Link:  Pharm/Biotech Resources

Title:  Polymer conjugates of interferon beta-1a and uses

United States Patent:  6,962,978

Issued:  November 8, 2005

Inventors:  Pepinsky; Blake (Arlington, MA); Runkel; Laura (Cambridge, MA); Brickelmaier; Margot (Boxford, MA); Whitty; Adrian (Hopkinton, MA); Hochman; Paula (Newton, MA)

Assignee:  Biogen, Inc. (Cambridge, MA)

Appl. No.:  832658

Filed:  April 11, 2001

An interferon beta polypeptide comprising interferon-beta 1a coupled to a polymer containing a polyalkylene glycol moiety wherein the interferon-beta-1a and the polyalkylene glycol moiety are arranged such that the interferon-beta-1a has an enhanced activity relative to another therapeutic form of interferon beta (interferon-beta-1b) and exhibits no decrease in activity as compared to non-conjugated interferon-beta-1a. The conjugates of the invention are usefully employed in therapeutic as well as non-therapeutic, e.g., diagnostic, applications.

SUMMARY OF THE INVENTION

We have exploited the advantages of glycosylated interferon-beta relative to non-glycosylated forms. In particular, we have developed an interferon-beta-1a composition with increased activity relative to interferon-beta-1b and that also has the salutory properties of pegylated proteins in general with no effective loss in activity as compared to interferon-beta-1a forms that are not conjugated. Thus, if modifications are made in such a way that the products (polymer-interferon-beta 1a conjugates) retain all or most of their biological activities, the following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution (e.g, ability to stay in the vasculature for longer periods of time), increased stability in solution, reduced immunogenicity, protection from proteolytic digestion and subsequent abolition of activity. Such a formulation is a substantial advance in the pharmaceutical and medical arts and would make a significant contribution to the management of various diseases in which interferon has some utility, such as multiple sclerosis, fibrosis, and other inflammatory or autoimmune diseases, cancers, hepatitis and other viral diseases. In particular, the ability to remain for longer periods of time in the vasculature allows the interferon beta 1a to be used to inhibit angiogenesis and potentially to cross the blood-brain barrier. Further, the thermal stability gained by creating polymer-interferon-beta-1a conjugates is an advantage when formulating interferon-beta-1a in powder form for use in subsequent administration via inhalation.

We used our knowledge of the crystallographic structure of interferon-beta-1a and developed an interferon-beta-1a-polymer conjugate in which the polymer is linked to those interferon-beta-1a site(s) that will allow the conjugate to retain full activity of the interferon-beta-1a as compared to interferon-beta-1a that is not conjugated.

One aspect of the invention is a conjugated interferon-beta-1a complex wherein the interferon-beta-1a is covalently bonded to a polymer incorporating as an integral part thereof a polyalkylene glycol.

In one particular aspect, the present invention relates to a physiologically active interferon-beta-1a composition comprising physiologically active interferon-beta-1a coupled with a polymer comprising a polyalkylene glycol moiety wherein the interferon-beta-1a and polyalkylene glycol moiety are arranged such that the physiologically active interferon-beta-1a in the composition has an enhanced half life relative to the interferon-beta-1a alone (i.e., in an unconjugated form devoid of the polymer coupled thereto).

Another aspect of the invention is an interferon-beta-1a composition comprising physiologically active interferon-beta-1a coupled with a polymer in which the interferon-beta-1a is a fusion protein, preferably an immunoglobulin fusion. In such a complex, the close proximity of the N-terminus (site of conjugation with polymer) and the C-terminus (site of fusion with the Ig moiety) suggests that polymer conjugation may reduce the immunogenicity of the fusion protein.

In another aspect, the present invention relates to a physiologically active interferon-beta-1a composition comprising physiologically active interferon-beta-1a coupled with a polymer comprising a polyalkylene glycol moiety wherein the interferon-beta-1a and polyalkylene glycol moiety are arranged such that the physiologically active interferon-beta-1a in the composition has an enhanced activity relative to interferon-beta-1b alone (i.e., in an unconjugated form devoid of the polymer coupled thereto).

Another embodiment of the invention is a conjugated interferon-beta-1a protein whose interferon-beta-1a moiety has been mutated to provide for muteins with selectively enhanced antiviral and/or antiproliferative activity relative to non-mutated forms of interferon-beta-1a.

The invention relates to a further aspect to a stable, aqueously soluble, conjugated interferon-beta-1a complex comprising a physiologically active interferon-beta-1a covalently coupled to a physiologically compatible polyethylene glycol moiety. In such complex, the interferon-beta-1a may be covalently coupled to the physiologically compatible polyethylene glycol moiety by a labile covalent bond at a free amino acid group of the interferon-beta-1a, wherein the labile covalent bond is severed in vivo by biochemical hydrolysis and/or proteolysis.

In another aspect, the present invention relates to a dosage form comprising a pharmaceutically acceptable carrier and a stable, aqueously soluble, interferon-beta 1a complex comprising interferon-beta coupled to a physiologically compatible polyethylene glycol.

In another aspect, covalently coupled interferon-beta-1a compositions such as those described above may utilize interferon-beta-1a intended for diagnostic or in vitro applications, wherein the interferon-beta-1a is for example a diagnostic reagent for immunoassay or other diagnostic or non-in vivo applications. In such non-therapeutic applications, the complexes of the invention are highly usefully employed as stabilized compositions which may for example be formulated in compatible solvents or other solution-based formulations to provide stable compositional forms which are of enhanced resistance to degradation.

Modification of interferon-beta 1a with a non-toxic polymer may offer certain advantages. If modifications are made in such a way that the products (polymer-interferon-beta 1a conjugates) retain all or most of their biological activities the following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution (e.g, ability to stay in the vasculature for longer periods of time), increased stability in solution, reduced immunogenicity, protection of the modified interferon-beta 1a from proteolytic digestion and subsequent abolition of activity; increased thermal stability leading to more effective formulation of powdered interferon-beta-1a for oral or inhaled use.

Interferon-beta-1a endowed with the improved properties described above may be effective as therapy following either oral, aerosol, or parenteral administration. Other routes of administration, such as nasal and transdermal, may also be possible using the modified interferon-beta 1a.

Another aspect of the invention is a method of inhibiting angiogenesis and neovascularization comprising subject an effective amount of the compositions of the invention. As a result of increasing the level and duration of the interferon in the vasculature, the pegylated product of the invention should be particularly effective as an angiogenesis inhibitor.

In non-therapeutic (e.g., diagnostic) applications, conjugation of diagnostic and/or reagent species of interferon-beta is also contemplated. The resulting conjugated agent is resistant to environmental degradative factors, including solvent- or solution-mediated degradation processes. As a result of such enhanced resistance and increased stability of interferon-beta-1a, the stability of the active ingredient is able to be significantly increased, with concomitant reliability of the interferon-beta-1a containing composition in the specific end use for which same is employed.

DETAILED DESCRIPTION OF THE INVENTION

The Interferon-beta

Interferon-beta-1a is useful as an agent for the treatment, remission or attenuation of a disease state, physiological condition, symptoms, or etiological factors, or for the evaluation or diagnosis thereof. The term also refers to interferon-beta-1a that is itself part of a fusion protein such as an immunoglobulin-interferon-beta-1a fusion protein, as described in co-pending applications Ser. Nos. 60/104,572 and 60/120,161. Preparation of fusion proteins generally are well within the knowledge of persons having ordinary skill in the art.

We found unique site(s) for polymer attachment that would not destroy function of the interferon-beta-1a. In addition, we also used site-directed mutagenesis methods to independently investigate site(s) for polymer attachment (See Example 1). Briefly, we undertook a mutational analysis of human interferon-beta-1a with the aim of mapping resides required for activity and receptor binding. The availability of the 3-D crystal structure of human interferon-beta-1a (see above and Example 1) allows us to identify, for alanine (or serine) substitutions, the solvent-exposed residues available for interferon beta receptor interactions, and to retain amino acids involved in intramolecular bonds. A panel of fifteen alanine scanning mutations were designed that replaced between two and eight residues along distinct regions of each of the helices (A (A1 (SEQ ID NO:26), A2 (SEQ ID NO:27)), B (B1(SEQ ID NO:31), B2(SEQ ID NO:32), C (C1 (SEQ ID NO:33), C2 (SEQ ID NO:34)), D (SEQ ID NO:37), E (SEQ ID NO:40)) and loops (AB1(SEQ ID NO:28), AB2 (SEQ ID NO:29), AB3 (SEQ ID NO:30), CD1 (SEQ ID NO:35), CD2 (SEQ ID NO:36), DE1 (SEQ ID NO:38), DE2 (SEQ ID NO:39) of interferon-beta-1a (SEQ ID NO:25). See Example 1.

An amino-terminal histidine tag ("his" tag) was included for affinity purification of mammalian cell expressed mutants (FIG. 10 and SEQ ID NOS: 1 and 2 for the cDNA and deduced amino acid sequences, respectively) Functional consequences of these mutations are assessed in antiviral and antiproliferation assays. A non-radioactive binding assay was developed to analyze these mutants for their binding to the interferon beta surface cell receptor (IFNAR½ cell surface receptor). In addition, an ELISA-based assay employing an IFNAR2-ectodomain/Ig fusion protein to bind interferon was used to map interactions of surfaces between interferon-beta-1a and IFNAR2 (See Example 1). These mutational analyses demonstrated that N- and C-termini lie in a portion of the interferon-beta molecule not important for receptor binding or biological function.

The mutants are further variants of the interferon beta 1a moiety of the invention that may be particularly useful inasmuch as they display novel properties not found in the wild type interferon-beta-1a (See Example 1). We have identified three types of effects that were caused by targeted mutagenesis. These effects may be advantageous for interferon drug development under certain circumstances. The three types of effect are as follows: (a) mutants with higher antiviral activity that of his-wild-type interferon-beta-1a (e.g. mutant C1); (b) mutants which display activity in both antiviral and antiproliferation assays, but for which antiproliferation activity is disproportionately low with respect to antiviral activity, compared to his-wild-type interferon-beta-1a (e.g., mutants C1, D and DE1); and (c) functional antagonists (e.g., A1, B2, CD2 and DE1), which show antiviral and antiproliferative activities that are disproportionately low with respect to receptor binding, compared to his-wild-type interferon-beta-1a.

The Polymer Moiety

Within the broad scope of the present invention, a single polymer molecule may be employed for conjugation with an interferon-beta 1a, although it is also contemplated that more than one polymer molecule can be attached as well. Conjugated interferon-beta 1a compositions of the invention may find utility in both in vivo as well as non-in vivo applications. Additionally, it will be recognized that the conjugating polymer may utilize any other groups, moieties, or other conjugated species, as appropriate to the end use application. By way of example, it may be useful in some applications to covalently bond to the polymer a functional moiety imparting UV-degradation resistance, or antioxidation, or other properties or characteristics to the polymer. As a further example, it may be advantageous in some applications to functionalize the polymer to render it reactive or cross-linkable in character, to enhance various properties or characterisics of the overall conjugated material. Accordingly, the polymer may contain any functionality, repeating groups, linkages, or other constitutent structures which do not preclude the efficacy of the conjugated interferon-beta 1a composition for its intended purpose. Other objectives and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.

Illustrative polymers that may usefully be employed to achieve these desirable characteristics are described herein below in exemplary reaction schemes. In covalently bonded peptide applications, the polymer may be functionalized and then coupled to free amino acid(s) of the peptide(s) to form labile bonds.

The interferon-beta-1a is conjugated most preferably via a terminal reactive group on the polymer although conjugations can also be branched from the non-terminal reactive groups. The polymer with the reactive group(s) is designated herein as "activated polymer". The reactive group selectively reacts with free amino or other reactive groups on the protein. The activated polymer(s) are reacted so that attachment may occur at any available interferon-beta-1a amino group such as the alpha amino groups or the epsilon-amino groups of lysines. Free carboxylic groups, suitably activated carbonyl groups, hydroxyl, guanidyl, oxidized carbohydrate moieties and mercapto groups of the interferon-beta-1a (if available) can also be used as attachment sites.

Although the polymer may be attached anywhere on the interferon-beta 1a molecule, the most preferred site for polymer coupling is the N-terminus of the interferon-beta-1a. Secondary site(s) are at or near the C-terminus and through sugar moieties. Thus, the invention contemplates as its most preferred embodiments: (i) N-terminally coupled polymer conjugates of interferon-beta-1a; (ii) C-terminally coupled polymer conjugates of interferon-beta-1a; (iii) sugar-coupled conjugates of polymer conjugates; (iv) as well as N-, C- and sugar-coupled polymer conjugates of interferon-beta-1a fusion proteins.

Generally from about 1.0 to about 10 moles of activated polymer per mole of protein, depending on protein concentration, is employed. The final amount is a balance between maximizing the extent of the reaction while minimizing non-specific modifications of the product and, at the same time, defining chemistries that will maintain optimum activity, while at the same time optimizing, if possible, the half-life of the protein. Preferably, at least about 50% of the biological activity of the protein is retained, and most preferably 100% is retained.

The reactions may take place by any suitable method used for reacting biologically active materials with inert polymers, preferably at about pH 5-7 if the reactive groups are on the alpha amino group at the N-terminus. Generally the process involves preparing an activated polymer (that may have at least one terminal hydroxyl group) and thereafter reacting the protein with the activated polymer to produce the soluble protein suitable for formulation. The above modification reaction can be performed by several methods, which may involve one or more steps.

As mentioned above, the most preferred embodiments of the invention utilize the N-terminal end of interferon-beta-1a as the linkage to the polymer. Suitable methods are available to selectively obtain an N-terminally modified interferon-beta-1a. One method is exemplified by a reductive alkylation method which exploits differential reactivity of different types of primary amino groups (the epsilon amino groups on the lysine versus the amino groups on the N-terminal methionine) available for derivatization on interferon-beta-1a. Under the appropriate selection conditions, substantially selective derivatization of interferon-beta-1a at its N-terminus with a carbonyl group containing polymer can be achieved. The reaction is performed at a pH which allows one to take advantage of the pKa differences between the epsilon-amino groups of the lysine residues and that of the alpha-amino group of the N-terminal residue of interferon-beta-1a. This type of chemistry is well known to persons with ordinary skill in the art.

We used a reaction scheme in which this selectivity is maintained by performing reactions at low pH (generally 5-6) under conditions where a PEG-aldehyde polymer is reacted with interferon-beta-1a in the presence of sodium cyanoborohydride. This results, after purification of the PEG-interferon-beta-1a and analysis with SDS-PAGE, MALDI mass spectrometry and peptide sequencing/mapping, resulted in an interferon-beta-1a whose N-terminus is specifically targeted by the PEG moiety.

The crystal structure of interferon-beta-1a us such that the N- and C-termini are located close to each other (see Karpusas et al., 1997, Proc. Natl. Acad. Sci. 94: 11813-11818). Thus, modifications of the C-terminal end of interferon-beta-1a should also have minimal effect on activity. While there is no simple chemical strategy for targeting a polyalkylene glycol polymer such as PEG to the C-terminus, it would be straightforward to genetically engineer a site that can be used to target the polymer moiety. For example, incorporation of a Cys at a site that is at or near the C-terminus would allow specific modification using a maleimide, vinylsulfone or haloacetate—activated polyalkylene glycol (e.g., PEG). These derivatives can be used specifically for modification of the engineered cysteines due to the high selectively of these reagents for Cys. Other strategies such as incorporation of a histidine tag which can be targeted (Fancy et al., (1996) Chem. & Biol. 3: 551) or an additional glycosylation site, represent other alternatives for modifying the C-terminus of interferon-beta-1a.

The glycan on the interferon-beta-1a is also in a position that would allow further modification without altering activity. Methods for targeting sugars as sites for chemical modification are also well known and therefore it is likely that a polyalkylene glycol polymer can be added directly and specifically to sugars on interferon-beta-1a that have been activated through oxidation. For example, a polyethyleneglycol-hydrazide can be generated which forms relatively stable hydrazone linkages by condensation with aldehydes and ketones. This property has been used for modification of proteins through oxidized oligosaccharide linkages. See Andresz, H. et al., (1978), Makromol. Chem. 179: 301. In particular, treatment of PEG-carboxymethyl hydrazide with nitrite produces PEG-carboxymethyl azide which is an electrophilically active group reactive toward amino groups. This reaction can be used to prepare polyalkylene glycol-modified proteins as well. See, U.S. Pat. Nos. 4,101,380 and 4,179,337.

We had previously discovered that thiol linker-mediated chemistry could further facilitate cross-linking of proteins. In particular, we generated homotypic multimers of LFA-3 and CD4 using a procedure such as generating reactive aldehydes on carbohydrate moieties with sodium periodate, forming cystamine conjugates through the aldehydes and inducing cross-linking via the thiol groups on the cystamines. See Pepinsky, B. et al., (1991), J. Biol. Chem., 266: 18244-18249 and Chen, L. L. et al., (1991) J. Biol. Chem., 266: 18237-18243. Therefore, we envision that this type of chemistry would also be appropriate for modification with polyalkylene glycol polymers where a linker is incorporated into the sugar and the polyalkylene glycol polymer is attached to the linker. While aminothiol or hydrazine-containing linkers will allow for addition of a single polymer group, the structure of the linker can be varied so that multiple polymers are added and/or that the spatial orientation of the polymer with respect to the interferon-beta-1a is changed.

In the practice of the present invention, polyalkylene glycol residues of C1-C4 alkyl polyalkylene glycols, preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycol residues of such glycols are advantageously incorporated in the polymer systems of interest. Thus, the polymer to which the protein is attached can be a homopolymer of polyethylene glycol (PEG) or is a polyoxyethylated polyol, provided in all cases that the polymer is soluble in water at room temperature. Non-limiting examples of such polymers include polyalkylene oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated glycols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Examples of polyoxyethylated polyols include, for example, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like. The glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, and triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body.

As an alternative to polyalkylene oxides, dextran, polyvinyl pyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like may be used. Those of ordinary skill in the art will recognize that the foregoing list is merely illustrative and that all polymer materials having the qualities described herein are contemplated.

The polymer need not have any particular molecular weight, but it is preferred that the molecular weight be between about 300 and 100,000, more preferably between 10,000 and 40,000. In particular, sizes of 20,000 or more are best at preventing protein loss due to filtration in the kidneys.

Polyalkylene glycol derivatization has a number of advantageous properties in the formulation of polymer-interferon-beta 1a conjugates in the practice of the present invention, as associated with the following properties of polyalkylene glycol derivatives: improvement of aqueous solubility, while at the same time eliciting no antigenic or immunogenic response; high degrees of biocompatibility; absence of in vivo biodegradation of the polyalkylene glycol derivatives; and ease of excretion by living organisms.

Moreover, in another aspect of the invention, one can utilize interferon-beta 1a covalently bonded to the polymer component in which the nature of the conjugation involves cleavable covalent chemical bonds. This allows for control in terms of the time course over which the polymer may be cleaved from the interferon-beta 1a. This covalent bond between the interferon-beta-1a drug and the polymer may be cleaved by chemical or enzymatic reaction. The polymer-interferon-beta-1a product retains an acceptable amount of activity. Concurrently, portions of polyethylene glycol are present in the conjugating polymer to endow the polymer-interferon-beta-1a conjugate with high aqueous solubility and prolonged blood circulation capability. As a result of these improved characteristics the invention contemplates parenteral, nasal, and oral delivery of both the active polymer-interferon-beta-1a species and, following hydrolytic cleavage, bioavailability of the interferon-beta-1a per se, in in vivo applications.

It is to be understood that the reaction schemes described herein are provided for the purposes of illustration only and are not to be limiting with respect to the reactions and structures which may be utilized in the modification of the interferon-beta-1a, e.g., to achieve solubility, stabilization, and cell membrane affinity for parenteral and oral administration. The reaction of the polymer with the interferon-beta 1a to obtain the most preferred N-terminal conjugated products is readily carried out using a wide variety of reaction schemes. The activity and stability of the interferon-beta-1a conjugates can be varied in several ways, by using a polymer of different molecular size. Solubilities of the conjugates can be varied by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition.

Utilities

The unique property of polyalkylene glycol-derived polymers of value for therapeutic applications of the present invention is their general biocompatibility. The polymers have various water solubility properties and are not toxic. They are believed non-immunogenic and non-antigenic and do not interfere with the biological activities of the interferon-beta-1a moiety when conjugated under the conditions described herein. They have long circulation in the blood and are easily excreted from living organisms.

The products of the present invention have been found useful in sustaining the half life of therapeutic interferon-beta 1a, and may for example be prepared for therapeutic administration by dissolving in water or acceptable liquid medium. Administration is by either the parenteral, aerosol, or oral route. Fine colloidal suspensions may be prepared for parenteral administration to produce a depot effect, or by the oral route while aerosol formulation may be liquid or dry powder in nature. In the dry, lyophilized state or in solution formulations, the interferon-beta-1a-polymer conjugates of the present invention should have good storage stability. The thermal stability of conjugated interferon-beta-1a (Example 3) is advantageous in powder formulation processes that have a dehydration step. See, e.g., PCT/US/95/06008 ("Methods and Compositions for Dry Powder of Interferons").

The therapeutic polymer conjugates of the present invention may be utilized for the prophylaxis or treatment of any condition or disease state for which the interferon-beta-1a constituent is efficacious. In addition, the polymer-based conjugates of the present invention may be utilized in diagnosis of constituents, conditions, or disease states in biological systems or specimens, as well as for diagnosis purposes in non-physiological systems.

In therapeutic usage, the present invention contemplates a method of treating an animal subject having or latently susceptible to such condition(s) or disease state(s) and in need of such treatment, comprising administering to such animal an effective amount of a polymer conjugate of the present invention which is therapeutically effective for said condition or disease state. Subjects to be treated by the polymer conjugates of the present invention include mammalian subjects and most preferably human subjects. Depending on the specific condition or disease state to be combated, animal subjects may be administered polymer conjugates of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, and without undue experimentation. Because of the species barriers of Type I interferons, it may be necessary to generate interferon-polymer conjugates as described herein with interferons from the appropriate species.

The anti-cell proliferative activity of interferon-beta-1a is well known. In particular, certain of the interferon-beta-1a polymer conjugates described herein are useful for treating tumors and cancers such as osteogenic sarcoma, lymphoma, acute lymphocytic leukemia, breast carcinoma, melanoma and nasopharyngeal carcinoma, as well as autoimmune conditions such as fibrosis, lupus and multiple sclerosis. It is further expected that the anti-viral activity exhibited by the conjugated proteins, in particular certain of the interferon-beta-1a mutein conjugates described herein, may be used in the treatment of viral diseases, such as ECM infection, influenza, and other respiratory tract infections, rabies, and hepatitis. It is also expected that immunomodulatory activities of interferon-beta-1a exhibited by the conjugated proteins described herein, may be used in the treatment of autoimmune and inflammatory diseases, such as fibrosis, multiple sclerosis. The ability of interferons to inhibit formation of new blood vessels (i.e., inhibit angiogenesis and neovascularization) enables conjugates of the invention to be used to treat angiogenic diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis and Osler-Webber Syndrome.

Moreover, the antiendothelial activity of interferon has been known for some time and one potential mechanism of interferon action may be to interfere with endothelial cell activity by inhibiting the production or efficacy of angiogenic factors produced by tumor cells. Some vascular tumors, such as hemangiomas, are particularly sensitive to treatment with interferon. Treatment with interferon-alpha is the only documented treatment for this disease. It is expected that treatment with the interferon-beta-1a conjugates of the invention will offer subtantial pharmaceutical benefits in terms of pharmacokinetics and pharmacodynamics, since the conjugate is expected to remain in the vasculature for a longer period of time than non-conjugated interferons, thus leading to more efficient and effective therapy for use as an anti-angiogenic agent. See Example 8.

The polymer-interferon-beta-1a conjugates of the invention may be administered per se as well as in the form of pharmaceutically acceptable esters, salts, and other physiologically functional derivatives thereof. In such pharmaceutical and medicament formulations, the interferon-beta-1a preferably is utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The interferon-beta-1a is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

The formulations include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration. Formulations suitable for oral, nasal, and parenteral administration are preferred.

When the interferon-beta-1a is utilized in a formulation comprising a liquid solution, the formulation advantageously may be administered orally or parenterally. When the interferon-beta-1a is employed in a liquid suspension formulation or as a powder in a biocompatible carrier formulation, the formulation may be advantageously administered orally, rectally, or bronchially.

When the interferon-beta-1a is utilized directly in the form of a powdered solid, the interferon-beta-1a may advantageously be administered orally. Alternatively, it may be administered nasally or bronchially, via nebulization of the powder in a carrier gas, to form a gaseous dispersion of the powder which is inspired by the patient from a breathing circuit comprising a suitable nebulizer device.

The formulations comprising the polymer conjugates of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the active ingredient(s) into association with a carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the active ingredient(s) into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active ingredient as a powder or granules; or a suspension in an aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a draught.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface active agent, or discharging agent. Molded tablets comprised of a mixture of the powdered polymer conjugates with a suitable carrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s). Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active conjugate, which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution). Such formulations may include suspending agents and thickening agents or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose form.

Nasal spray formulations comprise purified aqueous solutions of the active conjugate with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucus membranes.

Formulations for rectal administration may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acid.

Ophthalmic formulations such as eye drops are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.

Topical formulations comprise the conjugates of the invention dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.

Accordingly, the present invention contemplates the provision of suitable polymers for in vitro stabilization of interferon-beta 1a in solution, as a preferred illustrative application of non-therapeutic application. The polymers may be employed for example to increase the thermal stability and enzymic degradation resistance of the interferon-beta 1a. Enhancement of the thermal stability characteristic of the interferon-beta-1a via conjugation in the manner of the present invention provides a means of improving shelf life, room temperature stability, and robustness of research reagents and kits.
 

Claim 1 of 24 Claims

1. A composition comprising the glycosylated interferon-beta-1a of SEQ ID NO: 25 coupled to a non-naturally-occurring polymer at an N-terminal end of said glycosylated interferon-beta-1a, said polymer comprising a polyalkylene glycol moiety.

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