HK1176015B - Therapeutic use of protein-polymer conjugates - Google Patents

Abstract

This invention relates to use protein-polymer conjugates described in the specification to treat various diseases, including disease is idiopaic myelofibrsis, polycythaemia vera, and essential thromobocythaemia.

Description

Therapeutic uses of protein-polymer conjugates

Cross reference to related application paragraphs

Priority of U.S. provisional application No.61/285,411, filed on 10.12.2009 in this application, is incorporated herein by reference in its entirety.

Background

Advances in cell biology and recombinant protein technology have led to the development of protein therapies.

However, major obstacles still exist. Most proteins are susceptible to proteolytic degradation and therefore have a short half-life in the circulatory system. Other disadvantages include low water solubility and the induction of neutralizing antibodies.

Attaching polymers such as polyethylene glycol (PEG) to proteins prevents access of proteolytic enzymes to the protein backbone, resulting in increased protein stability. In addition, it also improves water solubility and minimizes immunogenicity. There is therefore a need for an efficient method of attaching polymers to proteins.

Brief description of the invention

One aspect of the invention relates to the use of protein-polymer conjugates for the treatment of a variety of diseases. The conjugate comprises at least one polymer moiety, an interferon alpha moiety, and a linker. In the conjugate, the total molecular weight is 2-200kD (preferably 40kD), and the number of polymer moieties in the conjugate is no greater than 10. The one or more polymer moieties are attached to a linker; the N-terminal nitrogen atom of the interferon alpha moiety is bound to a linker; and the linker is a covalent bond, C_1-10Alkylene radical, C_2-10Alkenylene or C_2-10Alkynylene radical. Preferably, the conjugate is substantially pure, e.g., greater than 70%, 80%, or 90% pure. Diseases that can be treated by the conjugates include multiple sclerosis, chronic viral infections (e.g., hepatitis b virus infection, hepatitis c virus infection, and human papilloma virus infection), cancer, primary myelofibrosis, polycythemia vera, and primary thrombocythemia.

Another aspect of the invention relates to the use of protein-polymer conjugates of formula I as shown below for the treatment of the above-mentioned diseases:

wherein R is₁、R₂、R₃、R₄And R₅Each independently is H, C_1-5Alkyl radical, C_2-5Alkenyl radical, C_2-5Alkynyl, aryl, heteroaryl, C_3-8Cycloalkyl or C_3-8A heterocycloalkyl group; a. the₁And A₂Each independently is a polymeric moiety; g₁、G₂And G₃Each independently is a bond or a linking functional group; p is an interferon alpha moiety; m is 0 or an integer from 1 to 10; and n is an integer from 1 to 10.

With respect to the above formula, the protein-polymer conjugate may have one or more of the following characteristics: g₃Is a bond and P is an interferon alpha moiety with the N-terminal amino group attached to G₃；A₁And A₂Is a polyalkylene oxide moiety having a molecular weight of 2-100kD, preferably 10-30kD, G₁And G₂Are each independently

(wherein O is attached to A)₁Or A₂And NH is bonded to a carbon atom as shown in formula I), or G₁And G₂Each is urea, sulfonamide, or amide, (wherein N is attached to a carbon atom as shown in formula I); m is 4, N is 2, and R₁、R₂、R₃、R₄And R₅Each is H; and P is a modified interferon alpha moiety containing 1-4 additional amino acid residues.

The term "alkyl" refers to a monovalent straight or branched chain hydrocarbon radical. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl and n-pentyl. Similarly, the term "alkenyl" or "alkynyl" refers to a monovalent straight or branched chain hydrocarbon radical comprising one or more C ═ C double bonds or one or more C ≡ C triple bonds.

The term "alkylene" refers to a divalent straight or branched chain hydrocarbon group. Similarly, the term "alkenylene" or "alkynylene" refers to a divalent straight or branched chain hydrocarbon radical comprising one or more C ═ C double bonds or one or more C ≡ C triple bonds.

The term "aryl" refers to a hydrocarbon ring system (mono-or bicyclic) having at least one aromatic ring. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and pyrenyl.

The term "heteroaryl" refers to a hydrocarbon ring system (mono-or bicyclic) having at least one aromatic ring that contains at least one heteroatom such as O, N or S as part of the ring system, with the remainder being carbon. Examples of heteroaryl moieties include, but are not limited to, furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, and indolyl.

The term "cycloalkyl" refers to a partially or fully saturated monocyclic or bicyclic ring system having only carbon ring atoms. Examples include, but are not limited to, cyclopropenyl, cyclopentenyl, and cyclohexenyl.

The term "heterocycloalkyl" refers to a partially or fully saturated monocyclic or bicyclic ring system having one or more heteroatoms (e.g., O, N or S) as ring atoms in addition to carbon. Examples include, but are not limited to, piperidine, piperazine, morpholine, thiomorpholine, and 1, 4-oxazepane.

Reference herein to alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl includes both substituted and unsubstituted moieties. Examples of the substituent include C₁-C₁₀Alkyl radical, C₂-C₁₀Alkenyl radical, C₂-C₁₀Alkynyl, C₃-C₈Cycloalkyl radical, C₅-C₈Cycloalkenyl radical, C₁-C₁₀Alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C₁-C₁₀Alkylamino radical, C₁-C₂₀Dialkylamino, arylamino, diarylamino, hydroxyamino, alkoxyamino, C₁-C₁₀Alkylsulfonamides, arylsulfonamides, hydroxy, halogen, thio, C₁-C₁₀Alkylthio, arylthio, cyano, nitro, acyl, acyloxy, carboxyl and carboxylic acid esters.

The term "polyalkylene oxide moiety" refers to a monovalent group derived from a linear, branched, or star-shaped polyalkylene oxide. The molecular weight of the polyalkylene oxide moiety may be from 2 to 100 kD. The polyalkylene oxide moiety is saturated or unsaturated. Examples of polyalkylene oxide moieties include, but are not limited to, polyethylene oxide, polyethylene glycol, polyisopropylene oxide, polybutylene oxide, and copolymers thereof. Other polymers such as dextran, polyvinyl alcohol, polyacrylamide or carbohydrate-based polymers may also be used in place of the polyalkylene oxide moieties, provided they are not antigenic, toxic or eliciting an immune response. The polyalkylene oxide moiety is substituted or unsubstituted. For example, it may be methoxy-terminated polyethylene glycol (mPEG).

The term "interferon alpha moiety" refers to a monovalent group derived from interferon alpha. "Interferon alpha" refers to a highly homologous family of species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune responses. See Bonnem et al, j.biol.response mod, 1984, 3 (6): 580-598; and Finter, j.hepatol, 1986, 3Suppl 2: s157-160. It may be in a naturally occurring form or in a modified form. The modified interferon alpha may be, for example, a protein comprising interferon alpha and having 1 to 4 additional amino acid residues at the N-terminus of the interferon. An example of such a modified interferon alpha is

IFN represents the interferon alpha-2 b part, its N terminal amino combined to the carbonyl.

Many types of interferon alpha proteins are commercially available, including Intron-A interferon provided by Schering Corporation, Kenilworth, N.J., Hoffmann-La Roche, Nutley, Roferon interferon provided by N.J., Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Berofor alpha 2 interferon provided by Conn., Sumitomo, Sumiferon provided by Japan, and Wellferon interferon alpha-n 1(INS) provided by London, Great Britain.

The following are the amino acid sequences of five typical human interferon alpha proteins in either the precursor or mature form:

maltfallva llvlsckssc svgcdlpqth slgsrrtlml laqmrrislf sclkdrhdfgfpqeefgnqf qkaetipvlh emiqqifnlf stkdssaawd etlldkfyte lyqqlndleacviqgvgvte tplmkedsil avrkyfqrit lylkekkysp cawevvraei mrsfslstnl qeslrskeSEQ ID NO.：1

(see Krasagakis et al, Cancer invest.26(6), 562-568, 2008) cdlpqthsrlslg srrrtlmllaq mrkisslfscl kdhdfpfpq eefgqfqka etplvlhelhemiqqifnfstkdssaawdetl ldfytelqlndeleacvi qgvvgvtplmkesvilflavdsilavr kfqrlylklyekyspcaw evalveimeisflslslslslslslqrselqrske SEQ ID NO: 2

(see Klaus et al, J.mol.biol.274(4), 661-675, 1997)

mcdlpqthsl gsrrtlmlla qmrrislfsc lkdrhdfgfp qeefgnqfqk aetipvlhemiqqifnlfst kdssaawdet lldkfytely qqlndleacv iqgvgvtetp lmkedsilav rkyfqritlylkekkyspca wevvraeimr sfslstnlqe slrske SEQ ID NO.：3

(see GenBank accession No. AAP20099, 30-month-4-2003 edition)

mallfpllaa lvmtsyspvg slgcdlpqnh gllsrntlvl lhqmrrispf lclkdrrdfrfpqemvkgsq lqkahvmsvl hemlqqifsl fhterssaaw nmtlldqlht elhqqlqhletcllqvvgeg esagaisspa ltlrryfqgi rvylkekkys dcawevvrme imkslflstnmqerlrskdr dlgss SEQ ID NO.：4

(see Capon et al, J.mol.cell.biol.5 (4): 768-779, 1985)

lsyksicslg cdlpqthslg nrralillaq mgrispfscl kdrhdfglpq eefdgnqfqktqaisvlhem iqqtfnlfst edssaaweqs llekfstely qqlnnleacv iqevgmeetplmnedsilav rkyfqritly ltekkyspca wevvraeimr slsfstnlqk rlrrkd SEQ ID NO.：5

(see Lund et al, J. Interferon Res.5(2), 229-

In one embodiment, the amino acid sequence of the interferon alpha protein used to prepare the conjugates of the invention has at least 80% (e.g., 85%, 90%, 95%, or 99%) identity to one of the amino acid sequences listed above, or to a fragment thereof corresponding to mature interferon alpha.

"percent identity" of two amino acid sequences was determined using Karlin and Altschul Proc.Natl.Acad.Sci.USA 87: 2264-68, 1990, in Karlin and Altschul Proc.Natl.Acad.Sci.USA 90: 5873-77, 1993. This algorithm is incorporated into Altschul et al, j.mol.biol.215: 403-10, 1990 in the NBLAST and XBLAST programs (version 2.0). BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. When a gap exists between the two sequences, as in Altschul et al, Nucleic Acids Res.25 (17): 3389 Toxoplasma 3402, 1997 describes the use of gapped BLAST. When BLAST and gapped BLAST programs are employed, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The term "linking functional group" refers to a divalent functional group, one end attached to a polymer moiety and the other end attached to a protein moiety. Examples include, but are not limited to, -O-, -S-, carboxylate, carbonyl, carbonate, amide, carbamate, urea, sulfonyl, sulfinyl, amino, imino, hydroxyamino, phosphonate, or phosphate.

The protein-polymer conjugates described above may be in free form or in salt form (if desired). For example, a salt may be formed between an anion and a positively charged group (e.g., an amino group) on a protein-polymer conjugate of the invention. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, and acetate. Similarly, salts can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a protein-polymer conjugate of the invention. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations, such as tetramethylammonium.

In addition, the protein-polymer conjugate may have one or more double bonds or one or more asymmetric centers. Such conjugates may exist as racemates, racemic mixtures, single enantiomers, single diastereomers, diastereomeric mixtures and isomeric forms of the n-or trans-or E-or Z-double bond.

An example of a protein-polymer conjugate of the invention is shown below:

wherein mPEG has a molecular weight of 20kD and IFN is an interferon alpha-2 b moiety.

The use of the conjugates in the manufacture of a medicament for the treatment of one of the above-mentioned diseases is also within the scope of the invention.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Detailed Description

Protein-polymer conjugates useful in the practice of the present invention may be prepared by synthetic methods well known in the chemical arts, such as the method described in U.S. serial No. 12/192,485. Scheme 1 shows an example of the preparation of a protein-polymer conjugate of the invention. The acetal group-containing diamine compound 1 is reacted with N-hydroxysuccinimidyl carbonate mPEG (i.e., compound 2) to form a di-pegylated compound 3, which is subsequently converted to an aldehyde 4. The aldehyde compound is subjected to a reductive alkylation reaction with a protein having a free amino group to produce the protein-polymer conjugate of the present invention.

Scheme 1

The protein-polymer conjugate thus synthesized can be further purified by, for example, ion exchange chromatography, gel filtration chromatography, electrophoresis, dialysis, ultrafiltration, or ultracentrifugation.

The chemical reactions described above include the use of solvents, reagents, catalysts, protecting and deprotecting reagents, and certain reaction conditions. They may also include steps to add or remove appropriate protecting groups before or after the steps specifically described herein to ultimately synthesize the protein-polymer conjugate. In addition, the various synthetic steps may be performed in an alternating sequence or order to form the desired protein-polymer conjugate. Synthetic chemical transformations and protecting group methods (protection and deprotection) for synthesizing suitable protein-polymer conjugates are known in the art and include, for example, those described in r.larock, Comprehensive organic transformations, VCH Publishers (1989); greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, second edition, John Wiley and Sons (1991); fieser and m.fieser, Fieser and Fieser's Reagents for Organic Synthesis, john wiley and Sons (1994); and those described in L.Patquette, ed., Encyclopedia of Reagents for organic Synthesis, John Wiley and Sons (1995) and subsequent versions thereof.

The conjugates of the invention can have very high purity. That is, 60% or more (e.g., 70%, 80%, or 90%) of the conjugate molecules are identical in all respects, including the sequence of the protein portion and its binding site to the polymer portion.

The conjugate forms of the conjugates of the invention may be pharmaceutically active. Alternatively, it may release the pharmaceutically active interferon alpha in vivo (e.g., by hydrolysis) by enzymatically cleaving the linkage between the protein moiety and the polymer moiety. Examples of enzymes involved in cleaving a bond in vivo include oxidases (e.g., peroxidases, amine oxidases, or dehydrogenases), reductases (e.g., ketoreductases), and hydrolases (e.g., proteases, esterases, sulfatases, or phosphatases).

The conjugates of the invention are useful for treating multiple sclerosis, chronic viral infections (e.g., hepatitis B virus infection, hepatitis C virus infection, and human papilloma virus infection), cancer, primary myelofibrosis, polycythemia vera, and primary thrombocytosis. Which have unexpectedly long in vivo half-lives, reduced drug dosages, and/or extended dosing intervals.

As used herein, the term "treating" or "treatment" is defined as administering or administering a composition comprising a protein-polymer conjugate to a subject (human or animal) having a disease, a disease symptom, a disease or condition secondary to the disease, or a predisposition to a disease, with the goal of curing, alleviating, remedying or ameliorating the disease, disease symptom, disease or condition secondary to the disease, or predisposition to a disease. By "effective amount" is meant an amount of protein-polymer conjugate that confers a therapeutic effect on the subject being treated. The therapeutic effect may be objective (i.e., measured by some test or marker) or subjective (i.e., the subject exhibits an indication of or feels an effect).

Pharmaceutical compositions comprising an effective amount of at least one of the above-described protein-polymer conjugates and a pharmaceutically acceptable carrier are also within the scope of the invention. In addition, the invention includes methods of administering an effective amount of one or more protein-polymer conjugates to a patient suffering from one or more diseases. As will be appreciated by those skilled in the art, the effective dose will vary depending, for example, on the rate of hydrolysis of the protein-polymer conjugate, the type of disease to be treated, the route of administration, excipient usage, and the potential for co-use with other therapies.

To practice the methods of the present invention, compositions containing one or more of the above compounds may be administered parenterally, orally, nasally, rectally, topically, or buccally. The term "parenteral" as used herein refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intraperitoneal, intratracheal, or intracranial injection, as well as any suitable infusion technique.

The sterile injectable composition may be a solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono-or diglycerides). Fatty acids (e.g. oleic acid) and glyceride derivatives thereof are useful in the preparation of injectable formulations, such as the natural pharmaceutically-acceptable oils, for example olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as tweens and spans or other similar emulsifying agents or bioavailability enhancers which are commonly used to prepare pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

Compositions for oral administration may be in any orally acceptable dosage form, including capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase supplemented with emulsifying or suspending agents. If desired, certain sweetening, flavoring or coloring agents may be added.

Nasal aerosol or inhalation compositions may be prepared according to techniques well known in the art of pharmaceutical formulation. For example, such compositions may be prepared as a salt solution using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Compositions containing one or more of the above compounds may also be administered in the form of suppositories for rectal administration of the drug.

Pharmaceutically acceptable carriers are generally employed with one or more of the active compounds described above. The carrier in the pharmaceutical composition must be "acceptable" in the sense that it is compatible with (and preferably capable of stabilizing) the active ingredients of the composition and not deleterious to the subject to be treated. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D & C Yellow # 10.

Appropriate assays can be used to initially evaluate the efficacy of the above conjugates in treating a variety of diseases. For example, the methods may be performed according to Kiladjian et al, Blood 2008; 112(8): 3065-72 and Langer et al, Haetatomagica 2005; 90: 1333-1338 for evaluating the effect of the conjugates in the treatment of polycythemia vera and essential thrombocythemia.

The following examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are incorporated herein by reference in their entirety.

Detailed Description

Preparation of di-PEG aldehyde

20kD PEGO (C ═ O) OSu was prepared from 20kDmPEGOH purchased from SunBio Inc., CA, USA according to the method described in Bioconjugate chem.1993, 4, 568-.

A solution of 6- (1, 3-dioxolan-2-yl) hexane-1, 5-diamine in dichloromethane (11.97 g solution containing 9.03mg of diamine, 47.8 μmol) was added to a flask containing 20kD PEGO (C ═ O) OSu (1.72g, 86.0 μmol). After complete dissolution of PEGO (C ═ O) OSu, N-diisopropylethylamine (79 μ L, 478 μmol) was added. The reaction mixture was stirred at room temperature for 24 hours, and then methyl t-butyl ether (200mL) was added dropwise with stirring. The resulting precipitate was collected and dried under vacuum to yield di-PEG acetal as a white solid (1.69g, 98%).

¹H NMR(400MHz，d₆-DMSO)δ7.16(t，J＝5.2Hz，1H)，7.06(d，J＝8.8Hz，1H)，4.76(t，J＝4.8Hz，1H)，4.10-3.95(m，4H)，1.80-1.65(m，1H)，1.65-1.50(m，1H)，1.48-1.10(m，6H)。

Di-PEG acetal (4.0g, 0.2mmol) was suspended in buffer pH 2.0 (citric acid, 40 mL). The reaction mixture was stirred at 35 ℃ for 24 hours and then extracted with dichloromethane (3 × 50 mL). The combined organic layers were dried over magnesium sulfate, concentrated, and then redissolved in dichloromethane (20 mL). The solution was added dropwise to methyl tert-butyl ether (400mL) with stirring. The resulting precipitate was collected and dried under reduced pressure to yield di-PEG aldehyde (3.8g, 95%) as a white solid.

¹H NMR(400MHz，d₆-DMSO)δ9.60(s，1H)，7.24(d，J＝8.4Hz，1H)，7.16(t，J＝5.2Hz，1H)，4.10-3.95(m，4H)，3.95-3.80(m，1H)，3.00-2.85(m，2H)，2.58-2.36(m，2H)，1.46-1.15(m，6H)。

Preparation of modified interferon

The modified recombinant human interferon alpha-2 b is cloned by a PCR method by using human genome DNA as a template. Oligonucleotides were synthesized based on flanking sequences of human interferon alpha-2 b (GenBank accession # J00207, 8.1.2008). The derived PCR product was subcloned into pGEM-T vector (Promega). IFN variants were PCR amplified again by pGEM-T cloning and subsequently subcloned into the protein expression vector pET-24a (Novagen), a T7RNA polymerase promoter driver vector, using NdeI/BamHI as cloning sites. The vector pET-24a was then transformed into the strain Escherichia coli BL21-CodonPlus (DE3) -RIL (Stratagene). High expressing clones were selected by maintaining transformed E.coli BL21-CodonPlus (DE3) -RIL in the presence of kanamycin (50. mu.g/mL) and chloramphenicol (50. mu.g/mL).

BL21-Codonplus (DE3) -RIL carrying the Pro-IFN gene was propagated in 1000mL flasks using Terrific medium (BD, 200 mL). The flask was shaken at 230rpm for 16 hours at 37 ℃. Batch and fed-batch fermentations were carried out in 5 liter fermentors (Bioflo 3000; New Brunswick Scientific Co., Edison, N.J.). The batch fermentation used 150mL of an overnight pre-incubated inoculum and contained kanamycin (50. mu.g/mL), chloramphenicol (50ug/mL), 0.4% glycerol, and 0.5% (v/v) trace elements (10g/L FeSO)₄·7H₂O、2.25g/L ZnSO₄·7H₂O、1g/L CuSO₄·5H₂O、0.5g/L MnSO₄·H₂O、0.3g/L H₃BO₃、2g/L CaCl₂·2H₂O、0.1g/L(NH₄)₆Mo₇O₂₄0.84g/LEDTA, 50ml/L HCl) 3L terrific medium. The dissolved oxygen concentration was controlled at 35% and the pH was maintained at 7.2 by the addition of 5N NaOH aqueous solution. Preparation of MgSO containing 600g/L glucose and 20g/L MgSO₄·7H₂Feed solution of O. When the pH rises to a value above the set point, an appropriate volume of feed solution is added to increase the glucose concentration in the medium. Expression of the Pro-IFN gene was induced by addition of IPTG to a final concentration of 1mM and the medium was harvested after 3 hours of culture.

The collected cell pellets were resuspended in TEN buffer (50mM Tris-HCl (pH 8.0), 1mM EDTA, 100mM NaCl) at a ratio of about 1: 10 (wet weight g/mL) and disrupted by a microfluidizer, and then centrifuged at 10,000rpm for 20 minutes. The pellet containing Inclusion Bodies (IB) was washed twice with TEN buffer and centrifuged as described above. The precipitate containing IB was then suspended in 150mL of 4M aqueous guanidine hydrochloride (GuHCl) and centrifuged at 20,000rpm for 15 minutes. IB was then dissolved in 50mL of 6MGuHCl solution. GuHCl solubilized material was centrifuged at 20,000rpm for 20 minutes. Renaturation was initiated by diluting the denatured IB with 1.5L of freshly prepared renaturation (reflding) buffer (100mM Tris-HCl (pH 8.0), 0.5M L-arginine, 2mM EDTA) with stirring only during the addition. The renaturation reaction mixture was incubated for 48 hours without stirring. Renatured recombinant human interferon alpha-2 b (i.e., Pro-IFN) was dialyzed against 20mM Tris buffer (containing 2mM EDTA and 0.1M urea, pH 7.0) for further purification by Q-Sepharose column chromatography.

The renatured recombinant human protein Pro-IFN was loaded onto a Q-Sepharose column (GE Amersham Pharmacia, Pittsburgh, Pa.). The column was pre-equilibrated with 20mM Tris-HCl buffer (pH 7.0) and washed. The product was eluted with a mixture of 20mM Tris-HCl buffer (pH 7.0) and 200mM NaCl. Pro-IFN containing fractions were collected based on their absorbance at 280 nm. Pro-IFN concentrations were determined by protein assay kits using the Bradford method (Pierce, Rockford, IL).

Preparation of protein-Polymer conjugates

To a solution of di-PEG aldehyde (1.2g, 0.03mmol) prepared above in water (72mL) was added 2M sodium phosphate buffer (pH 4.0, 5mL) and Pro-IFN (200mg in 22.2mL of pH 7.0 buffer containing 20mM Tris-HCl and 0.2M NaCl, 0.01 mmol). The reaction mixture was stirred at room temperature for 10 minutes; aqueous sodium cyanoborohydride (400mM, 1.25mL, 0.5mmol) was then added. The reaction mixture was stirred in the dark for 16 hours and purified by SP XL Sepharose chromatography. Fractions containing the desired polymer-protein conjugate were collected based on their retention time and absorbance at 280 nm. The concentration of the conjugate was determined by protein assay kit using the Bradford method (Pierce, Rockford, IL). The isolated yield of the conjugate was approximately 40% or higher.

Other embodiments

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of similar or analogous features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other implementations are within the scope of the following claims.

Claims (4)

1. Use of an effective amount of a conjugate of the formula:

wherein:

IFN represents an interferon alpha-2 b moiety, wherein the N-terminal amino group of IFN is bound to a carbonyl group;

the molecular weight of mPEG is 20 kD;

the disease is primary myelofibrosis, polycythemia vera or primary thrombocythemia.

2. The use of claim 1, wherein the disease is primary myelofibrosis.

3. The use of claim 1, wherein the disease is polycythemia vera.

4. The use of claim 1, wherein the disease is essential thrombocythemia.