WO1994028020A1

WO1994028020A1 - A method for the preparation of interferons

Info

Publication number: WO1994028020A1
Application number: PCT/DK1994/000196
Authority: WO
Inventors: Daniel Bar-Shalom; Yuen Shing; Judah Folkman
Original assignee: Buhk Meditec AS; Boston Childrens Hospital
Current assignee: Buhk Meditec AS; Boston Childrens Hospital
Priority date: 1993-05-18
Filing date: 1994-05-18
Publication date: 1994-12-08
Anticipated expiration: 1995-11-18
Also published as: AU6794194A

Abstract

The use of sucrose octasulfate compounds to prepare interferons is disclosed. Addition of sucrose octasulfate to interferon-containing solutions results in precipitation of interferons as substantially water-insoluble adducts that retain interferon activity, thereby effecting separation and/or purification of the interferons. The adducts are highly stable and, as sucrose octasulfates are not metabolized in vivo, the adducts can be used directly as pharmaceutical compositions for the treatment of, for example, infectious disease, inflammatory disease, neoplastic and proliferative disorders.

Description

A METHOD FOR THE PREPARATION OF INTERFERONS BY USING SUCROSE

OCTASULFATE COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to a method for the separation and/or purification and/or stabilization of an interferon. The invention further relates to a stable interferon- containing adduct and to a method for the treatment of a disease of an animal by the administration of such a stable interferon-containing adduct. In another aspect, the invention relates to a method for the separation and/or purification and/or stabilization of a heparin-binding protein.

BACKGROUND OF THE INVENTION

Interferons are proteins produced by cells in response to the action of specific inducers, such as viruses. Interferons are present in all living mammals. There are three main varieties of human interferon: interferon α (also known as leukocyte interferon) , interferon β (also known as fibroblast interferon) and interferon γ (also known as immune interferon) . Interferon α induces anti-viral activity and enhances NK cell and mixed lymphocyte reactions. Interferon a may be prepared from activated lymphocytes (helper T cells, B cells and macrophages) . Interferon β induces anti-viral activity, enhances NK cell activity and inhibits the growth of fibroblasts. Interferon β may be prepared from poly IC- induced fibroblasts, human foreskin cells, fetal muscle cells, epithelial cells, myeloblasts and lymphoblasts. Interferon γ enhances macrophage activation, activates lymphocytes, enhances class II antigen expression and induces anti-viral activity. Interferon γ may be prepared from activated lymphocytes and T-cell lines.

Interferon a and jS are not homogeneous. Many subtypes are found in one organism but they seem to bind to the same receptors within the organism. Commercial interferons for therapeutic use are predominantly produced by recombinant DNA techniques. Peptide synthesis may be another possibility. At the end of the production process, the interferon is present in admixture with a number of unwanted compounds or products, including proteins. To obtain a pure interferon product, separation and purification, including filtration through specific columns, must be performed.

Furthermore, the end product is susceptible to chemical alterations such as degradation (e.g. hydrolysis) . Various methods for the stabilization of interferon have been described in the literature. Thus, e.g., EP 0 420 049 discloses that a preparation of human interferon may be stabilized by adding a disaccharide such as lactose or saccharose in combination with gallic acid or a gallic acid derivative.

DESCRIPTION OF THE INVENTION

The present invention provides methods by which interferons can be separated from media in which they are present, e.g. media in which they have been produced, and/or interferon can be purified, by use of a relatively simple and fast, yet efficient method based on the use of sucrose octasulfate compounds. Also, the invention provides a highly effective stabilization of interferons in the form of adducts with sucrose octasulfate; furthermore these adducts are candidates as extremely valuable therapeutic agents.

One aspect of the invention provides a method for separation and/or purification and/or stabilization of an interferon, comprising adding a substantially water-insoluble compound of sucrose octasulfate to an aqueous solution containing the interferon, whereby sucrose octasulfate is combined with the interferon thus forming a stable substantially water- insoluble interferon-sucrose octasulfate adduct. This and other aspects of the invention are based on the discovery of interferon-sucrose octasulfate adducts showing valuable interferon activity and an extremely high stability, e.g. stability with respect to decomposition by hydrolysis in aqueous media and with respect to physical or chemical denaturation by heat. Details about the discovery are given below:

Heparin-binding proteins are defined as proteins which can be retained in a heparin-Sepharose^® column. Human interferon α has been found not to bind to an heparin-Sepharose^® column; its activity was not augmented by the presence of heparin in a murine model, while it was found that murine interferon activity was augmented by the presence of heparin (Sylvester, D.M. et al.: "Augmentation of antimetastatic activity of interferon and tumor necrosis factor by heparin" in Immunopharmacol.Immunotoxicol. (1990), 12., 161-180).

The inventors repeated the above-mentioned heparin-Sepharose^® column experiment. An aqueous solution of human recombinant interferon a 2 (provided by Hoffmann-la Roche, Switzerland) was passed through a standard heparin-Sepharose^® column. The interferon α was retained in the column, but was eluted by washing the column with 0.1 M sodium chloride solution, i.e. a sodium chloride solution having a osmotic pressure similar to the osmotic pressure of physiological fluids. The results obtained indicated that the retention was not physiologically significant. Surprisingly, however, the addition of sucralfate, i.e. the sucrose octasulfate complex with aluminum, to a clear solution of said interferon α, resulted in the formation of a precipitate and the disappearance of interferon α from the solution. The precipitate possessed interferon a activity. Furthermore, the addition of sodium sucrose octasulfate to an interferon a solution resulted in a clear solution, again retaining interferon α activity, and subsequent acidification of the solution produced first a clouding of the solution and subsequent precipitation of a sediment. At least 75% of the interferon α had disappeared from the solution by pH 4. The precipitate exhibited interferon α activity.

As demonstrated in Example 2 herein, the binding of interferon α to a sucrose octasulfate compound can modify the biological response to interferon α.

Furthermore, as described in Example 1 herein, the present inventors have found that interferon α bound to aluminum sucrose octasulfate did not dissociate in 2 M sodium chloride, indicating that the binding is very strong.

The results of the above-mentioned experiments performed by the inventors indicate that the binding of sucrose octasulfate to interferon a is a very specific binding. From EP 0 457 223 Al it is known that acidic and basic fibroblast growth factors form a so-called complex with sucrose octasulfate. Based on the assumption that sucrose octasulfate is able to bind to heparin-binding proteins such as fibroblast growth factors because it mimics heparin, the observation of the binding of sucrose octasulfate to interferon α together with the results from the above- mentioned heparin-Sepharose^® column experiments indicate that in nature the interferon α either binds to a very specific sequence of heparin, a segment which is under-represented or absent in the above-mentioned heparin-Sepharose^® column, or that it only binds to a free, most likely short heparin segment. It is highly suggestive that while interferon a exhibits high activity against certain tumors, it is totally without effect against other tumors. It is the impression of the present inventors that interferon α and β have an effect in tumors rich in heparin, but have no effect on tumors rich in heparin-degrading enzymes, whereas it seems justified to presume that adducts of interferon and sucrose octasulfate will have a very pronounced effect in both types of tumors; in this connection, it is worth pointing out that sucrose octasulfate is not metabolized in the body, in contrast to heparin which is degraded by enzymes present in the animal body.

In the following, details and definitions concerning the invention are given:

The term "interferon" in the present context encompasses interferon α, interferon β and interferon γ as well as any subtype thereof such as, e.g., interferon α 2 and interferon β 2. In preferred aspects of the present invention, interferon α or β are employed.

In preferred aspects of the above-mentioned method of the present invention a water-insoluble sucrose octasulfate is used in combining, complexing or otherwise binding the interferon to form an adduct, i.e an interferon-sucrose octasulfate adduct.

In the present context, the term "substantially water- insoluble compound" is defined as a compound having a water- solubility at 20°C of at the most 1% w/v determined as described below in the following.

Correspondingly, the term "substantially water-soluble" is used in the meaning that the water-solubility at a pH in a range of 5.5-8.5 such as 7.0, is higher than 1.0 g/100 ml when determined in an appropriate buffer solution, such as 0.05 M phosphate buffer solution, at 37°C with stirring for 1 hour.

The method according to the above-mentioned aspect of the present invention is very straightforward and may comprise the simple addition of the water-insoluble sucrose octasulfate compound in solid form, e.g. in the form of a powder or as a suspension, to an interferon-containing solution which may be any solution containing an interferon, e.g., obtained from a culture of recombinant bacteria. In the resulting mixture, an adduct comprising the interferon and sucrose octasulfate is formed. This adduct formation takes place at room temperature (but also at lower temperatures such as, e.g., about 5°C or at higher temperatures). The adduct formed under such conditions is substantially water- insoluble and can be isolated by filtration and/or centrifugation.

Due to the fact that the sucrose octasulfate employed in the above-mentioned method as well as the interferon-sucrose sulfate adduct formed are water-insoluble, the material isolated from the mixture may comprise the adduct as well as unbound sucrose octasulfate. By proper selection of the amount of sucrose octasulfate added to the interferon solution of known concentration, it will be possible to control the amount of unbound sucrose octasulfate in the material isolated from the mixture. It is normally preferred that the ratio on a molar basis between the water-insoluble sucrose octasulfate compound which is added to the interferon solution and the interferon contained in this solution is in the range of about 1:1-50:1 in order to secure that the material isolated from the mixture mainly comprises the above-mentioned adduct. In certain cases, however, the adduct may be formed by using a lower or higher ratio between the sucrose octasulfate compound and the interferon; thus, e.g., a ratio of 0.5:1 may be employed in those cases where an excess of unbound sucrose octasulfate is unwanted.

As mentioned above the water-insoluble sucrose octasulfate compound used in the method according to the present invention may be in solid form, e.g. in the form of a powder or as a suspension comprising a suitable carrier and/or suspending agents.

In a variant of the above-mentioned method, a substantially water-soluble sucrose octasulfate compound is employed. Thus, the invention also relates to a method for separation and/or stabilization and/or purification of an interferon, comprising adding a water-soluble compound of sucrose octasulfate to an aqueous solution containing the interferon, whereby sucrose octasulfate is combined with the interferon, thus forming a stable interferon-sucrose octasulfate adduct which precipitates at a pH of 6 or below, preferentially at a pH of 5 or below such as a pH of about 4, confer the details about the interferon α-sodium sucrose octasulfate adduct given in Example 3. The thus formed precipitated adduct can be isolated by filtration or centrifugation.

The water-insoluble or water-soluble sucrose octasulfate used in accordance with the present invention is a sucrose which is polysulfated or persulfated, which means that substantially all possible sulfur-containing moieties are present as substituents on hydroxy groups of the carbohydrate moiety.

The sucrose octasulfate may to a lesser extent comprise sucrose pentasulfate and/or sucrose hexasulfate.

The sucrose octasulfate salt or complex is a salt or complex with a metal selected from the group consisting of alkali metals and alkaline earth metals, e.g. Na, K, Ca, Mg, Ba, Al, Zn, Cu, Zr, Ti, Mn or Os or with an organic base (e.g. an amino acid) . The currently preferred water-insoluble salts or complexes are aluminum, barium or bismuth salts or complexes. The currently preferred water-soluble salts are sodium and potassium salts.

Irrespective whether a water-insoluble or a water-soluble sucrose octasulfate compound is employed in a method according to the invention, the material isolated from the mixture by filtration or centrifugation and comprising the interferon-sucrose octasulfate adduct may be used as such or it may be further processed to remove the cation or other contaminants which may have precipitated alongside the interferon. Also, it may be desirable to remove the sucrose octasulfate itself from the adduct in order to obtain a substantially pure interferon. Proteins in general are sensitive to degradation under storage conditions, mainly by hydrolysis and/or denaturation. Heparin-binding proteins in general become stabilized when bound to heparin or other sulfated polysaccharides. Potassium and aluminum sucrose octasulfate have been shown to stabilize acidic and basic fibroblast growth factor, cf. EP 0 457 233 Al. The present inventors have found that water-insoluble and water-soluble sucrose octasulfate compounds stabilize and protect interferon a against degradation; on this basis, it is justified to assume that also other interferons may be stabilized using a sucrose octasulfate compound. After administration to an animal, interferon α and 0 rapidly undergo glycosylation which may result in inactivation of the interferon activity. It is presumed that the interferon- sucrose octasulfate adduct will be glycosylated to a much lesser extent.

The strength of the binding of different heparin-binding proteins to sulfated polysaccharide depends upon the protein and the sulfated polysaccharide structures. In general, it is expressed in terms of the concentration of sodium chloride in the eluent needed to remove them from a heparin-Sepharose^® column or the concentration of sodium chloride in the medium needed to dissociate the protein from the polysaccharide.

An interferon-sucrose octasulfate adduct prepared according to the methods of the present invention has such a stability towards dissociation that when subjected to incubation for at least 5 hours in a 1.5 molar solution of sodium chloride, preferably a 2 molar solution, at 20°C, substantially no interferon is released from the adduct, cf. Example 3 herein.

The invention also relates to the novel interferon-sucrose octasulfate adduct per se. Such an adduct, which may either be formed by a method described above or simply by mixing of an appropriate amount of an interferon and a sucrose octasulfate compound, e.g., in a suitable medium such as, e.g., water, can be used as a therapeutically active substance, exhibiting qualitatively and quantitatively the same activity as the same interferon alone or a qualitatively broader and/or quantitatively higher activity compared to the same interferon alone, and exhibiting improved stability, and generally a better toxicological profile compared to the same interferon alone.

A pharmaceutical composition according to the present invention comprises a stable interferon-sucrose octasulfate adduct in admixture with a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition may be in the form of a pharmaceutical dosage form, such as suspensions or solutions for injections (ready mixed or as powder for reconstitution before use) , solid or semi-solid implants; suppositories, enemas, troches; suspensions or solutions for nasal application; suspensions, gels, emulsions, ointments or solutions for eye application; suspensions, solutions or powders for nebulization; tablets, capsules, syrups, solutions, suspensions, emulsions, granules, powders, sachets, cachets for oral administration and creams, ointments, gels, lotions, emulsions, suspensions, pastes, plasters for topical application to the skin or vagina.

Optionally, the dosage form comprising the adduct according to the invention further comprises another therapeutically active substance such as a active substance selected from the group consisting of hormones, antibacterials, antivirals, antifungals, antiparasitics, antineoplastics, antiinflammatory agents, enzymes, anticonvulsants, blood coagulation modifiers, vitamins and antihistamines. A particular interesting group of compounds in the present context is hormones such as steroids.

A dosage form according to the present invention may further comprise any appropriate pharmaceutically acceptable excipient (see e.g. Martindale The Extra Pharmacopoeia, 28th Ed., The Pharmaceutical Press, London, UK, 1982).

Formulations for oral use include tablets which contain the adduct in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium chloride, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, potato starch or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc. Other pharmaceutically acceptable excipients can be colorants, flavouring agents, plasticizers, humectants etc. The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as chewing tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Powders, dispersible powders or granules suitable for preparation of an aqueous suspension by addition of water are also convenient dosage forms of the present invention. Formulation as a suspension provides the adduct in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents are, for example, naturally-occurring phosphatides, as e.g. lecithin, or condensation products of ethylene oxide with e.g. a fatty acid, a long chain aliphatic alcohol or a partial ester derived from fatty acids and a hexitol or a hexitol anhydrides, for example, polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate etc. Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate etc.

For the rectal application, suitable dosage forms for a composition according to the present invention include suppositories (emulsion or suspension type) , and rectal gelatin capsules (solutions or suspensions) . In a typical suppository formulation, the adduct is combined with an appropriate pharmaceutically acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases like polyethylene glycols and polyoxyethylene sorbitan fatty acid esters. Various additives like e.g. enhancers or surfactants may be incorporated.

For parenteral use a pharmaceutical composition according to the invention may in addition to the adduct comprise solvents, such as water, alcohols, glycerin, vegetable or animal oils, polyethylene glycols; osmotic regulating agents, such as sodium chloride or sugars; preservatives; antioxidants; buffer substances; surface active agents etc.

For topical application, the composition may be formulated in accordance with conventional pharmaceutical practice with pharmaceutical excipients conventionally used for topical applications such as pectin, gelatin and derivatives thereof, polylactic acid or polyglycolic acid polymers or copolymers thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose or oxidised cellulose, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, polyvinylpyrrolidone, silica and derivatives thereof, xanthan gum, kaolin, talc, starch and derivatives thereof, paraffin, water, vegetable and animal oils, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol, propanol, propylene glycol, (glycols, alcohols), fixed oils, sodium, potassium, aluminium, magnesium or calcium salts (such as the chloride, carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartrate) .

The composition of the invention may also contain other additives such as emulsifiers, stabilizing agents, preservatives, etc.

Examples of emulsifying agents useful in a pharmaceutical composition of the invention are naturally occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and sorbitan monooleate derivatives.

Examples of antioxidants useful in a pharmaceutical composition of the invention are butylated hydroxy anisole (BHA) , ascorbic acid and derivatives thereof, tocopherol and derivatives thereof and cysteine.

Examples of preservatives useful in a pharmaceutical composition of the invention are parabens and benzalkonium chloride.

Examples of humectants useful in a pharmaceutical composition of the invention are glycerin, propylene glycol, sorbitol and urea.

Examples of chelating agents useful in a pharmaceutical composition of the invention are sodium EDTA, citric acid and phosphoric acid.

Examples of gel forming agents useful in a pharmaceutical composition of the invention are Carbopol, cellulose derivatives, bentonite, alginates, gelatin and PVP. The formulation and preparation of the above-mentioned compositions is well-known to those skilled in the art of pharmaceutical formulation. Specific formulation can be found in "Remington's Pharmaceutical Sciences".

In a further aspect, the invention provides a method for the treatment of a disease selected from viral diseases, neoplastic disorders, proliferative disorders, infectious diseases and inflammatory diseases in an animal, in particular a mammal, and especially a human, comprising administering to the animal an effective amount of a stable interferon-sucrose octasulfate adduct wherein the interferon is an interferon α or an interferon β .

Interferon α and/or β are used today to treat viral infections, neoplastic diseases, inflammatory diseases, bacterial, fungal and parasitic infections and as modulators of the immune response. Their usefulness is limited by unknown factors, e.g. interferon α might be useful in the early stages in AIDS but is useless in late stages of the disease, similarly it is highly efficient in the treatment of soft tumors, such as hemangiomas and some lymphomas but seems to have no effect on solid tumors. There might be a correlation between the activity and the presence or absence of a sulfated sugar chaperon.

In the present context infective diseases are those caused by organisms or vira such as, e.g., bacteria including filamentous bacteria, chlamydia, and rickettsias; mycoplasmata; chlamydia; spirochetes; protozoans; fungi; filaria and other nematodes; trematodes and cestodes.

In the present context, neoplastic diseases include carcinomas connected with viral infections; diseases caused by benign and malign neoplasms (neoplasm defined as an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and continues in the same manner after cessation of the stimuli which have initiated it) including neoplasia of the haemopoietic tissues.

In the present context, inflammatory diseases are degenerative processes of unclear etiology such as, e.g., arthritis, Alzheimer's disease, rheumatic disorders, Crohn's disease and ulcerative colitis.

In the present context, modulation of the immune response might be useful in the treatment of asthma, allergic diseases, lupus erythematosus, immunodefiency diseases (B cell, or T cell or combined B cell and T cell deficiencies) , congenital and acquired defects.

Based upon the above-discussed observations, it is contemplated that the phenomena characteristic to the present invention are representatives of a broader principle which is discussed in the following:

Various proteins with metabolic functions such as enzymes, growth factors and others are known to have the ability to bind to sulfated glycosaminoglycans present in the extracellular matrix of the cells and/or to circulating heparin (a sulfates mucopolysaccharide) . This binding serves as a storage mechanism and/or as a regulating mechanism, the protein being immobilized on the extracellular matrix and released by degradation at the appropriate site or point and/or by a configurational change induced by the binding to the heparin which enables the binding to the receptor.

Furthermore, many of those proteins are rapidly destroyed or metabolized when they are not bound to heparin.

Normally, heparin-binding proteins are characterized by their ability to be retained by an heparin-Sepharose^® column as described by Neufeld, G. et al.: "Bovine granulosa cells produce basic fibroblast growth factor" in Endocrinology (1987) , 121, 597-603. Over the years, a number of heparin-binding proteins have been identified including adhesive matrix proteins such as, e.g., fibronectin, vitronectin, laminin, collagens, thrombospondin; growth factors such as, e.g., acidic and basic fibroblast growth factor (acidic FGF and basic FGF, respectively), interleukin-2 (int-2) , hst/ks, FGF-5, FGF-6, keratinocyte growth factor (KGF) , heparin-binding epidermal growth factor (HB-EGF) , vascular endothelial growth factor (VEGF) , tumor growth factors (TGF) , platelet derived growth factor (PDGF) and interferon γ; proteins involved in lipid metabolism such as, e.g., lipoprotein lipase, hepatic triglyceride lipase, apolipoprotein B, apolipoprotein E; serine protease inhibitors such as, e.g., antithrombin III, heparin co-factor II, protease nexins; and other proteins such as, e.g., superoxide dismutase, elastase, platelet factor 4, N-CAM, viral coat protein, transcription fractions, coagulase enzymes.

It is contemplated that the same methods as described for the interferons a and β , in other words formation of adducts with compounds of sucrose octasulfate, may be employed in the separation and purification of any of the heparin-binding proteins mentioned above, and that the adducts will have similar advantages over the heparin-binding proteins alone as the interferon adducts show over interferon alone.

Thus, an aspect of the present invention provides a method for stabilization of heparin-binding proteins by combining them with sucrose octasulfate. Heparin-binding proteins are stabilized under storage and/or after administration to an animal against proteolytic degradation, denaturation, phosphorylation and glycosilation when they are bound to heparin or heparan sulfate. According to the present invention, sucrose octasulfate seems to form an extremely stable bond to heparin-binding proteins and therefore the combination of a heparin-binding protein with sucrose octasulfate is assumed to stabilize the heparin-binding protein in a pharmaceutical composition and/or after administration to an animal, in particular a mammal, such as a human. Furthermore, the binding will modulate the activity of the heparin-binding protein and/or prevent its being sequestrated by the extracellular matrix.

As will be understood, details and particulars concerning this aspect of the invention will be the same as or analogous to the details and particulars concerning the interferon adduct aspects discussed above, and this means that wherever appropriate, the statements above concerning the interferon adducts, their preparation, improved properties and uses apply mutatis mutandis to the heparin-binding protein aspect of the invention.

Leσends to figures

Figure 1 shows the affinity of interferon α for heparin and aluminum sucrose octasulfate. The results are obtained by Western blot analysis as described in Example l.

Figure 2 shows the effect of sodium sucrose octasulfate and an interferon-sodium sucrose octasulfate adduct, respectively, on DNA synthesis of human endothelial cell interferon α (see Example 2)

Figure 3 shows how a mixture of interferon α and sodium sucrose octasulfate turns cloudy at pH 4 as evident from the high optical density at 660 nm. Most (at least 75%) of the aggregates can be precipitated down with a table top centrifuge at 14,000 rpm for 5 minutes.

EXAMPLE 1

Binding of interferon α to a heparin-Sepharose^® column and to sucrose octasulfate compounds

The experiment was a repetition of the heparin-Sepharose column experiment described in Sylvester, D.M. et al.: "Augmentation of antimetastatic activity of interferon and tumor necrosis factor by heparin" in Immunopharmacol.Immuno- toxicol. (1990), 12., 161-180.

1 ml of an aqueous solution of human recombinant interferon containing 12 x 10⁶ units interferon α (provided by

Hoffmann-la Roche, Switzerland) was passed through a standard heparin-Sepharose^® 2 ml bed column. (Pharmacia, Sweden) and washed stepwise with 5 ml of each of purified water, 0.1 M, 0.6 M, 2 M and 2 M sodium chloride.

The eluted fractions were collected and analyzed by Western blot analysis followed by silver staining performed in accordance with the procedure described in Shing, Y. : "Heparin-copper bioaffinity chromatography of fibroblast growth factor" in J. Biological Chemistry (1988), 263. 9059- 9062 using an anti-interferon α antibody (supplied by Hoffmann-la Roche) . The results, shown in Fig. 1, demonstrated that, while the interferon was initially retained in the column and was not washed out by the purified water, it was eluted by washing the column with 0.1 M sodium chloride solution, i.e. a sodium chloride solution having a osmotic pressure similar to the osmotic pressure of physiological fluids. No further interferon was washed out by the following solutions. This indicated that the retention was not physiologically significant. Surprisingly, however, the addition of 0,1 mg sucralfate, i.e. the sucrose octasulfate complex with aluminum, to a 1 ml clear solution of said interferon a resulted in the formation of a precipitate and the disappearance of interferon from the solution. The precipitate contained interferon α, again, assayed by the same Western blot method. Furthermore, the addition of sodium sucrose octasulfate to an interferon solution resulted in a clear solution, retaining interferon α, the subsequent, gradual acidification of the solution with a 0.1 M HCl solution produced first, at pH 5.5 a clouding of the solution, measured as change in the optical density of the solution using a spectrophotometer operating at a wavelength of 660 nm and later, at pH 4 or lower, precipitation of a sediment. At least 75% of the interferon α- had moved from the solution into the precipitate at pH 4, confer figure 3.

EXAMPLE 2

Modification of interferon a activity by sodium sucrose octasulfate

Administration of interferon α bound to sodium sucrose octasulfate displayed an activity different from administration of interferon α alone. Human endothelial cells obtained from a woman undergoing liposuction were cultivated in identical media in eight different groups to which identical volumes of various solutions were added, cf. the following:

Group 1. saline

Group 2. saline plus sucrose octasulfate

Group 3. human serum

Group 4. human serum plus sucrose octasulfate

Group 5. saline plus interferon α- Group 6. saline plus sucrose octasulfate and interferon α

Group 7. human serum plus interferon a

Group 8. human serum plus sucrose octasulfate and interferon α

The results are shown in Fig. 2. The groups 1-4 had essentially the same rate of DNA synthesis. In group Nos. 5 and 7 the DNA synthesis was reduced to 50% compared to groups Nos. 1-4. Group No. 6 had essentially the same rate as groups Nos. 1-4 while in group No. 8, the synthesis was about 75% of groups Nos. 1-4. This indicated that sucrose octasulfate could modify the activity of interferon α. EXAMPLE 3

Test for the stability of the interferon-sucrose octasulfate adducts prepared according to the present invention

The isolated interferon-sucrose octasulfate adduct derived from a substantially water-insoluble sucrose octasulfate compound is incubated for 5 hours at 20°C in a 2 molar solution of sodium chloride.

After incubation the sediment is isolated by centrifugation with a table top centrifuge at 14,000 rpm for 5 minutes. The two fractions, i.e. the supernatant and the sediment, are separated and samples from both fractions are assayed by Western blot analysis followed by silver staining performed in accordance with the procedure described in Shing, Y. : "Heparin-copper bioaffinity chromatography of fibroblast growth factor" in J. Biological Chemistry (1988) , 263. 9059- 9062 using an anti-interferon a antibody.

An adduct is determined to be stable if no interferon α is detected in the supernatant fraction and interferon a is found in the sediment fraction.

An adduct made by adding 500 μg sucralfate to 100 μg interferon α (obtained from Hoffmann-la Roche, Switzerland) dissolved in 5 ml water, shaking the mixture in a Vortex mixer, centrifugating the mixture in a table top centrifuge at 14,000 rpm for 5 minutes, discarding the supernatant and replacing it with 5 ml of the incubation medium described above was tested in accordance with the procedure described above. No interferon a was detected in the supernatant fraction obtained after incubation. Thus, the adduct was stable.

Claims

1. A method for separation and/or purification and/or stabilization of an interferon, comprising adding a substantially water-insoluble compound of sucrose octasulfate to an aqueous solution containing the interferon, whereby sucrose octasulfate is combined with the interferon thus forming a stable substantially water-insoluble interferon- sucrose octasulfate adduct.

2. A method according to claim 1, wherein the adduct is separated from the solution by filtration or centrifugation.

3. A method according to claim 1, wherein the interferon- sucrose octasulfate adduct has a stability such that when subjected to incubation for at least 5 hours in a 1.5 molar solution of sodium chloride at 20°C, substantially no interferon is released from the adduct, as assessible by Western blot analysis with anti-interferon a antibody.

4. A method according to claim 3, wherein the interferon- sucrose octasulfate adduct has a stability such that when subjected to incubation for at least 5 hours in a 2 molar solution of sodium chloride at 20°C, substantially no interferon is released from the adduct, as assessible by Western blot analysis with anti-interferon α antibody.

5. A method according to claim 1, wherein the sucrose octasulfate compound is an aluminum complex or a bismuth complex.

6. A method according to claim 1, wherein the interferon is an interferon α or β .

7. A method for separation and/or purification and/or stabilization of an interferon, comprising adding a water- soluble compound of sucrose octasulfate to an aqueous solution containing the interferon, whereby sucrose octasulfate is combined with the interferon, thus forming a stable interferon-sucrose octasulfate adduct which precipitates at a pH of 6 or below.

8. A method according to claim 7, wherein the adduct is precipitated by adjusting the pH of the solution to a pH lower than 6.

9. A method according to claim 8, wherein the adduct is precipitated by adjusting the pH of the solution to a pH of at most 5.

10. A method according to claim 9, wherein the adduct is precipitated by adjusting the pH of the solution to a pH of at most 4.

11. A method according to claim 7, wherein the adduct is precipitated and isolated by filtration or centrifugation.

12. A method according to claim 7, wherein the interferon is an interferon α or β .

13. A method according to claim 7, wherein the sucrose octasulfate compound is a sodium or potassium salt.

14. A stable interferon-sucrose octasulfate adduct.

15. A stable adduct according to claim 14, wherein the interferon is interferon α or β .

16. A stable adduct according to claim 14, wherein the sucrose octasulfate is in a form selected from soluble and insoluble salts and complexes of sucrose octasulfate.

17. A stable adduct according to claim 16, wherein the sucrose octasulfate is in the form of a sodium or potassium salt.

18. A stable adduct according to claim 16, wherein the sucrose octasulfate is in the form of a complex with aluminum or bismuth.

19. A pharmaceutical composition comprising a stable interferon-sucrose octasulfate adduct in admixture with a pharmaceutically acceptable carrier or excipient.

20. A method for the treatment or prophylaxis of a disease selected from viral diseases, neoplastic disorders, proliferative disorders, infectious diseases and inflammatory diseases in an animal, comprising administering to the animal an effective amount of a stable interferon-sucrose octasulfate adduct wherein the interferon is an interferon α or an interferon β .

21. A method for separation and/or purification and/or purification of a heparin-binding protein, the heparin- binding protein being different from interferon α and β , comprising adding a substantially water-insoluble compound of sucrose octasulfate to an aqueous solution containing the heparin-binding protein to form a stable substantially water- insoluble adduct of the heparin-binding protein with the sucrose octasulfate compound.

22. A method according to claim 21, wherein the adduct is separated from the solution by filtration or centrifugation.

23. A method for separation and/or purification and/or stabilization of an heparin-binding protein, comprising adding a water-soluble compound of sucrose octasulfate to an aqueous solution containing the heparin-binding protein, the heparin-binding protein being different from interferon a and iS, whereby sucrose octasulfate is combined with the heparin- binding protein, thus forming a stable adduct of the heparin- binding protein with the sucrose octasulfate compound.

24. A method according to claim 23, wherein the adduct is precipitated by adjusting the pH.

25. A method according to claim 24, wherein the heparin- binding protein is recovered from the precipitate.

26. A pharmaceutical composition comprising a stable adduct of a heparin-binding protein, the heparin-binding protein being different from interferon α and β and different from acid fibroblast growth factor and basic fibroblast growth factor, with a sucrose octasulfate compound in admixture with a pharmaceutically acceptable carrier or excipient.

27. A method for the treatment or prophylaxis of conditions amenable to treatment with a heparin-binding protein in an animal, comprising administering to the animal an effective amount of a stable adduct of a heparin-binding protein, the heparin-binding protein being different from interferon a and β and different from acid fibroblast growth factor and basic fibroblast growth factor, with a sucrose octasulfate compound.