MXPA06006579A - Stabilized interferon liquid formulations - Google Patents

Abstract

Stabilized liquid pharmaceutical composition comprising an interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof, wherein said formulation is a solution that comprises a buffer, a cyclodextrin, an isotonicity agent and an anti-oxidant are described here. Preferably the interferon is interferon beta-1a and the cyclodextrin is HPBCD. These formulations are stable at room temperature, thus bringing the advantage of lower costs for formulation storage and increased safety for the patient with respect to possible"errors"during handling. As a matter of fact, having such formulations stable at room temperature reduces the risk of formation of degradation products potentially responsible for adverse events (e.g. immunogenicity).

Description

LIQUID FORMULATIONS OF 1NTERFERON STABILIZED FIELD OF THE INVENTION The invention relates generally to a liquid stabilized pharmaceutical composition comprising an interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof, wherein said formulation is a solution comprising a pH regulator , a cyclodextrin, an isotonicity agent and an antioxidant and use thereof.

BACKGROUND OF THE INVENTION Interferons are cytokines, that is, soluble proteins that transmit messages between cells and play an essential role in the immune system by helping to destroy microorganisms that cause infection and repair any resulting damage. The interferons are naturally secreted by infected cells and were first identified in 1957. Their name derives from the fact that they "interfere" with replication and viral production. Interferons have both antiviral and antiproliferative activity. Based on the biochemical and immunological properties, human interferons that occur naturally are grouped into three main classes: interferon-alpha (leukocytes), interferon-beta (fibroblasts) and interferon-gamma (immune). Alpha-interferon is currently approved in the United States and other countries for the treatment of hairy cell leukemia, venereal warts, Kaposi's sarcoma (a cancer that commonly affects patients suffering from acquired immunodeficiency syndrome (AIDS)), and non-A hepatitis, chronic non-B. In addition, interferons (IFNs) are glycoproteins produced by the body in response to a viral infection. They inhibit the multiplication of viruses in protected cells. Consisting of a lower molecular weight protein, the IFNs are remarkably non-specific in their action, ie, the IFN induced by a virus is effective against a broad spectrum of other viruses. However, they are species-specific, that is, the IFN produced by a species will only stimulate the antiviral activity in cells of the same species or a closely related species. The IFNs were the first group of cytokines to be exploited for their potential antitumor and antiviral activities. The three main IFNs are referred to as IFN-a, IFN-β and IFN-α. These main types of IFNs were initially classified according to their cells of origin (leukocyte, fibroblast or T cells). However, it became clear that several types could be produced by a cell. In this way, the leukocyte IFN is now called IFN-a, the fibroblast IFN is IFN-β and the IFN-T cell is IFN-α. There is a fourth type of IFN, lymphoblastoid IFN, produced in the "Namalwa" cell line (derived from Burkitt's lymphoma), which appears to produce a mixture of IFNs of both leukocytes as of fibroblasts. The interferon unit or international unit for interferon (U or IU, for international unit) has been reported as a measure of IFN activity defined as the amount needed to protect 50% of the cells against viral damage. The test that can be used to measure bioactivity is the inhibition test of cytopathic effect as described (Rubinstein, et al., 1981; Familletti, P. C, et al, 1981). In this antiviral test! for interferon approximately 1 unit / ml of interferon is the amount necessary to produce a cytopathic effect of 50%. The units are determined with respect to the international reference standard for Hu-IFN-beta provided by the National Institutes of Health (Pestka, S. 1986). Each IFN class contains several different types. IFN-ß and IFN-? they are each the product of a single gene. Proteins classified as IFNs-a are the most diverse group, containing approximately 15 types. There is a cluster of IFN-a genes on chromosome 9, which contains at least 23 members, of which 15 are active and transcribed. Mature IFNs-a are not glycosylated. The IFNs-a and IFN-β are all of the same length (165 or 166 amino acids) with similar biological activities. The IFNs-? they are 146 amino acids in length and resemble classes a and ß less closely. Only the IFNs-? they can activate macrophages or induce the maturation of killer T cells. These new types of therapeutic agents they can sometimes be called biological response modifiers (BRMs), because they have an effect on the body's response to the tumor, affecting recognition by immunomodulation. Human fibroblast interferon (IFN-β) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of approximately 20,000 Da induced by virus and double-stranded RNA. From the nucleotide sequence of the gene for fibroblast interferon, cloned by recombinant DNA technology (Derynk et al., 1980), the complete amino acid sequence of the protein was deduced. It is 166 amino acids in length. Shepard et al. (1981) describe a mutation in base 842 (Cys-Tyr at position 141) that abolishes its antiviral activity, and a variant clone with a nucleotide deletion 1119-1121. Mark et al. (1984) inserted an artificial mutation replacing the base 469 (T) with (A) causing an amino acid shift of Cys - »Ser at position 17. It was reported that the resulting IFN-β was active as the native IFN-β" "and stable during long-term storage (-70 ° C). Rebif® (Serono-recombinant human interferon-β), the most recent development in interferon therapy for multiple sclerosis (MS), is interferon (IFN) -beta-la, produced from mammalian cell lines. His name without international owner (INN) is "Interferon beta-1a". As with all pharmaceutical compounds based on protein, a major obstacle that must be overcome in the use of IFN-beta as a therapeutic agent, is the loss of pharmaceutical efficacy that can result in its instability in pharmaceutical formulations. Physical instabilities that threaten the activity and efficacy of the polypeptide in pharmaceutical formulations include denaturation and formation of soluble and insoluble aggregates, while chemical instabilities include hydrolysis, imide formation, oxidation, racemization and deamidation. It is known that some of these changes lead to the loss or reduction of the pharmaceutical activity of the protein of interest. In other cases, the precise effects of these changes are unknown, but it is still considered that the resulting degenerative products are pharmaceutically unacceptable due to the potential for undesirable side effects. Stabilization of polypeptides in pharmaceutical compositions remains an area in which trial and error play an important role (reviewed by Wang (1999) Int. J. Pharm. 185: 129-188; Wang and Hanson (1988) J. Parenteral Sci. Tech. 42: S3-S26). Excipients that are added to pharmaceutical formulations of polypeptide to increase their stability include pH regulators, sugars, surfactants, amino acids, polyethylene glycols and polymers, but the stabilizing effects of these chemical additives vary depending on the protein. Cyclodextrins are cyclic oligosaccharides. The most common cyclodextrins are alpha-cyclodextrin, which is composed of six rings of glucose residues; beta-cyclodextrin, which is composed of a ring of seven glucose residues; and gamma-cyclodextrin, which is composed of a ring of eight glucose units. The internal cavity of a cyclodextrin is lipophilic, while the exterior of the cyclodextrin is hydrophilic; that combination of properties has led to a widely disseminated study of cyclodextrins, particularly in connection with pharmaceutical compounds, and many inclusion complexes have been reported. Beta-cyclodextrin has been of special interest due to its cavity size, but its relatively low aqueous solubility (approximately p / v at 25 ° C) and consequent nephrotoxicity have limited its use in the pharmaceutical field. Attempts to modify the properties of natural cyclodextrins have resulted in the development of heptakis polymer (2,6-di-0-methyl) -beta-cyclodextrin, heptakis (2,3,6-tri-0-methyl) - beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, beta-cyclodextrin-epichlorohydrin and others. For a complete review of cyclodextrins and their use in pharmaceutical research, see Pitha et al, in Controlled Drug Deliver, ed. S. D. Bruck, Vol I, CRC Press, Boca Raton, Fia., Pp. 125-148 (1983). For a more recent overview, see Uekama et al, in CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1), 1-40 (1987); Uekama, in Topics in Pharmaceutical Sciences 1987, eds. D. Breimer and P. Speiser, Elsevier Science Publishers B.V. (Bíomedical Division), 181-194 (1987); and Pagington, Chemistry in Britain, pp. 455-458 (May 1987). The use of cyclodextrins specifically in the field of peptide and protein supply has been reviewed by T. Irle et al in Adv. Drug Deliv. Rev, Vol 36, 101-123 (1999) and examples in the case of sheep growth hormone, interleukin-2 and bovine insulin are described in Brewster et al., 1991, Pharmaceutical Research, 8 (6), 792- 795 WO 90/03784 and US 5,997,856 describe a method for the solubilization and / or stabilization of polypeptides, especially proteins, by means of cyclodextrins. However, no data are reported on the stabilization of interferons in this document. US 6,582,728 describes dry powder compositions for pulmonary administration containing interferon-beta which also contain human serum albumin, which may also contain cyclodextrins. However, even in this case no data are reported on the stabilization of the interferon composition which also contains cyclodextrins in this document. WO 2003/002152 describes stabilized compositions comprising an interferon molecule and a cyclodextrin specific derivative, ie, sulfoalkyl ether cyclodextrin. Accordingly, there is a need for additional IFN-beta pharmaceutical compositions comprising physiologically compatible stabilizers that improve the solubility of this protein and stabilize the protein against aggregate formation, thus instrumentation its pharmaceutical utility.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to stabilized pharmaceutical compositions comprising an interferon (IFN), methods for their preparation and use thereof. In particular, the main object of the invention is to provide a liquid stabilized pharmaceutical composition comprising an interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof, wherein said formulation is a solution that it comprises a pH regulator, a cyclodextrin, preferably an isotonicity agent and an antioxidant. The compositions are preferably prepared in the absence of human serum albumin (HSA) and therefore are free of this pharmaceutical excipient. Said compositions are referred to herein as IFN "HSA-free" pharmaceutical compositions and comprise an interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof. In accordance with one embodiment of the present invention, the compositions also comprise a bacteriostatic agent. An "interferon" or "IFN", as used herein, includes any molecule defined as such in the literature, comprising for example any types of IFNs mentioned in the preceding section "Background of the invention". In particular, IFN-a, IFN-β and IFN-α? they are included in the previous definition. IFN-β is the preferred IFN according to the present invention. Suitable IFN-β according to the present invention is commercially available, e.g., as Rebif® (Serono), Avonex® (Biogen) or Betaferon ® (Schering). The use of interferons of human origin is also preferred according to the present invention. The term interferon, as used herein, encompasses salts, functional derivatives, variants, analogs and active fragments thereof. The term "interferon-beta (IFN-beta or IFN-β)", as used herein, includes fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by immunoassay techniques. Recombinant DNA from prokaryotic or eukaryotic host cells, as well as their salts, functional derivatives, variants, analogs and active fragments thereof. IFN-beta preferably means interferon beta-1a. As used herein, the term "muteins" refers to IFN analogs in which one or more of the amino acid residues of a natural IFN are replaced by different amino acid residues, or are deleted, or one or more amino acid residues they are added to the natural IFN sequence, without significantly changing the activity of the resulting products compared to the wild-type IFN. These muteins are prepared by known synthesis and / or by site-directed mutagenesis techniques, or any other known technique suitable therefor. Preferred muteins include, e.g., those described by Shepard et al. (1981) or Mark et al. (1984).

Any of said muteins preferably has an amino acid sequence sufficiently duplicative of that of IFN, such that it has a substantially similar or even better activity to an IFN. The biological function of interferon is well known to one skilled in the art, and biological standards are established and are available, e.g., from the National Institute for Biological Standards and Control (http://immunoloqv.org/links/NIBSC). ). Bioassays have been described for the determination of IFN activity. An IFN test can be carried out, for example, as described by Rubinstein et al., 1981. Therefore, it can be determined whether any given mutein has substantially similar activity, or even better, than IFN by means of routine experimentation. The IFN muteins, which may be used in accordance with the present invention, or nucleic acid encoding the same, include a finite set of substantially corresponding sequences such as substitution peptides or polynucleotides that can be routinely obtained by one skilled in the art. , without additional experimentation, based on the teachings and guidance presented here. Preferred changes for muteins according to the present invention are what are known as "conservative" substitutions.

Conservative amino acid substitutions of polypeptides or proteins of the invention can include synonymous amino acids within a group, which have sufficiently similar physicochemical properties. That Substitution between members of the group will preserve the biological function of the molecule. It is clear that amino acid insertions and deletions can also be made in the previously defined sequences without altering their function, particularly if the insertions or deletions involve only a few as many amino acids, e.g., less than thirty, and preferably less than ten, and do not remove or displace amino acids that are critical to a functional conformation, e.g., cysteine residues. The proteins and muteins produced by said deletions and / or insertions are within the scope of the present invention. Preferably, the groups of synonymous amino acids are those defined in Table I. Most preferably, the groups of synonymous amino acids are those defined in Table II; and most preferably the groups of synonymous amino acids are those defined in Table III.

TABLE I Preferred amino acid groups Amino Acid Group Synonym Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu lie, Phe, Tyr, Met, Val, Leu Pro Gly, Wing, Thr, Pro Thr Pro, Ser, Wing, Gly, His, Gln, Thr Wing Gly, Thr, Pro, Wing Val Met, Tyr, Phe, Lie, Leu, Val Gly Wing, Thr, Pro, Ser, Gly lie Met, Tyr, Phe, Val, Leu, lie Phe Trp, Met, Tyr, He, Val, Leu, Phe Tyr Trp, Met, Phe, Lie, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr, Arg, Gln Asn Gin, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gin, His, Arg, Glu Met Phe, Lie, Val, Leu, Met Trp Trp TABLE II Most preferred amino acid groups Amino Acid Group Synonym Ser Ser Arg His, Lys, Arg Leu Leu, Lie, Phe, Met Pro Ala, Pro Thr Thr Ala Pro, Ala Val Val, Met, Lie Gly Lie, Met, Phe, Val, Leu Phe Met, Tyr, He, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser His Hls, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gln Met Met, Phe, He, Val, Leu Trp Trp TABLE III Even more preferred amino acid groups Amino Acid Group but Being Ser Arg Arg Leu Leu, He, Met Pro Pro Thr Thr Wing Wing Val Val Gly Gly He lile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, lie , Leu Trp Met Examples of production of amino acid substitutions in proteins that can be used to obtain IFN muteins, for use in the present invention include any known method steps, such as as those presented in the patents of E.U.A. 4,959,314, 4,588,585 and 4,737,462, Mark et al; 5,116,943 to Koths et al., 4,965,195 to Ñamen et al; 4,879,111 to Chong et al; and 5,017,691 to Lee et al; and lysine substituted proteins presented in the US patent. No. 4,904,584 (Shaw et al). Specific IFN-beta muteins have been described, for example, by Mark et al. al., 1984. The term "fused protein" refers to a polypeptide comprising an IFN, or a mutein thereof, fused to another protein, which, eg, has an extended residence time in body fluids. An IFN can therefore be fused to another protein, polypeptide or the like, eg, an immunoglobulin or a fragment thereof. "Functional derivatives" as used herein, cover IFN derivatives, and their muteins and fused proteins, which can be prepared from the functional groups that occur as side chains in the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention so long as they remain pharmaceutically acceptable, that is, they do not destroy the activity of the protein that is substantially similar to the activity of IFN, and do not confer toxic properties in compositions containing them. . These derivatives can include, for example, polyethylene glycol side chains, which can cover antigenic sites and extend the residence of IFN in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. carbocyclic alkanoyl or aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example, that of seryl or threonyl residues) formed with acyl moieties. As "active fractions" of IFN, or muteins and proteins fused, the present invention covers any fragment or precursor of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein or sugar residues by themselves, provided that the fraction does not have significantly reduced activity compared to the corresponding IFN. The term "salts" herein refers to both salts of carboxyl groups and acid addition salts of amino groups of the above-described proteins or analogs thereof. Salts of a carboxyl group can be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any of these salts must retain the biological activity of the proteins (IFN) relevant to the present invention, that is, the ability to bind to the corresponding receptor and initiate receptor signaling. In accordance with the present invention, the use of recombinant human IFN-beta and the compounds of the invention is particularly preferred.

A special type of interferon variant has been recently described. The so-called "consensus interferons" are variants of IFN that do not occur naturally (US 6,013,253). In accordance with a preferred embodiment of the invention, the compounds of the invention are made in combination with a consensus interferon. As used herein, human consensus interferon (IFN-con) means a polypeptide that does not occur naturally, which predominantly includes those amino acid residues that are common to a subset of IFN-alpha representative of most human leukocyte interferon subtype sequences that occur naturally and that include, at one or more of those positions in where there is no amino acid common to all subtypes, an amino acid that predominantly occurs in that position and in no case includes any amino acid residue that is not existing in that position in at least one subtype that occurs naturally. IFN-con encompasses but is not limited to the amino acid sequences designated as IFN-con1, IFN-con2 and IFN-con3 which are described in U.S. 4,695,623, 4,897,471 and 5,541, 293. DNA sequences encoding IFN-con can be produced as described in the aforementioned patents, or by other standard methods. In a further preferred embodiment, the fused protein comprises an Ig fusion. The fusion can be direct or by a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues of length. Said linker can be a tripeptide of the sequence EFM (Glu-Phe-Met) (SEQ ID: 1), for example, or a 13 amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val -Leu-GIy-Gly-Gln-Phe-Met (SEQ ID: 2) introduced between the sequence of IFN and the immunoglobulin sequence. The resulting fusion protein may have improved properties, such as extended residence time in body fluids (half-life), increased specific activity, increased expression level, or purification of the fusion protein is facilitated. In a further preferred embodiment, IFN is used for the constant region of an Ig molecule. Preferably, it is used for heavy chain regions, such as the CH2 and CH3 domains of human IgG1, for example. Other forms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as the IgG, IgG3 or IgG4 forms, or other Ig classes, such as IgM or IgA, for example. The fusion proteins can be monomeric or multimeric, heterologous or homomultimeric. In a further preferred embodiment, the functional derivative comprises at least a portion attached to one or more functional groups, which is presented as one or more side chains in the amino acid residues. Preferably, the portion is a polyethylene (PEG) portion. PEGylation can be carried out by known methods, such as, for example, those described, in WO 99/55377. The dose administered, as a single dose or multiple doses, to an individual will vary depending on a variety of factors, including pharmacokinetic properties, the route of administration, the conditions and characteristics of the patient (sex, age, body weight, health, size), degree of symptoms, concurrent treatments, frequency of treatment and desired effect. The standard doses of human IFN-beta vary from 80,000 lU / kg and 200,000 lU / kg per day or 6 MIU (millions of international units) and 12 MIU per person per day or 22 to 44 μg (microgram) per person. In accordance with the present invention, IFN can be administered preferably at a dose of about 1 to 50 μg, most preferably about 10 to 30 μg or about 10 to 20 μg per person per day. The administration of active ingredients according to the present invention can be intravenously, intramuscularly or subcutaneously. The preferred route of administration for IFN is the subcutaneous route. The IFN can also be administered daily or a day yes and a day no, or less frequently. Preferably, IFN is administered one, two or three times per week. The preferred route of administration is subcutaneous administration, administered, e.g., three times a week. An even more preferred route of administration is intramuscular administration, which may be applied, e.g., once a week. The dosage of IFN-β in the treatment of recurrent MS- re-emitter according to the invention depends on the type of IFN-β used. In accordance with the present invention, wherein IFN is recombinant IFN-β1β produced by E. coli, commercially available under the tradename Betaseron®, it can be preferably administered subcutaneously every other day at a dose of about 250 to 300 μg. u 8 MIU at 9.6 MIU per person. In accordance with the present invention, wherein IFN is recombinant IFN-β1a, produced by Chinese hamster ovary cells (CHO cells) commercially available under the trade name Avonex®, it can preferably be administered intramuscularly once a week to a dose of approximately 30 μg to 33 μg or 6 MIU to 6.6 MIU per person. In accordance with the present invention, when IFN is recombinant IFN-β1a, produced in Chinese hamster ovary cells (CHO cells), commercially available under the tradename Rebif®, it can preferably be administered subcutaneously three times a week ( TIW) at a dose of 22 to 44 μg or 6 MIU at 12 MIU per person. The term "stability" refers to physical stability, chemistry and conformation of the interferon formulations of the present invention (including maintenance of biological potency). The instability of a protein formulation can be caused by chemical degradation or aggregation of the protein molecule to form polymers of higher order, deglycosylation, glycosylation modification, oxidation or any other structural modification that reduces at least one biological activity of an interferon polypeptide included in the present invention. A "stable" or "stabilized" solution or formulation is one in which the degree of degradation, modification, aggregation, loss of biological and similar activity of the proteins therein is acceptably controlled, and does not increase unacceptably with time. Preferably, the formulation retains at least about 60%, most preferably at least or about 70%, most preferably at least about 80% of the labeled interferon activity over a period of 24 months. The compositions of Stabilized IFNs of the invention preferably have a shelf life of at least about 6 months, 12 months, 18 months, most preferably at least 20 months, most preferably still at least about 22 months, most preferably at least about 24 months. months when stored at 2-8 ° C. Methods for monitoring the stability of the IFN pharmaceutical compositions of the invention are available in the art, including those methods described in the examples described herein. Therefore, the formation of IFN aggregate during storage of a liquid pharmaceutical composition of the invention can be easily determined by measuring the change in soluble IFN in solution over time. The amount of soluble polypeptide in solution can be quantified by a number of analytical tests adapted for detection of IFN. Said tests include, for example, size exclusion chromatography (SEC) - HPLC and UV absorption spectroscopy, as described in the following examples. The determination of both soluble and insoluble aggregates during storage in liquid formulations can be achieved, for example, using analytical ultracentrifugation as indicated in the examples below to distinguish between that portion of the soluble polypeptide that is present as soluble aggregates and that portion which is present in the non-aggregated biologically active molecular form. The term "multiple dose use" is intended to include the use of a single vial, vial or cartridge of an interferon formulation for more than one injection, for example 2, 3, 4, 5, 6 or more injections. The injections are preferably made over a period of at least about 12 hours, 24 hours, 48 hours etc., preferably up to a period of about 12 days. The injections can be separated in time, for example, for a period of 6, 12, 24, 48 or 72 hours. The term "pH regulator" or "physiologically acceptable pH regulator" refers to solutions of compounds that are known to be safe for pharmaceutical or veterinary use in formulations and which have the effect of maintaining or controlling the pH of the formulation in the formulation. desired pH range of the formulation. Acceptable pH regulates to control pH at a moderately acidic pH at a moderately basic pH include, but are not limited to compounds such as phosphate, acetate, citrate, arginine, TRIS and histidine. "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol, and to any pharmacologically acceptable salt thereof. Preferred pH regulators are acetate pH regulators with saline or an acceptable salt. The "cyclodextrins" contemplated for use herein are derivatives of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl of beta-cyclodextrin and the corresponding derivatives of gamma-cyclodextrin. The hydroxyalkyl groups may contain one or more hydroxyl groups, e.g., hydroxypropyl (2-hydroxypropyl, 3-hydroxypropyl), dihydroxypropyl, and the like. The glucosyl, maltosyl and maltotriosyl derivatives may contain one or more sugar residues, e.g., glucosyl or diglucosyl, maltosyl or dimaltosyl. Various mixtures of the cyclodextrin derivatives can also be used, e.g., a mixture of maltosyl and dimaltosyl derivatives. Specific cyclodextrin derivatives to be used herein include hydroxypropyl-beta-cyclodextrin (HPCD or HPBCD), hydroxyethyl-beta-cyclodextrin (HEBCD), hydroxypropyl-gamma-cyclodextrin (HPGCD), hydroxyethyl-gamma-cyclodextrin (HEGCD), dihydroxypropyl-beta- ciciodextrin (2HPBCD), glucosyl-beta-cyclodextrin (G beta-CD or GiBCD), diglucosyl-beta-cyclodextrin (2G G beta-CD or 2 G-iBCD), maltosyl-beta-cyclodextrin (G2-beta-CD or G2BCD) ), maltosyl-gamma-cyclodextrin (G2-gamma-CD or G2GCD), maltotriosil-beta-cyclodextrin (G3-beta-CD or G3BCD), maltotriosil-gamma-cyclodextrin (G3-gamma-CD or G3GCD) and dimaltosil-beta -cyclodextrin (2 G2-beta-CD or 2 G2BCD), and mixtures thereof such as maltosyl-beta- Cyclodextrin / dimaltosyl-beta-cyclodextrin. Hydroxypropyl-beta-cyclodextrin for use in the compositions of the present invention is commercially available and is the preferred cyclodextrin according to the invention. Alternatively, it can be prepared by known methods, especially by using the optimized procedure of Pitha et al, International Journal of Pharmaceutics, 29.73-82 (1986). An "isotonicity agent" is a compound that is physiologically tolerated and imparts a proper tonicity to a formulation to prevent the net flow of water through the cell membranes that are in contact with the formulation. Compounds such as glycerin are commonly used for those purposes at known concentrations. Other suitable isotonicity agents include, but are not limited to, amino acids or proteins (e.g., glycine or albumin), salts (e.g., sodium chloride), and sugars (e.g., dextrose, mannitol , sucrose and lactose). Preferably, the isotonicity agent is mannitol. The term "antioxidant" refers to a compound that prevents oxygen free radicals or oxygen derivatives from interacting with other substances. Antioxidants are among a number of excipients commonly added to pharmaceutical systems to increase physical and chemical stability. Antioxidants are added to minimize or slow the oxidative processes that occur to some drugs or excipients under oxygen exposure or in the presence of free radicals.

These procedures can often be catalyzed by light, temperature, hydrogen on the concentration, presence of trace metals or peroxides. Sulfites, bisufites, thiourea, methionine, salts of ethylenediaminetetraacetic acid (EDTA), butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA) are frequently used as antioxidants in drugs. It has been found that sodium EDTA increases the activity of antioxidants by metal chelating ions that would otherwise catalyze the oxidation reaction. The most preferred antioxidant is methionine. The term "bacteriostatic" refers to a compound or compositions added to a formulation to act as an antibacterial agent. A conserved interferon-containing formulation of the present invention preferably satisfies statutory or regulatory guidelines for conservative effectiveness to be a commercially viable multi-use product. Examples of bacteriostats include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl-, ethyl-, propyl-, butyl- and the like), benzalkonium chloride, benzethonium chloride, dehydroacetate sodium and thimerosal. Preferably, the bacteriostatic agent is benzyl alcohol. In a preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition comprising an interferon (IFN) or a soforma, mutein, fused protein, functional derivative, active fraction or salt thereof, wherein said formulation is a solution comprising a pH regulator, 2-hydroxypropyl-beta-cyclodextrin, an agent of isotonicity and an antioxidant. In a preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein said interferon is IFN-beta such as recombinant human IFN-beta. In another preferred embodiment, the invention provides a liquid stabilized pharmaceutical composition wherein said pH regulator is present in an amount sufficient to maintain the pH of the composition within plus or minus 0.5 units of a specified pH, wherein the specified pH is about 3 to about 6, such as a pH value of from about 3.0 to about 6.0, including a pH value of or about 3.8. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the pH regulator is present at a concentration of about 5 M to 500 mM such as a concentration of or about 50 mM. In another preferred modality, the invention provides a stabilized liquid pharmaceutical composition wherein the pH regulator is an acetate pH regulator. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the isotonicity agent is mannitol. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the isotonicity is present at a concentration of about 0.5 mg / ml to about 500 mg / ml, such as a concentration of or about 50 mg / ml. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the antioxidant is methionine. In another preferred embodiment, the invention provides a liquid stabilized pharmaceutical composition wherein the antioxidant is present at a concentration of about 0.01 to about 5 mg / ml, including concentrations of about 0.01 to about 5.0 mg / ml, such as a concentration of or approximately 0.1 mg / ml. In another preferred embodiment, the invention provides a liquid stabilized pharmaceutical composition wherein the interferon is present at a concentration of about 10 μg / ml to about 800 μg / ml, such as a concentration of or about 44, 88 or 276 μg / ml . In another preferred embodiment, the invention provides a liquid stabilized pharmaceutical composition wherein the cyclodextrin is present at a molar vs. Interferon approximately 500 times the molar excess up to approximately 700 times the molar excess. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the composition is an aqueous solution. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the composition of any of the preceding claims, further comprises a bacteriostatic agent such as benzyl alcohol. In another preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the composition of any of the preceding claims, further comprises a bacteriostatic agent and wherein the bacteriostatic agent is present at a concentration of from about 0.1% to about 2%, including concentrations of or about 0. 1% to about 2.0%, such as concentrations of or about 0.2 or about 0.3%. In a further preferred embodiment, the invention provides a stabilized liquid pharmaceutical composition wherein the isotonicity agent is mannitol, the antioxidant is methionine and the interferon is interferon beta. In still another preferred embodiment, the invention provides a liquid stabilized pharmaceutical composition wherein the composition is the following liquid formulation: In another embodiment, the invention provides a method for preparing a liquid pharmaceutical composition stabilized according to the invention, wherein the method comprises adding calculated amounts of 2-hydroxypropyl-beta-cyclodextrin, antioxidant and isotonicity agent to the regulated solution in its pH and then adding the interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof. In another embodiment, the invention provides a hermetically sealed container under sterile conditions and suitable for storage before use, comprising the liquid pharmaceutical formulation according to the invention. Examples of said container are a vial or a cartridge for auto-injector. The containers according to the invention are for administration of a single dose or multiple doses. In a preferred embodiment, the invention provides a container according to the invention wherein said container is a pre-filled syringe for administration of a single dose. In another embodiment, the invention provides a device for administering multiple doses of a pharmaceutical composition according to the invention, wherein the equipment comprises a first container filled with a pharmaceutical composition according to the invention and a second cartridge filled with a solution of the bacteriostatic agent. Preferably, the concentration of IFN-beta in the formulation is from or about 10 μg / ml to or about 800 μg / ml, most preferably at or about 20 μg / ml up to or about 500 μg / ml, very particularly preferably from or about 30 to about 300, very particularly still from or about 44, 88 or 264 μg / ml. Preferably, the formulations of the present invention have a pH between about 3.0 and about 4.5, very particularly about or about 3.8. A preferred pH regulator is acetate, with preferred counterions being sodium or potassium ions. Saline acetate pH regulators are well known in the art. The pH regulator concentrations in the total solution may vary between or about 5 mM, 9.5 mM, 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM and 500 mM. Preferably the concentration of the pH regulator is about 10 M. Particularly preferred is a 50 mM pH buffer in acetate ions with a pH of 3.8. Preferably, in the composition of the invention the antioxidant, for example methionine, is present at a concentration of from about 0.01 to about 5 mg / ml, most preferably from about 0.05 to about 0.3. mg / ml, most preferably still at or about 0.1 mg / ml. Preferably, the concentration of the isotonicity agent (e.g., mannitol) in liquid formulations is from or about 0.5 mg / ml up to or about 500 mg / ml, most preferably from or about 1 mg / ml up to or about 250 mg / ml, very particularly preferably from or about 10 mg / ml up to or about 100 mg / ml, most preferably still at or about 5 mg / ml. In a further preferred embodiment, the invention provides a composition according to the invention wherein the isotonicity agent is mannitol, the antioxidant is methionine and the interferon is interferon beta. In another preferred embodiment, the invention provides a composition according to the invention wherein the liquid composition is as follows: The invention includes liquid formulations. The preferred solvent is water for injection. The liquid formulations can be a single dose or multiple doses. Those liquid formulations of interferon of the invention that are designed for multiple dose use preferably comprises a bacteriostatic, such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl-, ethyl-, propyl-, butyl- and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal. Particularly preferred are phenol, benzyl alcohol and m-cresol, most preferred is benzyl alcohol. The bacteriostatic agent is used in an amount that will give a concentration that is effective to keep the formulation essentially free of bacteria (suitable for injection) during the multiple dose injection period, which can be from or about 12 or 24 hours up to or about 12 days, preferably from or about 6 up to about 12 days. The bacteriostatic is preferably present in a concentration of about 0-1% (bacteriostatic mass / mass of solvent) up to or about 2.0%, most preferably from or about 0.2% or up to or about 1.0%. In the case of benzyl alcohol, particularly preferred concentrations are 0.2 or 0.3%). The bacteriostatic may also be present in single-dose formulations. The interferon range in the formulations of the invention includes amounts that give, upon reconstitution, concentrations of about 1.0 μg / ml to about 50 mg / ml, although lower or higher concentrations are operable and depend on the intended delivery vehicle, v .gr., solution formulations will differ from Transdermal, pulmonary, transmucosal, or osmotic or micro-pump patch methods. The interferon concentration is preferably at or about 5 μg / ml to or about 2 mg / ml, most preferably at or about 10 μg / ml up to or about 1 mg / ml, most preferably still at or about 30 μg / ml up to or about 100 μg / ml. Preferably, the formulations of the invention retain at least about 60%, most preferably at least about 70%, most preferably at least about 80% of the interferon activity at the time of packaging during a period of 24 months. In a further preferred embodiment, the invention provides a method for manufacturing a liquid pharmaceutical composition as described above. In still another preferred embodiment, the invention provides a method for manufacturing a packaged pharmaceutical composition comprising placing a solution comprising the active ingredient and the excipients as described above. In yet another preferred embodiment, the invention provides an article of manufacture for human pharmaceutical use, comprising a vial comprising the pharmaceutical compositions as described above, and written material that reports that the solution may be contained for a period of about twenty-four. hours or more after the first use. Preferably the written material says that the solution can be contained up to or about 12 days. After the first use of a multiple dose formulation, it can be maintained and used for at least about 24 hours, preferably at least about 4, 5 or 6 days, most preferably up to 12 days. After the first use, the formulation is preferably stored at below room temperature (i.e., below or about 25 ° C), most preferably below or about 10 ° C, most preferably at or about 2-8. ° C, most preferably still at or about 5 ° C. The formulations of the present invention can be prepared by a process comprising adding the calculated amounts of the excipients to the pH regulated solution and then adding the interferon. The resulting solution is then placed in vials, ampoules or cartridges. Variations of this procedure would be recognized by one skilled in the art. For example, the order in which the components are added, if additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used. In the case of using a multiple dose formulation, the bacteriostatic agent can be added to the solution containing the active ingredient (interferon) or alternatively can be kept in a separate vial or cartridge and subsequently mixed with the solution containing the active ingredient at the time of use. The formulations of the invention can be administered using recognized devices. Examples comprising these individual vial systems include self-injector devices or pen-type injector for delivery of a solution such as Rebiject®. The products currently claimed include packaging material. The packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used. The packaging material of the present invention provides instructions for the patient, if necessary, to prepare the final solution and use said final solution for a period of twenty-four hours or more for the product of two vials, wet / dry. For the solution product of a single vial, the label indicates that said solution can be used for a period of twenty-four hours or more. The products currently claimed are useful for the use of human pharmaceutical products. Stable preserved formulations can be provided to patients as clear solutions. The solution may be for single use or it may be reused multiple times and may be sufficient for a single cycle or multiple cycles of treatment of a patient and therefore provides a more convenient treatment regimen than those currently available. Interferon either in formulations or stable solutions or preserved herein described, can be administered to a patient in accordance with the present invention by a variety of delivery methods including subcutaneous or intramuscular injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro-pump, oral or other means appreciated by the person skilled in the art, as is known in the art. The term "vial" refers broadly to a suitable reservoir for retaining interferon in solid or liquid form in a contained sterile state. Examples of a vial, as used herein, include ampoules, cartridges, bubble packings, or other suitable reservoir for delivering the interferon to the patient by syringe, pump (including osmotic), catheter, transdermal patch, pulmonary or transmucosal spray. Vials suitable for packing products for parenteral, pulmonary, transmucosal or transdermal administration are well known and recognized in the art. The term "treatment", within the context of this invention, refers to any beneficial effect on the progression of the disease, including attenuation, reduction, reduction or decrease in pathological development after the onset of the disease. Pharmaceutical compositions of the invention comprising IFN or a soforma, mutein, fused protein, functional derivative, active fraction or salt are useful in the diagnosis, prevention and treatment (local or systemic) of clinical indications that respond to therapy with this polypeptide . These clinical indications include, for example, disorders or diseases of the central nervous system (CNS), brain, and / or spinal cord, including multiple sclerosis; autoimmune diseases, including rheumatoid arthritis, psoriasis, Crohn's disease; and cancers, including cancers of the breast, prostate, bladder, kidney and colon. In one embodiment, the invention includes the use of compositions of the invention for the preparation of a pharmaceutical formulation for the treatment of disorders or diseases affecting the central nervous system (CNS), brain, and / or spinal cord, including multiple sclerosis; autoimmune diseases, including rheumatoid arthritis, psoriasis, Crohn's disease; and cancers, including cancers of the breast, prostate, bladder, kidney and colon. In another embodiment, the invention provides a method of treating disorders or diseases that affect the central nervous system (CNS), brain, and / or spinal cord, including multiple sclerosis; autoimmune diseases, including rheumatoid arthritis, psoriasis, Crohn's disease; and cancers, including cancers of the breast, prostate, bladder, kidney and colon, which comprises the administration of the composition of the invention in a patient in need thereof. In another embodiment of the invention, the compositions of the invention are useful for the treatment of disorders or diseases affecting the central nervous system (CNS), brain, and / or spinal cord, including multiple sclerosis; autoimmune diseases, including rheumatoid arthritis, psoriasis, Crohn's disease; and cancers, including cancers of breast, prostate, bladder, kidney and colon. All references cited herein, including articles or abstracts of specialized journals, patent applications of E.U.A. or foreign published or unpublished, patents of E.U.A. or foreign issued or any other references, are hereby incorporated by reference in their entirety, including all the data, tables, figures and text presented in the cited references. In addition, the entire contents of the references cited within the references herein are also incorporated herein by reference in their entirety. Reference to steps of known methods, steps of conventional methods, known methods or conventional methods is not a way of admitting that any aspect, description or embodiment of the present invention is described, taught or suggested in the pertinent art. The above description of the specific modalities will fully reveal the general nature of the invention that others, by applying the knowledge within the scope of the technique (including the content of the references cited herein), can easily modify and / or adapt for the various applications of said specific embodiments, without further experimentation, without departing from the general concept of the present invention. Therefore, it is intended that said adaptations and modifications are within the meaning of a range of equivalents of the described modalities, based on the teachings and guidelines presented herein. It is understood that the phraseology and terminology herein is for the purpose of description and not limitation, in such a way that the phraseology and terminology of the present specification should be interpreted by the person skilled in the art in light of the teachings and guidelines presented herein, in combination with the knowledge of a person skilled in the art.

DESCRIPTION OF THE FIGURES Figure 1: shows the aggregation kinetics of interferon beta-1a 0.116 mg / ml (5.16, μM) in PEG / PBS, after incubation at 62 +/- 2 ° C 10 min, in the presence of different concentrations of HPBCD: 1.19 mg / ml (150 times the molar excess with respect to the molar amount of interferon beta-1a), 3.97 mg / ml (500 times the molar excess with respect to the molar amount of interferon beta-1a), 5.56 mg / ml ( 700 times the molar excess with respect to the molar amount of interferon beta-1a) and 7.94 mg / ml (1000 times the molar excess with respect to the molar amount of interferon beta-1a). In the Y axis, the optical density measured at 360 nm is reported, which is directly proportional to the turbidimetry (Cancellíeri et al., BIOPOLYMERS, Vol 13, 735-743, 1974). Figure 2: shows the effect of 10,000, 20,000 and 40,000 molar excess of mannitol on aggregation of 0.116 mg / ml (5.1 pM) interferon beta-l a in PBS / PEG10000 (after incubation at 62 + 3 ° C 10 min) . Figure 3: shows the kinetic aggregation of interferon beta-1a in the presence of different concentrations of L-methionine: 0.077 mg / ml (100 times the molar excess with respect to the molar amount of interferon beta-1 a), 0.158 mg / ml (205 times the molar excess with respect to the molar amount of interferon beta-1 a), 0.308 mg / ml (400 times the molar excess with respect to the molar amount of interferon beta-1 a), 0.769 mg / ml (1000 times the molar excess with respect to the molar amount of interferon beta-1 a) and 7.69 mg / ml (10,000 times the molar excess with respect to the molar amount of interferon beta-1 a). Figure 4: shows the kinetic aggregation of interferon beta-1 to alone and in the presence of methionine 400 times the molar excess with respect to the molar amount of interferon beta-1 a (0.308 mg / ml) and / or HPBCD 700 times the excess molar with respect to the molar amount of interferon beta-1 a (5.56 mg / ml). Figure 5: shows the effect of L-ascorbate 50 times the molar excess with respect to the molar amount of interferon beta-1 a (0.045 mg / ml), 150 times the molar excess with respect to the molar amount of interferon beta-1 a (0.136 mg / ml), 500 times the molar excess with respect to the molar amount of interferon beta-1 a (0.453 mg / ml) and 10,000 times the molar excess with respect to the molar amount of interferon beta-1 a ( 9.07 mg / ml) on the aggregation of interferon beta-1a 0.116 mg / ml (5.16 μM) in PEG / PBS, after incubation at 62 +/- 2 ° C, 10 minutes. Figures 6A and 6B: report thermal denaturation (ie, the effect of temperature on the CD signal at 222 nm) of interferon beta-1 to bulky (approximately 44 μg / ml) in Figure 6A and the spectrum of relative CD before (solid line) and after (broken line) of the fusion transition in Figure 6B. From here on, the deconvolutions of CDNN (neural network of circular dichroism) are reported (average of four analyzes). N.B .: The melting transition curve or thermal denaturation represents the effect of temperature on the CD signal at 222 nm. All the CD graphs have on the axis of Y the molar elipicity as deg M_1 crrf1 (Y axis). In the table, the residual alpha helix in the 200-260 nm interval has been considered for comparison of conformational stability of IFN in the deconvolutions of pre / post-fusion CDNN (average of four analyzes). Figure 7A and 7B: the thermal denaturation curve is reported for a solution containing interferon beta-1 at approximately 44 ug / ml and HPBCD 2.11 mg / ml (700 times the molar excess with respect to the molar amount of interferon beta-1) a), that is, the effect of temperature on the CD signal at 222 nm, expressed as molar elipicity, deg M "1 cm" 1. In Figure 7A (solid line) compared to the protein alone (dashed line) and the relative CD spectrum before (solid line) and after (dashed line) the fusion transition in Figure 7B. From here on, the deconvolutions of CDNN (neural network of circular dichroism) are reported (average of three analyzes). Figure 8: shows the thermal denaturation of interferon beta- 1a only (discontinuous curve) and in the presence of L-ascorbate Na 500 times the molar excess with respect to the molar amount of interferon beta-1 a (solid curve). Figure 9: shows the CD spectrum [interferon beta-1a / ascorbate 500x] before (solid curve) and then (dashed curve) the fusion transition and the respective CDNN deconvolutions (see above). Figure 10: shows the kinetic aggregation of interferon beta-1 to 0-116 mg / ml (5.16 pM) in PEG / PBS (final pH = 4.7) after incubation at 62 +/- 2 ° C, 10 minutes, and the effect of an excess of 700x M HPBCD (5.56) mg / ml). In the Y axis, the optical density measured at 360 nm is reported, which is directly proportional to turbidimetry. Figure 11: shows the kinetic aggregation of interferon beta-1 at 0.116 mg / ml (5.16, uM) in PEG / PBS (final pH = 5.1) after incubation at 62 +/- 2 ° C, 10 minutes, and the effect of an excess of 700x M of HPBCD (5.56 mg / ml) and excess of 500x M of RMBCD (3.38 mg / ml). Figure 12: shows the kinetic aggregation of interferon beta-1 at 0.116 mg / ml (5.16 μM) in PEG / PBS (final pH = 5.7) after incubation at 62 +/- 2 ° C, 10 minutes, and the effect of an excess of 700x M HPBCD (5.56 mg / ml).

EXAMPLES The following abbreviations refer respectively to the following definitions: cm (centimeter), mg (milligram), μg (microgram), min (minute), mM (millimolar), ml (milliliter), nm (nanometer), BHT (butylated hydroxytoluene) ), CD (circular dichroism), EDTA (ethylenediaminetetraacetic acid), HPBCD (hydroxypropyl-beta-cyclodextrin), HPLC (high performance liquid chromatography), IFN (interferon), IM (intramuscular), OD (optical density), PBS (salt solution regulated in its pH with phosphate), PEG (polyethylene glycol), RMBCD (methyl-beta-cyclodextrin randomly substituted), SC (subcutaneous), TFA (trifluoroacetic acid), TRIS (2-amino-2-hydroxymethyl-1, 3-propanediol, UV (ultraviolet), WFI (water for injection).

Methods Turbidimetry measurements Protein aggregation was monitored for 30 minutes at 360 nm using a UV-visible light spectrophotometer system (Perkin Elmer Lambda 40). Preliminary studies established the operating conditions under which the protein displays appropriate aggregation behavior, ie, volume dilution of interferon beta-1 to 1: 2 with a PEG 10,000 30 mg / ml solution in PBS (0.2 μm or filtered), in a polypropylene vial, followed by incubation in a thermostatic water bath at T = 62.2 ° C for 10 min. Heating and PEG were used to increase the protein association process through thermal denaturation and volume exclusion effect, respectively. The UV-visible analysis was performed in a cell containing a sample volume of 3 ml (final concentration of interferon beta-1 a = 0.116 mg / ml). Each turbidity analysis was repeated at least in triplicate and the average curve of optical density (OD) 360 nm versus time is reported. Aggregation of the protein alone was compared with interferon beta-1a in the presence of excipients at different concentrations.

Measurements of circular dichroism CD measurements were made with a spectropolarimeter Jasco J810 equipped with a Peltier temperature controller. The samples were contained in a 1 cm quartz cell with lid and, for thermal scrutiny, the magnetic stirring speed was approximately 150 rpm. For the far-UV spectrum (260-185 nm), a protein concentration of approximately 44 μg / ml, a resolution of 0.2 nm and a scanning speed of 2 nm / min were used with a response time of 2 seconds. and 3 accumulations.

In order to monitor the thermal perturbation of interferon beta-1a technical structure, CD signal at 222 nm as a function of temperature was followed between intervals of 25 ° C and 85 ° C to 0.2 ° C, using a rate of temperature ramp of 1 ° C / min and a delay time of 60 seconds. Each measurement was performed at least in triplicate on the volume of interferon beta-1 a, as a control, and on interferon beta-1 a solutions containing different concentrations of excipients.

Size exclusion chromatography analysis (Sec) The liquid formulations of interferon beta-1 a at predetermined time points of the stability studies were analyzed by SE-CLAR in order to determine purity and interferon beta-1 test at (expressed as% recovery). The operating conditions used were: • chromatographic column: TSK G2000 SWxL (7.8 mm ID x 30 cm, μ, 125 A); injection volume: 100 μl; Column temperature: room temperature; • sample temperature: room temperature; flow rate: 0.5 ml / min; mobile phase: 70% v / v of purified water (MILLIQ-Millipore) -30% v / v of acetonitrile- 0.2% v / v of TFA; • operating time: 27 min; • equilibrium time: 3 min; • wavelength: 214 nm; • A calibration curve, which ranged from 25 μg to 100 μg, was used to quantify the interferon beta-1 a test.

Materials • Volume of interferon beta-1 a (Serono SA, lot G4D024) • PEG 10000 (polyethylene glycol) • Lutrol F68 (polyoxyethylene-polyoxypropylene block copolymer) • L-Methionine • D-Mannitol • L-ascorbic acid • Regulator of pH of saline regulated in its pH with phosphate, pH 7.4 ± 0.1 (composition: KH2P04 0.19 g / l, Na2HP04.12H20 2.38 g / l, NaCl 8 g / l) • Hydroxypropyl-beta-cyclodextrin Equipment • Systems of CLAR (Waters and PE) equipped with TSK column.

G2000 UV-visible light spectrophotometer system (Perkin Elmer Lambda 40) • Jasco J810 spectropolarimeter equipped with a Peltier temperature controller. Osmometer (OSMOMAT 030D, Gonotech) Conductivity meter-PH MPC 227-Mettler Toledo Analytical balance AG245 and AG 285 (Mettler Toledo) Calibrated pipettes (Gilson) Hot plate of magnetic stirrer (Stuart Scientific) Ultrasonic bath, Fair, Thermometers Results and Discussion Turbidimetry test The effect on aggregation of interferon beta-1 to HPBCD, mannitol and L-methionine, detected by the turbidity method, is reported below. Sodium ascorbate salt is also included as an example of excipient having an aggregation enhancing effect. Figure 1 shows the kinetics of aggregation of interferon beta-1a in the presence of different concentrations of HPBCD: 1.19 mg / ml (150 times the molar excess with respect to the molar amount of interferon beta-1 a), 3.97 mg / ml (500 times the molar excess), 5.56 mg / ml (700 times the molar excess) and 7.94 mg / ml (1,000 molar excess). It should be noted that in the rate of At the investigated concentration this excipient does not completely avoid the destabilization of interferon beta-1a and the molar ratios of the intermediate show the best inhibitory effect. 700 times the molar excess was chosen as reference concentration for preparation of interferon beta-1 formulation and subsequent physico-chemical characterization (eg, circular dichroism). The concentration of cyclodextrin is best expressed as the molar ratio vs. interferon beta-1 a (fold the molar excess) as the concentration varies depending on the amount of interferon beta-1 a used in the preparation and can be calculated accordingly . The influence of mannitol on aggregation of interferon beta-1 a was then monitored but no significant effect was found even at 40,000 times the molar excess (corresponding to 37.35 mg / ml) under the conditions used (62 ° C in PEG / PBS), as shown in Figure 2. However, mannitol was used for liquid formulations of interferon beta-1 in order to achieve the isotonicity necessary for parenteral administration. Finally, L-methionine was tested in a turbidimetry experiment. Figure 3 shows the kinetics of aggregation of interferon beta-1a (0.116 mg / ml, in PEG / PBS, after incubation at 62 +/- 2 ° C, 10 ') in the presence of different concentrations of L-methionine. It should be noted that L-methionine has an inhibitory effect on protein aggregation than HPBCD: even at the concentration investigated maximum does not completely prevent destabilization of interferon beta-1a and the curve reaches a "plateau" similar to interferon beta-1 a control. In Figure 4, the aggregation kinetics of interferon beta-1 is reported alone and in the presence of methionine 400 times the molar excess (0.308 mg / ml) and / or HPBCD 700 times the molar excess (5.56 mg / ml). This experiment was carried out in order to eventually evaluate a synergistic effect or interference of these two excipients. It is clear that methionine has no protective effect against protein aggregation in addition to the activity of cyclodextrin. Following the previous considerations, it was confirmed that the HPBCD played an important role in the stabilization of interferon beta-1a towards aggregation and the interaction between protein and cyclodextrin was further investigated by circular dichroism. As an example of an excipient that has a "negative effect" on the aggregation of proteins in Figure 5, the kinetics of turbidity of interferon beta-1a in the presence of different concentrations of ascorbate salt is reported. The effect of L-ascorbate 50 times the molar excess (0.045 mg / ml), 150 times the molar excess (0.136 mg / ml), 500 times the molar excess (0.453 mg / ml) and 10,000 times the molar excess (9.07 mg) / ml) on the aggregation of interferon beta-1a 0.116 mg / ml (5.16 uM) in PEG / PBS, after incubation at 62 +/- 2 ° C, 10 minutes is shown in figure 5. It is interesting to note that the effect on the aggregation of Protein varies in a concentration-dependent manner: at high concentration, the negative charges appear to exhibit a destabilizing effect, while at lower molar ratios it seems to have an inhibitory influence. However, it was possible to identify a concentration (0.453 mg / ml corresponding to a molar ratio of excipient / interferon beta-1 equal to 500) that seems to impede protein aggregation. Ascorbate was then chosen as a possible excipient for formulations of interferon beta-1 a, with the purpose of combining its antioxidant action with a specific anti-aggregation effect.

Circular Dichroism In Figures 6A and 6B, the thermally induced splitting, between 25 ° C and 85 ° C, and far-UV spectra of a volumetric sample of interferon beta-1a (approximately 44 μg / ml) were reported. It should be noted that the estimated Tm value is 64.97 ± 0.31 ° C, while the spectrum deconvolution shows a remarkable difference in the helix content before and after denaturation, such as 48.0 vs 41.2% respectively. For comparison, an analogous sample containing hydroxypropyl-beta-cyclodextrin was analyzed by circular dichroism at 700 times the molar excess with respect to the molar amount of interferon beta-1a, being the concentration at which protein aggregation is partially inhibited. the turbidimetry experiment. In Figures 7A and 7B, the thermal denaturation curve is reported for a solution containing interferon beta-1a approximately 44 μ / mL and HPBCD 2.11 mg / mL (700 molar excess). The estimated melting temperature value was 65.46 + 0.50 ° C, identical to that concerning the protein alone (64.97 ° C) under the same operating conditions (see Figure 5). Although the melting temperature of the protein is constant in the presence of the tested additive, the reversibility of the thermal splitting is very different. It should be noted that in the presence of HPBCD there is clearly an interferon beta-1 alpha helix gap to smaller between the pre- and post-denaturation situation (approximately 4% difference versus approximately 7% for the protein alone), which suggests that cyclodextrin could prevent irreversible reactions of interferon beta-1 a, such as heat-induced aggregation after cleavage, or help re-doubled the protein. These observations are very interesting because the finding of formulation compositions that make the reversible splitting can really be very important for long-term stability (shelf life) than to increase the melting temperature (see also Arakawa et al, in Adv. Drug Deliv Rev. 46 (1-3) .307-326 (2001)). In order to better characterize the interaction of interferon beta-1a with sodium ascorbate salt, a CD analysis was performed on a sample of interferon beta-1a containing 500 times the molar excess of sodium salt. ascorbate. In fact, studies of kinetic turbidity revealed that an ascorbate-interferon beta-1 a solution, at a molar ratio equal to 500, does not exhibit protein aggregation. In Figure 8, the effect of temperature on the CD signal (222 nm) of interferon beta-1 a (approximately 44 g / ml) alone and in the presence of 500 times the molar excess of ascorbate (0.194 mg / ml) shows. In the case of the sample containing ascorbate, it was not possible to adjust the experimental curve to estimate the Tm value, which suggested a denaturing effect of the excipient towards the protein. In fact, the comparison of the second structure of the previous sample, before and after the fusion transition confirmed a very low residue of alpha helix already in the pre-melting situation, such as 35.0 (vs 48.0 volume of interferon beta-1 a under the same conditions). The secondary structure of interferon beta-1 a was even more altered by the presence of ascorbate after the fusion transition, decreasing to 28.8% (see Figure 9). This result contrasts with the protective effect shown in turbidimetry and suggests that ascorbate could induce conformational changes of interferon beta-1 that could destabilize the protein over time. For this reason, a liquid formulation containing sodium ascorbate was prepared, maintained in stability at 25 ° C and analyzed at pre-set time points by SE-CLAR.

Stability Study Following the above results, two interferon beta-1 a liquid formulations were prepared, which had the compositions shown in Table 1 and Table 2, and were maintained at 25 ° C and 50 ° C over time in the case of formulation 1 and at 25 ° C in the case of formulation 2. As a control, the volume of interferon beta-1 a (50 mM in pH buffer of acetate at the same concentration) was also tested.

TABLE 1 Composition of liquid formulation of interferon beta-1 a containing HPBCD (formulation 1) TABLE 2 Liquid formulation composition of interferon beta-1 a containing sodium ascorbate (formulation 2) The stability study samples were analyzed by SE-CLAR as described in the method section and the results are reported in table 3 (formulation 1), table 4 (formulation 2) and table 5 (volume of interferon beta-1) a / control).

TABLE 3 Results of the stability study by SE-CLAR on formulation 1 at 25 ° C v 50 ° C TABLE 4 Results of the stability study by SE-CLAR on Formulation 2 at 25 ° C TABLE 5 Results of the SE-CLAR stability study on the control formulation of interferon beta-1 at 25 ° C v 50 ° C From the results in Table 3, it can be clearly seen that the liquid formulation of interferon beta-1 a containing HPBCD is stable at 25 ° C and 50 ° C for 1 month: the level of aggregation is very low, being the monomer content above 90% even after 1 week at 50 ° C. In addition, the recovery of mass is above 90% which suggests that also the formation of insoluble aggregates could be minimized by the presence of cyclodextrin. In comparison, the volume of interferon beta-1 by itself leads to aggregation at both considered temperatures (see Table 5): at 50 ° C after 1 week, the monomer content decreases up to 83% and in parallel, recovery of dough was only about 73%. The latest results are consistent with turbidimetry and circular dichroism measurements that have suggested a beneficial effect of HPBCD on the aggregation of interferon beta-1a and conformational stability. Finally, the results shown in table 4 related to formulation 2 (containing ascorbate salt) indicate that the formulation is not stable at 25 ° C: a significant increase in dimers and aggregates and a parallel decrease in mass recovery were recorded after 1 month at 25 ° C. Moreover, an unknown peak appeared in the chromatogram, which suggested the presence of possibly changed conformation, as also suggested by the CD results. This was the reason why the stability of the formulation containing ascorbate was not carried out at higher temperatures (e.g., 50 ° C). In the latter case, it was important to verify the initial promising result obtained by turbidimetry analysis with alternative methods such as circular dichroism that is able to monitor the effect of a known excipient on the conformational stability of the protein.

Turbidimetry test at higher pH The effect on the aggregation of interferon beta-1 a of HPBCD was investigated over a wider pH range of the liquid formulation (ie, pH from about 3.0 to about 40). The method consisted of the turbidimetric test described above: volume of interferon beta-1 a was diluted 1: 2 with a PEG solution ,000 30 mg / ml in PBS (0.2 μm 0 filtered and appropriately basified by adding a small volume of 1 N NaOH) and then incubated in a thermostatic water bath at T = 62 + 2 ° C for 10 minutes. The aggregation protein was monitored for 30 minutes at 360 nm using a UV-visible light spectrophotometer system (Perkin Elmer Lambda 40). Each turbidity analysis was repeated in duplicate and the optical density (OD) curve 360 nm versus time average was reported. Aggregation of the protein alone was compared with interferon beta-1 a in the presence of cyclodextrin. The dilution of 50 mM acetate buffer (ie, medium volume of IFN) 1: 1 by PBS leads to a final solution pH of 4.4. The purpose, therefore, was to investigate the protein aggregation at the highest final pH. Figure 10 shows the aggregation kinetics of interferon beta-la in the absence and in the presence of an excess of 700x M HPBCD (5.56 mg / ml), with a pH of the solution analyzed equal to 4.7 at room temperature. It should be noted that an increase in pH increases the degree of aggregation of 1FN, but the presence of cyclodextrin still partially inhibits protein destabilization. The relative percentage of DO (ie, the percentage ratio between OD of 360 nm after 30 minutes in the presence and absence of the excipient) calculated for this experiment is 66%, not so far from what was observed under operating conditions usual at lower pH (52. 7%). The study was extended to a higher pH, that is, 5.1, as shown in figure 11: the effect of HPBCD was investigated at an excess of 700 M (5.56 mg / ml) and of RMBCD at an excess of 500 M ( 3.38 mg / ml) on the IFN aggregation. Note a more marked protein destabilization due to the increase in pH and the almost total absence of a kinetic tendency (ie, the plateau region at the beginning of the analysis). The interesting finding is that the aggregation of IFN is still partially inhibited by cyclodextrins, with a relative percentage of OD equal to 69.7% in the case of HPBCD (no advantage can be seen from the use of the methyl derivative in this case). A third pH value was investigated. Figure 12 shows kinetics of aggregation of IFN in PEG / PBS at pH 5.7 and the effect of addition of a molar excess of 700x of HPBCD. The excipient does not prevent protein destabilization, but significantly reduces its extension with respect to IFN control (relative percentage of OD 62-5%). The above considerations indicate that the use of HPBCD as a stabilizing excipient could be extended to the liquid formulation at a pH higher than the characteristic value of the protein volume (ie, pH 3.8 0. 5).

Conclusions • Some liquid formulations of interferon beta-1 a were prepared and maintained in stability at room temperature (25 ° C) and under accelerated conditions (50 ° C). • The most stable formulation contains L-methionine, HPBCD and mannitol. The SE-CLAR results show a monomer content per up to 90% after 1 week at 50 ° C or 1 month at 25 ° C. • The positive result of HPBCD was predicted and confirmed by measurement of turbidimetry that showed an inhibition effect dependent on the concentration of this excipient, (and partially also of L-methionine), towards aggregation of interferon beta-1 a. In addition, the CD analysis showed that in the presence of HP-beta-cyclodextrin there is clearly a loss of helix a from interferon beta-1 to smaller after fusion transition. • The volume of interferon beta-1 a, maintained under the same storage conditions, exhibits a different stability profile; the monomer content decreased to 83% after 1 week at 50 ° C. It should be noted that at 25 ° C the monomer content after 1 month is still equal to 97%, which is surprisingly high. This result could be explained by the fact that the volume contains acetate pH regulator at pH 3.8. This condition has by itself a certain degree of stabilizing effect for interferon beta-1 a. • A clear negative result in the stability study was found with the formulation containing ascorbate salt. SEC shows a remarkable monomer loss (up to 60%) at 25 ° C after 1 month. In parallel, a negative effect on the conformation of interferon beta-1a was also shown by CD analysis.

Manufacturing of the pharmaceutical substance Preparation of a 9M solution of sodium hydroxide A 1M solution of sodium hydroxide was prepared in WFI.

Preparation of 0.05 M sodium acetate pH regulator, pH 3.8 (100 ml) 0.286 ml of acetic acid (glacial) is added to a volumetric flask containing 80 ml of MilliQ water, and after stirring, 0.500 ml of acetic acid (glacial) is added. 1 M NaOH and water up to 100 ml; pH = 3.8 ± 0.05.

Preparation of excipient solution A concentrated solution (10-fold) of HPBCD and L-methionine in acetate pH buffer is prepared in a polypropylene volumetric flask. The appropriate amount of 50 mM acetate buffer containing 5 g of mannitol is added to the polypropylene flask. The solution is brought to homogeneity by flipping from top to bottom three times.

Combination of the drug substance solution The required amount B (g) of Interferon beta-1 a drug substance is added to the required amount of excipient solution V (g) and gently stirred until homogeneous.

Filling syringes 1 ml glass syringes can be filled aseptically with the final solution.

References 1. Arakawa, Prestrelski, Kenney and Carpenter (2001), "Factors affecting short-term and long-term stabilities of proteins", Adv. Drug Deliv. Rev. 46 (1-3): 307-326; 2. Brewster et al., 1991, Pharmaceutical research, New York, 8 (6), 792-795; 3. Cancellierí et al., Biopolymers, Vol 13,735-743, 1974; 4. Clegg and Bryant, Exp. Opin. Parmacother 2001; 2 (4): 623-639; 5. Derynk R. et al., Nature 1980; 285,542-547; 6. Familletti, P.C., Rubinstein, S., and Pestka, S. 1981"A Convenient and Rapid Cytopathic Effect Inhibition Assay for Interferon, "in Methods in Enzymology, Vol. 78 (S. Pestka, ed.), Academic Press, New York, 387-394; 7. Hultgren C, Milich DR, Weiland O, Sallberg M. (1998) The antiviral compound ribavirin modulates the T helper (Th) 1 / Th2 subset balance in hepatitis B and C virus-specific immune responses J Gen Virol 1998; 79: 2381-2391; 8. Irie et al., 1999, Adv. Drug Deliv. Rev, Vol 36, 101-123; 9. McCormick JB, King IJ, Webb PA, Scribner CL, Craven RB, Johnson KM, Elliott LH, Belmont-Williams R. Lassa fever. Ribavirin N Engl J Med. 1986 Jan 2; 314 (1): 20-6; 10. Mark DF et al, Proc. Nati Acad. Sci. US A, 81 (18) 5662-5666 (1984); 11. Pagington, Chemistry in Britain, pp. 455-458 (1987); 12. Pestka, S. (1986) "Interferon Standards and General Abbreviations, in Methods in Enzymology (S. Pestka, ed.), Academic Press, New York 119, 14-23, 13. Pitha et al, International Joumal of Pharmaceutics , 29.73-82 (1986); 14. Pitha et al, in Controlled Drug Deliver, ed. S. D. Bruck, Vol. I, CRC Press, Boca Raton, Fia., Pp. 125-148 (1983); 15. Rubinstein, S., Familletti, P.C., and Pestka, S-Convenient Assay for Interferons. J. Virol 1981; 37, 755-758; 16. Shepard H. M. et al., Nature 1981; 294,563-565; 17. T. Irie et al., Cyclodextrins in peptide and protein delivery, Adv. Drug Deliv. Rev, Vol 36,101-123 (1999); 18. Study Group. The Lancet 1998; 352.1498-1504; 19. Uekama et al, ¡n CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 3 (1), 1-40 (1987); 20. Uekama, in Topics in Pharmaceutical Sciences 1987, eds.

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Claims (31)

NOVELTY OF THE INVENTION CLAIMS

1. - A stabilized liquid pharmaceutical composition comprising an interferon (IFN) or an isoform, mutein, fused protein, functional derivative, active fraction or salt thereof, wherein said formulation is a solution comprising a pH regulator, 2-hydroxypropyl -beta-cyclodextrin, an isotonicity agent and an antioxidant.

2. The composition according to claim 1, further characterized in that the interferon is IFN-beta.

3. The composition according to claim 1 or 2, further characterized in that the IFN-beta is recombinant human IFN-beta.

4. The composition according to any of the preceding claims, further characterized in that the pH regulator is present in an amount sufficient to maintain the pH of the composition within plus or minus 0.5 units of a specified pH, wherein the pH specified is approximately 3 to approximately 6.

5. The composition according to any of the preceding claims, further characterized in that the pH is 3.8.

6. The composition according to any of the preceding claims, further characterized in that the pH regulator it is present at a concentration of about 5 mM to 500 mM.

7. The composition according to any of the preceding claims, further characterized in that the pH regulator is present at a concentration of approximately 50 mM.

8. The composition according to any of the preceding claims, further characterized in that the pH regulator is an acetate pH regulator.

9. The composition according to any of the preceding claims, further characterized in that the isotonicity agent is mannitol.

10. The composition according to any of the preceding claims, further characterized in that the isotonicity agent is present at a concentration of about 0.5 mg / ml to about 500 mg / ml.

11. The composition according to any of the preceding claims, further characterized in that the isotonicity agent is present at a concentration of about 50 mg / ml.

12. The composition according to any of the preceding claims, further characterized in that the antioxidant is methionine.

13. The composition according to any of the preceding claims, further characterized in that the antioxidant is present at a concentration of about 0.01 to about 5 mg / ml.

14. The composition according to any of the preceding claims, further characterized in that the antioxidant is present at a concentration of approximately 0.1 mg / ml.

15. The composition according to any of the preceding claims, further characterized in that the interferon is present at a concentration of about 10 μg / ml to about 800 μg / ml.

16. The composition according to any of the preceding claims, further characterized in that the cyclodextrin is present at a molar vs. Interferon 500 times the molar excess up to 700 times the molar excess.

17. The composition according to any of the preceding claims, further characterized in that the interferon is present at a concentration of approximately 44, 88 or 276 μg / ml.

18. The composition according to any of the preceding claims, further characterized in that the composition is an aqueous solution.

19. The composition according to any of the preceding claims, further characterized in that it comprises a bacteriostatic agent.

20. The composition according to any of the preceding claims, further characterized in that the agent Bacteriostatic is benzyl alcohol.

21. The composition according to any of the preceding claims, further characterized in that the bacteriostatic agent is present at a concentration of from about 0.1% to about 2%.

22. The composition according to any of the preceding claims, further characterized in that the bacteriostatic agent is present at a concentration of approximately 0.2 or 0.3%.

23. The composition according to any of the preceding claims, further characterized in that the isotonicity agent is mannitol, the antioxidant is methionine and the interferon is interferon beta.

24. The composition according to any of the preceding claims, further characterized in that the composition is the following liquid formulation: interferon beta-1 at 44 pg / ml; HPBCD 1.9 mg / ml; methionine 0.1 mg / ml; mannitol 50 mg / ml; acetate pH regulator up to 1 ml.

25. A method for preparing a liquid pharmaceutical composition stabilized according to any of claims 1 to 24, wherein the method comprises adding calculated amounts of 2-hydroxypropyl-beta-cyclodextrin, antioxidant and isotonicity agent to the pH-regulated solution and then adding the interferon (IFN) or a isoform, mutein, fused protein, functional derivative, active fraction or salt thereof.

26. A container hermetically sealed under sterile conditions and suitable for storage before use, comprising the liquid pharmaceutical formulation according to any of claims 1 to 24. 27.- The container according to claim 26, further characterized in that the container is a pre-filled syringe for administration of a single dose. 28. The container according to claim 26, further characterized in that the container is a vial. 29. The container according to claim 26, further characterized in that the container is a cartridge for a self-injector. 30. The container according to any of claims 26 to 29, further characterized in that the container is for administration of a single dose or multiple doses. 31. A device for administration of multiple doses of a pharmaceutical composition according to any of claims 26 to 29, wherein said equipment comprises a first container filled with a pharmaceutical composition according to any of claims 1 to 24 and a second cartridge filled with a solution of the bacteriostatic agent.