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WO2011077067A1 - Conjugués polymères d'érythropoïétine non glycosylée - Google Patents

Conjugués polymères d'érythropoïétine non glycosylée Download PDF

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Publication number
WO2011077067A1
WO2011077067A1 PCT/GB2010/001633 GB2010001633W WO2011077067A1 WO 2011077067 A1 WO2011077067 A1 WO 2011077067A1 GB 2010001633 W GB2010001633 W GB 2010001633W WO 2011077067 A1 WO2011077067 A1 WO 2011077067A1
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Prior art keywords
epo
conjugate
polymer
erythropoietin
group
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Ji-Won Choi
Anthony Godwin
George Badescu
Keith Powell
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Abzena UK Ltd
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Polytherics Ltd
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    • 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
    • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • This invention relates to novel polymer conjugates, and a process for their preparation.
  • EPO Erythropoietin
  • the native human protein has 165 amino acids, 4 glycosylation sites, and a molecular weight of about 34,000 Da.
  • Non-glycosylated EPO has a molecular weight of about 1 ,000 Da.
  • EPO has been used for the treatment of a variety of conditions, including anaemia, the reduction of allogeneic blood transfusion in patients undergoing surgery, and pruritis associated with renal failure.
  • One major disadvantage of EPO therapy is that the protein has a short circulation half-life, and frequent dosing is necessary.
  • EPO EPO
  • mammalian cell culture EPO
  • EPO EPO
  • microorganisms such as E. coli
  • it is highly prone to aggregation, making it difficult to formulate as a stable medicine.
  • PEGylation The process of covalently conjugating water-soluble, synthetic polymers, particularly polyethylene glycol, PEG, to proteins is well known.
  • PEGylation The conjugation of PEG is commonly known as "PEGylation".
  • PEGylating certain proteins can improve certain of their properties, and many PEGylating reagents are known, for example from WO 99/45964, WO 2005/007197, and WO 2009/047500.
  • PEGylation can prolong the half-life of certain therapeutic proteins.
  • PEGylation can reduce the intrinsic in vivo activity of some proteins, and this has been reported with EPO.
  • US 6,340,742 discloses conjugates of glycosylated EPO and PEG, the PEG being linked to the glycoprotein via a specific linker. One, two or three PEG groups may be present.
  • EP 1 219 636 (US 2002/0081734) seeks to address the problem of lack of activity of non- glycosylated EPO by modifying the amino acid structure of the peptide chain in order to provide alternative attachment sites for PEG or other modifiers.
  • Int. J. Pharmaceutics describes producing non-glycosylated EPO from E coli and then PEGylating it.
  • WO 2006/061853 describes the attachment of a branched PEG to nonglycosylated EPO.
  • WO 2006/089228 discloses a number of possible EPO-polymer conjugates, the claimed conjugates being of the formula POLY-Q-(CH 2 ) m -CHZ-CO.NH.EPO
  • POLY is a polyalkylene oxide
  • Q is an optional linking group having a length of from 1 to 10 atoms
  • m is an integer from 0 to 20
  • Z is alkyl, substituted alkyl, aryl or substituted aryl
  • EPO is a residue of erythropoietin
  • "NH-EPO" represents an amino group of EPO.
  • POLY is preferably PEG which may be linear, branched, and/or forked.
  • EP 1 333 036 (US 2006/0276634) describes PEG-EPO conjugates obtained by reacting recombinant human EPO produced in animal host cells - i.e. glycosylated EPO - with an amino reactive derivative of PEG.
  • EP 1 333 036 explain that "The general recognition of the relationship between the molecular weight of PEG used for conjugation and in vivo activity of EPO was that EPO conjugated with higher molecular weight PEG showed more improved plasma retention and hence greater and more sustained efficacy.”
  • the inventors of the present invention have clarified that such general recognition does not apply to PEG conjugation for native (recombinant) EPO with sugar chains because conjugation with extremely high molecular weight PEG also reduces the in vivo activity of EPO.
  • the present invention provides a conjugate of erythropoietin with a polymer, characterised in that the erythropoietin is non-glycosylated and is conjugated to two separate polymer chains, each of said polymer chains being bonded to the erythropoietin at two amino acid residues.
  • erythropoietin, EPO should be understood to include any polypeptide having the amino acid sequence of human EPO, and any polypeptide substantially homologous thereto, whose biological properties relate to the ability, in vivo, to stimulate red blood cell production as well as the division and differentiation of committed erythroid progenitors in the bone marrow.
  • NCBI Reference Sequence: NM_000799.2 The nucleotide sequence of human EPO (NCBI Reference Sequence: NM_000799.2) was reported in 1985 [Jacobs, et al. Nature 313 (6005), 806-810 (1 85)] and encodes a polypeptide chain containing 193 amino acids (NCBI Reference Sequence: NP_000790.2).
  • a 166 amino acid peptide SEQ ID NO: 2; Figure 2 is initially generated following the cleavage of a 27 amino acid hydrophobic secretory leader at the amino-terminal.
  • a carboxy-terminal arginine in position 166 is removed by post-translational modification both in the mature human and recombinant human EPO (rhEPO) resulting in a circulatory mature protein of 165 amino acids (SEQ ID NO: 1, Figure 1).
  • additional terminal amino acid(s) may be present, and such sequences should be understood to be included in the definition of human EPO.
  • Met is a common terminal amino acid often found in E. coli derived EPO.
  • the EPO used in the present invention comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • Polypeptides are understood to be substantially homologous to EPO if they differ from each other by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity. In the present invention, sequences having greater than 95% homology are considered substantially homologous.
  • the EPO used in the present invention has a peptide chain which contains 0 to 6, preferably 0, modifications of amino acids within the polypeptide chain, and which may optionally be modified by the addition of one or more amino acids at the N or C terminus of the chain.
  • 6 or fewer additional terminal amino acids are present, unless the additional amino acids form a polyhistidine tag, in which larger number of histidine residues, for example up to 12, preferably up to 9, histidine residues may be introduced.
  • the polypeptide chain of EPO is unmodified from the human sequence, or is unmodified from the human sequence other than by the addition of a polyhistidine tag, for example at the N or C terminus, or elsewhere along the main chain.
  • Non-glycosylated EPO should be understood to include EPO which has been produced in non-glycosylated form, or EPO which has been produced in glycosylated form and has had the glycosylating groups partially or fully removed (sometimes referred to as "de- glycosylated” EPO).
  • the EPO has been produced in non-glycosylated form, for example by expression in bacterial cells, e.g. E. coli.
  • the conjugates of the invention contain two separate polymer chains, each of which is bonded to the EPO at two amino acid residues.
  • the two chains may be the same or different.
  • the conjugate may also if desired contain additional polymer chains. Each of these additional chains may be bonded to the EPO via one or two amino acid residues. Preferably each additional chain, if present, is bonded to the EPO via two amino acid residues.
  • Each of said two polymer chains may be bonded via amine groups in the EPO (e.g. via lysine or histidine residues), via the N-terminal group, and/or via thiol groups (i.e. via cysteine residues). Said residues may be present in the unmodified EPO sequence, or may have been artificially introduced.
  • the bonding is via amine groups, these are preferably artificially introduced as histidine residues in the form of a polyhistidine tag.
  • the attachment can be on the N-terminal group of native EPO; such attachment may be achieved for example by reductive amination.
  • the bonding is via thiol groups, these groups are preferably provided by reduction of one or both of the disulfide bridges found in native EPO, at residues 29/33, and residues 7/161 , as shown in Figures 1 and 2.
  • the conjugate comprises one polymer chain bound via two cysteine residues of the first disulfide bridge of EPO, e.g. at positions 29 and 33, and one polymer chain bound via the two cysteine residues of the second disulfide bridge, e.g. at positions 7 and 161.
  • the conjugate comprises one polymer chain bound via one of the disulfide bridges, e.g. either the 29 and 33 cysteine residues of EPO or the 7 and 161 cysteine residues of EPO, and one polymer chain bound via a polyhistidine tag.
  • the conjugate comprises two (or more) polymer chains bound via a polyhistidine tag.
  • the conjugate may contain one or more additional polymer chains bound to the EPO at any desired point or points.
  • a polymer in a conjugate according to the invention may for example be a poly(alkylene glycol), a polyvinylpyrrolidone, a polyacrylate, for example poly(acryloyl morpholine), a polymethacrylate, a polyoxazoline, a polyvinylalcohol, a polyacrylamide or polymethacrylamide, for example
  • polycarboxymethacrylamide or a HPMA copolymer.
  • the polymer may be one that is susceptible to enzymatic or hydrolytic degradation.
  • Such polymers include polyesters, polyacetals, poly(ortho esters), polycarbonates, poly(imino carbonates), and polyamides, such as poly(amino acids).
  • the polymer may be a homopolymer, random copolymer or a structurally defined copolymer such as a block copolymer. For example it may be a copolymer, e.g.
  • a block copolymer derived from two or more alkylene oxides, or from poly(alkylene oxide) and either a polyester, polyacetal, poly(ortho ester), or a poly(amino acid).
  • Polyfunctional polymers that may be used include copolymers of divinylether-maleic anhydride and styrene-maleic anhydride.
  • Naturally occurring polymers may also be used, for example polysaccharides such as chitin, dextran, dextrin, chitosan, starch, cellulose, glycogen, poly(sialylic acid) and derivatives thereof.
  • a protein may be used as the polymer. This allows conjugation of EPO to a second protein.
  • Poly(amino acid)s such as polyglutamic acid or polyglycine may also be used, as may hybrid polymers derived from natural monomers such as saccharides or amino acids and synthetic monomers such as ethylene oxide or methacrylic acid.
  • the polymer used in the present invention is a hydrophilic or water-soluble, synthetic polymer.
  • the polymer is a poly(alkylene glycol)
  • this is preferably one containing C 2 and/or C 3 units, and is especially a poly( ethylene glycol) (PEG).
  • PEG poly( ethylene glycol)
  • any reference to a polymer in this specification should be understood to include a specific reference to PEG.
  • a polymer, particularly a poly(alkylene glycol) may contain a single linear chain, or it may have branched morphology composed of many chains either small or large.
  • Pluronics are an important class of PEG block copolymers. These are derived from ethylene oxide and propylene oxide blocks. Substituted polyalkylene glycols, for example
  • methoxypolyethylene glycol may be used.
  • a single-chain poly(ethylene glycol) is initiated by a suitable group, for example an alkoxy, e.g. methoxy, aryloxy, carboxy or hydroxyl group, and is ultimately connected at the other end of the chain, via appropriate linker groups, to the EPO.
  • the polymer chains may have any suitable molecular weight, and each chain may have the same or different molecular weight as any other.
  • each chain may have a molecular weight of at least 5, 10, 15, 20, 30, or 40 kDa.
  • the preferred maximum molecular weight of each chain is 60 kDa.
  • each of said two chains are PEG, they preferably have the same molecular weight as each other.
  • the novel conjugates of the present invention may be prepared by reacting non-glycosylated EPO with one or more appropriate polymer conjugating reagents. Depending upon the conjugate to be prepared, two polymer chains may be conjugated to the EPO in either a one- or a two-step process.
  • any suitable di-functional polymer conjugation reagent may be used, for example one of the reagents described in WO 99/45964 or WO 2005/007197.
  • the reagent is of the formula I, II or III below: in which one of X and X' represents a polymer and the other represents a hydrogen atom;
  • Q represents a linking group
  • W represents an electron-withdrawing group, for example a keto group, an ester group -O-CO- or a sulfone group -S0 2 -; or, if X' represents a polymer, X-Q-W together may represent an electron withdrawing group;
  • A represents a Ci -5 alkylene or alkenylene chain
  • B represents a bond or a C alkylene or alkenylene chain
  • each L independently represents a leaving group
  • X, X', Q, W, A and L have the meanings given for the general formula I, and in addition if X represents a polymer, X' and electron-withdrawing group W together with the interjacent atoms may form a ring, and m represents an integer 1 to 4; or
  • X-Q-W-CR 3 C4 2 -L (III) in which X, Q and W have the meanings given for the general formula I, and either R represents a hydrogen atom or a Ci ⁇ alkyl group and L represents a leaving group, or R L together represent a bond; and R 4 represents a hydrogen atom or a C alkyl group.
  • An especially preferred polymer conjugation reagent has the formula:
  • the first step of the process will involve reducing the disulfide bond and subsequently reacting the reduced product with the polymer conjugation reagent, preferably one of those of formulae I, II or III given above. If it is desired to link both polymer chains across the two disulfide bonds of EPO, both bonds may be reduced in a single step, following which the conjugation reaction takes place. If it is desired to attach two polymer chains to a
  • polyhistidine tag this may be done in a single-step process. If it is desired to attach one polymer chain across a disulfide bridge and a second polymer chain to a polyhistidine tag, this is generally carried out as a two-step process, appropriate reaction steps being used for each process.
  • the immediate product of the process described using one of the reagents I, II or III above is a conjugate which contains an electron- withdrawing group W.
  • the process of the invention is reversible under suitable conditions. This may be desirable for some
  • the process described above may comprise an additional optional step of reducing the electron withdrawing group W in the conjugate.
  • a borohydride for example sodium borohydride, sodium
  • cyanoborohydride potassium borohydride or sodium triacetoxyborohydride
  • reducing agent is particularly preferred.
  • Other reducing agents which may be used include for example tin(II) chloride, alkoxides such as aluminium alkoxide, and lithium aluminium hydride.
  • a sulfone may be reduced to a sulfoxide, sulfide or thiol ether.
  • a group X-Q-W- which is a cyano group may be reduced to an amine group.
  • a key feature of using polymeric conjugation reagents of the formulae I, II and III is that an ci-mefhylene leaving group and a double bond are cross-conjugated with an electron withdrawing function that serves as a Michael activating moiety. If the leaving group is prone to elimination in the cross-functional reagent rather than to direct displacement and the electron-withdrawing group is a suitable activating moiety for the Michael reaction then sequential intramolecular bis-alkylation can occur by consecutive Michael and retro Michael reactions.
  • the leaving moiety serves to mask a latent conjugated double bond that is not exposed until after the first alkylation has occurred and bis-alkylation results from sequential and interactive Michael and retro-Michael reactions as described in J. Am. Chem. Soc. 1979, 101, 3098-3110 and J. Am. Chem. Soc. 1988, 1 10, 5211-5212.).
  • the electron withdrawing group and the leaving group are optimally selected so bis-alkylation can occur by sequential Michael and retro-Michael reactions. It is also possible to prepare cross-functional alkylating agents with additional multiple bonds conjugated to the double bond or between the leaving group and the electron withdrawing group as described in J. Am. Chem. Soc. 1988, 1 10, 5211-5212.
  • a linking group Q may for example be a direct bond, an alkylene group (preferably a Ci.io alkylene group), or an optionally-substituted aryl or heteroaryl group, any of which may be terminated or interrupted by one or more oxygen atoms, sulfur atoms, -NR groups (in which R represents a hydrogen atom or an alkyl (preferably Ci.
  • aryl preferably phenyl
  • alkyl-aryl preferably Ci -6 alkyl -phenyl
  • keto groups -O-CO- groups, -CO-O- groups, -O-CO-O, -0-CO-NR-, -NR-CO-0-, -CO-NR- and/or -NR.CO- groups.
  • aryl and heteroaryl groups Q form one preferred embodiment of the invention.
  • Suitable aryl groups include phenyl and naphthyl groups
  • suitable heteroaryl groups include pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, primidine and purine.
  • linking groups Q are heteroaryl or, especially, aryl groups, especially phenyl groups, terminated adjacent the polymer X by an -NR.CO- group.
  • the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • W may for example represent a keto group CO, an ester group -O-CO- or a sulfone group -S0 2 -; or, if X-Q-W- together represent an electron withdrawing group, this group may for example be a cyano group.
  • substituents include for example CN, N0 2 , -OR, -OCOR, -SR, -NHCOR, -NR.COR, -NHOH and -NR.COR.
  • Typical structures in which W and X' together form a ring include
  • n' is an integer from 1 to 4
  • a leaving group L may for example represent -SR, -S0 2 R, -OS0 2 R,-N + R 3 ,-N + HR 2 ,-N + H 2 R, halogen, or -00, in which R has the meaning given above, and 0 represents a substituted aryl, especially phenyl, group, containing at least one electron withdrawing substituent, for example -CN,-N0 2 , -C0 2 R, -COH, -CH 2 OH, -COR, -OR, -OCOR, -OC0 2 R, -SR,-SOR, -
  • the EPO may be allowed to react directly with the polymer conjugation reagent in an aqueous reaction medium.
  • This reaction medium may also be buffered, depending on the pH requirements of the nucleophile.
  • the optimum pH for the reaction will generally be at least 4.5, typically between about 5.0 and about 8.5, preferably about 5.0 to 7.0.
  • the optimal reaction conditions will of course depend upon the specific reactants employed.
  • Reaction temperatures between 3-37°C are generally suitable. Reactions conducted in organic media (for example THF, ethyl acetate, acetone) are typically conducted at temperatures up to ambient.
  • organic media for example THF, ethyl acetate, acetone
  • the EPO can be effectively conjugated with the desired reagent using a stoichiometric equivalent or an excess of reagent.
  • Excess reagent and the product can be easily separated by standard chromatography methods, e.g. ion exchange chromatography during routine purification of proteins, or, when a polyhistidine tag is present, by separation using metal affinity chromatography, e.g. based on nickel.
  • Conjugates according to the present invention may be used for the treatment of a number of conditions, especially anaemia, for example anaemia associated with chronic renal failure, zidovudine therapy in HIV-infected patients, or in cancer patients undergoing
  • conjugates can be used to reduce allogeneic blood transfusion in surgery patients.
  • the present invention therefore also provides a conjugate according to the invention for use in therapy, specifically for use in the treatment of anaemia; the use of a conjugate according to the invention for the manufacture of a medicament for the treatment of anaemia; and a method of treating anaemia which comprises the administration to a patient, especially a human patient, of a conjugate according to the invention.
  • the conjugate can be administered to the patient prior to, simultaneously with, or after administration of another active agent, such as a chemotherapy agent.
  • the conjugate can be administered to an anemic patient prior to undergoing surgery.
  • conjugates will be administered by similar routes to that of currently administered EPO, e.g., by intravenous, subcutaneous or intramuscular routes.
  • conjugates according to the present invention may be formulated into the desired dosage form such as solutions, prefilled syringe-, painless needle- or needleless-systems for subcutaneous administration.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a conjugate according to the invention together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for formulating the products of the invention are human serum albumin, human plasma proteins, etc.
  • the conjugates may be formulated in 10 mM sodium/potassium phosphate buffer at pH 7 containing a tonicity agent, e.g. 132 mM sodium chloride.
  • the pharmaceutical composition may contain a preservative.
  • Alternative compositions may comprise sustained release preparations for intracutaneous implantation (e.g., microcapsules, polymeric micelles, polymer-based gelatinous
  • composition may if desired further comprise a second therapeutic material.
  • the actual dose of the conjugate to be administered will vary depending upon the age, weight, and general condition of the individual as well as the severity of the condition being treated, the erythropoietin sensitivity of the individual, and the conjugate being administered.
  • the dosage may be adjusted to achieve a haemoglobin increase of >lg/dL from the baseline or a haemoglobin concentration of >1 Ig/dL.
  • Suitable dosages may include from 1 to 1000 ⁇ g.
  • the dose main comprise from 0.01 to 10 ⁇ g per kg body weight, such as 0.1 to 1 ⁇ g per kg body weight, for intravenous administration, e.g. once weekly.
  • the percentage of reticulocytes (erythrocyte precursors) in total erythrocytes can be used as an indicator for EPO activity.
  • activity of native EPO observed as the percentage of reticulocytes will reach a peak 3 to 5 days after administration by any route mentioned above, followed by a rapid decline.
  • native EPO generally needs to be injected twice or three times per week to ensure sufficient therapeutic effects.
  • conjugates according to the present invention thus allows an extended interval for erythropoietin administration.
  • the interval can be extended to once per week from 2 to 3 times per week, or extended to every 10 days to every 2 weeks from every week. Therefore, it is possible not only to ease the physical and time burden on patients by reducing the numbers of hospital visits and injections, but also to save medical costs by reducing the load on medical staff members.
  • the present invention makes possible for the first time, the viable pharmacological use of non-glycoslylated EPO, including EPO produced in bacterial cells rather than mammalian cells.
  • PEGylation of proteins including EPO is expected to increase the circulation half-life of the protein in vivo.
  • this increase in half-life is frequently at the expense of inherent activity.
  • Introduction of more than one PEG chain generally decreases the inherent activity dramatically, as demonstrated for example in EP 1 333 036.
  • non-glycosylated EPO diPEGylated in accordance with the present invention not only has a much better half-life than either native EPO or mono-PEGylated non-glycosylated EPO, it also has an intrinsic level of activity which is as high as mono- PEGylated EPO.
  • This combination of long half-life and high intrinsic activity is most unusual in polymer conjugates of proteins: it is not normally obtained on di-PEGylation of proteins, and could not have been predicted for EPO.
  • Figure 1 shows the sequence listing of human EPO with 165 amino acids (SEQ ID No: 1).
  • Figure 2 shows the sequence listing of human EPO with 166 amino acids including a terminal Arg (SEQ ID No: 2).
  • Figure 3 shows the SDS-PAGE analysis of the initial products of Example 1.
  • Figure 4 shows the results of the in vitro proliferative assay of Example 1.
  • Figure 5 shows SDS-PAGE analysis of mono and di 20kDa PEG-EPO products of Example 2.
  • Figure 6 shows the results of the in vitro proliferative assay of Example 2.
  • Figure 7 shows the results of the testing of Example 11.
  • Example 1 PEGylation of /j.co/Z-expressed, non-glycosylated erythropoietin (EPO) with a PEG reagent with a molecular weight of 20 kDa
  • the dialysed PEG stock solution (56 ⁇ , 1 molar equivalent) was added to 5 ml of reduced EPO solution (0.2 mg ml) and the reaction mixture was vortexed for several seconds and then placed at 4°C for 3 h, whereafter a sample was taken for SDS-PAGE analysis.
  • the SDS- PAGE gel was stained both with InstantBlue (Expedeon cat. No. ISB1 L) for protein visualisation and with barium iodide for PEG visualisation. The result is shown in Figure 3b.
  • Lane 1 shows the protein markers used to estimate MW (Novex sharp protein standards, Invitrogen cat. No. LC5800)
  • lane 2 shows the reaction solution of reduced EPO with 20 kDa PEG reagent (1 molar equivalent).
  • the unreacted PEG reagent was removed by cation exchange chromatography.
  • the reaction mixture was diluted 10-fold with 50 mM sodium acetate buffer (pH 4.5) and loaded onto a 1 ml HiTrap SP FF cation exchange column (GE Healthcare cat. no. 17-5054-01).
  • the column was washed with 20 ml of 50 mM sodium acetate buffer (pH 4.5) and the bound protein was eluted with a buffer of 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA and 1 M NaCl (pH 7.0). Elution fractions were collected (0.5 ml each) and analysed by SDS-PAGE.
  • the fractions containing PEGylated EPO were pooled together and loaded onto a HiLoad Superdex 200 16/60 (GE Healthcare cat. no. 17-5175-01) size exclusion chromatography column, pre-equilibrated with running buffer (23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, (pH 7.0) at 1 ml/min flow rate and detection at 280 nm). The fractions collected (1 ml each) were analysed by SDS- PAGE. Fractions containing either di-PEGylated EPO or mono-PEGylated EPO were combined and quantified by measuring the UV absorbance at 280 ran and by the Bradford assay, using BSA as the standard.
  • EPO-sensitive UT-7 cells cultured in growth medium consisting of MEM alpha (PAA, cat. no. E15-832), 2% penicillin/streptomycin (PAA, cat. no. PI 1-010), 10% foetal calf serum (FCS) (Invitrogen, cat. no. 10099-141) and 5 ng/ml of EPO, were used for the in vitro EPO- dependent proliferative assay.
  • Required amount of UT-7 cells were centrifuged at 500 g for 5 min and resuspended in assay medium (MEM alpha, 2% penicillin/streptomycin, 10% FCS) at a concentration of 3xl0 6 cells/ml.
  • the plates were then centrifuged at 500 g for 5 min and the media was carefully removed as not to disturb the formazan crystals.
  • DMSO 200 ⁇ was then added to each well to dissolve the formazan crystals that form an intense purple colour, and the absorbance was measured spectrophotometrically at 570 nm.
  • Example 2 PEGylation of iT.co/i-expressed, non-glycosylated erythropoietin (EPO) with a PEG reagent with molecular weight of 5, 10, 20, 30 and 40 kDa.
  • EPO erythropoietin
  • PEG reagent with molecular weight of 5, 10, 20, 30 and 40 kDa.
  • EPO solution 0.9 mg/ml, in 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 7.0
  • DTT stock solution (1 M in deionised water
  • Reduced EPO solution 1.5 ml was loaded onto a PD-10 column (GE Healthcare cat.
  • reaction buffer 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 6.0
  • reaction buffer 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 6.0
  • PEG reagents of the formula shown in Example 1 with molecular weights of 5, 10, 20, 30 and 40 kDa were dissolved in 5 mM phosphate buffer (pH 7.8) to give 5, 10, 20, 30 and 40 mg/ml solution concentrations respectively. Each solution was dialysed against 35 ml 5 mM phosphate buffer (pH 7.8) overnight at 4°C.
  • the fractions containing PEGylated EPO were pooled together and loaded onto a HiLoad Superdex 200 16/60 (GE Healthcare cat. no. 17-5175-01) size exclusion chromatography column, pre-equilibrated with running buffer (23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, (pH 7.0) at 1 ml/min flow rate and detection at 280 nm). The fractions collected (1 ml each) were analysed by SDS- PAGE. The results for mono- and di- 20 kDa PEG-EPO are shown in Figure 5. Fractions containing either di-PEGylated EPO or mono-PEGylated EPO from each purification run were combined and quantified by measuring the UV absorbance at 280 nm and by the Bradford assay, using BSA as standard.
  • EPO-sensitive UT-7 cells cultured in growth medium consisting of MEM alpha (PAA, cat. no. E15-832), 2% penicillin/streptomycin (PAA, cat. no. PI 1-010), 10% foetal calf serum (FCS)(Invitrogen, cat. no. 10099-141) and 5 ng/ml of EPO, were used for the in vitro EPO- dependent proliferative assay.
  • Required amount of UT-7 cells were centrifuged at 500 g for 5 min and resuspended in assay medium (MEM alpha, 2% penicillin/streptomycin, 10% FCS) at a concentration of 3xl0 6 cells/ml.
  • the in vitro UT-7 proliferative assay was performed on each of the 10 kDa, 20 kDa, 30 kDa and 40 kDa mono- and di-PEGylated EPO, and the results are shown in Figure 6.
  • the mono- and the di-PEGylated EPO showed similar activity at equimolar concentration, based on the 50% effective dose (ED 5 o).
  • attachment of 10 kDa, 20 kDa, 30 kDa and 40 kDa PEG did not affect the in vitro activity of the PEGylated EPO.
  • PVP poly(l-vinyl-2-pyrrolidone)
  • PVP-amine 500 mg
  • 4-[2,2- bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid 125 mg
  • 4-dimethylaminopyridine 6 mg
  • anhydrous dichloromethane 10 ml
  • 1 ,3- diisopropylcarbodiimide 80 ⁇
  • the resulting mixture was allowed to stir for 20 h at room temperature and then filtered though non-absorbent cotton- wool.
  • diethyl ether 20 ml
  • the resulting precipitate isolated by centrifugation (2,000 rpm, 2 min, 2 °C).
  • a fraction that eluted at the same time as a 20 kDa PEG run under the same conditions was used for protein conjugation and therefore, the PVP fraction had a PEG equivalent molecular weight of 20 kDa.
  • a sufficient volume of the fraction was buffer exchanged to pH 7.8 phosphate using a protein spin desalting column (Pierce) and immediately frozen until required for protein conjugation.
  • E.co 7-expressed non-glycosylated EPO (1 mg, 1.0 mg/ml) was reduced with DTT (1.54 mg) for 90 min at room temperature and then the DTT was removed and the buffer exchanged to pH 7.8 buffer (5 M urea, 0.4 M guanidine hydrochloride, 10 mM EDTA, 100 mM Gly-Gly) using a PD-10 column (GE Healthcare).
  • pH 7.8 buffer 5 M urea, 0.4 M guanidine hydrochloride, 10 mM EDTA, 100 mM Gly-Gly
  • a PD-10 column GE Healthcare
  • Example 4 Preparation of di-PEGylated EPO using a 10 kDa PEG reagent with a carbonate linking group, Q, and reduction of the PEG reagent electron withdrawing group, W, with sodium borohydride.
  • DTT (10 ⁇ , 1 M solution in deionised water) was added to an E.co/z ' -expressed non- glycosylated EPO solution (1 ml, 1 mg/ml solution in 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 7.0) and the resulting solution was allowed to stand at room temperature for 30 min. The solution was then loaded onto a PD- 10 column (GE Healthcare cat. no.
  • the eluted fraction was then diluted with 1.5 ml of freshly prepared and argon-purged reaction buffer (23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 6.0) to give a final concentration of 0.2 mg/ml reduced EPO. Reduction of EPO was confirmed by SDS-PAGE analysis.
  • the unreacted PEG reagent was removed by cation exchange chromatography.
  • the reaction mixture was diluted 10-fold with 50 mM sodium acetate buffer (pH 4.5) and loaded onto a 1 ml HiTrap SP FF cation exchange column (GE Healthcare cat. no. 17-5054-01).
  • the column was washed with 20 ml of 50 mM sodium acetate buffer (pH 4.5) and the bound protein was eluted with a buffer of 23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM
  • fractions collected (1 ml each) were analysed by SDS-PAGE and fractions containing di-PEGylated EPO were combined which gave a single band when reanalysed by SDS-PAGE, running close to the 60 kDa protein marker.
  • Example 5 Structural study demonstrating the linkage of PEG polymer chains to cysteine residues which form the two disulfide bridges in native .E.co/i-expressed, non- glycosylated erythropoietin (EPO)
  • the di-PEGylated E.co/z ' -expressed non- glycosylated EPO product of Example 4 was subjected to tryptic digestion analysis of peptide fragments by LC-MS MS (Q-TRAP) and the results compared to masses obtained by in silico digestion of EPO.
  • the PEG Prior to tryptic digestion, the PEG was cleaved from the EPO and PEG reagent linker by hydrolysis of the carbonate bond between PEG and linker under basic conditions (pH 8.9 for 16 h at 4°C) resulting in the almost complete cleavage of PEG when analysed by SDS- PAGE.
  • the PAGE band corresponding to de-PEGylated EPO was excised from the gel to provide the sample for analysis.
  • Digestion with trypsin was performed by adding a trypsin solution and incubating at 37°C overnight. After treating with formic acid aqueous solution, samples were analysed by LC-MS/MS using a 5500 QTRAP instrument (AB Sciex UK Ltd). Some of the samples were also treated with DTT and iodoacetamide prior to analysis. For each run, 10 ⁇ of sample was injected using 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) as mobile phases and a gradient elution method (flow rate 0.4 ml/min).
  • A acetonitrile
  • B 0.1% formic acid in water
  • Example 7 Use of bifurcated PEG as the polymer component: Conjugation of a 20 kDa bifurcated PEG to disulfide reduced £.co/i-expressed non-glycosylated (EPO).
  • the PEG reagent stock solution (81.5 ⁇ ) was added to a solution of reduced EPO (1 ml, 0.5 mg/ml) and the reaction mixture was vortexed for several seconds and incubated at room temperature for 3 h, whereafter a sample was taken for SDS-PAGE analysis.
  • the SDS- PAGE gel was stained with InstantBlue (Expedeon cat. No. ISB1 L) for protein visualisation and with barium iodide for PEG visualisation. A band was visible next to the 260 kDa protein marker, which corresponding to di-PEGylated EPO conjugate.
  • Example 8 Preparation and in vitro activity of di-PEGylated non-glycosylated EPO using a 20 kDa PEG reagent with non-aromatic leaving groups based on succinic acid, L, and reduction of the PEG reagent electron withdrawing group, W, with sodium borohydride.
  • DTT 25 ⁇ , 1 M solution in deionised water
  • E.co/ -expressed non- glycosylated EPO solution 2.5 ml, 1.17 mg/ml, in 50 mM Tris-HCl, 5 M urea, 0.4 M guanidinium hydrochloride, pH 8.2
  • the solution was then loaded onto a PD-10 column (GE Healthcare cat. no.
  • the eluted fraction was then diluted with freshly prepared and argon-purged reaction buffer (23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 6.0) to give a final concentration of 0.15 mg/ml reduced EPO. Reduction of EPO was confirmed by SDS-PAGE analysis.
  • Example 9 Preparation and in vitro activity of di-PEGylated non-glycosylated EPO using a 20 kDa PEG reagent with alkyl leaving groups, L.
  • DTT 25 ⁇ , 1 M solution in deionised water
  • E.co/ -expressed non- glycosylated EPO solution 2.5 ml, 1.17 mg/ml, in 50 mM Tris-HCl , 5 M urea, 0.4 M guanidinium hydrochloride, pH 8.2
  • the solution was then loaded onto a PD-10 column (GE Healthcare cat. no.
  • the eluted fraction was then diluted with freshly prepared and argon-purged reaction buffer (23 mM imidazole, 5 M urea, 0.4 M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl, pH 6.0) to give a final concentration of 0.2 mg/ml reduced EPO. Reduction of EPO was confirmed by SDS-P AGE analysis.
  • Example 1 In vitro activity of PEGylated EPO conjugates was determined using the protocol described in Example 1. Both mono- and di-PEGylated EPO were active and showed similar activity at equimolar concentration based on protein amount (9-22 ng/ml and 5-50 ng/ml, respectively).
  • Example 10 Preparation of diPEG EPO using a N-terminally octahistidine-tagged ii.c0//-expressed non-glycosylated EPO (N 8xH i S -EPO) with a 20 kDa PEG reagent.
  • Example 11 Pharmacokinetic study demonstrating half- life of diPEGylated E.coli- expressed, non-glycosylated EPO from Example 9.
  • glycosylated EPO (Erythropoietin BRP batch 3, E1515000, European Directorate for the Quality of Medicines & HealthCare, European Pharmacopoeia) was compared to mono- and di-PEGylated E. co/7-expressed non-glycosylated EPO (20 kDa PEG-EPO and 2x20 kDa PEG-EPO).
  • Mono-PEGylated and di-PEGylated EPO were prepared according to the procedure described in Example 8.
  • the test compounds were first radiolabelled with 125 I, purified by RADIO-HPLC and specific activity was determined before bolus injection into male CD-I mice. At each time point, plasma samples were collected and activity in the samples was determined by using gamma counting. Plasma clearance of the molecules was determined by calculating area under the curve (AUC) and half-life (Ti /2 ) for each of the compounds.
  • Molecules were labelled using IODO-GEN tubes (Cat no: 28601, Thermo Scientific). One tube was used for each radiolabelling reaction. Tubes were first rinsed with a buffer containing 100 tnM gly-gly, 5 M urea, 0.4M guanidinium hydrochloride, 5 mM EDTA, 18 mM NaCl (pH 7.2) (1 ml), and then a small aliquot of the same buffer (100 ⁇ ) was added to the tubes. Radioactive Iodine ( l25 I) was added and incubated for 6 min at room temperature (RT) shaking gently every minute.
  • RT room temperature
  • Oxidized iodine was removed from the IODO-GEN tubes and added to fresh tubes containing solutions of the test compounds and incubated for 15 min at RT shaking every two min. Then the samples were diluted 1 :10 with a buffer containing 100 mM gly-gly, 5 M urea, 0.4 M guanidinium hydrochloride, 50 mg/ml NV-10 (Expedeon), 5 mM EDTA and 18 mM NaCl (pH 7.2) and then incubated at 4°C for 3 hours.
  • Free iodine was removed by gel filtration (HiTrap 5 ml, cat no: 17-1408-01, GE Healthcare) column using phosphate saline buffer (PBS) (pH 7.4) supplemented with 1 mg/ml NV-10 as running buffer. Both UV absorbance (280 nm) and radioactivity were monitored and fractions were collected (1 ml). The concentration of the fractions containing radiolabelled compounds was adjusted to 50 g/ml using the gel filtration running buffer.
  • PBS phosphate saline buffer
  • I labelled molecule 100 ⁇ was injected intravenously (IV) using three mice for each time point.
  • the animals were sacrificed by C0 2 asphyxiation and immediately after that blood samples were taken via cardiac puncture into K 2 EDTA tubes (BD, REF 365 955).
  • the tubes were then centrifuged (13,000 RPM, 3 min) and plasma samples (150-200 ⁇ ) were collected into gammacounter tubes. All plasma samples were measured with an automated gammacounter (Gammamaster 1277, Wallac, Finland) using 60 s pulse collection time and physical decay corrections were made during the analysis.
  • % ID / ml of plasma values were calculated from all the time points measured and these values are shown in Figure 7, in which there are compared the % ID / ml plasma values of glycosylated EPO (closed circle), mono-PEGylated (20 kDa) non-glycosylated E.coli- expressed EPO (open square) and di-PEGylated (20 kDa) non-glycosylated E.co/i-expressed ⁇ (closed triangle). Standard deviation for each time point is presented as error bars.
  • Table 1 T1 ⁇ 2 and AUC values for glycosylated EPO and for mono- and di-PEGylated(20 kDa) non-glycosylated E.co/i-expressed ⁇ .
  • mice Eight male or female B6D2F1 mice, purchased from RCC Ltd (Switzerland), were used for each dosing solution.
  • the test compounds were glycosylated EPO (Erythropoietin BRP batch 3, El 515000, European Directorate for the Quality of Medicines & HealthCare, European Pharmacopoeia), mono-PEGylated E. co/7-expressed non-glycosylated EPO (20 kDa, PEG- EPO), and di-PEGylated E. co/ -expressed non-glycosylated EPO (2x20 kDa PEG-EPO).
  • the vehicle buffer was also administered to mice as a negative control/placebo.
  • Mono- and di-PEGylated EPO were prepared according to the procedure described in Example 1. Each PEGylated EPO sample was buffer exchanged into a
  • PBS/BSA vehicle buffer PBS, pH 7.2/ 0.1% BSA
  • PD-10 column GE Healthcare cat. no. 17-0851-01
  • the samples were then diluted to the required concentration with PBS/BSA vehicle buffer.
  • Lyophilised EPO-BRP was reconstituted with PBS/BSA buffer to a concentration of 1000 IU/ml and then this stock solution was diluted to the required concentration with PBS/BSA buffer.
  • Three doses (high, medium, low) of each compound were administered to mice via subcutaneous injection with a volume of 0.5 ml. Each dose was injected into eight mice, and compared to the vehicle buffer/ placebo.

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Abstract

La présente invention concerne de nouveaux conjugués d'érythropoïétine avec un polymère, qui se caractérisent en ce que l'érythropoïétine est non glycosylée et est conjuguée à deux chaînes de polymère séparées. Chacune desdites chaînes de polymère est liée à l'érythropoïétine au niveau de deux résidus d'aminoacide.
PCT/GB2010/001633 2009-12-21 2010-08-27 Conjugués polymères d'érythropoïétine non glycosylée Ceased WO2011077067A1 (fr)

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WO2016059377A1 (fr) * 2014-10-14 2016-04-21 Polytherics Limited Procédé pour la conjugaison d'un peptide ou d'une protéine avec un réactif comprenant un groupe partant comportant une partie de peg
WO2016063006A1 (fr) * 2014-10-24 2016-04-28 Polytherics Limited Conjugués et réactifs de conjugaison

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130338231A1 (en) * 2012-06-18 2013-12-19 Polytherics Limited Novel conjugation reagents
US9650331B2 (en) 2012-06-18 2017-05-16 Polytherics Limited Conjugation reagents
US10174125B2 (en) 2012-06-18 2019-01-08 Polytherics Limited Conjugation reagents
WO2016059377A1 (fr) * 2014-10-14 2016-04-21 Polytherics Limited Procédé pour la conjugaison d'un peptide ou d'une protéine avec un réactif comprenant un groupe partant comportant une partie de peg
CN106794259A (zh) * 2014-10-14 2017-05-31 宝力泰锐克斯有限公司 采用包含含有peg部分的离去基团的试剂缀合肽或蛋白质的方法
US10835616B2 (en) 2014-10-14 2020-11-17 Polytherics Limited Process for the conjugation of a peptide or protein with a reagent comprising a leaving group including a portion of PEG
CN106794259B (zh) * 2014-10-14 2021-07-16 宝力泰锐克斯有限公司 采用包含含有peg部分的离去基团的试剂缀合肽或蛋白质的方法
WO2016063006A1 (fr) * 2014-10-24 2016-04-28 Polytherics Limited Conjugués et réactifs de conjugaison
CN107073131A (zh) * 2014-10-24 2017-08-18 宝力泰锐克斯有限公司 缀合物和缀合试剂
CN107073131B (zh) * 2014-10-24 2021-05-25 宝力泰锐克斯有限公司 缀合物和缀合试剂

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