniques, followed by mixing for thirty minutes.
  • a reducing agent sodium cyanoborohydride (NaCNBH 3 )
  • NaCNBH 3 sodium cyanoborohydride
  • the reaction solution is stirred for five hours at room temperature in the dark and is thereafter stirred overnight at 3-8 °C in a cold room in the dark, thereby resulting in a conjugate solution.
  • the reaction is quenched with glycine.
  • the conjugate solution is characterized.
  • the degradable PEG reagent mPEG SBC having a weight average molecular weight of 30,000 Daltons, is warmed from -20 °C to room temperature in a dessicator.
  • Factor VII is measured and is dissolved in a suitable buffer, pH 6.5) to make a 2 mg/mL solution.
  • mPEG SBC (1.67g) is measured and is dissolved in 37.5 mL DMSO to form an mPEG SBC solution.
  • the mPEG SBC solution (37.5 mL) is infused into the stirred Factor VII solution at a rate of 4.6 mL/min.
  • the molar ratio of mPEG SBC to protein is 5: 1.
  • the reaction proceeds at room temperature for 10 minutes with stirring.
  • the reaction is diluted 1 :3 with the purification buffer, 20 mM sodium acetate, pH 5.3, in preparation for purification.
  • a composition comprising a degradable conjugate is formed.
  • the warmed mPEG-MAL reagent (4.4 mg) is dissolved in 0.044 ml of HEPES buffer [50 mM HEPES (or other suitable buffer) pH 8.0] to make a 10% mPEG-MAL solution.
  • HEPES buffer 50 mM HEPES (or other suitable buffer) pH 8.0]
  • the mPEG-MAL solution is quickly added to the Factor VII solution [HEPES (or other suitable formulation) pH 8.0] and is mixed well. After 30 minutes of reaction at room temperature and the pH is determined (pH 8.0 ⁇ 0.2). The reaction is allowed to proceed overnight at 4 °C on Rotomix (slow speed, Thermolyne).
  • treatment with TCEP [tra(2-carboxyethyl)phosphine)] can be used to enhance conjugation at cytseine residues
  • mPEG2-MAL 20K, stored at -20 °C under argon, is warmed to ambient temperature.
  • the warmed mPEG2-MAL reagent (4.4 mg) is dissolved in 0.044 ml of HEPES buffer [50 mM HEPES (or other suitable buffer) pH 8.0] to make a 10% mPEG2-MAL solution.
  • HEPES buffer 50 mM HEPES (or other suitable buffer) pH 8.0
  • the mPEG2-MAL solution is quickly added to 4 ml of Factor VII solution [0.4324 mg/ml in 50 mM HEPES (or other suitable formulation) pH 8.0] and is mixed well. After 30 minutes of reaction at room temperature and the pH is determined (pH 8.0+0.2).
  • reaction is allowed to proceed overnight at 4 °C on Rotomix (slow speed, Thermolyne).
  • treatment with TCEP [im(2-carboxyethyl)phosphine)] can be used to enhance conjugation at cytseine residues.
  • SEQ. ID. NO. 1 (single letter abbreviation): Amino acid sequence of human Factor VII

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Abstract

La présente invention concerne des conjugués associant une fraction de facteur VII et un ou plusieurs polymères solubles dans l'eau. De façon générale, ledit polymère soluble dans l'eau est un poly(éthylèneglycol) ou un dérivé de celui-ci. L'invention concerne également (et entre autres choses) des compositions contenant lesdits conjugués, des procédés de fabrication desdits conjugués et des procédés d'administration à un patient de compositions contenant lesdits conjugués.
PCT/US2011/066176 2010-12-22 2011-12-20 Conjugués polymère-fraction de facteur vii WO2012088123A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156488A3 (fr) * 2012-04-16 2014-01-16 Leverton Licence Holdings Limited Agents thérapeutiques sous-cutanés optimisés

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090227504A1 (en) * 2002-06-21 2009-09-10 Novo Nordisk A/S Pegylated Factor VII Glycoforms
US20090247459A1 (en) * 2007-11-09 2009-10-01 Baxter International Inc. Modified recombinant factor viii and von willebrand factor and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090227504A1 (en) * 2002-06-21 2009-09-10 Novo Nordisk A/S Pegylated Factor VII Glycoforms
US20090247459A1 (en) * 2007-11-09 2009-10-01 Baxter International Inc. Modified recombinant factor viii and von willebrand factor and methods of use

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156488A3 (fr) * 2012-04-16 2014-01-16 Leverton Licence Holdings Limited Agents thérapeutiques sous-cutanés optimisés
GB2516388A (en) * 2012-04-16 2015-01-21 Cantab Biopharmaceuticals Patents Ltd Optimised subcutaneous therapeutic agents
EA033469B1 (ru) * 2012-04-16 2019-10-31 Cantab Biopharmaceuticals Patents Ltd Подкожное введение конъюгатов факторов крови с полиэтиленгликолем
US11351112B2 (en) 2012-04-16 2022-06-07 Cantab Biopharmaceuticals Patents Limited Optimised subcutaneous therapeutic agents

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