Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/36—Blood coagulation or fibrinolysis factors
• • 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
  • A61K47/62—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 a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
  • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
  • A61K47/62—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 a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

• the present invention relates to materials and methods for conjugating water soluble polymers, in particular polysialic acid, to carbohydrate-containing compounds, in particular glycoproteins other than a blood coagulation proteins, and to the conjugates obtained.
• PEG molecules have a large hydrodynamic volume (5-10 times the size of globular proteins), are highly water soluble and hydrated, non-toxic, non-immunogenic and rapidly cleared from the body. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half- life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability, and reduced aggregation.
• the first PEGylated drugs were approved by the FDA in the early 1990s. Since then, the FDA has approved several PEGylated drugs for oral, injectable, and topical administration.
• Sialic acids also called N-acetyl neuraminic acids
• polysialic acids are found widely distributed in animal tissues and to a lesser extent in other species ranging from plants and fungi to yeasts and bacteria, mostly in glycoproteins and gangliosides.
• PSA polysialic acid
• mPSA modified polysialic acid
• PSAs consist of polymers (generally homopolymers) of N-acetylneuraminic acid.
• the secondary amino group normally bears an acetyl group, but it may instead bear a glycolyl group.
• Possible substituents on the hydroxyl groups include acetyl, lactyl, ethyl, sulfate, and phosphate groups.
• sialic acid N-acetylneuraminic acid
• PSAs and mPSAs generally comprise linear polymers consisting essentially of N- acetylneuraminic acid moieties linked by 2,8- or 2,9- glycosidic linkages or combinations of these (e.g. alternating 2,8- and 2,9- linkages).
• the glycosidic linkages are ⁇ -2,8.
• Such PSAs and mPSAs are conveniently derived from colominic acids, and are referred to herein as "CAs" and "mCAs”.
• Typical PSAs and mPSAs comprise at least 2, preferably at least 5, more preferably at least 10 and most preferably at least 20 N-acetylneuraminic acid moieties.
• PSAs and CAs can be polymers comprising different sugar moieties. They can be copolymers. PSAs and CAs preferably are essentially free of sugar moieties other than N- acetylneuraminic acid. PSAs and CAs preferably comprise at least 90%, more preferably at least 95% and most preferably at least 98% N-acetylneuraminic acid moieties.
• PSAs and CAs comprise moieties other than N-acetylneuraminic acid (as, for example in mPSAs and mCAs) these are preferably located at one or both of the ends of the polymer chain.
• Such "other" moieties may, for example, be moieties derived from terminal N-acetylneuraminic acid moieties by oxidation or reduction.
• WO-A-0187922 describes such mPSAs and mCAs in which the non- reducing terminal N-acetylneuraminic acid unit is converted to an aldehyde group by reaction with sodium periodate.
• WO 2005/016974 describes such mPSAs and mCAs in which the reducing terminal N-acetylneuraminic acid unit is subjected to reduction to reductively open the ring at the reducing terminal N-acetylneuraminic acid unit, whereby a vicinal diol group is formed, followed by oxidation to convert the vicinal diol group to an aldehyde group.
• Sialic acid rich glycoproteins bind selectin in humans and other organisms. They play an important role in human influenza infections.
• sialic acid can hide mannose antigens on the surface of host cells or bacteria from mannose-binding lectin. This prevents activation of complement.
• Sialic acids also hide the penultimate galactose residue thus preventing rapid clearance of the glycoprotein by the galactose receptor on the hepatic parenchymal cells.
• CAs are produced, inter alia, by particular strains of Escherichia coli possessing the Kl antigen. CAs have many physiological functions. They are important as a raw material for drugs and cosmetics.
• the preparation of conjugates by forming a covalent linkage between the water soluble polymer and the therapeutic protein can be carried out by a variety of chemical methods.
• One approach for coupling PSA to therapeutic proteins is the conjugation of the polymers via the carbohydrate moieties of the protein.
• Vicinal hydroxyl (OH) groups of carbohydrates in proteins can be easily oxidized with sodium periodate (NaIO 4 ) to form active aldehyde groups (Rothfus and Smith, J Biol Chem 1963; 238:1402-10; van Lenten and Ashwell, J Biol Chem 1971 ;246: 1889-94).
• the polymer can be coupled to the aldehyde groups of the carbohydrate by use of reagents containing, for example, an active hydrazide group (Wilchek M and Bayer EA, Methods Enzymol 1987; 138:429-42).
• reagents containing, for example, an active hydrazide group Wang M and Bayer EA, Methods Enzymol 1987; 138:429-412.
• a more recent technology is the use of reagents containing aminooxy groups which react with aldehydes to form oxime linkages (WO 96/40662, WO2008/025856).
• Additional examples describing conjugation of a PSA to a therapeutic protein are described in US Publication No. 2009/0076237 which teaches the oxidation of rFVIII and subsequent coupling to PSA and other water soluble polymers (e.g.
• the present invention provides materials and methods for conjugating a water soluble polymer to a carbohydrate-containing compound other than a blood coagulation protein that improve the compound's pharmacodynamic and/or pharmacokinetic properties while minimizing the costs associated with the various reagents.
• a method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a carbohydrate-containing compound other than a blood coagulation protein comprising contacting the oxidized carbohydrate moiety with water soluble polymer under conditions that allow conjugation, wherein said water soluble polymer contains an aminooxy group and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the water soluble polymer, or wherein said water soluble polymer contains a hydrazide group and a hydrazone linkage is formed between the oxidized carbohydrate moiety and the hydrazide group on the water soluble polymer.
• the compound may be (1) a glycoprotein other than a blood coagulation protein, (2) a ganglioside, or (3) a drug delivery system comprising a carbohydrate group.
• the carbohydrate moiety may be oxidized using a sugar-specific oxidizing enzyme (e.g. galactose or glucose oxidase) or by incubation with a buffer comprising an oxidizing agent selected from sodium periodate (NaIO 4 ), lead tetraacetate (Pb(OAc) 4 ) and potassium perruthenate (KRuO 4 ).
• a sugar-specific oxidizing enzyme e.g. galactose or glucose oxidase
• a buffer comprising an oxidizing agent selected from sodium periodate (NaIO 4 ), lead tetraacetate (Pb(OAc) 4 ) and potassium perruthenate (KRuO 4 ).
• the carbohydrate moiety may be oxidized at a sialic acid, mannose, galactose or glucose residue.
• the water-soluble polymer used in the invention can be, but is not limited to, polyethylene glycol (PEG), branched PEG, PEG derivative, PSA, mPSA, CA, mCA, hydroxyethyl cellulose (HEC), dextrin, polyoxazoline, carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG) polyoxazoline, poly acryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, poly(l-
• the water soluble polymer is PEG or branched PEG.
• the water soluble polymer is polysialic acid (PSA) or a modified PSA (mPSA).
• PSA or mPSA may have a molecular weight range of 350 Da to 120,000 Da, 500 Da to 100,000 Da,1000 Da to 80,000 Da, 1500 Da to 60,000 Da, 2,000 Da to 45,000 Da or 3,000 Da to 35,000 Da.
• the PSA or mPSA may be coloniinic acid or modified colominic acid.
• the PSA or mPSA is comprised of about 2- 500 or 10 - 300 sialic acid units.
• the aforementioned method is provided wherein the oxidizing agent is sodium periodate (NaIO 4 ).
• the method of the invention may comprise oxidizing the water soluble polymer to form an aldehyde group on a terminal sialic acid unit of the water soluble polymer, and reacting the oxidized water soluble polymer with an aminooxy linker.
• the aforementioned method is provided wherein the water soluble polymer is prepared by reacting an activated aminooxy linker with oxidized water soluble polymer wherein the linker is a homobifunctional or heterobifunctional linker.
• the linker may specifically be selected from:
• PSA or mPSA may be oxidized by incubation with an oxidizing agent to form a terminal aldehyde group at the non-reducing end of the PSA.
• the method may comprise oxidizing the water soluble polymer to form an aldehyde group on a terminal unit of the water soluble polymer, e.g. a terminal sialic acid unit of the PSA or mPSA, and reacting the oxidized water soluble polymer with an aminooxy linker.
• a terminal unit of the water soluble polymer e.g. a terminal sialic acid unit of the PSA or mPSA
• an aminooxy linker is 3-oxa-pentane-l,5-dioxyamine.
• the oxidizing agent is NaIO 4 .
• the aforementioned method is provided wherein the contacting of the oxidized carbohydrate moiety with the activated water soluble polymer occurs in a buffer comprising a nucleophilic catalyst selected from the group consisting of aniline and aniline derivatives.
• a hydrazide group may be formed on the water soluble polymer by reacting oxidized water soluble polymer with a hydrazide linker.
• the hydrazide linker can suitably be adipic acid dihydrazide or hydrazine.
• an aforementioned method is provided further comprising the step of reducing an oxime or hydrazone linkage in the conjugated protein, for example by incubating the conjugated protein in a buffer comprising a reducing compound selected from the group consisting of sodium cyanoborohydride (NaCNBH 3 ) and ascorbic acid (vitamin C).
• the reducing compound is sodium cyanoborohydride (NaCNBH 3 ).
• a conjugated glycoprotein produced by any aforementioned method comprises (a) the said glycoprotein, ganglioside or drug delivery system; and (b) at least one aminooxy water soluble polymer bound to the glycoprotein of (a), wherein said aminooxy water soluble polymer is attached to the glycoprotein, ganglioside or drug delivery system via one or more carbohydrate moieties.
• a conjugated glycoprotein other than a blood coagulation protein, a ganglioside or a drug delivery system comprises (a) the said glycoprotein, ganglioside or drug delivery system; and (b) at least one hydrazide water soluble polymer bound to the glycoprotein of (a), wherein said hydrazide water soluble polymer is attached to the glycoprotein ganglioside or drug delivery system via one or more carbohydrate moieties.
• Figure 1 shows the synthesis of the water soluble di-aminoxy linkers 3-oxa- pentane-l,5-dioxyamine and 3,6,9-trioxa-undecane-l,l 1-dioxyamine.
• Figure 2 shows the preparation of aminooxy-PSA.
• carbohydrate-containing compounds such as glycoproteins other than blood coagulations proteins
• water soluble polymer in particular PEG or PSA or mPSA.
• the properties of the resulting conjugates generally strongly depend on the structure and the size of the polymer. Thus, polymers with a defined and narrow size distribution are usually preferred.
• PSA and mPSA used in specific examples, can be purified in such a manner that results in a final PSA preparation with a narrow size distribution.
• glycoproteins other than blood coagulation proteins including, but not limited to cytokines such as interleukins, alpha-, beta-, and gamma-interferons, colony stimulating factors including granulocyte colony stimulating factors, fibroblast growth factors, platelet derived growth factors, phospholipase-activating protein (PUP), insulin, plant proteins such as lectins and ricins, tumor necrosis factors and related alleles, soluble forms of tumor necrosis factor receptors, interleukin receptors and soluble forms of interleukin receptors, growth factors, tissue growth factors, transforming growth factors such as TGF ⁇ s or TGF ⁇ s and epidermal growth factors, hormones, somatomedins, pigmentary hormones, hypothalamic releasing factors, antidiuretic hormones, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, and immunoglobul
• cytokines such as
• the glycoprotein is EPO. In a further embodiment the glycoprotein is a galactosidase. In a still further embodiment the glycoprotein is a DNAse. In a yet further embodiment the glycoprotein is fetuin. Lastly, in a still yet further embodiment the glycoprotein is a granulocyte colony stimulating factor.
• biologically active derivative or “biologically active variant” includes any derivative or variant 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.
• an "analog,” “variant” or “derivative” is a compound substantially similar in structure and having the same biological activity, albeit in certain instances to a differing degree, to a naturally-occurring molecule.
• a polypeptide variant refers to a polypeptide sharing substantially similar structure and having the same biological activity as a reference polypeptide.
• Variants or analogs differ in the composition of their amino acid sequences compared to the naturally-occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or one or more internal regions of the naturally- occurring polypeptide sequence (e.g., fragments), (ii) insertion or addition of one or more amino acids at one or more termini (typically an "addition” or “fusion") of the polypeptide and/or one or more internal regions (typically an "insertion") of the naturally-occurring polypeptide sequence or (iii) substitution of one or more amino acids for other amino acids in the naturally-occurring polypeptide sequence.
• a “derivative” refers to a polypeptide sharing the same or substantially similar structure as a reference polypeptide that has been modified, e.g., chemically.
• Variant or analog polypeptides include insertion variants, wherein one or more amino acid residues are added to a protein amino acid sequence of the invention. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the protein amino acid sequence. Insertion variants, with additional residues at either or both termini, include for example, fusion proteins and proteins including amino acid tags or other amino acid labels.
• the protein molecule optionally contains an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.
• deletion variants one or more amino acid residues in a protein or polypeptide as described herein are removed.
• Deletions can be effected at one or both termini of the protein or polypeptide, and/or with removal of one or more residues within the protein amino acid sequence.
• Deletion variants therefore, include fragments of a protein or polypeptide sequence.
• substitution variants one or more amino acid residues of a protein or polypeptide are removed and replaced with alternative residues.
• the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.
• the invention embraces substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp.71-77] and are set out immediately below.
• Non-polar (hydrophobic) A. Aliphatic ALIVP
• gangliosides are conjugated to water soluble polymers, e.g. PEG or PSA or mPSA.
• Gangliosides are known to provide cells with distinguishing surface markers that can serve in cellular recognition and cell-to-cell communication. They are useful as therapeutic agents.
• Conjugates of the invention may comprise a ganglioside and a water soluble polymer, in which the ganglioside comprises a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids linked on the sugar chain.
• Gangliosides can be classified according to how many sialic acid units are present on the molecule. Examples of gangliosides are GMl, GM2 and GM3 (monosialo-gangliosides), GDIa, GDIb, GD2 and GD3 (disialo-gangliosides), GTIb (trisialo-ganglioside) and GQl (tetrasialo-ganglioside).
• preferred gangliosides comprise a ceramide linked to glucose, which is linked to a first galactose, which is linked to N-acetylgalactosamine, which is linked to a second galactose.
• This second galactose can be linked to one sialic acid.
• the first galactose can be linked to one, two, three or four sialic acids.
• Sialic acids may be linked either as monomers (one on each of the galactose molecules), or as oligosialic acids (2-4 sialic acids) to the first galactose.
• gangliosides can be polysialylated, for example, by the method of the invention.
• drug delivery systems are conjugated to a water soluble polymer, e.g. PEG or PSA or mPSA.
• a drug delivery system is any molecular or particulate entity which can control the fate and effect of drugs associated with the entity. DDSs can be separated into two general types. The first type comprises macromolecules (MDDSs), for instance antibodies, neoglycoproteins as well as synthetic polymers, such as poly(hydroxypropylmethacrylamide), polylysine and polymerised alkyl cyanoacrylates.
• MDDSs macromolecules
• synthetic polymers such as poly(hydroxypropylmethacrylamide), polylysine and polymerised alkyl cyanoacrylates.
• PDDSs particulate DDSs
• the second type is particulate DDSs (PDDSs), which comprises for instance nanospheres or microspheres, which comprise biodegradable materials such as albumin or semibiodegradable materials such as dextran and alkylcyanoacrylate polymers, or vesicles formed of nonionic surfactants or liposomes - for details of which see for example Gregoriadis in NIPS, 4, 146-151 (1989).
• Drugs can either be covalently linked to, or passively entrapped into, the DDS.
• PDDS comprising surfactant vesicles or liposomes may entrap hydrophilic or hydrophobic pharmaceutically active compounds by being formed of an appropriate combination of layers of surfactant or lipid molecules.
• Pharmaceutically active compounds are usually covalently linked to MDDSs, by a bond which may or may not be Iysed in the body, for instance before or after the active compound performs its function.
• MDDSs have an intrinsic (e.g. antibodies) or acquired (e.g. neoglycoproteins) ability to be recognised by target cells or tissues through receptors on the latter's surface.
• DDSs are taken up specifically by the target upon injection. Specific uptake is, however, limited with the bulk of the DDSs being taken up by other, irrelevant (to therapy) tissues. The reason for this is that antibodies and other DDS proteins (regardless of their specificity for the target) must be, like other proteins, catabolised at the end of their biological life.
• Synthetic polymers used in the macromolecular type MDDSs are for instance poly(hydroxypropylmethacrylamide) polylysine and polymerised alkyl cyanoacrylates. These may be catabolised in the reticuloendothelial system (RES) or other tissues by appropriate lysosomal enzymes. It would be desirable to reduce the rate of catabolism of such biodegradable macromolecular type DDS by some means, for instance by reducing uptake of the DDS by the RES or other tissues, or by reducing degradation by lysosomal enzymes once taken up by the RES.
• RES reticuloendothelial system
• Particulate DDSs are, as a rule, removed from the circulation by the RES. Because of their propensity for the RES, PDDSs are often used for the delivery of drugs to these tissues. It is often desirable however, that PDDSs are directed to tissues other than those of the RES. To achieve this goal, one must block or delay RES interception of PDDSs.
• DDSs for use in the invention may not initially contain glycons.
• An option is to add or otherwise incorporate a glycon into the DDS structure. Examples of such cases are liposomes incorporating a mannosylated or a galactosylated lipid. These glycoliposomes will target actives to tissues which express a mannose or galactose receptor respectively.
• DDSs need to circulate in the blood for long periods so that e.g. uptake by target tissues is more effective (as with hepatic parenchymal cells), they are advantageously polysialylated by the methods of the invention.
• a conjugated compound of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.
• the compositions may be useful as therapeutic, diagnostic and/or similar agents
• compositions comprising a conjugated compound of the present invention to human or test animals
• the compositions comprise one or more pharmaceutically acceptable carriers.
• pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, including those agents disclosed above.
• ⁇ ел ⁇ ество includes a dose suitable for treating a mammal having a clinically defined disorder.
• compositions may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well.
• compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.
• compositions can be carried out with the dose levels and pattern being selected by the treating physician.
• the appropriate dosage will depend on the type of disease to be treated, as described above, the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of a conjugated compound or protein as defined herein.
• the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient.
• the pharmaceutical composition can be used for treating clinically-defined disorders.
• the pharmaceutical composition of the invention may be a solution or a lyophilized product. Solutions of the pharmaceutical composition may be subjected to any suitable lyophilization process.
• kits which comprise a composition of the invention packaged in a manner which facilitates its use for administration to subjects.
• a kit includes a compound or composition described herein (e.g., a composition comprising a conjugated protein), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method.
• the kit contains a first container having a composition comprising a conjugated protein and a second container having a physiologically acceptable reconstitution solution for the composition in the first container.
• the compound or composition is packaged in a unit dosage form.
• the kit may further include a device suitable for administering the composition according to a specific route of administration.
• the kit contains a label that describes use of the therapeutic protein or peptide composition.
• the derivative retains the full functional activity of native therapeutic compounds, and provides an extended half-life in vivo, as compared to native therapeutic compounds. In another embodiment, the derivative retains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44.
• sialic acid moieties includes sialic acid monomers or polymers ("polysaccharides") which are soluble in an aqueous solution or suspension and have little or no negative impact, such as side effects, to mammals upon administration of the PSA-protein conjugate in a pharmaceutically effective amount.
• PSA and mPSA are characterized, in one aspect, as having 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. In certain aspects, different sialic acid units are combined in a chain.
• the sialic acid portion of the PSA or mPSA compound is highly hydrophilic, and in another embodiment the entire compound is highly hydrophilic. Hydrophilicity is conferred primarily by the pendant carboxyl groups of the sialic acid units, as well as the hydroxyl groups.
• 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.
• the PSA and mPSA used in the methods and conjugates of the invention may be further characterized as described above in the Background of the Invention.
• the naturally occurring polymer PSA is available as a polydisperse preparation showing a broad size distribution (e.g. Sigma C-5762) and high polydispersity (PD). Because the polysaccharides are usually produced in bacteria carrying the inherent risk of copurifying endotoxins, the purification of long sialic acid polymer chains may raise the probability of increased endotoxin content. Short PSA molecules with 1-4 sialic acid units can also be synthetically prepared (Kang SH et al., Chem Commun. 2000;227-8; Ress DK and Linhardt RJ, Current Organic Synthesis. 2004; 1 :31 -46), thus minimizing the risk of high endotoxin levels.
• Polysaccharide compounds of particular use for the invention are, in one aspect, those produced by bacteria. Some of these naturally-occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compounds are substantially free of terminal galactose units.
• the compound is linked to or associated with the PSA or mPSA compound in stoichiometric amounts (e.g., 1:1, 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :7, 1 :8, 1 :9, or 1 :10, etc.).
• 1-6, 7-12 or 13-20 PSA and/or mPSA units are linked to the compound.
• 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more PSA and/or mPSA units are linked to the compound.
• the compound is modified to introduce glycosylation sites (i.e., sites other than the native glycosylation sites).
• glycosylation sites i.e., sites other than the native glycosylation sites.
• Such modification may be accomplished using standard molecular biological techniques known in the art.
• the compound, prior to conjugation via one or more carbohydrate moieties may be glycosylated in vivo or in vitro.
• the reaction of hydroxylamine or hydroxylamine derivatives with aldehydes (e.g., on a carbohydrate moiety following oxidation by sodium periodate) to form an oxime group is applied to the preparation of conjugates of compound.
• a glycoprotein is first oxidized with a oxidizing agent such as sodium periodate (NaIO 4 ) (Rothfus JA et Smith EL., J Biol Chem 1963, 238, 1402- 10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246, 1889-94).
• a oxidizing agent such as sodium periodate (NaIO 4 )
• NaIO 4 sodium periodate
• glycoproteins is based on the classical Malaprade reaction described in 1928, the oxidation of vicinal diols with periodate to form an active aldehyde group (Malaprade L., Analytical application, Bull Soc Chim France, 1928, 43, 683-96).
• oxidizing agent a mild oxidizing compound which is capable of oxidizing vicinal diols in carbohydrates, thereby generating active aldehyde groups under physiological reaction conditions is meant.
• the second step is the coupling of the polymer containing an aminooxy group to the oxidized carbohydrate moiety to form an oxime linkage.
• this step can be carried out in the presence of catalytic amounts of the nucleophilic catalyst aniline or aniline derivatives (Dirksen A et Dawson PE, Bioconjugate Chem. 2008; Zeng Y et al., Nature Methods 2009;6:207-9).
• the aniline catalysis dramatically accelerates the oxime ligation allowing the use of very low concentrations of the reagents.
• the oxime linkage is stabilized by reduction with NaCNBH 3 to form an alkoxyamine linkage.
• reaction steps to conjugate PSA or mPSA to a protein are carried out separately and sequentially (i.e., starting materials (e.g., protein, polymer, etc), reagents (e.g., oxidizing agents, aniline, etc) and reaction products (e.g., oxidized carbohydrate on a protein, activated aminooxy polymer, etc) are separated between individual reaction steps).
• starting materials e.g., protein, polymer, etc
• reagents e.g., oxidizing agents, aniline, etc
• reaction products e.g., oxidized carbohydrate on a protein, activated aminooxy polymer, etc
• Advantages of the invention include high recovery of conjugate, high retention of activity of the conjugated glycoprotein compared to unconjugated protein and high conjugation efficiency.
• the conjugate was eluted within 2.4 column volumes (CV) with 5OmM HEPES, 6.7mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5ml/min.
• the preparation was analytically characterized by measuring total protein (BCA) and FIX chromogenic activity.
• BCA total protein
• FIX FIX chromogenic activity
• the conductivity of the cooled reaction mixture was raised to 180mS/cm and loaded onto a 5ml HiTrap Butyl FF (GE Healthcare, Fairfield, CT) HIC column (1.6 x 2.5cm), pre-equilibrated with 5OmM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween 80, pH 6.9.
• the conjugate was eluted with a linear gradient to 5OmM HEPES, 6.7mM calcium chloride, 0.005% Tween 80, pH 7.4 in 20CV with at a flow rate of 5ml/min.
• the excess PSA reagent was removed by means of HIC.
• the conductivity of the cooled reaction mixture was raised to l ⁇ OmS/cm and loaded onto a 5ml HiTrap Butyl FF (GE Healthcare, Fairfield, CT) HIC column (1.6 x 2.5cm), pre- equilibrated with 5OmM HEPES, 3M sodium chloride, 6.7mM calcium chloride, 0.01% Tween 80, pH 6.9.
• the conjugate was eluted within 2.4CV with 5OmM HEPES, 6.7mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5ml/min.
• the ionic strength was increased by adding a buffer containing 8M ammonium acetate (8M ammonium acetate, 5OmM Hepes, 5mM CaCl 2 , 35OmM NaCl, 0.01% Tween 80, pH 6.9) to get a final concentration of 2.5M ammonium acetate.
• a buffer containing 8M ammonium acetate 8M ammonium acetate, 5OmM Hepes, 5mM CaCl 2 , 35OmM NaCl, 0.01% Tween 80, pH 6.9
• equilibration buffer 2.5M ammonium acetate, 5OmM Hepes, 5mM CaCl 2 , 35OmM NaCl, 0.01% Tween 80, pH 6.9.
• the product was eluted with elution buffer (5OmM Hepes, 5mM CaCl 2 , 0.01% Tween 80, pH 7.4), and the eluate was concentrated by centrifugal filtration using Vivaspin (Sartorius, Goettingen, Germany) devices with 30,000 MWCO.
• elution buffer 5OmM Hepes, 5mM CaCl 2 , 0.01% Tween 80, pH 7.4
• An Aminooxy-PSA reagent is prepared by use of a maleimido/aminooxy linker system (Toyokuni et al., Bioconjugate Chem 2003; 14, 1253-9).
• PSA-SH (2OkD) containing a free terminal SH - group is prepared using a two step procedure: a) Preparation of PSA- NH 2 by reductive animation of oxidized PSA with NH 4 Cl according to WO05016973 Al and b) introduction of a sulfhydryl group by reaction of the terminal primary amino group with 2- iminothiolane (Traut's reagent / Pierce, Rockford, IL) as described in US7645860.
• PSA-SH is coupled to the maleimido-group of the linker at pH 7.5 in PBS - buffer using a 10-fold molar excess of the linker and a PSA-SH concentration of 50 mg/ml.
• the reaction mixture is incubated for 2 hours under gentle shaking at room temperature. Then the excess linker reagent is removed and the aminooxy-PSA is buffer exchanged into oxidation buffer (5OmM sodium phosphate, pH 6.0) by diafiltration.
• the buffer is exchanged 25 times employing a Pellicon XL5kD regenerated cellulose membrane (Millipore, Billerica, MA).
• rFIX is oxidized in 50 mM sodium phosphate buffer, pH 6.0 employing 100 ⁇ M sodium periodate in the buffer. The mixture was shaken in the dark for Ih at 4 0 C and quenched for 15 min at room temperature by the addition of glycerol to a final concentration of 5mM. Low molecular weight contaminates were removed by means of size exclusion chromatography (SEC) employing PD-10 desalting columns (GE Healthcare, Fairfield, CT). Oxidized rFIX is then spiked with aniline to obtain a final concentration of 1OmM and mixed with the aminooxy-PSA reagent to achieve a 5 fold molar excess of PSA. The reaction mixture was incubated for 2 hours under gentle shaking in the dark at room temperature.
• SEC size exclusion chromatography
• PSA-rFIX containing fractions are collected and subjected to UF/DF by use of a 30 kD membrane made of regenerated cellulose (Millipore).
• the preparation is analytically characterized by measuring total protein (BCA) and FIX chromogenic activity.
• BCA total protein
• FIX chromogenic activity For the PSA-rFIX conjugates prepared with both variants a specific activity of > 50 % in comparison to native rFIX was determined.
• An aminooxy - PSA reagent was prepared according to Example 3. The final product was diafiltrated against buffer, pH 7.2 (50 mM Hepes) using a 5 kD membrane (regenerated cellulose, Millipore), frozen at -8O 0 C and lyophilized. After lyophilization the reagent was dissolved in the appropriate volume of water and used for preparation of PSA- protein conjugates via carbohydrate modification.
• the Dichloromethane layer was dried over Na 2 SC ⁇ and then evaporated to dryness under reduced pressure and dried in high vacuum to give 64.5 g of 3-oxapentane- l,5-dioxy-endo-2',3'-dicarboxydiimidenorbornene as a white-yellow solid (intermediate 1).
• Step2 [0087] To a solution of intermediate 1 (64.25 g; 1.OOeq) in 800 ml anhydrous Ethanol, 31.0 ml Hydrazine hydrate (4.26eq) were added. The reaction mixture was then refluxed for 2 hours. The mixture was concentrated to the half of the starting volume by evaporating the solvent under reduced pressure. The occurring precipitate was filtered off. The remaining ethanol layer was evaporated to dryness under reduced pressure. The residue containing the crude product 3-oxa-pentane-l,5-dioxyamine was dried in vacuum to yield 46.3g.
• the crude product was further purified by column chromatography (Silicagel 60; isocratic elution with Dichloromethane/Methanol mixture, 9+1) to yield 1 1.7 g of the pure final product 3-oxa- pentane -1,5-dioxyamine.
• oxidized colominic acid 23kDa was dissolved in 18ml of 5OmM sodium acetate pH 5.5 ⁇ 0.02. 20 fold molar excess of 1, l l-diamino-3,6,9-trioxaundecane (also referred to as 3,6,9-trioxa-undecane-l,l 1 -dioxyamine) was dissolved in minimum amount of 5OmM sodium acetate (pH 5.5 ⁇ 0.02) and was added to the PSA solution. The final colominic acid concentration was 62.5mg/ml. This reaction mixture was incubated for 2 ⁇ 0.1hr at 22 ⁇ 1.0°C on a gentle mixer (22 oscillations per minute).
• ⁇ -Galactosidase For oxidation of ⁇ -Galactosidase ( ⁇ -Gal), different concentrations of NaIO 4 (ranging from 0.157 mM to 2 mM) were used. 0.5 mg of ⁇ -Gal was oxidized under acidic pH of 5.75 at 4° C for 30 minutes in the dark. Oxidation was stopped by adding NaHSO 3 to a final concentration of 5 mM. The conjugation reaction was carried out using the oxidized ⁇ - GaI with diaminooxy PSA polymer (22kDa). The final concentration of polymer in the reaction mixture was 1.25 mM whereas the concentration of ⁇ -Gal ranged from 0.125 mg/ml to 0.76 mg/ml. All the reactions were done at pH5.75.
• ⁇ -Gal was oxidized with 1.5 mM OfNaIO 4 for 30 minutes at 4°C and then oxidation was stopped by adding NaHSO 3 to a final concentration of 5 mM.
• the conjugation reaction was carried out using the oxidized ⁇ -Gal with diaminooxy PSA polymer.
• the final concentrations of polymer and protein in the reaction mixture were 1.25 mM and 0.76 mg/ml respectively.
• the final pH of the reaction mixture was around 5.75.
• Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17mg/ml. The reaction was carried out at 4°C for 2 hours.
• 0.2 mg of EPO was oxidized with 10 mM of NaIO 4 for 30 minutes at 4 0 C. The oxidation was stopped by adding NaHSO 3 to a final concentration of 5 mM.
• the conjugation reaction was carried out using the oxidized EPO with diaminooxy polymer of 23kDa. The final concentration of polymer in the reaction mixture was 1.25 mM. The final concentration of EPO in the reaction mixture was 0.125 mg/ml. The final pH of the reaction mixture was around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 5OmM or 3.17mg/ml. The reaction was carried out at 4°C for 24 hours. Unpurified conjugate was characterized using SDS PAGE. A shift in the band was seen for the conjugate in SDS PAGE.
• the oxidation was stopped by adding NaHSO 3 to a final concentration of 2 mM.
• the conjugation reaction was carried out by addition of diaminooxy PSA polymer to a final concentration of 1.25 mM.
• NaCNBH 3 was added to the reaction mixture to a final concentration of 50 mM or 3.17 mg/ml and the polysialylation of the DNAse was preformed 4.0 ⁇ 1.0° C for at least 2 hours.
• the reactions were stopped with 25 molar excess of Tris over polymer.
• the conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and positive result was obtained from western blotting. Activity was measured as 95% (compared with the less than 50% observed in comparable conjugates made using aldehyde linker chemistry).
• colominic acid-hydrazide is subjected to 1 cycle of ultrafiltration, 1 cycle of diafiltration using 2OmM TEA, pH 8.0 ⁇ 0.02 and at least 3 cycles of diafiltration using 2mM TEA, pH 8.0 ⁇ 0.02. This can be done using 3kDa vivaflow cassettes.
• EPO erythropoietin
• NaIO 4 was used at a concentration of 10 mM.
• EPO (1 mg) was oxidized at pH 5.75 at 4°C for 30 minutes then oxidation was stopped by adding NaHSO 3 to a final concentration of 5 mM.
• the conjugation reaction was carried out using the oxidized EPO with hydrazide-PSA polymer.
• the molecular weight of the hydrazide-PSA used for conjugation was 24.34 kDa.
• the final concentration of hydrazide- PSA in the reaction mixture was 1.25 mM.
• the final concentration of EPO in the reaction mixture was 0.125 mg/ml.
• the final pH of the reaction mixture was around 5.75.
• ⁇ -Galactosidase (0.5 to 4.5 mg) was oxidized with 0.625 to 2 mM of NaIO 4 for 30 minutes at 4 0 C. The oxidation was stopped by adding NaHSO 3 to a final concentration of 5 mM.
• the conjugation reaction was carried out using the oxidized ⁇ -galactosidase with hydrazide-PSA ranging from 24.34 kDa to 27.9 kDa.
• the final concentration of hydrazide- PSA in the reaction mixture was 1.25 mM.
• the final concentration of ⁇ -galactosidase in the reaction mixture was in a range from 0.125 mg/ml to 0.76 mg/ml.
• the final pH of reaction mixture should be around 5.75.
• Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml.
• the reaction was carried out at 4 0 C and samples were collected at 1, 2 and 24 hours.
• Purified and unpurified conjugate was characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE and a positive result was obtained from western blotting. Activity was measured as 84%. Less than 50% activity was observed in comparable conjugates made using aldehyde linker chemistry.
• Fetuin (0.25 mg) was oxidized with NaI04 (5 or 10 mM) for 30 or 60 minutes at 4°C.
• the oxidation was stopped by adding NaHSO 3 to a final concentration of 5 or 10 mM as appropriate to match the concentration of NaIO 4 used for oxidation.
• the conjugation reactions were carried out using the oxidized Fetuin with adipic acid dihydrazide-PSA polymer.
• the final concentration of the polymer in the reaction mixture was between 1.25 and 2.5 mM.
• the final pH of reaction mixture was around 5.75.
• Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml.
• the reaction was carried out at 4°C for 1 hour to 4 hours.
• the conjugates were characterized using SDS PAGE and western blotting. A shift in the band was seen for the conjugate in SDS PAGE for each set of reaction conditions and a positive result was obtained from western blotting.
• a scaled-up reaction for 5 mg Fetuin followed by purification of the resulting conjugate was carried out. 5 mg Fetuin was oxidized with 10 mM NaIO 4 for 60 minutes at 4°C and then oxidation was stopped by adding NaHSO 3 to a final concentration of 10 mM. The conjugation reaction was carried out using the oxidized Fetuin with adipic acid dihydrazide-PSA polymer. The final concentration of polymer in the reaction mixture was 2.5 mM. The final pH of reaction mixture was around 5.75.
• DNAse was oxidized with NaIO4 to a final concentration ranging from 0.2 mM to 2 mM for 30 minutes at 4 0 C.
• the oxidation reaction was stopped by adding NaHSO 3 to a final concentration of between 2 and 5 mM depending upon the concentration Of NaIO 4 used for oxidation.
• Glycopolysialylation of oxidized DNAse was carried out by addition of hydrazide-PSA polymer to a final concentration of 1.25 mM to the oxidized DNAse.
• ⁇ -Galactosidase (1 mg) was oxidized with 1.5 mM Of NaIO 4 for 30 minutes at 4°C. The oxidation was stopped by adding NaHSO 3 to a final concentration of 1.5 mM.
• the conjugation reaction was carried out using the oxidized ⁇ -Galactosidase with diaminooxy-PEG polymer (20 kDa). The final concentration of the polymer in the reaction mixture was 1.25 mM. The final concentration of ⁇ -Galactosidase in the reaction mixture was 1 mg/ml. The final pH of reaction mixture should be around 5.75. Sodium cyanoborohydride was added to the reaction mixture to a concentration of 50 mM or 3.17 mg/ml. The reaction was carried out at 4°C for 2 hours. Unpurified conjugate was characterized using SDS PAGE and a shift in the band was seen for the conjugate in SDS PAGE. The activity was measured as 59%.
• EPO Erythropoietin
• the invention thus provides conjugates of compounds other than blood coagulation proteins with water soluble polymers, in particular PSA and mPSA.

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