MXPA06009863A - Interferon-beta polymer conjugates - Google Patents

Abstract

Biologically-active, interferon-beta lb-polymer conjugate compositions are disclosed. The polymer portion is preferably a polyalkylene oxide polymer having a molecular weight of at least about 12 kDa. Methods of making and using the same are also disclosed.

Description

CONJUGATES OF INTERFERON-BETA POLYMER Field of the Invention The present invention is directed to interferon beta-polymer conjugates. In particular, the present invention is directed to interferon beta 1 b polymer conjugates and to substantially non-antigenic polymers, such as PEG. BACKGROUND OF THE INVENTION Many proteins or polypeptides are known to hold great hope for use in the treatment of a wide variety of diseases or conditions. Unfortunately, protein-based therapeutics suffer from a number of drawbacks, including poor solubility in water and body fluids, rapid clearance from the bloodstream after administration, and high potential to elicit an immune response from the person or animal. treated. A proposed solution to address these drawbacks, is to conjugate said proteins or polypeptides to substantially non-antigenic polymers, in order to improve the life in circulation, solubility in water and / or reduce antigenicity. For example, some of the initial concepts of coupling peptides or polypeptides to polyethylene glycol (PEG) and water soluble polymers and the like is described in US Patent No. 4,179,337, the disclosure of which is incorporated herein by reference.

Interferons, also referred to in the present invention as I FNs, are a class of therapeutic proteins that will benefit from improved circulation life, water solubility and / or reduced antigenicity. Interferons are relatively small polypeptide proteins that are secreted by most of the cells of animals, in response to a variety of inducers. Due to their antiviral, antiproliferative and immunomodulatory properties, the interferons are of great interest as therapeutic agents. They exert their cellular activities by binding to specific membrane receptors on the cell surface. Once bound to the cell membrane, interferons initiate a complex sequence of intracellular events. In vitro studies have shown that these include the induction of certain enzymes, suppression of cell proliferation, immunomodulation of activities such as increased phagocytic activity of macrophages and increased lymphocyte-specific cytotoxicity for target cells, and inhibition of virus replication in cells infected with viruses. Therefore, interferon proteins are functionally defined, and a wide variety of natural and synthetic or recombinant interferons are known. There are three types of I major FNs. These are: I FN of leukocyte or I FN-alpha, an I FN Type 1 produced in vivo by leukocytes. I FN fibroblast or I FN-beta, an I FN Type 1 produced in vivo by fibroblasts. I Immune FN or I FN-gamma, an I FN Type 2 produced in vivo by the immune system. The I FN-beta is of particular interest for the treatment of a number of diseases or conditions, and especially in the treatment of multiple sclerosis or MS. Natural human IFN-beta is a glycoprotein of 166 amino acids, and the coding gene has been sequenced by Taniguchi, and associates, 1980, Gene 10: 1-15, and R. Derynck, et al. , supra. The natural I FN-beta has three cysteine residues (cys), located at amino acid positions 17, 31 and 141, respectively. In addition, numerous recombinant variants of IFN-beta are known. Three recombinant IFN-beta products are licensed in Europe and the United States for the treatment of MS. These are interferon beta-1 a ("I FN-beta-1 a") or Avonex® (Biogen, Inc., Cambridge, Massachusettes), another product is I FN-beta-1 marketed as Rebif® (Ares-Serono , Norwood, Massachusettes) and interferon-beta-1 b Ser17 ("I FN-beta-1 bSeri7") or Betaseron® (Berlex, Richmond, California). IFN beta-1 a is produced in mammalian cells, for example, Chinese Hamster Ovary cells ("CHO") using the natural human gene sequence, and the protein produced is glycosylated. See for example, U.S. Patent Nos. 5,795,779, 5,376,567 and 4,966,843, incorporated herein by reference. I FN beta-1 b Ser17 differs structurally from I FN-beta 1 a (Avonex® and Rebif®), because it is produced in Escherichia coli ("E. coli") using a modified human gene sequence that has a substitution constructed from cysteine to serine at amino acid position 17, so that the protein is non-glycosylated. See, for example, U.S. Patent Nos. 4,588,585 and 4,737,462, the disclosures of which are incorporated herein by reference. Both Rebif® and Avonex® are manifested by their package inserts that have specific activities, through differentiation methods, of at least 2-3 x 108 international units (IU) / mg. The Betaseron® package insert reports a specific activity of approximately 3 x 107 l U / mg, which indicates a difference of ten times in power. Although these activities are determined by somewhat different methods, the order of magnitude differences in antiviral antiviral activities are also reflected in the recommended doses, which are measured in micrograms (60-130 mcg / week) for IFN- products. beta 1 with glycosylated Rebif® and Avonex®, and 0.25 milligrams and more for the non-glycosylated IFN beta-1 b Betaseron®. IFN-beta, in each of its recombinant formulations, has multiple effects on the immune system, including the ability to inhibit viral replication. IFN-beta-1 b is described by the manufacturer (Berlex, Richmond, California) as a suppressor of T cell activity improvement, which reduces the production of proinflammatory cytokine, deactivates the presentation of antigens and inhibits lymphocyte trafficking in the central nervous system. Other sources have reported that IFN-beta reduces IFN-gamma production by T-lymphocytes. Other beneficial therapeutic effects are also expected. However, as therapeutic protein controls, the drug is rapidly cleared from the bloodstream by non-specific mechanisms, including renal filtration. In addition, patients injected with I FN-beta develop anti-I neutralizing antibodies FN-beta ("Nabs"). Nabs are a subset of binding antibodies that work to inhibit the normal biological effects of the production antigen, and if produced through a therapeutic protein, can reduce the effectiveness of the treatment. The risk of the development of an anti-I FN-beta Nab and the subsequent effects in the treatment, can make the early treatment of MS with interferon drugs impossible - a consequence that could restrict the therapeutic promise of these agents. Each of the interferon beta drugs marketed is associated with the development of Nabs in clinical trials during the treatment of MS. In a two-year study of Betaseron®, almost half of the patients treated, in groups with both high and low doses, developed Nabs at some point in the study. Some additional disadvantages of interferon therapeutics are physical instability, for example, protein aggregation, denaturation and precipitation, to name a few, and chemical instability, ie, deamidation, hydrolysis, disulfide exchange, oxidation, etc. The protein aggregation, as used in the present invention, refers to the formation of dimers, trimers, tetramers or multimers from monomers which may or may not be precipitated in the formulation regulators and under the conditions of the present invention. The formulation of Ribif® and Avonex® and Betaseron®, currently includes the use of HSA that can contribute to viral contamination, as well as to the aggregation of the protein. The polymer conjugates of I FN-beta's are known. US Pat. Nos. 4, 766, 106 and 4,917,888, incorporated herein by reference, describe, inter alia, conjugates of I FN-beta 1 b linked by amide using mPEG-N-succinimidyl glutarate or mPEG-N-succinimidyl succinate. The patents describe that PEGylation of the protein is carried out using relatively high molar excesses of the activated polymer. Although the linkage of the polymer to the Lys residues is preferred, the N-terminal polymer linkages are also described, as well as those comprising Cys, Glu and Asp. See column 8, lines 34 to 40 of the '888 patent, for example. The published PCT patent application, No. WO99 / 55377 which describes the selective modification at the site of IFN-beta 1 a in Cys-17, using a thiol-reactive PEGylating agent, describes, however, drawbacks with the results of '106 and' 888. Specifically, page 4, line 5 to 18 of the published PCT application, states that although non-specific PEGylation using a large molar excess of activated PEG, provided conjugates with improved solubility, "a major problem was the reduced level of activity and production " Commonly assigned US Patent No. 5,738,746 describes the preparation of several PEG-interferon conjugates. Column 14, line 1 thereof, mentions I FN-beta as an interferon suitable for conjugation with various forms of activated PEG. Fractionation of the PEGylated product to recover specific species, including mono-PEGylated conjugates, is also described. US Patent No. 5, 109, 120, incorporated herein by reference, discloses methods for making PEG conjugates having an imidoester linker, generally including, IFN-beta. US Patent No. 6,531,122 describes variants of IFN-beta or muteins other than IFN-beta 1 b, optionally conjugated to polymers such as PEG, including binding through constructed Cys or Lys residues. Pepinsky and associates, in the published US Patent Application No. 20030021765, describe polymer conjugates of IFN-beta 1 a, including PEG conjugates and uses thereof. However, an I-N-terminal PEGylated FN-beta 1 conjugate of 20 kDa failed to provide prolonged effects in a biological marker with respect to IFN-beta activity, despite the prolonged presence in the serum of the animals of test (Pepinsky and associates, 2001, The Journal of Pharm.v Exper. Ther 297 (3): 1059-1066). In addition, Pepinsky's report observed that a conjugate of IFN-beta 1 to PEGylated by N-terminus of 30 kDa, retained only one sixth of the activity of the conjugate from I FN-beta 1 to PEGylated N-terminal of 20 kDa, and an I-FN-beta 1 to PEGylated N-terminal 40 kDa conjugate, lost all interferon activity. In spite of the above, it should be noted that the different types of IFN proteins exhibit significant homology differences. For example, IFN-alpha and I FN-beta exhibit an average homology of only 3% in the domain of the signal sequence, and only 45% in the IFN polypeptide sequence, for example, as described by Derynck, 1980 Nature, 285: 542-547. Furthermore, even though there is a greater homology between the IFN-beta's, there are nevertheless some significant differences between the two, both in terms of therapeutic use, indications, etc. Despite the descriptions described above, there remains a permanent and hitherto unresolved need in the art for improved polymer conjugated I FN-beta compositions, particularly those containing IFN-beta 1 b. There also continues to be a need for improved compositions containing polymerized conjugated FN-beta 1 b, wherein the polymer has a molecular weight of about 30 kDa (average number), or more, which are free of human serum albumin (" HSA "). Brief Description of the Invention The needs described above are addressed, and other advantages are provided through the polymer conjugated I FN-beta compositions described herein. In one aspect of the present invention, an improved biologically active interferon-polymer conjugate composition is provided. The composition includes an interferon-beta 1 b conjugated to a polyalkylene oxide (PAO) polymer having a molecular weight of at least about 12 kDa. Preferably, the PAO is a polyethylene glycol (PEG) having a molecular weight of from about 12 kDa to about 60 kDa. More preferably, the PEG has a molecular weight from about 30 kDa to about 60 kDa. In one aspect of the present invention, the polymer is linked to the amino terminus of I FN-beta 1 b, although in other separate and preferred aspects of the present invention, the polymer is adhered through an epsilon amino group of a Lys of IFN-beta 1 b. Depending on the adhesion site and molecular weight of the selected polymer, the anti-viral activities retained from the conjugates will range from at least about 65% for 30 kDa polymer conjugates., and at least about 15% for the 40 kDa polymer conjugates. In both cases, the amount of activity retained is significantly greater than what was expected. In certain optional embodiments, more than one polymer is linked to each molecule of I FN-beta 1 b. Preferably, the number of polymers linked to each molecule of IFN-beta 1 b ranges from 1 to about 4, and more preferably from 1 to about 3. The composition of the present invention incorporates the polymer conjugate described above in the presence of certain regulators and excipients, to increase physical and chemical stability. A further improvement comprises providing a formulation that is free of human serum albumin ("HSA"), in order to reduce the risk of viral contamination and protein aggregation. In still further improvement, the present invention provides an improved process for conjugating a protein with a non-antigenic polymer, such as a polyalkylene oxide in the presence of Zwittergent®. Previously, the removal of Zwittergent® from said reaction mixture has proven to be impractical. However, the present invention provides an economical method for separating Zwittergent® from a separate protein with polymer, for example, IFN-beta 1 b, under acidic conditions. Other aspects of the present invention include methods for making the conjugate compositions or formulations, as well as methods of treatment using them. As a result of the present invention, improved IFN-beta 1 b polymer conjugate compositions are provided. The anti-viral activity retained from the conjugates of the present invention is surprisingly high, especially by virtue of the fact that the polymer portion thereof, in most aspects of the present invention, is at least about 30. kDa The prior art, see the publication by Pepinsky and associates, 2001, The Journal of Pharm. v Exper. Ther. 297 (3): 1059-1066, supra, which reports that IFN-beta 1 a, a form of glycosylated IFN-beta and more potent compared to IFN-beta 1 b, was substantially less active or inactive when types of PEG with the same molecular weight. In a related aspect of the present invention, improved methods are provided for preparing a polyalkylene oxide-protein conjugate with a soluble protein in a deficient form, wherein the methods comprise the steps of (a) solubilizing a protein of interest in a solution compatible aqueous in the presence of a compatible protein-detergent solubilization amount; (b) reacting the solubilized protein of interest with an activated polyalkylene oxide polymer, to produce a solution comprising a polyalkylene oxide-protein conjugate and the detergent; (c) adjusting the reactivated solution from step (b) to an effective pH to dissociate the detergent from the polyalkylene oxide-protein conjugate; (d) separating the dissociated detergent from the polyalkylene oxide-protein conjugate, and recovering the polyalkylene oxide-protein conjugate. The method of the present invention optionally applies any protein, and preferably a protein that is poorly soluble in aqueous solution. More preferably, the protein is a interferon, such as interferon beta. Preferably, the pH is adjusted in step (c) to a range from about pH 3 to about pH 4. The activated polyalkylene oxide polymer is, for example, a polyethylene glycol polymer that ranges in size from about 12 kDa to about 60 kDa. The detergent is optionally selected from an ionic detergent, a non-ionic detergent, a zwitterionic detergent, and combinations thereof. Preferably, the detergent is a zwitterionic detergent. Brief Description of the Drawings Figure 1 is a graph that shows comparative data that are described in example 7. The bars are represented as follows: D week 2,? week 3, = week 4 and m week 6. Figure 2 is a graph that illustrates the comparative data described in example 8. The curves are represented as follows: - * - I FN-beta 1 b, -__ ? -PEG2-40k, - A - PEG-UA-40k and - • - Di PEG-20k. Figure 3, a graph showing the average concentrations of serum I FN-beta, by ELI SA, in male and female Cynomolgus monkeys, after injection of 15 μg / kg EZ-2046, as described in example 9. Example 9. The curves are represented by the following symbols, where "IM" is intramuscular, "IV" is intravenous, "SC" is subcutaneous, " F "is female and" M "is male. - A - IMF -or- IV-M -? - I M_M - «- SC_F ... | V_F -p- SC_M Figure 4 shows the purity of the final product of PEG-I FN-beta 1 b, as is determined by RP-H PLC using an ELSD detector, as described in Examples 2 and 3. Detailed Description of the Invention Accordingly, the present invention provides a composition comprising: (a) an interferon conjugated to a polymer of polyalkylene oxide having a molecular weight of at least about 12 kDa, or alternatively, at least about 20 kDa; and optionally (b) a surfactant; (c) an excipient, and (d) a regulator, wherein the pH range of the solution is from about 3.0 to about 1 1. In the embodiments of the present invention, the compositions have a pH from about 3.0 to about 8.0 In other embodiments, the compositions of the present invention have a pH from about 3.0 to about 5.0, with a pH from about 3.0 to about 4.0, with the most preferred It has been found that the ionic strength of the compositions provided herein affects the stability, for example, they prevent aggregation. Low ionic strength in low pH regulators is preferred, while high ionic resistance in high pH regulators is preferred. In one embodiment, the ionic strength in a composition of the present invention having a pH of from about 3.0 to about 4.0 is less than 10 mM. In another embodiment, the ionic strength of a composition of the present invention having a pH from about 5.5 to about 7.5 is from about 100 to about 150 mM. The interferon used in the compositions of the present invention is preferably interferon-beta 1 b and more preferably IFN-beta-1 bSe.-i7-A. Interferons Beta The term "interferon-beta" or "I FN-beta" as used in the present invention, refers to I FN-beta isolated from natural sources and / or produced by recombinant DNA technology as described in the art, which has sequence homology with, and functionality, including bioactivity of the native I FN-beta. The term "interferon-beta 1 b" or "I FN-beta 1 b" as used in the present invention, refers to a mutein of I FN-beta having the residue Cys 17 is replaced by the residue Ser-t 7, and expressed in a non-glycosylated form, with the N-terminal amino acid, Methionine, removed post-translationally, and represented in the present invention as SEQ ID NO: 1. As noted in greater detail, supra, the interferon beta (IFN-beta) part and the polymer conjugate, can be prepared or obtained from a variety of sources, including recombinant techniques such as those using synthetic genes expressed in suitable eukaryotic or prokaryotic host cells, by example, see U.S. Patent No. 5,814,485, incorporated herein by reference. In addition, IFN-beta can also be a mammalian source extract such human, ruminant or bovine I FN-beta. A particularly preferred IFN-beta, is I FN-beta-1 b seri7, recombinant available from Berlex, (Richmond, California), as described in U.S. Patent No. 4,737,462, incorporated herein by reference. The I FN-beta proteins used to produce the conjugates of the present invention were either commercially obtained, for example, IFN-beta 1 b was obtained from Berlex, Inc. (Richmond, California) or produced and isolated as exemplified later. Native human IFN-beta is optionally employed, although it is preferred to use an optimized FN-beta mutein I for optimization and solubilization in a prokaryotic host. A preferred prokaryotic host cell is Escherichia coli. Many muteins of the human or native animal FN-beta are known and contemplated to be employed in the practice of the present invention. Preferred muteins are described in greater detail in U.S. Patent Nos. 4, 588,585, 4,959,314, 4,737,462 and 4,450, 103, incorporated herein by reference. In synthesis, as noted above, a preferred mutein is one in which the Cys-7 residue of native human IFN-beta is replaced by serine, threonine, glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine. , tryptophan or methionine. Most preferred is the non-glycosylated mutant Ser 7 of IFN-beta, also referred to in the present invention as IFN-beta 1 b. Various methods are known for expressing and isolating I FN-beta proteins from prokaryotic host systems, and vectors suitable for expression through prokaryotic host cells. For example, many of the IFN-beta employed in the examples provided herein were produced through the following method. A synthetic gene encoding an IFN-beta, for example, IFN-beta 1 b, was synthesized after codon optimization for bacterial expression. Other methods and reagents for the production and purification of IFN-beta are disclosed, for example, in US Patent Nos. 6, 107,057, 5,866,362, 5,814,485, 5,523,215, 5,248,769, 4,961, 969, 4,894,334, 4,894,330, 4,748,234, 4,656, 132 , which are all incorporated herein by reference, as well as through other references too numerous to be mentioned. Methods for expressing and isolating I FN-beta proteins, and vectors suitable for expression through eukaryotic host cells, such as Chinese Hamster Ovary cells ("CHO"), are described in detail, for example, in U.S. Pat. Nos. 4,966, 843, 5,376,567 and 5,795,779, incorporated herein by reference. B. Non-Antigenic Polymers The polymeric part of the conjugate useful in the compositions of the present invention can be linear and is preferably selected from the group consisting of: A-O- (CH2CH2O) x-. AO- (CH2CH2O) x-CH2C (O) -O-, AO- (CH2CH2O) x-CH2CH2 N R7-, AO- (CH2CH2O) x-CH2CH2 SH, -OC (O) CH2-O- (CH2CH2O) x -CH2C (O) -O-, -NR7CH2CH2-O- (CH2CH2O) x-CH2CH2 N R7-, -SHCH2CH2-O- (CH2CH2O) x -CH2CH2 SH-, wherein A is a cover group; R7 is selected from hydrogen, Ci-e alkyls, C3 .-? 2 branched alkyl, C3-8 cycloalkyls, C1-6 substituted alkyls, C3.8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C1-6 alkenyls, C3_? 2 branched alkenyl, C 1-6 alkynyl, C 3-12 branched alkynyl, C- | 6 heteroalkyl, C? -6 hetero-substituted alkyls, C- | .6 alkoxyalkyl, phenoxyalkyl and C-? 6 heteroalkoxy, and x is the degree of polymerization. The variable x is preferably a positive integer selected such that the molecular weight of the polymer is within the ranges described herein, for example, from about 20 to about 60 kDa, as preferred. Alternatively, the polymer part of the conjugate useful in the compositions of the present invention may be branched, and preferably, is selected from the group consisting of: cn-PEG- -NH \ (CH2) to HC (ZCH2) pC (0) -, / (CH2) 3 m-PEG C- or II wherein: (a) is an integer of from about 1 to about 5; Z is O, NR8, S, SO or SO2; wherein R8 is H, C? _8 alkyl, C? -8 branched alkyl, C-? 8 substituted alkyl, aryl or aralkyl; (n) is 0 or 1; (p) is a positive integer, preferably from about 1 to about 6, and m-PEG is CH3-O- (CH2CH2O) x-. Preferably, the cover group A is selected from the group consisting of OH, CO2H, N H2, -SH and C- | .6 alkyl portions. More preferably, interferon-beta 1 b is conjugated to the polyalkylene oxide polymer selected from the group selected from: A-O- (CH 2 CH 2 O) x-. AO- (CH2CH2O) x-CH2C (O) -O-, AO- (CH2CH2O) x-CH2CH2 N R7-, AO- (CH2CH2O) x-CH2CH2 SH, -OC (O) CH2-O- (CH2CH2O) x -CH2C (O) -O-, -N R7CH2CH2-O- (CH2CH2O) x-CH2CH2 N R7-, -SHCH2CH2-O- (CH2CH2O) x-CH2CH2 SH-, wherein A is a cover group; R7 is selected from hydrogen, C? .6 alkyls, C3-12 branched alkyls, C3-8 cycloalkyls, Ci.β substituted alkyls, C3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C? -6 alkenyls, C3- ? 2 branched alkenyls, C1-6 alkynyl, C3.12 branched alkynyl, C? -6 heteroalkyls, C1-6 heteroalkyls substituted, C?? 6 alkoxyalkyl, phenoxyalkyl and C- | .6 heteroalkoxy, and x is the degree of polymerization . The variable x is preferably a positive integer selected so that the molecular weight of the polymer is within the ranges described herein, e.g., 20-60 kDa, as preferred. Alternatively, the polymer part of the conjugate useful in the compositions of the present invention may be branched and is preferably selected from the group consisting of: (ZCH2) nC (0) - m-PEG- -NH \ (CH2) to HC (ZCH2) "C (0) - ^ (CH2) to m-PEG C- or II wherein: (a) is an integer from about 1 to about 5; Z is O, N R8, S, SO or SO2; wherein R8 is H, C- | 8 alkyl, C-, 8 branched alkyl, C- | 8 substituted alkyl, aryl or aralkyl; (n) is 0 or 1; (p) is a positive integer, preferably from about 1 to about 6, and m-PEG is CH 3 -O- (CH 2 CH 2 O) x-. Preferably, the cover group A is selected from the group consisting of OH, CO2H, NH2, -SH and C-? 6 alkyl portions. More preferably, interferon-beta 1 b is conjugated to a polyalkylene oxide polymer selected from the group selected from: A-O- (CH 2 CH 2 O) x-. A-O- (CH 2 CH 2 O) x-CH 2 C (O) -O-, A-O- (CH 2 CH 2 O) x-CH 2 CH 2 N R 7 -, A-O- (CH 2 CH 2 O) x -CH 2 CH 2 SH, wherein the molecular weight of the polyalkylene oxide polymer ranges from about 20 to 40 and preferably from 30 kDa to about 40 kDa. In order to conjugate the IFN-beta to polymers, such as poly (alkylene oxides), one of the polymer hydroxyl end groups is converted into a reactive functional group which allows conjugation. This process is often referred to as an "activation" and the product is referred to as an "activated" polymer or activated poly (alkylene oxide). Other substantially non-antigenic polymers are similarly "activated" or functionalized. Polyethylene glycol (PEG) is the most preferred PAO. The general formula for PEG and its derivatives is, for example, A'-O- (CH2CH2O) xA where (x) represents the degree of polymerization or number of repeating units (up to about 2300) in the polymer chain and depends of the molecular weight of the polymer. (A) is an activated linking group such as those described below, while A 'is the same as (A), an alternate activated binding group, H or a cover group such as CH3. Said mono-activated PEG derivatives are commonly referred to as mPEG derivatives. In addition to mPEG, it should generally be understood that PEGs terminated at one end with any d-4 alkyl group are also useful. In alternative aspects, the polymer is a poly (propylene glycol) or PPG. Branched PEG derivatives, such as those described in the commonly assigned U.S. Patent Nos. 5,643,575, 5,919,455 and 6,11,906, "Star-PEG's", multi-arm PEG's, Trench PEG's or terminal branched PEG's, such as those described in the Nektar catalog "Polyethylene Glycol and Derivatives for Advanced PEGylation 2003". The description of each of the above publications is incorporated by reference into the present invention. A list without limitation of PEG derivatives is provided, including: mPEG-A, A-PEG-A, and A list without limitation of suitable PEG activated linkage groups is provided below. Activated linkage groups correspond to A in the formula given above.

CH2CH2CH2-CHO-CHO (PEG-Butylaldehyde) (PEG-aldehyde) The above can be attached to an alpha and / or omega terminal of PEG, it being understood that when both linking groups are used, the resulting conjugates can have two (2) ) equivalents of I FN-beta per polymer unit. As appreciated by those skilled in the art, the aldehyde derivatives are used for the N-terminal adhesion of the I FN polymer. For example, the polyalkylene oxide (PAO) aldehydes react only with amines and pass through reductive amination reactions with primary amines in the presence of sodium cyanoborohydride to form a secondary amine. Suitable polyethylene glycol (PEG) aldehydes are available from Nektar of San Carlos, CA. In other aspects of the present invention, the other activated linkers shown above, will allow non-specific binding of the polymer to amino groups Lys which form carbamate (urethane) or amide bonds. In some preferred aspects of the present invention, when Lys adhesion is desired, the activated linker is an oxycarbonyl-oxy-N-dicarboximide group, such as a succinimidyl carbonate group. Alternative activating groups include N-succinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide and N-norborene-2,3-dicarboxy. These urethane-forming groups are described in commonly assigned US Patent No. 5, 122,614, the disclosure of which is incorporated herein by reference. Other activated urethane-forming polymers can also be used, such as activated benzotriazole carbonate (PEG activated by BTG, available from Nektar). See also the commonly assigned US Patent No. 5,349,001, with respect to the aforementioned T-PEG. It will be appreciated that the heterobifunctional polyalkylene oxides are also contemplated for IFN-beta crosslinking purposes, or that they provide a means for adhering other portions such as targeting agents for the convenient detection or localization of the polymer-IFN-beta conjugate in particular, areas of testing, research or diagnostic purposes. In many aspects, suitable polymers will vary in some part by weight, although preferably they are at least about 20,000 (average molecular weight). Alternatively, the polymers can range from about 20,000 to about 60,000, with from about 30,000 to about 40,000 being preferred. In some aspects of the present invention, when bifunctional PEG is used, the molecular weight can be as low as 20,000. As an alternative to the preferred PAO-based polymers, other terminally functionalized, effectively non-antigenic polymers, such as dextran, polyvinyl alcohols, polyvinyl pyrrolidones, polyacrylamides, such as HPMA's-hydroxypropylmethacrylamides, polyvinyl alcohols, can be used. carbohydrate-based polymers, copolymers of the above and the like, if the same type of activation as described in the present invention is employed for PAO's such as PEG. Those skilled in the art will consider that the list that follows is merely illustrative and that polymeric materials having the qualities described herein are also contemplated. For purposes of the present invention, the "effectively non-antigenic" and "substantially non-antigenic" should be understood to include all polymeric materials considered in the art, such as being substantially non-toxic and which do not produce an appreciable immune response in mammals. Activated polymers are reacted with I FN-beta under suitable conditions to allow adhesion of protein sites that do not significantly interfere with biological activity, for example, so that the conjugated IFN-beta retains the antiviral activity and other desirable biological activities. Histidine groups, free carboxylic acid groups, appropriately activated carbolino groups, portions of oxidized carbohydrate and mercapto groups, are available in the IFN-beta of interest, can also be used as supplementary adhesion sites, when appropriate . In one embodiment, the PEG-lFN-beta 1 b conjugate of the composition is in a concentration from about 0.01 mg / ml to about 4.0 mg / ml. In other embodiments, the protein conjugate is in a concentration from about 0.05 mg / ml to about 3.0 mg / ml. C. Solubilization of Proteins for Conjugation Reaction by Detergent In order for a polyalkylene oxide polymer to pass through a useful conjugation reaction with a protein of interest, the protein must be in solution. Unfortunately, many of the proteins that are desirable to be reacted with polyalkylene oxide polymers are difficult to maintain in aqueous solution under conditions that are compatible with conjugation reaction conditions. This is a problem with many insoluble proteins, including IFN-beta 1 b. A non-destructive method to solubilize proteins is to include a surfactant or detergent in the solution. A detergent will solubilize an otherwise insoluble protein in an aqueous solution, associating it with the protein and preventing precipitation and / or aggregation. A number of ionic, non-ionic and zwitterionic detergents are well adapted for protein solubilization, although prior to the present invention, the effective separation of the associated detergent from the reaction product, ie, the protein conjugated by polymer produced, had been a significant obstacle. The present invention provides a new and efficient method for separating a detergent from conjugated proteins. In synthesis, the protein of interest is solubilized with a compatible detergent and subjected to a conjugation reaction. After the conjugation reaction is completed, the pH of the reaction solution is decreased sufficiently to dissociate the detergent from the protein. The decreased pH fluctuates, for example, from about pH 3 to about pH 4. Accordingly, the detergent is physically separated from the conjugated protein, for example, by centrifugation and / or filtration. Preferably, the separation step is by diafiltration, so that the conjugated protein is retained by the diafilter, while at the same time the detergent is removed by washing with an excess of compatible regulator. The diafilter is preferably 10 K in size, although experts will appreciate that this may vary with the size of the conjugated protein per polymer of interest.

Preferred detergents for stabilization or solubilization of a relatively insoluble protein in an aqueous solution include ionic, nonionic and zwitterionic detergents. Ionic detergents are those that contain a head group with a net charge. These contained a straight hydrocarbon (alkyl) chain, such as sodium dodecyl sulfate (SDS), or a rigid steroidal structure, such as in deoxycholate-based detergents, for example, sodium deoxycholate. Nonionic detergents contain non-charged hydrophilic head groups consisting of any polyoxyethylene portions. Exemplary nonionic detergents include polyoxyethylene derivatives, such as polyoxyethylene lauryl ether (for example BRIJ®), Glylucamide-based detergents, such as octyl dodecanol (TRITONX-100®, ethoxylated fatty acid esters (e.g. , TWEENs®) or glycosidic groups, such as octyl glucoside and dodecyl maltoside.Zwiterionic detergents do not contain a net charge.Zwiterionic detergents include those provided by Anatrace in the form of Anzergent® or Calbiochem in the form of ZWITTERGENT®. Preferred zwitterionic detergents are ZWITTERGENT®, 3-X series and CHAPS, ZWITTERGENT® 3-14 is most preferred, as described in examples 2 and 3, below. , additional particular detergents contemplated to be used in the processes described above are described.

* Reference: Guide to the Properties and Uses of Detergents in Biology and Biochemistry of Calbiochem, incorporated in its entirety to the present invention as a reference. The optimum concentration of the detergent or surfactant will vary with the protein of interest and the particular detergent or surfactant that is selected, although it will preferably be determined to have the lowest concentration necessary to safely hold the protein of interest in the aqueous solution. It is contemplated that this detergent removal process has activity to support the polymer conjugation of a range of useful proteins with otherwise limited aqueous solubility. Such proteins generally include lipoproteins or membrane-bound proteins, such as interleukin-2 (IL-2). Preferably, the protein is an I FN, such as I FN beta 1 b.

D. Regulators, Surfactants and Excipients The compositions of the present invention contain a regulator which can be selected from the group consisting of Glycine-HCl, acetic acid, sodium acetate, sodium aspartate, sodium citrate, sodium phosphate and succinate. of sodium. Preferably, the regulator is selected from sodium acetate, sodium citrate and glycine HCl. In addition, the regulator preferably has an ionic strength of about 10 mM, and is in a concentration of about 1 mM to about 10 mM. Preferably, the regulator is in a concentration of from about 3 mM to about 5 mM. The compositions of the present invention also contain an excipient, wherein the excipient is non-ionic and is selected from the group consisting of monosaccharides, disaccharides and alditols. Preferably, the excipient is selected from the group consisting of monosaccharides such as glucose, ribose, galactose, D-mannose, sorbose, fructose, xylulose and the like, disaccharides such as sucrose, maltose, lactose, trehalose and the like, polysaccharides such as raffinose. , maltodextrins, dextrans and the like and alditols such as glycerol, sorbitol, mannitol, xylitol and the like. More preferably, the excipient is selected from the group consisting of sucrose, trehalose, mannitol and glycerol or a combination thereof, with the group consisting of mannitol and sucrose or a combination thereof as preferred.

For the compositions of the present invention, mannitol may be present in a concentration of between 1% to about 6%, sucrose may be present in a concentration of about 8% to about 10%, and trehalose may be present in a concentration of about 8% up to approximately 1 0%. Preferably, the compositions contain about 5% mannitol or about 9% sucrose or 9% trehalose. The compositions of the present invention further contain a surfactant, wherein the surfactant is nonionic and is selected from the group consisting of polysorbate 80 (Tween 80), polysorbate 20 (Tween 20) and polyethylene glycol. In one embodiment, the surfactant is polysorbate 80. In one embodiment, the Tween 80 surfactant is in a concentration from about 0.01% to about 0.5%.

Preferably, for compositions of the present invention, Tween 80 is in a concentration of about 0.5%. Reaction Conditions In the examples below, details are given with respect to the specific reaction conditions, which are suitable for making monoPEG ions. However, the processes of the present invention generally include reacting interferon-beta 1 b with an activated polyalkylene oxide polymer having a molecular weight of at least about 30 kDa under conditions sufficient to cause conjugation of the oxide polymer. activated polyalkylene for interferon-beta 1 b, and the retention of at least a part of the antiviral activity relative to native interferon-beta 1 b, using the standard assay measurements. A non-denaturing surfactant, such as a non-ionic detergent or a zwitterionic detergent, was found as a component in the PEGylation reaction. The preferred surfactant is a zwitterionic detergent. Most preferred is sulfobetaine, such as Zwittergent® 3-14. The reaction conditions to effect conjugation additionally include carrying out the adhesion reaction in an approximately equimolar excess to about a relatively small molar excess of the activated polymer with respect to the IFN. In this regard, the process can be carried out with approximately 1 to 15 times of molar excess; preferably 2 to 12 times of molar excess and most preferably about 3 to 10 times of molar excess. The conjugation reaction can be carried out at about room temperature, 20-25 ° C. It is also preferred that the coupling reaction be allowed to proceed rather for short periods of time, ie 0.5 to 2 hours, before extinction. It was determined that the reaction with the aldehyde-activated polymers was best carried out with a pH of about 5.2, with a subsequent addition of reducing agent, sodium cyanoborohydride. In practice, polymers not activated by aldehyde result in the formation of a mixture of position I isomers FN-polymer. Preferably, each isomer contains a single strand of polymer adhered to the interferon through an amino acid residue. In alternative embodiments, there may be more than one strand of polymer adhered to the IFN as a result of the directed Lys processes. The solutions containing these conjugates are also useful as such, or they can be further processed to separate the conjugates on the molecular weight bases. Due to the nature of solution-based conjugation reactions, the Lys-adhered compositions are a heterogeneous mixture of species containing the polymer strand (s) attached at different sites in the interferon molecule. In any solution containing the conjugates, it is likely that a mixture of at least about 2, preferably about 6 and more preferably about 8 positional isomers will be found. Methods of Treatment Another aspect of the present invention provides methods of treatment for various medical conditions in mammals, preferably humans. The methods include administering an effective amount of a pharmaceutical composition comprising an interferon-beta-polymer conjugate prepared as described in the present invention, to a mammal in need of such treatment. The conjugates are useful, inter alia, for treating susceptible conditions and interferon or conditions that could respond positively or favorably, since these terms are known in the medical arts as interferon-based therapy. The conditions that can be treated according to the present invention are generally those that are susceptible to treatment with I FN-beta. For example, susceptible conditions include those that could respond positively or favorably, since these terms are known in the medical arts as therapy based on IFN-beta. Exemplary conditions that can be treated with I FN-beta include, but are not limited to, multiple sclerosis and other autoimmune conditions, cell proliferation conditions, cancer, viral infections and other medical conditions known to those skilled in the art, to benefit from interferon-beta therapy and / or interferon-beta 1 b. In a preferred aspect of the present invention, polymer conjugated IFN-beta is administered to patients in effective amounts to treat multiple sclerosis. A further aspect of the present invention provides for the treatment of conditions that can be treated with polymer conjugated IFN-beta, and preferably polymerized conjugated FN-beta 1 b, which have not hitherto completely responded to said treatment due to the Negative side effects that have previously exceeded the benefits of treatment at a given dose. For example, IFN-beta has been tested to treat Kaposi's sarcoma with poor diagnosis, related to VI H / SI DA infection (Miles et al., 1990 Ann Intern Med. 1 12 (8): 582-9 and the data suggested a minimum potential benefit The practice of the present invention can allow the treatment of this condition, and others, in higher doses in combination with other therapeutic agents known in the art Administration Methods The administration of the doses described can be every third day, although preferably it is once or twice a week.The doses are usually administered for at least a period of 24 weeks by injection or infusion.The administration of the dose can be intravenous, subcutaneous, intramuscular, or any other acceptable systemic method, including Subdermal or transdermal injection through a medical syringe and / or a conventional pressure system, based on the judgment of the attending physician, the The amount of drug administered and the treatment regimen used will, of course, depend on the age, sex and medical history of the patient being treated, the stage or severity of the specific disease condition and the patient's tolerance to treatments, such as it can be evidenced by local toxicity and by systemic side effects. The amount of dosage and frequency can be determined during initial classifications of neutrophil counts. The amount of IFN-beta-polymer conjugate composition administered to treat the conditions described above is based on the I FN activity of the polymer conjugate. It is an amount that is sufficient to significantly affect a positive clinical response. Although the clinical dose will cause some level of side effects in some patients, the maximum dose for mammals, including humans, is the highest dose that can not cause clinically important unmanageable side effects. For purposes of the present invention, such clinically important side effects are those that may require termination of therapy due to flu-severe symptoms, central nervous system pressure, severe gastrointestinal complaints, alopecia, pruritis or severe rash. Conditions such as substantial abnormalities of white and / or red blood cells and / or liver enzymes or anemia also limit the dose. Naturally, the doses of the various I FN-beta conjugate compositions will vary to some extent depending on the I FN-beta portion and selected polymer. However, in general the conjugates are administered in amounts ranging from about 1 00,000 to about 1 to 50 million I U / m2 per day, based on the condition of the treated human or mammalian patient. The range set forth above is illustrative and those skilled in the art will determine the optimal dosage of the conjugate selected based on clinical experience and indication of treatment. EJ EM PLOS The following examples serve to provide an additional appreciation to the present invention., although they do not mean in any way that they restrict the effective scope of the present invention. EXAMPLE 1 PRODUCTION OF I FN-beta 1 b RECOMMENDED A. Optimized Gene Coding I FN-beta 1 b A cDNA gene (SEQ ID NO: 2) was synthesized that encodes the reported sequence of 165 amino acids of interferon-beta- 1 b human (SEQ ID NO: 1). This gene has codons optimized for expression in E. coli, and was synthesized using standard chemical synthesis of overlapping oligonucleotide segments. The flanking restriction sites, Ndel and BamHI, were included at the end of the gene. After digestion of synthetic DNA with restriction enzymes Ndel and BamHI, the 0.5 kilobase gene was ligated through DNA ligase T4 into the plasmid vector pET-27b (+) (Novagen Corporation), which had also been restricted with these two enzymes. The recombinant plasmid was introduced into the E. coli strain BLR (DE3), by electroporation, using a BTX 600 Electro Cellular Manipulator, according to the manufacturer's instructions. The transformation mixture was plated on LB agar plates containing kanamycin (15 μg / ml) allowing the selection of colonies containing the plasmid pET-27b (+) / IFN-beta-1 b (plasmid pEN831 designated in strain EN834 ). Additional isolated colonies were purified by plating and analyzed for inducible I PTG gene expression by standard methods, such as those described in the Novagen pET System Manual, Ninth Edition. B. Expression of IFN-beta 1 b The gene optimized by E. coli codon described above for IFN-beta-1 b, was expressed in the BLR / pET system which uses the expression control of T7 RNA polymerase. The IFN-beta-1 b protein was expressed in inclusion bodies comprising approximately 30% total cell protein. After solubilization and extraction of butanol, the protein was purified almost to homogeneity by DEAE and SP ion exchange chromatography (Amersham) in the presence of Zwittergent® 3-14. Other standard recovery steps were employed. The expression of betaseron was achieved by inducing the culture growth in the presence of I PTG, 1.0mM, for 2 to 3 hours at a temperature of 37 ° C. I FN-beta 1 b accumulated in inclusion bodies. C. Purification of Interferon-beta-1b from the Inclusion Bodies The purification of IFN-beta-1b from the inclusion bodies was achieved almost until homogeneity after modifications and amalgamations of previously published protocols. In synthesis, IFN-beta-1 b of the inclusion bodies was solubilized in SDS, extracted in the butanol phase and subsequently precipitated with acid. Butanol extraction offered twice the advantages to achieve purification with greater advantage in one step, and eliminate most of the free SDS from the preparation. The acid precipitated protein was subsequently suspended again in Zwittergent® and solubilized by temporary pH shock, from pH 12.0 to pH 8.0, carefully avoiding the process of amino-terminal deamination. IFN-beta-1 b was subsequently subjected to a critical renaturation step, and two ion exchange chromatographies to achieve maximum purity. Alternatively, the protein I FN-beta-1 b can be obtained commercially from Berlex Laboratories. EXAMPLES 2-3 PREPARATION OF PEG2-40k-I FN and PEG-UA-40k-I FN In these examples, PEG2-40k-IFN and alanine-activated NHS PEG2-40k-beta obtained from Nektar Therapeutics, Huntsville, AL, and Enzon Pharmaceuticals, Inc., respectively, were each incubated separately with the I FN-beta of Example 1. With rapid agitation, each amine-activated PEG powder was added separately at 0.3-0.8 mg / mL IFN-beta (>; 95% purity) in ~ 100 mL of 50-100 mM sodium phosphate, pH 7.8, 2 mM EDTA, and 0.05% Zwittergent® at 0.5-1.0 g / min. Alternatively, the PEG powder was previously dissolved in a tenth volume of solution I FN-beta, in 1 mM HCl and the PEG solution was added to the IFN-beta solution. The molar ratio of PEG: I FN reaction was 5-10: 1. After 60 minutes of reaction at a temperature of 25 ° C, each reaction was quenched by lowering the pH to 6.5 with 2 N HAc. The conjugation production of mono PEG-IFN was 40 to 60%, as analyzed by RP-HPLC.

The reaction mixture was diluted with 0.03% Zwittergent® in H2O for a conductivity of 5.8 mS / cm. A cation exchange resin, such as SP FF resin (Amersham Biosciences, NJ), was packed on a Waters AP-2 column for a bed height of 6 cm, ID 2 cm, CV 1 8.85 ml and equilibrated with 10 mM sodium phosphate, pH 6.5, 20 mM NaCl, 0.05% Zwittergent® 3-14. A sample of the reaction mixture was loaded into the column at 50 cm / hr (~ 4 mg of proteins were loaded per mL of resin), washed with 1 -1.5 column volume (CV) of the equilibrium regulator from column to baseline, and subsequently with 5 CV of 10 mM sodium phosphate, pH 6.5, 60 mM NaCl to eliminate high level MW conjugates. The product was extracted with solvents with 10 mM sodium phosphate, pH 6.5, 200 mM NaCl. Zwittergent® was eliminated in exhaustion solution by diafiltration using the Millipore Labscale TFF system (two 1K regenerated cellulose membrane cartridges "Pellicon XL cartridges", PLCTK 1 0 50 cm2, cat # PXC030C50, Lot # C3SN75289-023, LFL Tygon with 6 mm (1/4") OD, 3 mm (1/8") ID pipe (Masterflex 06429-1 6, mfg by Saint-Gobain) The system settings were pP = 4 psi, the power supply the pump was set to "1" with stirring speed at "2" to measure = 1 8 psi, retention = 14 psi The product that was extracted with solvents from an HS (Applied Biosystems) or SP (Amersham) column it was immediately diluted with 1 0 folds of diafiltration regulator (5 mM HAc, pH 3.7) and then concentrated ten times in the diafiltration system The process consumed 50 folds of the sample volume of the diafiltration regulator for a complete elimination of Zwittergent ® The formulation was subsequently carried out in the same system. Purity was confirmed by RP-H PLC chromatography. The parameters of the RPHPLC analysis were as indicated below. Column: Jupiter C5, 5μ, 300A, 4.6x150mm (Phenomenex, CA) Column temperature: 45 ° C Auto-sample thermostat: 4 ° C Mobile phase A: 0.1% Trifluoroacetic acid (TFA) and 1 0% of 1,4-Dioxane in Water Mobile Phase B: 0.1% Trifluoroacetic Acid (TFA) and 1.0% of 1,4-Dioxane in methanol; Flow Range: 1.0 mL / min The RP-HPLC results are shown in Figure 4, confirming that the purity of the product was > 95% mono PEG-I FN-beta 1 b pure. EXAMPLE 4 di PEG-20k-I FN The same PEGylation conditions employed in Examples 2 and 3 as the above were employed, except that the molar ratio of reaction was about 1: 20. After 60 minutes of reaction with PEG-20k-SPA, obtained from Nektar Therapeutics, di PEG-20k-I FN was purified through a size exclusion column, followed by a cation exchange column. EXAMPLE 5 METHODS FOR DETECTING AGGREGATION Samples were exchanged with regulator to the regulators described in the table below, using Centricon YM-30 (Millipore Corp., Bedford, MA). To accelerate the study, the samples were placed at a temperature of 37 ° C and under N2 for 24 hours. The stability was monitored in SEC-HPLC. Aggregation of the sample particles was determined by HPLC size exclusion chromatography (Superdex 200, HR, Amersham Biosciences, Piscataway, NJ), using 0.1 M sodium phosphate, regulator system with pH 6.8. RP-HPLC was used to detect degradation. SDS-PAGE without reduction and antiviral activities and antiproliferation were also used. The aggregation in the present invention was defined as a physical linkage of one or more protein monomers to form dimers, trimers, tetramers or multimers, which may or may not be precipitated from the solution in the regulators of the formulation, and conditions were reviewed. The soluble aggregate was converted to monomer in SDS gel without reduction, and will be inverted to monomer at the time of dilution. Liquid Formulation Example 5A. A lower pH is preferred for the formulation regulator. Organic and inorganic regulators were tested, with a pH that fluctuates from 3.0 to 1 1 .0. Glycine-HCl, pH 3.0, acetic acid, pH 3.7, sodium acetate, pH 4.5, sodium succinate, pH 4.4, sodium aspartate, pH 5.4, sodium citrate, pH 3-6 and sodium phosphate, pH were used. 6.0-7.4, as basic regulators to review the effects of excipients. In the presence of 3 mM Hac, pH 3.7, the conjugate was stable at a temperature of 37 ° C for at least 17 days. PH effect of the Regulator in the Aggregation * * The percentage of aggregation was analyzed by SEC-HPLC. ** Concentration As summarized in the above table, the preferred regulators are acetate (acid or free salt), citrate (acid or free salt), and glycine-HPI. Citrates have a dual role as chelating agents. The preferred pH is acid, more specifically between pH 3.0 and 4.0. The preferred concentrations of the regulators at pH 3.0-4.0 are below 10 mM. The citrate regulators in > 50 mM, will result in excessive pain in the subcutaneous injection and toxic effects due to the chelation of calcium in the blood.

EX EMPLOE 5B. Carbohydrate Excipients Nonionic tonicity modifying agents were reviewed as volume generating agents, to stabilize the conjugate and to make the compositions isotonic with body fluids. As classified in textbooks, monosaccharides include glucose, ribose, galactose, D-mannose, sorbose, fructose, xylulose and the like; the disaccharides are sucrose, maltose, lactose, trehalose and the like, and the polysaccharides comprise raffinose, maltodextrins, dextrans and the like. Alditoles contain glycerol, sorbitol, mannitol, xylitol and the like. Preferred nonionic agents are sucrose, trehalose, mannitol and glycerol or a combination thereof. The most preferred nonionic volume generating agents are mannitol and sucrose, or a combination thereof. Preferred compositions of the tonicity enhancing agents were 4-6% mannitol, 8-1 0% sucrose, or 8-1 0 trehalose. The most preferred compositions were 5% mannitol or 9% sucrose or trehalose. It was observed that negatively charged polysaccharides, such as heparin and chondroitin sulfate at 0.5 mg / ml at 20 mg / ml did not help to prevent aggregation of the conjugate at neutral pH. Example 5C. A lower ionic strength with a lower pH is preferred, and at the same time a higher ionic strength with a high pH is preferred. The effects of increasing the ionic resistance with reagents, such as NaCl, KCl, CaCl2 facilitated the aggregation of the conjugates at an acidic pH. In particular, these salts at a concentration of 140 mM, pH 3.7, facilitated the aggregation. At a pH of 5.5 to 7.5, the highest ionic strength was preferred over the lowest ionic strength to prevent the protein from going through aggregation. For example, 100 mM sodium phosphate, at a pH of 7.4, is better than its 10 mM concentration to prevent aggregation. The ionic strength of a solution is expressed as half the sum of CiZi2, where C is the concentration, Z is the charge and i represents the ion. A low ionic strength is preferred in low level pH regulators, while a high ionic strength in high level pH regulators is preferred. The preferred ionic strength in regulators with a pH of 3.0-4.0 is less than 10 mM, and the preferred ionic strength in regulators with a pH of 5.5-7.5 is 100-150 mM. Example 5D. Effect of Surfactants Nonionic surfactants include esters of polyoxyethylene sorbitol, such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20) and polyethylene glycol. The Zwitterionic surfactant, such as Zwittergent® was used to solubilize the unmodified protein. Preferred nonionic surfactants are polysorbate 80, polysorbate 20 and polyethylene glycol. Polysorbate 20 from Sigma prevented the aggregation of protein, while Polisorbate 20 from Calbiochem and J. T. Baker did not.

Example 5E. Low storage temperature The stability of the protein at different temperatures in various regulators, pH and excipients, was investigated. The stability of the protein decreased with elevated temperatures: -20 ° C, 40C > 25 ° C > 37 ° C. The temperature of 37 ° C, was used to accelerate the stability of the study. The preferred temperatures are -20 ° C and 2-8 ° C. At a temperature of 2-8 ° C, the conjugates were stable even in the presence of unfavorable components, such as high salt content (140 mM NaCl) or high pH (pH 7.4) for at least a few weeks. In regulators with a low pH (3.0-4.0) and low ionic strength, ten freeze-thaw cycles at temperature from -80 ° C to + 20 ° C, originated approximately 2% aggregation. Each freeze-thaw cycle at a temperature of -80 ° C to + 37 ° C caused approximately 3% freezing). In regulators with a pH of 5.0-6.5, for example, 10 mM sodium acetate, pH 5.0, 150 mM NaCl, or 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, in the presence or absence of Polysorbate 80, Five freeze-thaw cycles at a temperature of -80 ° C or -20 ° C to + 20 ° C, did not cause any aggregation or loss of antiviral activity. Example 5F. Protein Concentration The concentrations of PEG-IFN-beta-1 b between 0.125-4 mg / ml were reviewed. The samples were incubated in 5% mannitol, 3 mM HAc (pH 3.7), at a temperature of 37 ° C during the storage stability testing period. The integrity of the conjugate was monitored by SEC-H PLC. The decrease in protein concentration decreased the aggregation of the same. Preferred protein concentrations are between 3.0 mg / ml to 0.05 mg / ml.

Storage Results of Different Concentrations Example 5G. Neutralization of pH of solution for administration Since the acid solution could cause skin irritation, it was observed that the acidity can be neutralized by adding a solution or powder of sodium phosphate before administration. For example, when adding 1/10 volume of 1 x PBS or powder with the same components to 1% mannitol, 3 mM HAc, pH 3.7, 0.30 mg / ml PEG-protein, the pH increased to 6.5. The sample after neutralization should be administered at 2 hours at a temperature of 25 ° C or 20 hours at a temperature of 4 ° C. The effects of said pH neutralization on the aggregation of the tested PEG-I FN-beta 1 b, was minimal, as summarized in the following table. Neutralization effect in Aggregation * Aggregation Percentage was analyzed by SEC HPLC Example 5H. Antiviral Activity The antiviral activity of PEG2-40k-I FN-beta-1 b in 3 mM acetic acid, 5% mannitol, 0.3 mg / mL and various temperatures was reviewed with respect to A549 cells / EMCV virus. The antiviral activity was expressed as the percentage of native IFN-beta-1 b activity in side-by-side assays. The data in the table shows that the conjugate was stable for at least eight months, when it was stored at a temperature of 4 ° C.

Stability and Antiviral Activity of PEG-2-40k-lFN-beta-1b EXAMPLE 6 DELIVERY Example 6a: Addition of Mannitol in Freeze Drying Regulator This example confirms that the inclusion of mannitol in the lyophilization regulator reduced aggregation and allowed the antiviral activity retained after reconstitution. The ratio of PEG2-40k-I FN-beta-1 b to mannitol was 0.5-2.5% by weight. A concentration of 1% mannitol was preferred.

* The protein concentration was 0.3-0.4 mg / ml and pH 3.7 and the lyophilization regulators contained 3 mM HAc, pH 3.7 and mannitol as indicated. The reconstitution regulator was 3 mM Hac, pH 3.7.

Example 6b: Addition of Polysorbate to Lyophilization Regulators The example confirms that the addition of polysorbate 80 to the lyophilization regulators allowed the retained antiviral activity of PEG-I FN-beta 1 b tested after reconstitution, as summarized in next table.

Addition of Polysorbate 80 in Lyophilization Regulator * The lyophilization regulator contained 5% mannitol, 3 mM, and polysorbate 80 at the indicated concentration. The reconstitution buffer was 10 mM sodium phosphate, pH 7.4. ** Vero cell assay Example 6c: Effect of Regulator of Reconstitution in aggregation of PEG-protein This example compares the efficacy of three different reconstitution regulators at a pH of 7.4 (10 mM sodium phosphate), pH . 0 (1.0 mM sodium acetate), and 3.7 (3 mM acetic acid) for the incidence of aggregation in the PEG-I FN-beta 1 b tested after lyophilization and reconstitution. The lower the pH of the reconstitution regulators, the lower the amount of aggregation. The preferred lyophilization regulator contained 0.1 -2 mg / ml of the tested PEG-I FN-beta 1 b, 0.1 -5% mannitol, 3 mM Hac, pH 3.7 and 0.02-0.5% polysorbate 80 of J.T.

Baker. The lyophilized powder was reconstituted in a reconstitution buffer of 1.0 mM sodium acetate or sodium phosphate, pH . 0-7.4, 0-140 mM NaCl. As an alternative, the reconstitution regulator was 3 mM Hac, pH 3.7, to make 0.1 -2 mg / ml PEG-protein. Subsequently, 1.0 mM sodium phosphate pH 7.4 was added to neutralize the pH before administration. The effects of these two regulators on the aggregation of PEG-I FN-beta 1 b are summarized in the following table.

Reconstruction Regulator Effect * * The percentage of aggregation was analyzed by SEC HPLC CONCLUSION On the basis of the foregoing, the characteristics of the polymer conjugate I FN-beta 1 b of the present invention, in solution, they can be summarized. The preferred regulators (at a temperature of -20 ° C, -80 ° C or + 40 ° C), are composed of glycine-HCl, citrate, acetate or aspartate with a pH between 3.0 and 5.0 and the concentration between 5-1 0 mM. The ionic resistance of the regulator of the regulators described above was preferably less than 10 mM. Also preferred are the glycine-HCI, citrate, acetate, aspartate, phosphate and carbonate regulators, with a pH ranging from 3 to 8. Preferably, the acid regulators are neutralized with sodium phosphate before administration. Preferred carbohydrate excipients include, mannitol, sorbitol, sucrose, trehalose and glycerol, and / or a combination thereof. When the regulator is used in the form of a lyophilization regulator, the preferred carbohydrate excipients include mannitol, sucrose or trehalose or a combination thereof, in a concentration ranging from 0.1 to 5% (w / v). Preferred surfactants employed as excipients include polysorbate 80, polysorbate 20, and / or polyethylene glycol. When the regulator is used as a lyophilization regulator, the preferred surfactant excipients include polysorbate 80 or polysorbate 20 at a concentration of 0.002-0.5% (w / v). The preferred reconstitution buffer was sodium acetate or sodium phosphate, with pH 5.0-7.4, plus NaCl added until the isotonicity was achieved. Other preferred reconstitution regulators were glycine-HCl, citrate, acetate or aspartate prepared with a pH ranging from 3.0 to 4.0, followed by neutralization with sodium acetate or sodium phosphate to a final pH ranging from 5.0 to 7.4 for the administration . EXAMPLE 7 IMMUNOGENICITY AND STABILITY IN VITRO Experimental design: Sprague-Dawley rats (Harian) weighing from 1 50 to 300 g (three in a group) were administered intramuscularly or subcutaneously, with conjugates I FN- beta-1 b or PEG-I FN-beta-1 b native at 0.1 mg / kg, once a week for 3 to 6 weeks. Plasma samples were collected 7 days after the previous injection, and just before the next injection.

Assay design: The antibodies produced against the conjugates IFN-beta-1 b or PEG-beta-1 b, were analyzed by direct ELISA, when the capture reagent was I FN-beta-1 b and the detection antibody was IgG from rabbit against rat conjugated with horseradish peroxidase. The results are described in the table below. Analysis of Anti-hIFN-beta-1 b antibodies by ELISA (μg / ml) * * Mouse hlFN-beta monoclonal antibody (R & D, # 21405-1, clone # MMHB-3, lgG 1, kappa), was used as a standard. See also figure 1. CONCLUSIONS From the foregoing, it was concluded that the polymer conjugate lFN-beta-1 b of the present invention provides a number of advantages, including: • immunogenicity of the protein largely reduced by PEGylation. • the immunogenicity (IgG titrators) of IFN-beta-1 b was reduced by 94 to 98% after PEGylation with PEG-40k mono and PEG-20k di. • the rat immune system was more tolerant to the PEG protein than the native protein, as confirmed by the determination that there was no significant increase in antibodies from the first to the sixth dose. • the antibodies were neutralizing antibodies, when they were analyzed through antiviral bioassays. • there was no increased production of antibodies after 4 doses with IM administration. • PEG-IFN-beta compounds were found to be more resistant to proteases in mouse liver and liver extracts at the time of PEGylation. The half-life of IFN-beta-1b increased sixfold in kidney and liver extracts after PEGylation. The stability was analyzed by ELISA. • it was further discovered that PEG-IFN-beta compounds were more resistant to oxidation by hydrogen peroxidase after PEGylation.

EXAMPLE 8 IMPROVED PHARMACOKINETIC PROFILE Pharmacokinetic Parameters in Rats * By Vero cell assay. See also figure 2. The results of the pharmacokinetic studies can be summarized as indicated below. • The AUC of I FN-beta-1 b was improved more than 90 times by SC or IM administration and the clearance range was prolonged by more than 80 times after monoPEGylation with PEG-40k. • The bioavailability of PEGylated IFN-beta-1 b was better when administered by route I M than when administered by SC route, both in rats and in mice.

• The bioavailability of PEGylated IFN-beta-1 b was better than native I FN-beta.

Bioavailability of Conjugate IFN-beta-1 b and PEG-IFN-beta-1 b in Rats and Mice * Average numbers of Vero cell assays and antivirals A549 (EMC). EXAMPLE 9 PHARMACEUTICAL PROFILES IN MONKEYS This example provides the following information. The serum kinetics of EZ-2046 PEG-I FN-beta in Cynomolgus monkeys after administration of the EZ-2046 polymer conjugate. EZ-2046 is a conjugate linked to recombinant IFN-beta-1β amides with a single branched 40 kDa PEG. The effect of different routes of administration on pharmacokinetics and pharmacodynamics. The bioavailability EZ-2046 after SC or IM administration; The relationship between the EZ-2046 administration and the pharmacodynamic marker neopterin. MATERIALS AND METHODS Female and male Cynomolgus monkeys were administered a single dose by intravenous ("IV"), subcutaneous ("SC"), or intramuscular ("IM") administration with EZ-2046 PEG-I FN-beta, in a dose level of 15 μg / kg or 480,000 μL / kg IFN-beta of IFN-beta equivalents (specific activity of I FN-beta 32MI U / mg). The in vitro antiviral activity of the conjugate indicated that 32% to 34% of the native IFN-beta activity was retained, therefore, the conjugate dose adjusted to the activity was approximately 160,000 IU / kg. Since these methods can not differentiate free IFN-beta from PEGylated IFN-beta, the equivalence of IFN-beta, including both forms of the drug, is actually measured. For simplicity, IFN-beta and the equivalent of IFN-beta are interchangeable in this report. The pharmacokinetic parameters for EZ-2046 were evaluated by ELISA or analysis of serum bioactivity. The pharmacodynamic effects were evaluated using an ELISA assay to check plasma neopterin levels as a marker for biological activity EZ-2046 in vivo. Neopterin is a pteridine derivative derived from guanosine triphosphate and is produced by lymphocytes and / or macrophages in response to stimulation of the immune system. It is a well-known biomarker for the bioactivity of interferon in vivo in primates and humans. The maximum plasma concentrations of EZ-2046 ("Cmax"), the plasma terminal elimination half-life ("t? 2"), the area under the serum-time concentration curve over a period of 0 to infinity ("AUC0."), and bioavailability were determined using either first-order pharmacokinetic models of one compartment (SC, IM) or two compartments (IV). Neopterin E0 (serum baseline level), T? Ag (lag time after administration), Tmax (time to reach maximum concentration), Emax (net maximum effect), K10_HL (the range of neopterin that leaves the half-life of the serum compartment), and AUC (area under the serum concentration versus time curve), using a first-order elimination model, lag, first-order uptake from a single compartment. Collection of Samples Blood samples were collected from three (3) animals / sex / group at 10 and 30 minutes and at 1, 3, 6, 24, 72, 120, 168, and 240 hours after the injection. Samples were collected in tubes and allowed to clot for 20 minutes at room temperature before being placed on ice in a vertical position. After separation of the serum, the serum was distributed in five tubes and frozen at a temperature of about -70 ° C (or less) 2 hours after collection until analysis. Animals were bled before dosing for baseline levels. Serum Determination Two different methods were used to determine serum EZ-2046. In method A, the serum EZ-2046 concentration was determined by an ELISA assay. In method B, the bioactivity of serum EZ-2046 was determined by means of an antiviral assay using A549 cells stimulated with virus from Encephalomyocarditis (murine). * Quantitative ELISA of IFN-beta Serum. Serum IFN-beta concentrations were determined using a commercially available one-step sandwich ELISA assay kit (Immuno-Biological Laboratories, Cat.

# MG53221). The test was carried out as described by the manufacturer. 1 . Equipment. (a) Polypropylene microtiter tubes. Catalog No. 29442-608, VWR, S. Plainfield, NJ or equivalent source. (b) Precision repeat pipettes were used to deliver 100 μl and 1,000 μl, 100 μl fixed-volume pipette and 100 μl adjustable pipette. Eppendorf or equivalent, VWR, S. Plainfield, NJ 07080 or equivalent. (c) Absorbent paper. (d) Molecular Device Versamex plate reader, Sunnyvale, CA. (e) Automatic plate agitator: Lab-Line Instruments, Inc., I L, USA. (f) Bio-Tek Píate washer, ELx405, Winooski, VT. 2. Materials (a) Human interferon-beta ELISA kit: Immuno-biological Laboratories, Cat. No. MG 53221, Lot # GL40502, Minneapolis, MN. 3. Preparation of Regulators / Solutions, (a) Washing solution. (i) Concentrate of 50 ml wash solution (Bottle 4) diluted to 450 ml with distilled water. It was used at room temperature before use. It is stored at 4 ° C. (a) Dilution regulator. (i) Ready to use "Bottle 5". Use and storage at a temperature of 4 ° C. (b) Antibody solution marked with enzymes. (i) Dissolved vial (# 2) of mouse monoclonal antibody labeled with H RP for I FN-beta Hu), Fab 'in 6 ml of dilution buffer. Using and stored at a temperature of 4 ° C. (a) Substrate solution. (i) Before use, 1 0 ml "Substrate A" (bottle 6) was combined with 0.5 ml of "Substrate B" (bottle 7). It was used immediately at room temperature, (a) Stop reagent. • Stop solution (Bottle 8) ready to be used. It was used at room temperature. 4. Caliber standards. (a) Standard of I FN-beta lyophilized Hu reconstituted with 1 ml of ice-cold water with gentle agitation until a working solution with a concentration (1 U / ml) described on the vial label is obtained. The working solution was diluted with an ice cooled dilution regulator to elaborate standard at 200, 1 00, 50, 20, 1 0, 5, and 2.5 l U / ml. The dilution regulator was used as a standard solution for 1 U / ml. The dilutions were made on ice with gentle mixing. 5. Immunoassay procedure. (a) The antibody was left on a covered micro-tank test plate until it reached room temperature before use. (b) 400 μL of wash buffer per tank was added to the assay plate. Completely eliminated the regulator by aspiration. (c) 50 μl was added per deposit of antibody labeled with enzymes. (d) 100 μl of standards or test samples were added per deposit in duplicate using a grid map. (e) The plate was sealed and incubated 2 hours at room temperature (20-30 ° C) with orbital shaking (350-450 rpm). (f) The solutions were removed and the plate was washed four times with incubation for 1 minute between wash intervals. (g) 1 00 μl of substrate solution was added per tank. (h) The plate was sealed and incubated 30 minutes at room temperature with shaking. (i) 1 00 μl of stop solution was added, (j) The plate was read at 450 nm with 570 nm as reference. (k) The serum concentrations were determined from the calibration curve generated using a 4-parameter curve fitting method. B. Bioactivity assay for I FN-beta in Cynomolgus monkey leather. 1 . Equipment. (a) 96-well polyethylene tissue culture plate: Catalog No. 29442-054, VWR, S. Plainfield, NJ, 07080 or equivalent source. (b) Polypropylene microtiter tubes, Catalog No. 29442-608, VWR, S. Plainfield, NJ or equivalent source. (c) 250 μl and adjustable multichannel pipettes of 1,000 μl. Finnpipette or equivalent, VWR, S. Plainfield, NJ 07080. (d) Sterile paper towel. (e) Incubator, CO2, humidified form, USA. (f) Versamax molecular apparatus plate reader, Sunnyvale, CA 2. Materials. (a) IFN-beta calibration standards, Catalog No. 1-4151, Sigma, Lot # 082K16781, storage temperature from 2 to 8 ° C. (b) Ham's Medium F12K, Catalog # 30-2004, ATCC, Lot # 3000144, storage temperature from 2 to 8 ° C. (c) MEM, Catalog # 20-2003, Lot # 3000302. (d) Fetal bovine serum, Catalog No. SH30071, Hyclone, Logan, UT. (e) Penicillin and streptomycin, Catalog # 1 5140-1 22, Gibco, USA. (f) Phosphate regulated saline solution (PBS), Catalog # 1 7-51 6F, Lot # 01 1 04281, Bio Whittaker, USA. (g) A549 cells, Catalog # CCL-1 85, ATCC. (h) Encephalomyocarditis murine virus (EMCV), Enzon Pharmaceutical Lot # V6, produced in Vero cells (CCL-81, ATCC) from EMCV, Catalog number VR-1 29B, ATCC. (i) Solution of 3-84,5-dimethyloxy-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide ("MTT"), Catalog # G41 02, Lot 1 8264601, Promega, USA . (j) Solubilization / detention solution, Catalog # G41 01, Lot # 1 78861, Promega, USA. 3. Bioassay procedure. The bioactivity of serum EZ-2046 was determined by examining its antiviral activity, for example, the amount of protection provided by EZ-2046 to A549 cells stimulated with Encephalomyocarditis virus (EMCV) infection. Serial dilutions of serum samples or standard I FN-ß were added in triplicate to deposits in a 96-well plate. A549 cells were added (104 / deposit) in Ham's-F12K containing 1 0% fetal bovine serum (FBS), to plaque deposits and incubated overnight at a temperature of 37 ° C in 5% CO2. The growth medium was removed and 50 μl / EMCV deposit (1.1 x 1 05 PFU / ml) was added to the plate and incubated for 2 hours at a temperature of 37 ° C under 5% CO2. The virus inoculum was removed and the cells were fed Ham's-F12K containing 5% FBS. The plates were incubated for 40 hours at a temperature of 37 ° C in 5% CO2. 15 microliters of MTT solution (Promega Corporation) was added to each plate tank, and the plate was incubated for 4 hours at a temperature of 37 ° C with 5% CO2. The deposits were solubilized with 100 μl of solubilization / arrest solution (Promega) overnight at room temperature in the dark. The optical density of the deposits was determined at 570 nm with a reference of 630 nm and the serum concentrations of the samples were determined from the standard calibration curve generated using a 4-parameter adjustment. Determination of Serum Neopterin through Immunoassays in the Form of a Biomarker for activity I FN-beta. Serum neopterin concentrations were determined using a commercially available competitive ELISA assay kit (Immuno-Biological Laboratories, Catalog # RE59321). The test was carried out as described by the manufacturer as indicated below. 1. Team a. Polypropylene microtiter tubes. Catalog No. 29442-608, VWR, S. Plainfieid, NJ. or equivalent source. b. Precision repeat pipettes to deliver 100 μl and 1,000 μl, 100 μl fixed volume pipette and 100 μl adjustable pipette. Eppendorf or equivalent, VWR, S. Plainfield, NJ 07080 or equivalent. c. Absorbent paper. d. Versamax molecular plate reader, Sunnyvale, CA. and. Automatic plate agitator: Lab-Line Instruments, Inc., IL, USA. F. Bio-Tek plate washer, ELx405, Winooski, VT. 2. Materials. Neopterin ELISA Kit HU: IBL Immuno-Biological Laboratories, Catalog No. RE59321, Lot # ENO187, Minneapolis, MN. 3. Preparation of Regulators / Solutions. to. Wash solution. 50 ml of wash solution concentrate was diluted to 450 ml with distilled water. It was left at room temperature before use. It was stored at a temperature of 4 ° C for up to a month. b. Test regulator. Ready to use. It was used at room temperature and stored at a temperature of 4 ° C. c. Antibody solution labeled with enzymes. 25 μl of antibody concentrate is added in 5 ml of assay buffer (1: 201). It is used at room temperature and stored at a temperature of 4 ° C for 24 hours protected from light. d. Substrate solution. ' Before use, 300 μl of TMB substrate is added to 9 ml of substrate regulator. It is used immediately at room temperature. Store at a temperature of 4 ° C for up to 48 hours. and. Detention reagent. The detention solution is ready to be used. Use at room temperature. 4. Calibration Standards. to. The calibration standards that are ready to be used contain neopterin in phosphate buffer with stabilizers. The assay regulator was used as the zero standard. b. It is provided ready to use control serum for quality control. 5. Immunoassay procedure. c. Microdeposition test plates coated with antibodies are left at room temperature before use. d. 10 μl of each sample of standard, control or monkey serum is added in duplicate to the deposits of the microassay plate using a grid map. and. 100 μl of Enzyme Conjugate was added per tank. F. 50 μl of neopterin antibody was added. g. The plate is sealed with black film and incubated in the dark for 90 minutes at room temperature (20-30 ° C) with orbital shaking (350-450 rpm). h. The solutions are removed and the plate is washed four times with 1 minute of incubation between wash intervals. i. 200 μl of substrate solution is added per tank. j. The plate is sealed and incubated 10 minutes at room temperature with shaking. k. 100 μl of stop solution is added. I. The plate is read at 450 nm with reference 650 nm. m. Serum concentrations were determined from the calibration curve generated using a 4-parameter curve fitting method. Software Analysis of descriptive statistics (average and standard deviation or SD) and of compartment and non-compartment statistics were carried out using WinNonlin Professional version 4.1 (Pharsight Corporation). Calculations of ELISA serum concentrations and bioassays were performed using Microsoft® Excel 2002. Graphic presentations were made using SPSS® SigmaPlot version 8.0. The comparative statistics were carried out using SigmaStat version 3.01.

RESULTS Concentration Profile-Time EZ2046 Tabulated Determined by ELISA Average pharmacokinetic parameters for EZ-2046 as determined by ELISA ELISA data Conclusions * The average EZ-2046 serum concentration values are similar in males and females, resulting in similar EZ-2046 concentration-time profiles. The pharmacokinetic values EZ-2046 for monkeys receiving subcutaneous and intramuscular administrations were similar to each other. The monkey that had intravenous administration had a Cma? 6 to 8 times higher, a slower terminal elimination of 1.4 to 2.5 times and an AUC greater than 1.4 to 1.6 times compared to administration SC to I M. The bioavailability of EZ-2046, after SC administration and IM was approximately 65%. * It was measurable in serum 168 hours after administration. B. Concentration-time profile EZ-2046 determined by bioactivity assay The ELISA analysis provides a protein concentration profile of EZ-2046 kinetics. It was also used to determine the kinetics of EZ-2046 bioactive. These data are summarized in the form of the ratio between the estimates of the pharmacokinetic ELISA parameter and the pharmacokinetic parameter of the bioactivity assay. Proportion of EIA-2046 ELISA Pharmacokinetic Estimates with Respect to Bioactivity Test Estimates EZ-2046 Conclusions: The EZ-2046 bioactivity assay showed a decrease of 8.1 to 13.1 times in the average Cmax EZ-2046 activity, compared to the Cmax concentration determined by ELISA, after intramuscular and subcutaneous administration of EZ-2046 and a decrease of 2.8 times after intravenous administration. All administration routes showed similar bioactivity decreases in the terminal elimination half-life (t -? / 2) with a fluctuation of 2.9 to 3.2 times compared to the serum elimination half-life determined by ELISA The AUC of bioactivity EZ-2046 likewise, decreased 14.7, 20.1 and 26.1 times after IV, SC, and IM administration compared to ELISA values. EZ-2046 administered subcutaneously and intramuscularly, showed a decrease of 1.4 to 1.8 times in bioavailability by the IFN-beta bioactivity assay compared to estimated ELISA. C. EZ-2046 Pharmacodynamic Effect Determined by Neopterin Synthesis The pharmacological effect of EZ-2046 administration was determined by checking the serum neopterin response using the methods described supra.

Pharmacodynamic parameters Average for Neopterin E0: The average serum baseline for neopterin was 1.4 ng / ml. Conclusions: Regardless of the route of administration, pharmacodynamic parameters for neopterin synthesis were similar. Neopterin synthesis occurred after 4 to 7 hours after the administration of EZ-2046 (T? Ag). • The maximum effect time (Tmax) fluctuates from 28 to 34 hours. The maximum effect of I FN-beta-1 b (Emax) ranges from 2.0 to 3.2 ng / ml above the baseline neopterin levels of 1.4 ng / ml. The effect of serum neopterin decreased with a half-life elimination ranging from 43.2 to 60.7 hours. The neopterin exposure above the previous background levels fluctuates from 236 to 332 hng / ml. Additional Conclusions: After administration of the PEG-IFN-β EZ2046 conjugate to Cynomolgus monkeys, the pharmacokinetic and pharmacodynamic parameters for IFN-β and neopterin were similar between genders. Subcutaneous and intramuscular administration of EZ-2046 showed a Cmax, terminal elimination half-life (t1 / 2), and comparable bioavailability. The elimination half-life of serum EZ-2046 SC and I M was approximately two times lower compared to intravenous administration. The bioactivity of EZ-2046 showed a three-fold decrease in Cmax after administration compared to ELISA values, most likely due to the lower specific activity of the pegylated IFN-beta compared to the unconjugated drug. The neopterin response was similar regardless of the route of administration of EZ-2046. • Neopterin increased slowly twice above the baseline after the administration of EZ-2046, and the neopterin response slowly decreased and remained detectable one week after the EZ-2046 administration.

Claims (54)

1. R E I V I N D I C A C I O N S 1. A composition comprising: a) interferon conjugated to polyalkylene oxide polymer having a molecular weight of at least about 12 kDa; and optionally b) a surfactant; (c) an excipient, and (d) a regulator wherein the pH range of the solution is from about 3 to about 11. The composition according to claim 1, characterized in that the interferon is interferon-beta-1b . 3. The composition according to claim 1, characterized in that the surfactant is selected from the group consisting of sorbitol esters of polyoxyethylene and polyethylene glycol. 4. The composition according to claim 1, characterized in that the pH range is from about 2.5 to about 8.5. 5. The composition according to claim 1, characterized in that the pH range is from about 3.0 to about 5.0. 6. The composition according to claim 1, characterized in that the pH range is from about 3.0 to about 4.0. The composition according to claim 1, characterized in that the regulator is selected from the group consisting of glycine-HCl, acetic acid, sodium acetate, sodium aspartate, sodium citrate, sodium phosphate, and sodium succinate. The composition according to claim 1, characterized in that the regulator is selected from sodium acetate, sodium citrate, and glycine-HCl. 9. The composition according to claim 1, characterized in that the regulator has an ionic strength of about 1.0 mM. The composition according to claim 1, characterized in that the regulator is in a concentration of about 3 mM to about 10 mM. eleven . The composition according to claim 1, characterized in that the excipient is nonionic and is selected from the group consisting of monosaccharides, disaccharides and alditols. The composition according to claim 7, characterized in that the excipient is selected from the group consisting of glucose, ribose, galactose, D-mannose, sorbose, fructose, xylulose, sucrose, maltose, trehalose, raffinose, maltodextrins, dextrans, glycerol, sorbitol, mannitol, and xylitol. The composition according to claim 8, characterized in that the excipient is selected from the group consisting of sucrose, trehalose, mannitol, and glycerol or a combination thereof. The composition according to claim 9, characterized in that the excipient is selected from the group consisting of mannitol and sucrose or a combination thereof. 5. The composition according to claim 1, characterized in that the surfactant is non-ionic and is selected from the group consisting of polysorbate 80 polysorbate 20 and polyethylene glycol. 16. The composition according to claim 1, characterized in that the polyalkylene oxide polymer is linear or branched. 17. The composition according to claim 1, characterized in that the linear polyalkylene oxide polymer is of the formula: AO- (CH2CH2O) x- AO- (CH2CH2) x -CH2C (O) -O-, AO- ( CH2CH2O) x-CH2CH2N R7-, AO- (CH2CH2O) x-CH2CH2SH, -OC (O) CH2-O- (CH2CH2O) x -CH2C (O) -O-, -NR7CH2CH2-O- (CH2CH2O) x-CH2CH2N R7, -SHCH2CH2-O- (CH2CH2O) x -CH2CH2SH-, wherein: A is a cover group; R7 is selected from hydrogen, C? .6 alkyls, C3-? 2 branched alkyl, C3-? Cycloalkyls, Ci.e substituted alkyls, C3.8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C- | .4 alkynes , C3 .-] 2 branched alkenyl, C? _6 alkynyl, C3 .-? 2 branched alkynyl, C- | .6 heteroalkyls, C- | .6 hetero-substituted alkyls, C-? - 6 alkoxyalkyl, phenoxyalkyl and C? -6 heteroalkoxyls, and x is the degree of polymerization. The composition according to claim 5, characterized in that the cover group is selected from the group consisting of O H, CO2H, NH2, S H, and C-? 6 alkyl portions. 9. The composition according to claim 1, characterized in that the branched polyalkylene oxide polymer is selected from the group consisting of: -NH m-PEG-0 C N. \ (GH2) a (CH2) a N - (CH2) PC (0) - HC (ZCHzMO) -, < CH2) to iCH2) _ m-PEG-O-pt-PEG-0-C- or ll -NH (CH2). HC (ZCH2) "C (0) - wherein: (a) is an integer of from about 1 to about 5; Z is O, N R8, S, SO, or SO2; wherein R8 is H, C-, 8 alkyl, C-, 8-branched alkyl, C-i-s substituted alkyl, aryl or aralkyl; (x) is the degree of polymerization; (n) is 0 or 1; (p) is a positive integer; preferably from about 1 to about 6; m-PEG is CH3-O- (CH2CH2O) x-; and the ligand is interferon-beta-1 b. The composition according to claim 1, characterized in that the interferon-beta 1 b comprises the amino acid sequence of SEQ I D NO: 1. twenty-one . The composition according to claim 20, characterized in that the interferon-beta 1 b is conjugated to a polyalkylene oxide polymer selected from the group consisting of: AO- (CH 2 CH 2 O) x- AO- (CH 2 CH 20) x -CH 2 C (0 ) -, AO- (CH2CH2O) x -CH2CH2NR7-, AO- (CH2CH2O) x -CH2CH2SH, 22. The composition according to claim 21, characterized in that the molecular weight of the polyalkylene oxide polymer ranges from about 12 kDa to about 60 kDa. 23. The composition according to claim 21, characterized in that the molecular weight of the polyalkylene oxide polymer is about 30 kDa. 24. The composition according to claim 21, characterized in that the molecular weight of the polyalkylene oxide polymer is about 40 kDa. The composition according to claim 1, characterized in that the polyalkylene oxide polymer is conjugated to interferon-beta 1 b through a ligature selected from the group consisting of urethane, secondary amine, amide or thioether. 26. The composition according to claim 1, characterized in that interferon-beta 1 b is conjugated to a polyalkylene oxide polymer through the alpha-amino-terminal of interferon-beta 1 b. 27. The composition according to claim 1, characterized in that the interferon-beta 1 b is conjugated to a polyalkylene oxide polymer through an amino-epsilon group of a Lys of interferon-beta 1 b. 28. The composition according to claim 1, characterized in that the interferon conjugate is in a concentration from about 0.01 mg / ml to about 4 mg / ml. 29. The composition according to claim 28, characterized in that the interferon conjugate is in a concentration from about 0.05 mg / ml to about 3 mg / ml. 30. A composition comprising: a) from 0.05 to 3.0 mg / ml of interferon-beta 1 b conjugated to a polyalkylene oxide polymer and having a molecular weight of at least about 12 kDa, b) 1% -5% mannitol , and c) 3-10 mM acetic acid, where the pH is about 3.7. 31. A biologically active polymer-interferon conjugate composition according to claim 1, characterized in that at least about 65% of the antiviral activity is retained relative to the native interferon-beta 1 b, using the EMC / Vero antiviral bioassay. or EMC / A549. 32. A biologically active polymer-interferon conjugate composition according to claim 1, characterized in that at least about 20% of the antiviral activity is retained relative to the native interferon-beta 1 b, using the EMC / Vero or EMC antiviral bioassay. / A549. 33. A method for preparing the biologically active polymer-interferon conjugate composition according to claim 1, characterized in that it comprises reacting the interferon-beta 1 b with an activated polyalkylene oxide polymer having a molecular weight of less than about 30 kDa under conditions sufficient to cause conjugation of the activated polyalkylene oxide polymer for interferon-beta 1 b, purifying the resulting conjugate and again suspending the conjugate in a regulated solution having a pH range of from about 3.0 to about 8.0, wherein the solution optionally contains an excipient and a surfactant, and wherein the composition retains at least about 20% of the antiviral activity that is retained relative to native interferon-beta 1 b, using the antiviral bioassay EMC / Vero or EMC / A549. 34. The method according to claim 33, characterized in that the conditions are sufficient to cause conjugation of the activated polyalkylene oxide polymer to the amino-terminal of interferon-beta 1 b. 35. The method according to claim 33, characterized in that the conditions are sufficient to cause the conjugation of the alkylated polyethylene oxide polymer to an epsilon amino group of a Lys of interferon-beta 1 b. 36. The method according to claim 33, characterized in that the molecular weight of the activated polyalkylene oxide polymer ranges from about 30 kDa to about 40 kDa. 37. The method according to claim 33, characterized in that the molecular weight of the activated polyalkylene oxide polymer is about 30 kDa. 38. The method according to claim 33, characterized in that the molecular weight of the activated polyalkylene oxide polymer is about 40 kDa. 39. The method according to claim 33, characterized in that the molecular weight of the activated polyalkylene oxide polymer is an activated polyethylene glycol. 40. The method according to claim 39, characterized in that the molecular weight of the activated polyalkylene oxide polymer comprises a portion of the terminal reactive aldehyde. 41 The method according to claim 40, characterized in that the activated polyethylene glycol is selected from the group consisting of mPEG-CH2CH2CHO, mPEG2CH2CH2CHO, mPEG-CH2CH2CH2CH2CHO, and mPEG2-CH2CH2CH2CH2CHO. 42. The method according to claim 39, characterized in that the activated polyethylene glycol is selected from the group consisting of: m-PEG- -NH \ (CH,) B HC - '. { ZCH2) "CHO. (CH2) to m-PEG- wherein: (a) is an integer from about 1 to about 5; Z is O, N R8, S, SO, or SO2; wherein R8 is H, C .. 8 alkyl, branched alkyl, C-? 8 substituted alkyl, aryl or aralkyl; (x) is the degree of polymerization; (n) is 0 or 1; (p) is a positive integer, preferably from about 1 to about 6, and mPEG is CH3-O- (CH2CH2O) x-. 43. The method according to claim 33, characterized in that the activated polyethylene glycol comprises a terminal reactive portion selected from the group consisting of: 44. A method for administering a composition according to claim 1, characterized in that it comprises a first step to neutralize acidic regulators followed by administration of the composition to a patient in need of such administration. 45. The method according to claim 44, characterized in that the acid buffer is neutralized with sodium phosphate. 46. The method according to claim 44, characterized in that the composition is administered orally, intravenously, subcutaneously, or intramuscularly. 47. A method for treating a mammal having a disease or condition responsive to interferon-beta, wherein the method comprises administering an amount of the pharmaceutical composition according to claim 1 effective to treat the disease or condition. 48. A method for preparing a polyalkylene oxide-protein conjugate comprising the steps of: (a) solubilizing a protein of interest in a compatible aqueous solution in the presence of a protein solubilizing amount of a compatible detergent; (b) reacting the solubilized protein of interest with an activated polyalkylene oxide polymer, to produce a solution comprising a polyalkylene oxide-protein conjugate and the detergent; (c) adjusting the reactivated solution from step (b) to an effective pH to dissociate the detergent from the polyalkylene oxide-protein conjugate; (d) separating the dissociated detergent from the polyalkylene oxide-protein conjugate, and recovering the polyalkylene oxide-protein conjugate. 49. The method according to claim 48, characterized in that the pH is adjusted in step (c), to a range of from about pH 3 to about pH 4. 50. The method according to claim 48, characterized in that the activated polyalkylene oxide polymer is a polyethylene glycol polymer that ranges in size from about 12 kDa to about 60 kDa. 51 The method according to claim 48, characterized in that the detergent is selected from the group consisting of an ionic detergent, a non-ionic detergent, a zwitterionic detergent and combinations thereof. 52. The method according to claim 51, characterized in that the detergent is a zwitterionic detergent. 53. The method according to claim 48, characterized in that the protein is an interferon. 54. The method according to claim 53, characterized in that the protein is an lFN-beta. R E S U M N Biologically active interferon-beta Ib-polymer conjugate compositions are described. The polymer part is preferably a polyalkylene oxide polymer having a molecular weight of at least about 12 kDa. Methods to elaborate and use them are also described.