MXPA00010223A - Polyol-ifn-beta conjugates - Google Patents

Abstract

PEG-IFN-&bgr;conjugates, where a PEG moiety is covalently bound to Cys17 of human IFN-&bgr;, are produced by a process of site specific PEGylation with a thiol reactive PEGylating agent. A pharmaceutical composition and a method for treating infections, tumors and autoimmune and inflammatory diseases are also provided. The invention further relates to a method for the stepwise attachment of PEG moieties in series to a polypeptide, and more particularly to IFN-&bgr;.

Description

INTERNATIONAL APPUCA? ON PBBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) WO 99 / S5377 A61K 47/48 A3 CONJUGATES OF POL IOL- I FN-BETA CROSS REFERENCE TO THE RELATED APPLICATION This application claims priority according to 35 U.S.C. § 119 (e) of the United States Provisional Patent Application Serial No. 60 / 083,339, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The invention relates to polyol-IFN-β conjugates wherein a polyol unit is covalently bound to Cys17. The additional objects of the present invention are the process for their specific production at the site as well as their use in the therapy, prognosis or diagnosis of bacterial infections, viral infections, autoimmune diseases and inflammatory diseases. The present invention further relates to a method for the gradual binding of two or more portions of PEG to a polypeptide.

BACKGROUND OF THE INVENTION Human fibroblast interferon (IFN-β) * "- •" «- *« * - It has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of approximately 20,000 Da induced by virus and double-stranded RNA. From the nucleotide sequence of the gene for inferred fibroblasts, cloned by recombinant DNA technology, Deryn et al., (Nature, 285: 542-547, 1980) deduced the complete amino acid sequence of the protein. It is 166 amino acids long. Shepard et al., (Nature, 294: 563-565, 1981) described a mutation in base 842 (Cys -. Tyr at position 141) that abolished its antiviral activity, and a variant clone with a deletion of nucleotides 1119 -1121. Mark et al., (Proc. Nati. Acad.? USA, 81 (18): 5662-5666, 1984) inserted an artificial mutation by replacing the base 469 (T) with (A) causing an amino acid change of Cys? Being in position 17. The resulting IFN-β was reported to be as active as "native" IFN-β and stable during long-term storage (-70 ° C). The covalent attachment of the hydrophilic polymer polyethylene glycol (PEG), also known as oxide Polyethylene (PEO), to molecules has important applications in biotechnology and medicine. In its most common form, PEG is a linear polymer that has hydroxyl groups in each term: HO-CH2-CH2? (CH2CH2?) NCH2CH2-OH This formula can be represented in summary as HO-PEG-OH, where it is meant that -PEG- represents the polymer structure without the terminal groups: "-PEG-" means "-CH2CH20 (CH2CH20) nCH2CH2-" PEG is commonly used as methoxy-PEG-OH, (m-PEG), in which one term is the relatively inert methoxy group, while the other term is a hydroxyl group that is subjected to chemical modification.

CH30- (CH_CH20) n-CH2CH2-OH Branched PEGs are also in common use. The branched PEGs can be represented as R (-PEG-OH) m in which R represents a portion of the central group such as pentaerythritol or glycerol, and m represents the number of branch arms. The number of branch arms (m) may vary from three to one hundred or more. The hydroxyl groups are subjected to chemical modification.

Another branched form, such as that described in PCT Patent Application WO 96/21469, has an individual term that is subject to chemical modification. This type of PEG can be represented as (CH3O-PEG-) PR-X, where p is equal to 2 or 3, R represents a central core such as lysine or glycerol, and X represents a functional group such as carboxyl which is undergoes chemical activation. Yet another branched form, the "hanging PEG", has reactive groups, such as carboxyl, together with the PEG structure instead of being at the end of the PEG chains. In addition to these forms of PEG, the polymer can also be prepared with weak or degradable bonds in the structure. For example, Harris showed in the application of United States of America Patent Serial No. 06 / 026,716 that PEG can be prepared with ester linkages in the polymer structure which is subjected to hydrolysis.

This hydrolysis results in the cleavage of the polymer into low molecular weight fragments, according to the reaction scheme: -PEG-C02-PEG- + H20 -PEG-C02H + HO-PEG- In accordance with the present invention, the term "polyethylene glycol" or "PEG" is meant to mean that it comprises all the derivatives described above. The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and can be used in place of PEG in many of their applications. They have the following General Formula: HO-CH2CHRO (CH2CHRO) nCH2CHR-OH where R is H or CH3. PEG is a useful polymer that has the property of high solubility in water as well as high solubility in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically bound (PEGylation) to a water-insoluble compound, the conjugate resulting in general is soluble in water, as well as soluble in many organic solvents. PEG-protein conjugates are currently being used in protein replacement therapies and for other therapeutic uses. For example, PEGylated adenosine deaminase (ADAGEN®) is being used to treat severe combined immunodeficiency disease (SCIDS), PEGylated L-asparaginase (ONCAP? PAR®) is being used to treat acute lymphoblastic leukemia (ALL ), and the inner PEGylated feron-a (INTRON® A) is in Phase III trials to treat hepatitis C.

For a general review of PEG-protein conjugates with clinical efficacy see N.L. Burnham, Am. J. Hosp. Pharm. , 15: 210-218, 1994. A variety of methods for PEGylating proteins have been developed. The binding of PEG to reactive groups found in the protein is typically done using activated PEG derivatives electically and activated. The binding of PEG to the a- and e-amino groups found in the lysine residues and the N-term results in a conjugate consisting of a mixture of products.

In general, these conjugates consist of a population of the various PEG molecules attached per protein molecule ("PEGmeros") that go from zero to the number of amino groups in the protein. For a protein molecule that has been individually modified, the PEG unit can be attached to a number of different amine sites. This type of non-specific PEGylation has resulted in several conjugates that become almost inactive. The reduction in activity is typically caused by protecting the active binding domain of the protein as is the case with many cytokines and antibodies. For example, Katre et al., In U.S. Patent No. 4,766,106 and U.S. Patent No. 4,917,888 describe the PEGylation of IFN-β and IL-2 with a large excess of methoxy-polyethylene glycol 1- N-succmimidyl-glutarate and metoxy-polyethylene glycolyl-N-succinimidyl-succinate. Both proteins are produced in microbial host cells, which allow the specific mutation in the sequence of the free cistern to a serma. The mutation was necessary in the microbial expression of IFN-β to facilitate the folding of the protein. In particular, the IFN-β used in these experiments is the commercial product Betaseron®, in which the Cys residue is replaced by a serine. Additionally, the absence of glycosylation reduced its solubility in aqueous solution. The non-specific PEGylation resulted in increased solubility, but a major problem was the reduced level of activity and yield. European Patent Application EP 593 868, entitled PEG-inter feron conjugates, describes the preparation of PEG-IFN-a conjugates. However, the PEGylation reaction is not specific to the site or sequence, and therefore a mixture of positional isomers of the PEG-IFN-a conjugates is obtained (see also Monkarsh et al., AC? Sy. Ser. , 680: 207-216, 1997). Kinstler et al., In the Patent Application European EP 675 201 demonstrates the selective modification of the N-terminal residue of the growth factor and development of megakaryocytes (MGDF) with mPEG-propionaldehyde. This allowed for reproducible PEGylation and assembly of one batch to another batch. Gilbert et al., In U.S. Patent No. 5,711,944 showed that PEGylation of IFN-a could be produced with an optimum level of activity. In this case, a laborious purification step is needed to obtain the optimal conjugate. Most cytokines, as well as other proteins, do not possess a specific PEG binding site and apart from the above-mentioned examples, it is very likely that some of the isomers produced through the PEGylation reaction are partially or totally inactive, thus causing a loss of activity of the final mixture. Mono-site-specific PEGylation or sequence in this manner is a desirable objective in the preparation of these protein conjugates. Oghiren et al. , in Bioconjuqate Chem., 4 (5): 314-138, 1993, synthesized a thiol selective PEG derivative for this specific site or sequence PEGylation. A stable thiol-protected PEG derivative in the form of a parapyridyl disulfide reactive group was shown to be specifically conjugated to the free cysteine in the protein, papain. The newly formed disulfide bond between papain and PEG could be cleaved under mild reducing conditions to regenerate the native protein. The citation of any document herein is not intended as an admission that that document is the relevant prior art, or material considered for the patent ability of any claim of the present application. Any statement regarding the content or date of any document is based on information available to applicants at the time of filing and does not constitute an admission as to the correctness of this statement. 3REVE DESCRIPTION OF THE INVENTION In the present invention, polyol-IFN-β conjugates and particularly PEG-IFN-β conjugates are provided, wherein a polyol unit is covalently linked to Cys17. Specific conjugation is obtained by allowing a thiol reactive polyol agent to react with the Cys17 residue in IFN-β These conjugates are expected to show increased effectiveness m vi vo. The purpose is to obtain increased solubility at neutral pH, increased stability (decreased aggregation), decreased immunogenicity and no loss of activity with respect to "native" IFN-β. The results of this conjugation will decrease the number of doses for a proposed effect, simplify and stabilize the formulation of a pharmaceutical composition, and possibly increase long-term efficacy. The present invention further provides a method for the gradual binding of portions of PEG in series to a polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a graph of capillary electrophoresis (CE) of the PEG-IFN-β conjugate before purification. Figures 2A-2C show the purification of the PEG-IFN-β conjugate carried out by size exclusion chromatography (Super 12): Figure 2A.- first pass; Figure 2B.- second pass; Figure 2 C. - third pass. Figure 3 shows the SDS-PAGE chromatography of the PEG-IFN-β conjugate purified from the third pass of the chromatography. Lanes 1 and 4 are protein molecular weight standards, lane 2 is the "native" IFN-β and lane 3 is the PEG-IFN-β conjugate. Figure 4 reports the capillary electrophoresis (CE) graph of the purified PEG-IFN-β conjugate in which IFN-β is PEGylated with Mpeg-OPSS5k.

Figure 5 reports the MALDI MS spectrum of the purified PEG-IFN-β conjugate. Figure 6 shows a comparison between the anti-viral activity of the "native" IFN-β and the PEG-IFN-β conjugate. WISH cells were incubated with indicated concentrations of IFN-β samples for 24 hours before stimulation with the cytopathic dose of vesicular stomatitis virus. The cytopathic effect was determined after an additional 48 hours by MTT conversion. Figure 7 shows the binding profile of IFN-β and PEG-IFN in Daudi cells. Figure 8 shows the armacokinetic f-profile of IFN-β and PEG-IFN in mice after intravenous administration. The dashed lines indicate the LOQ test for each normal curve. Figure 9 shows the pharmacokinetic profile of IFN-β and PEG-IFN in mice after subcutaneous administration. The dashed lines indicate the LOQ test for each normal curve.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery that the binding of a polyol portion more specifically to a portion of PEG, the Cys17 residue of human IFN-β unexpectedly increases (or at least retains and does not as a result the decrease) of the biological activity of IFN-β from that of native human interferon-β. Thus, not only IFN-β with a polyol moiety bound to the Cys 17 residue exhibits the same activity or increased biological activity of IFN-β but this polyol-IFN-β conjugate also provides the desirable properties conferred by the of polyol, such as increased solubility. "IFN-β", as used herein, means human fibroblast interferon, as obtained by isolation from biological fluids or as obtained by recombinant DNA techniques from prokaryotic or eukaryotic host cells as well as their salts , functional derivatives, propellants and active fractions, with the condition that they contain the cysteine residue that appears in position 17 in the form that occurs naturally.

The polyol portion in the polyol-IFN-β conjugate according to the present invention can be any water soluble mono- or di-functional poly (alkylene oxide) having a straight or branched chain. Typically, the polyol is a poly (alkylene glycol) such as poly (ethylene glycol) (PEG). However, those skilled in the art will recognize that other polyols, such as, for example, poly (propylene glycol) and copolymers of polyethylene glycol and polypropylene glycol, can be used suitably. As used herein, the term "PEG portion" is intended to include, but is not limited to, linear and branched PEG, methoxy-PEG, hydrolyzed or enzymatically degradable PEG, pendant PEG, dendimer PEG, copolymers of PEG and one or more polyols, and copolymers of PEG and PLGA (poly (lactic acid / glycolic acid)). The definition "salts" as used herein refers to both salts of the carboxyl groups and salts of the amino functions of the compounds obtainable by known methods. The salts of the carboxyl group include inorganic salts such as, for example, sodium, potassium, calcium salts and salts with bases organic such as those formed with an amine such as t ret anolamine, argmam or lysine. The salts of the amino groups include, for example, salts with inorganic acids such as hydrochloric acid and with organic acids such as acetic acid. The definition "functional derivatives" as used herein refers to derivatives that can be prepared from functional groups present in the side chains of the amino acid portions or in the N- or C-terminal groups according to known methods and are included in the present invention when they are pharmaceutically acceptable, that is, they do not destroy the protein activity or impart toxicity to the pharmaceutical compositions containing them. These derivatives include, for example, aliphatic esters or amides of the carboxyl groups and the N-acyl derivatives of the free amino groups or the 0-acyl derivatives of the free hydroxyl groups and are formed with acyl groups such as, for example, alkanoyl groups or aroyl. "Precursors" are compounds that are converted to IFN-β in the human or animal body. As "active fractions" of the protein, the present invention refers to any fragment or precursor of the polypeptide chain of the compound itself, alone or in combination with related molecules or residues bound thereto, eg, residues of sugars or phosphates, or aggregates of the polypeptide molecule when these fragments or precursors exhibit the same activity of the IFN-ß as a medicine. The conjugates in the present invention can be prepared by any of the methods known in the art. According to one embodiment of the invention, IFN-β is reacted with a PEGylating agent in a suitable solvent and the desired conjugate is isolated and purified, for example, by applying one or more chromatographic methods. "Chromatographic method" means any technique that is used to separate the components of a mixture by its application in a support "stationary phase" through which a solvent flows "mobile phase". The separation principles of chromatography are based on the different physical nature of the stationary phase and the mobile phase. Some particular types of chronic methods, which are well known in the Literature includes: liquid chromatography, high pressure liquid, ion exchange, absorption, affinity, partition, hydrophobic, inverted phase, gel filtration, ultrafiltration or thin layer chromatography. The "thiol reactive PEGylation Agent", as used in the present application, means any PEG derivative that is capable of reacting with the thiol group of the cysteine residue. It may be for example PEG containing a functional group such as ortho-pyridinium disulphide, vinylsulphone, maleimide, iodoacetimide, and others. According to a preferred embodiment of the present invention, the thiol reactive PEGylation agent is the ortho-pyridyl disulfide derivative (OPSS) of PEG. The PEGylating agent is used in its mono-methoxylated form when only one term is available for conjugation, or in a bifunctional form where both terms are available for conjugation, such as for example in the formation of a conjugate with two IFN- β bound covalently to an individual portion of PEG. Preferably, it has a molecular weight between 500 and 100,000. A typical reaction scheme for the preparation of the conjugates of the invention is presented below: Protein-SH + mPEG-S-S- Protein ß-Mercaptoethanol Protein -S-H + mPEG-S-H + HOCH2CH2-S-S-CH2CH2OH The second line of the previous scheme reports a method to cleave the PEG-protein link. The mPEG-OPSS derivative is highly selective for free sulfhydryl groups and reacts rapidly under acid pH conditions where IFN-β is stable. The high selectivity can be shown from the reduction of the conjugate to the native form of IFN-β and PEG. The sulfide bond that occurs between the protein and PEG portions has been shown to be stable in the circulation, but can be reduced upon entering the cellular environment. Therefore, it is expected that this conjugate, which does not enter the cell, will be stable in circulation until it is depure It should be noted that the above reaction is site specific because of the other two Cys residues that appear at positions 31 and 141 in the naturally occurring form of human IFN-β does not react with the PEGylating agent reactive with thiol since it forms a disulfide bridge. The present invention is also directed to a method for the gradual binding of two or more portions of PEG to a polypeptide. This method is based on the recognition that an activated PEG of low molecular weight reacts more completely with a spherically impeded reaction site in a protein than does an activated PEG of high molecular weight. In the PEG modification of costly therapeutic proteins it must be cost-effective in order to make the production of the PEG conjugate practical. In addition, in order to reduce glomerular filtration and optimize the pharmacological properties of the PEG-protein conjugate, the conjugate must have an effective size equivalent to that of a protein with a molecular weight of 70 kDa. This means that for a specific modification of the where a PEG will be attached, a PEG derivative having a molecular weight of more than 20 kDa binds preferentially. If the modification site is sterically occupied, the reactive group in the giant portion of PEG may have difficulty in reaching the modification site and will thus lead to low yields. A preferred method for PEGylating a polypeptide according to the present invention increases the yield of PE (site-specific dilation by first attaching a small, homogenous or homobifunctional PEG moiety which, due to its relative size) Smaller vameiite may react with physically occupied sites Subsequent binding of a high molecular weight PEG derivative to small PEG results in high yield of the PEGylated protein desired, ie, method for gradual binding of two or more by serial PEGs to a polypeptide according to the present invention includes the binding of a portion of heterobi functional PEG or low molecular weight homobi fune, first to the id i polypeptide and then binding to a portion of PEG monofunctional or bifunctional to the free term of the portion of PEG of low molecular weight that binds to the polypeptide 3. Following the gradual binding of two or more portions of PEG in series to a polypeptide, the id3 polypeptide which is preferably IFN-β and where Cys17, located at a spherically occupied site, is the preferred binding site. of PEG, the PEG-pol conjugate can be purified using one or more of the purification techniques such as ion exchange chromatography, size exclusion chromatography, interaction and hydrophobic chromatography, affinity chromatography and chromatography. of inverted phase. The portion of PEG of low molecular weight has the formula: W-CH2CH20 (CH2CH20) nCH2CH2-X, where W and X are groups that react independently with a functional group of amine, sulphide, carboxyl or hydroxyl to bind the < e) Low molecular weight PEG to polypeptide i. W and X are preferably independently selected from ortho-iridyl disulfide, maleimides, vinylsulfonates, iodoacetamides, amines, thiols, carboxyl, active esters, benzotriazole bicarbonates, p-nit rof enol, isocyanates and biotin. The portion of PEG of low molecular weight preferably has a molecular weight in the range of about 100 to 5,000 daltons. The monofunctional or bifunctional PEG portion for binding to the free terminus of a low molecular weight PEG that binds to the polypeptide preferably has a molecular weight in the range of about 100 daltons to 200 kDa and is preferably a methoxy-PEG, branched PEG , Hydrolytic or enzymatically degradable PEG, hanging PEG, or dendrimer PEG. The monofunctional or bifunctional PEG also has the formula: Y-CH2CH20 (CH2CH20) MCH2CH2-Z, Y is reactive to a terminal group in the free term of the low molecular weight PEG portion that can be linked to the Z polypeptide is-OCH3 or a reactive group to form a bifunctional conjugate. The PEG-polypeptide conjugate produced by the above method for the gradual binding of two or more portions of PEG can be used to produce a medicament or pharmaceutical composition for treating diseases or disorders for which the polypeptides are effective as an active ingredient. Another object of the present invention is to provide the conjugates in substantially purified form in order to make them suitable for use in pharmaceutical compositions, as the active ingredient for the treatment, diagnosis or prognosis of bacterial and viral infections as well as autoimmune, inflammatory diseases. and tumors. These pharmaceutical compositions represent a further object of the present invention. Non-limiting examples of the diseases mentioned above include: septic shock, AIDS, rheumatoid arthritis, lupus erythematosus and multiple sclerosis. Additional embodiments and advantages of the invention will be apparent in the following description. One embodiment of the invention is the administration of a pharmacologically active amount of the conjugates of the invention to subjects at risk of developing one of the diseases previously reported or to subjects that already show these pathologies. Any administration route compatible with the active principle can be used. Parenteral administration, such as subcutaneous, intramuscular or intravenous injection is preferred. The dose of the active ingredient to be administered depends on the basis of the medical prescriptions according to the patient's age, weight and individual response. The dose may be between 10 μg and 1 mg daily for an average body weight of 75 kg, and the preferable daily dose is between 20 μg and 200 μg. The pharmaceutical composition for parenteral administration can be prepared in an injectable form comprising the active ingredient and a suitable vehicle. Vehicles for parenteral administration are well known in the art and include for example water, saline, Ringer's solution and / or dextrose. The vehicle may contain small amounts of excipients in order to maintain the stability and stability of the pharmaceutical preparation. The preparation of the solution can be carried out according to the ordinary modalities. The present invention has been described with distinction to specific embodiments, but the content of the description includes all modifications and substitutions that can be made by a person skilled in the art without extending beyond the meaning and purposes of the claims. The invention will now be described by means of the following examples, which are not to be construed as limiting the present invention in any way.

EXAMPLE 1: Preparation of PEG-IFN-β Conjugate Modification of IFN-ß with mPEG5k-OPSS Recombinant human IFN-β, stable at a concentration of 0.37 mg / ml in 50 mM sodium acetate buffer, pH 3.6, was used for the preparation of a PEG-conjugate IFN-β. Approximately 1.0 ml of 6 M urea was added to 2 ml of IFN-β at a concentration of 0.37 mg / ml (0.74 mg, 3.7 xl "8 moles) mPEG5k-OPSS was added in a molar excess of 50 moles to a mole of IFN-β and the two were allowed to react in a flask of polypropylene for either 2 hours at 37 ° C or 1 hour at 50 ° C. The reaction mixture was analyzed with a capillary electrophoresis (CE) graph to determine the degree of formation of the PEG-IFN-β conjugate by the PEGylation reaction before any purification (Figure 1). A typical yield for this reaction is 50% PEG-IFN-β. The reaction products were filtered from the reaction mixture with a 0.22 mm syringe filter and the filtered solution was then loaded onto a size expression column (either Super 12 or Superdex 75, Pharmacia) and eluted with Sodium phosphate in 50 mM, 150 mM NaCl, pH 7.0 buffer. Figure 2A shows the elution profile from the purification of the PEG-IFN-β conjugate on an oversize size 12 exclusion chromatography column. The peaks were collected and analyzed with SDS-PAGE (Figure 3). The fractions containing the PEG-IFN-β conjugate were mixed together and the concentrate was then reloaded to the same size exclusion column to further purify the PEG-IFN-β conjugate due to the close proximity of the peak of PEG-IFN-ß "native" (Figure 2B). This procedure was repeated (third pass) for ensure purity (Figure 2C). Figure 4 and Figure 5 show the capillary electrophoresis graph and the MALDI MS spectrum, respectively, of the purified PEG-IFN-β conjugate.

Modification of IFN-ß with mPEG3ok-OPSS Stable recombinant human IFN-ß was provided in solution at 0.36 mg / ml in a 50 mM sodium acetate buffer, pH 3.6. about 36 mg of mPEG.ok-OPSS in 3 ml of deionized H20 were added to 3 ml of IFN-β at 0.36 mg / ml (1.08 mg, 4.9 x 10"8 moles) and the two were allowed to react in a flask of polypropylene for 2 hours at 50 ° C. The reaction mixture was analyzed with capillary electrophoresis for the degree of modification.The typical yields for this reaction were <30% .The solution was then loaded onto a size exclusion column (Superio 12, Pharmacia) and eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer, the peaks were collected and analyzed with SDS-PAGE for their contents.

EXAMPLE 2: Biological Activity of PEG-IFN-β Conjugate To assess the effects of PEGylation on the antiviral activity of human recombinant IFN-β, human WISH amniotic cells were preincubated with either freshly prepared IFN-β (same batch as was used for PEGylation) or the IFN-β conjugate. The anti-viral activity mediated by IFN-β, as measured by the WISH-VSV cytological assay, was determined according to an anti-viral WISH bioassay developed based on the protocol of Novick et al., J. Immunol. , 129: 2244-2247 (1982). The materials used in this WISH assay are as follows: WI? H cells (ATCC CCL 25). Vesicular stomatitis virus materials (ATCC V-520-001-522), stored at -70 ° C. IFN-ß, recombinant, human, InterPharm Laboratories LTD (32, 075-type, Lot number 205035), 82 x 106 IU / ml, specific activity: 222 x 106 IU / mg. PEG-IFN-β conjugate prepared as in Example 1 and maintained in PBS, pH 7.4. WISH growth medium (MEM high glucose content with Earls salts + 10% of FBS + 1.0% L-glutamine + penicillin / streptomycin (100 U / ml, 100 μg / ml). WISH assay medium (MEM high glucose content with Earls salts + 5% FBS + 1.0% L-glutamine + penicillin / streptomycin (100 U / ml, 100 μg / ml) MTT at 5 mg / ml in PBS , stored at minus 70 ° C. The protocol for the WISH assay is as follows: Dilute the IFN-β samples to 2X at the start concentration in the WISH assay medium, make three-fold dilutions of the IFN samples -β in the WISH assay medium in a 96-well flat-bottomed plate so that each well contains 50 μl of the diluted IFN-β sample (some control wells received 50 μl of the WISH assay medium only) Collect the WISH cells in logarithmic growth phase with trypsin / EDTA solution, wash in the WISH assay medium, and put to a final concentration of 0.2 x 106 cells / ml.

Add 50 μl of WISH cell suspension (4 X 104 cells per well) to each well. The final concentration of IFN-β exposed to the cells is now IX. After incubation for 24 hours in a humidified incubator with 5% C02, 50 μl of a 1:10 dilution (in WISH assay medium) of the VSV material (a predetermined dose to lyse 100 percent of the cells) is added. WISH in the space of 48 hours) to all the cavities except for the control cavities without virus (these received an equal volume of the assay medium only). After an additional 48 hours, 25 μl of MTT solution is added to all the wells, after which the plates are incubated for an additional 2 hours in an incubator. The contents of the cavities are removed by plaque inversion, and 200 μl of 100% ethanol is added to the cavities. After 1 hour, the plates are read at 595 nm using the Soft max Pro computer program package and the Spectramax spectrophotometer system (Molecular Devices) Table 1 Antiviral activity of PEGylated and PEGylated IFN-beta samples in false * concentrations of IFN-ß material from samples determined by amino acid analysis ** EC50 (+/- S.D.) was determined by Microcal Origin 4.1 computer program As demonstrated in Figure 6 and Table 1 above, the PEG-IFN-β conjugate maintained a higher level of antiviral activity than that of the freshly prepared parenteral lot of IFN-β. The observation that the PEG-IFN-β conjugate has approximately a 4-fold higher bioactivity than that of the newly prepared IFN-β may also be a consequence of the increased stability of the PEG-IFN-β conjugate with respect to IFN-β "native" after the addition of the WISH cell assay medium.

EXAMPLE 3: In Vitro Tests of the Relative Activity of PEG-IFN Samples The relative bioactivity of PEG [30 kD] -IFN-β and PEG [2 X 20 kD] -IFN-β was determined by WISH assay using the protocol normal described in Example 2 (Table 2). Three independent trials were performed by three different individuals at separate times. Table 2 Relative Antiviral Activity of PEG-IFN-β * EC50 dose compared to normal IFN-ß included in each test ** Comparison based on IFN-ß 300 μg / ml concentration. Concentrations of the material of PEG [30 kD] -IFN-β (5.41 μg / ml) and PEG [2 X 20 kD] -IFN-β (6.86 μg / ml) were determined by AAA.

The binding of PEG-IFN-β to its receptor in cells was evaluated in the presence of a fixed amount of 125 I-FN-a2a. The IFN-a2a was radiolabelled with 125 I using the chloramine T method. The 125 I-bound IFNa2a was removed from the free iodine by running the reagents through a Sephadex G25 column and mixing the fractions containing the protein (Pharmacia). The 1 5I-IFN-2a was quantified by an ELISA assay of IFN-a2a (Biosource, USA) and the specific activity was determined. Daudi cells grown in exponential growth phase were harvested and incubated 2 x 106 cells with 0.5 nM 125 I-1 FN-a2a for 3 hours at room temperature in the presence of different concentrations of PEG-IFN-β or diluted IFN-α2a in a test buffer that is RPMI 1640 containing 2% fetal bovine serum and 0.1% sodium azide. At the end of the incubation, the cells were rotated through a layer of phthalate oil and the r adioctivity bound to the cells was counted in a gamma counter. In addition, the binding of PEG [30 kD] -IFN-β and PEG [2 X 20 kD] -IFN-β the receptor was very similar or very close to the binding activity of IFN-β as shown in Figure 7. In addition, the relative activity was determined in an anti-proliferation assay of Daudi cells (human B-cell lymphoma) (Table 3). All IFN were made at a concentration of 2 X of 200 ng / ml. The samples were diluted three times throughout the plate to a final volume of 100 ul. 1 x 105 cells / well (100 μls) were added to each well and incubated for a total of 72 hours at 37 ° C in a humidified incubator with C02. After 48 hours, tritiated thymidine (3H) was added at 1 μCi / cavity in 20 ul. At the end of the 72-hour incubation period, the plate was harvested with a Tomtek plate harvester. The results shown in Table 3 indicate that no detectable loss of I FN activity was observed from PEGylation. Actually the activity was found to be somewhat higher than free IFN-β. This may be due to the formation of inactive aggregates in the free IFN or differences in the quantification methods (amino acid analysis for the PEG-IFN and RP-HPLC samples for IFN-β).

Table 3 Anti-proliferation test of Daudi pg / ml EXAMPLE 4: Pharmacokinetic Studies in Mice Intravenous Administration Mice were injected with 100 ng of IFN-β, PEG [30 kD] -IFN-β or PEG [2 X 20 kD) -IFN-β and blood was removed at the times indicated below. Serum IFN-β levels were determined by ELIAS specific for IFN-β (Toray Industries) and the results are shown in Figure 8. Twenty-eight female mice of the B6D2F1 strain (6-8 weeks) (approximately 20 grams each) ) They separated in four groups as follows: Group 1 contained nine mice injected with an individual 20 ul bolus of 500 ng / ml human IFN-β (final dose of 100 ng / mouse); Group 2 (nine mice) received 200 ul of an equivalent mass of PEG30 kD-IFN-β; Group 3 received 200 ul of an equivalent mass of PEG (2 x 20 kD) -IFN-β; and Group 4 is a group of three non-injected mice that serve as a negative control. Blood samples (approximately 200 ul / sample) were collected in the nine times indicated by interruption of the retro-orbital venous plexus with a capillary tube. The blood samples were allowed to clot for 1 hour at room temperature, rinsed and micro-centrifuged. The serum removed from them was stored at -70 ° C until all the samples were collected. Sera were analyzed for the presence of bioactive human IFN-β using the Toray assay. The results indicate the area under the curve (AUC) markedly intensified in the PEG-IFN samples against the free IFN-beta and that the PEG-IFN samples against the IFN-β and that the PEG [2 x 20 kD ] -IFN-β is superior to PEG [30 kD] -IFN-β.

Subcutaneous administration Mice were injected subcutaneously with IFN-β and PEG-IFN (100 ng / mouse). Figure 9 shows that the total area under the curve (AUC) is dramatically improved for the PEG-IFN samples compared to the free IFN-β. The pharmacokinetic studies are consistent with the PEG-IFN samples that have a longer half-life and an increased AUC.

EXAMPLE 5. Binding of PEG Portion of Low Molecular Weight to Polypeptide Marking Interferon-beta with OPSS-PEG2? hydrazide Protein -S-S-PEG2K-C-NH-NH2 Recombinant human interferon-ß is provided in solution at 0.33 mg / ml in 50 mM sodium acetate buffer, pH 3.8.

Approximately 3.6 mg (excess of 40 mol to mol of protein) of the heterobi functional PEG reagent, OPSS-PEG2? -hydrazide in 2 ml of deionized water is added to 3 ml of IFN-ß at 0.33 mg / ml (0.99 mg) and the two are allowed to react in a polypropylene bottle for 1 hour at 45 ° C. The reaction mixture was then analyzed by capillary electrophoresis to determine the degree of modification. Typical yields varied from 90-97% depending on the purity of the interferon-β and the PEG reagent. The solution was then loaded onto a size exclusion column (Superdex 75, Pharmacia) and eluted with 5 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The peaks were collected and analyzed with SDS-PAGE. The monoPEGi-interferon-ß fractions were mixed together, then used in a further modification step with high molecular weight PEG.

Marking of interferon-ß with (OPSS) 2-PEG3 Protein -SH > Recombinant human interferon-ß was provided in solution at 0.33 mg / ml in 50 mM sodium acetate buffer, pH 3.8. Approximately 6.1 mg (40 moles in excess of moles of protein) of the homobifunctional PEG reagent, (OPSS) 2PEG34oo, in 2 ml of deionized water was added to 3 ml of interferon-β at 0.33 mg / ml (0.99 mg) and the two were allowed to react in a polypropylene bottle for 2 hours at 50 ° C. The reaction was monitored with non-reducing SDS-PAGE and the final reaction mixture was analyzed by capillary electrophoresis to determine the degree of modification. Typical modifications for this reaction with interferon-ß were > 95% The solution was then loaded onto a size exclusion column (Superdex 75, Pharmacia) and eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The peaks were collected and analyzed with SDS-PAGE for their contents. The monoPEGylated interferon-β fractions were combined.

EXAMPLE 6: Union of the Second Portion of PEG to the PEG Polypeptide of Low Molecular Weight Ilation Modification of IFN-S-S-PEG2 -Hydrazine with mPEG3o ?; Aldehyde (ALD) OR II Protein -S-S-PEG2K-C-NH-NH_ O CA > proton-S-S-PEQI? -C- H-N = CH-mPEG30? mPEG30K-CH2CH_CH To the combined fractions of IFN-S-S-PEG2? -hydra zin in Example 5 mPEG3ok-ALD was added in an excess of 20 moles to the protein.

The reaction was carried out at room temperature (25 ° C) for 4 hours and the sample was added to a size exclusion column (Super6se 6, Pharmacia) to determine the modification yield. The modification yield to this reaction was typically > 80% depending on the purity of the PEG reagent and the reaction conditions. Having now fully described this invention, it will be appreciated by those skilled in the art that it can be performed within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without excessive expe- mentation. While this invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is proposed to cover any variation, use or adaptations of the invention following, in general, the principles of the invention and including the items of the present description as they come within the common and known practice within the technique to which it corresponds the invention and as may be applicable to the essential features set forth hereinbefore as follows in the scope of the appended claims. All references cited herein, including articles of publications or extracts, foreign or US patent applications, published or unpublished, foreign or US patents issued, or any other reference, are hereby incorporated by reference in their entireties, including all data, tables, figures and text present in the cited references. Additionally, the complete contents of the references cited within the references cited herein are also fully incorporated by reference. With reference to the steps of known methods, the steps of conventional methods, known methods or conventional methods is in no way an admission that any aspect, description or embodiment of the present invention is described, taught or suggested in the relevant art. The foregoing description of the specific embodiments will fully disclose the general nature of the invention that others may in applying knowledge of the skill of the art (including the contents of the references cited herein) easily modify and / or adapt to various applications of these specific modalities, without undue experimentation, without departing from the general concept of the present invention. Therefore, these adaptations and modifications are proposed to be within the meaning and range of equivalents of the described modalities, based on to the teaching guide presented in the present. It is to be understood that the phraselogy or terminology herein is for the purpose of description and not limitation, such that the terminology or phraselogy of the present specification is to be interpreted by the person skilled in the art in view of the teachings and guide presented herein, in combination with the knowledge of one skilled in the art.

Claims (25)

1. CLAIMS 1. Polyol-interferon-β conjugate having a polyol portion covalently linked to Cys17 of human interferon-β.
2. 2. The polyol-interferon-β conjugate according to claim 1, wherein the polyol portion is a polyalkylene glycol moiety.
3. 3. The conjugate of polyol-interferon-ß according to claim 2, wherein the polyalkylene glycol portion is a polyethylene glycol (PEG) moiety.
4. 4. The polyol-inter feron-ß conjugate according to any of the above indications 1-3, wherein the conjugate of polyol-interf eron-β has the same or greater activity of interferon-β as the native interferon-β, human .
5. A process for producing the polyol-interferon-β conjugate of claim 1, comprising the steps of: reacting interferon-β with a thiol-reactive polyol agent to specifically and covalently link to the site a portion polyol to Cys17 of human interferon-ß to produce a polyol-interferon-β conjugate; and recover the polyol conjugate interferon-ß produced.
6. 6. The process according to claim 5, wherein the thiol reactive polyol agent is a thiol reactive PEGylation agent.
7. 7. The process according to either claim 5 or claim 6, wherein the thiol reactive polyol agent is mono-methoxylated.
8. 8. The process according to either the rei indication 5 or claim 6, wherein the thiol reactive polyol agent is bifunctional.
9. 9. The process according to either claim 5 or claim 6, wherein the thiol reactive polyol agent is a polyol derivative having a functional group selected from the group consisting of orthopyridyl bisulfide, vinyl sulphone, maleimide, and Iodoacetamide The process according to either claim 5 or claim 6, wherein the thiol reactive polyol agent is an orthopyridyl disulfide derivative of a mono-methoxylated polyol. 11. The process according to rei indication 5, wherein the step of reacting is brought to out at an acidic pH where interferon-β is stable. 12. A pharmaceutical composition, comprising the polyol-interferon-β conjugate according to any of claims 1-3, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or auxiliary agent. 13. A method for treating infections, tumors and inflammatory autoimmune diseases, which comprises administering an effective amount of the pharmaceutical composition according to claim 12 to a subject in need thereof. 14. A method for the gradual joining of polyethylene glycol (PEG) portions in series to a polypeptide, comprising the steps of: reacting a polypeptide with a low molecular weight homobi functional or homobi functional PEG portion having the following formula : W-CH2CH20 (CH2CH20) nCH2CH2-X, where W and X are groups that react independently with an amine functional group, sulfhydryl, carboxyl or hydroxyl to link the low molecular weight PEG portion to the gone polypeptide; and reacting the low molecular weight PEG portion bound to the polypeptide with a monofunctional or bifunctional PEG portion to bind the monofunctional or bifunctional PEG portion to a free term of the low molecular weight PEG portion and to form a conjugate. of PEG-polypept gone. 15. The method according to claim 14, wherein the portion of PEG monofunctional or bifunctional has the following formula: Y-CH2CH20 (CH2CH20) mCH_CH2-Z, wherein Y is reactive to a terminal group on the free end of the low molecular weight PEG portion bound to the polypeptide and Z is -OCH3 or an X-reactive group to form a bi-functional conjugate. The method according to claim 15, wherein the monofunctional or bifunctional PEG portion is methoxy-PEG, branched PEG, hydrolytic or enzymatically degradable PEG, PEG Pendant or dendrimer PEG. 17. The method according to claim 14, wherein W and X are independently selected from the group consisting of disulfide of topir idyl, maleimides, vinylsufones, iodoacetamides, hydrazides, aldehydes, succmimidi es es, epoxides, amines, thiols, carboxyl, active esters, carbonates of benzotriazole, p-nit rofenol carbonates, isocyanates and biotin 18. The method according to claim 14, wherein the low molecular weight PEG portion has a molecular weight in a range of about 100 to 5,000 daltons. The method according to claim 14, wherein the unofunctional or bifunctional monofunctional PEG portion has a molecular weight in a range of about 100 daltons to 200 kilodaltons.The method according to claim 14, wherein the portion of PEG of low molecular weight. and / or the monofunctional or bifunctional PEG portion is a polyethylene glycol copolymer 21. The method according to claim 20, wherein the polyethylene glycol copolymer is select from a group that consists of copolymers of polyethylene glycol / polypropylene glycol and polyethylene glycol / poly (lactic acid / glycolic acid) copolymers. 22. The method according to claim 14, further comprising a step of purifying the PEG-polypeptide conjugate after the gradual joining of the two portions of PEG in series to a polypeptide. The method according to claim 22, wherein the purification step comprises one or more purification techniques selected from a group consisting of ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography and reversed phase chromatography. The method according to any of claims 14-23, wherein the polypeptide is interferon-β. 25. The use of PEG-polypeptide conjugates has been produced by methods according to any of claims 14-23 as a medicament.