[go: up one dir, main page]

WO1994028020A1 - Procede de preparation d'interferons - Google Patents

Procede de preparation d'interferons Download PDF

Info

Publication number
WO1994028020A1
WO1994028020A1 PCT/DK1994/000196 DK9400196W WO9428020A1 WO 1994028020 A1 WO1994028020 A1 WO 1994028020A1 DK 9400196 W DK9400196 W DK 9400196W WO 9428020 A1 WO9428020 A1 WO 9428020A1
Authority
WO
WIPO (PCT)
Prior art keywords
interferon
adduct
sucrose octasulfate
heparin
binding protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DK1994/000196
Other languages
English (en)
Inventor
Daniel Bar-Shalom
Yuen Shing
Judah Folkman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Buhk Meditec AS
Boston Childrens Hospital
Original Assignee
Buhk Meditec AS
Boston Childrens Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Buhk Meditec AS, Boston Childrens Hospital filed Critical Buhk Meditec AS
Priority to AU67941/94A priority Critical patent/AU6794194A/en
Publication of WO1994028020A1 publication Critical patent/WO1994028020A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]

Definitions

  • 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.
  • the invention relates to a method for the separation and/or purification and/or stabilization of a heparin-binding protein.
  • 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 ⁇ (also known as leukocyte interferon) , interferon ⁇ (also known as fibroblast interferon) and interferon ⁇ (also known as immune interferon) . Interferon ⁇ 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 ⁇ induces anti-viral activity, enhances NK cell activity and inhibits the growth of fibroblasts.
  • Interferon ⁇ may be prepared from poly IC- induced fibroblasts, human foreskin cells, fetal muscle cells, epithelial cells, myeloblasts and lymphoblasts. Interferon ⁇ enhances macrophage activation, activates lymphocytes, enhances class II antigen expression and induces anti-viral activity. Interferon ⁇ 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.
  • 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.
  • 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 ⁇ 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 ⁇ 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.
  • sucralfate i.e.
  • the sucrose octasulfate complex with aluminum to a clear solution of said interferon ⁇ , resulted in the formation of a precipitate and the disappearance of interferon ⁇ from the solution.
  • the precipitate possessed interferon a activity.
  • the addition of sodium sucrose octasulfate to an interferon a solution resulted in a clear solution, again retaining interferon ⁇ 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 ⁇ had disappeared from the solution by pH 4.
  • the precipitate exhibited interferon ⁇ activity.
  • the binding of interferon ⁇ to a sucrose octasulfate compound can modify the biological response to interferon ⁇ .
  • Example 1 the present inventors have found that interferon ⁇ bound to aluminum sucrose octasulfate did not dissociate in 2 M sodium chloride, indicating that the binding is very strong.
  • 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 ⁇ together with the results from the above- mentioned heparin-Sepharose ® column experiments indicate that in nature the interferon ⁇ 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.
  • 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 ⁇ and ⁇ 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.
  • interferon in the present context encompasses interferon ⁇ , interferon ⁇ and interferon ⁇ as well as any subtype thereof such as, e.g., interferon ⁇ 2 and interferon ⁇ 2. In preferred aspects of the present invention, interferon ⁇ or ⁇ are employed.
  • 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.
  • 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.
  • 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.
  • 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.
  • the material isolated from the mixture may comprise the adduct as well as unbound sucrose octasulfate.
  • 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.
  • 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.
  • 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.
  • a substantially water-soluble sucrose octasulfate compound is employed.
  • 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 ⁇ -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) .
  • 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.
  • 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.
  • 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.
  • interferon ⁇ and 0 After administration to an animal, interferon ⁇ 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.
  • 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.
  • 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, e
  • 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 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.
  • 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.
  • 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.
  • 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.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • 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.
  • suitable dosage forms for a composition according to the present invention include suppositories (emulsion or suspension type) , and rectal gelatin capsules (solutions or suspensions) .
  • 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.
  • 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.
  • additives like e.g. enhancers or surfactants may be incorporated.
  • 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.
  • 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 such as sodium chloride or sugars
  • 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), cellulose derivative
  • composition of the invention may also contain other additives such as emulsifiers, stabilizing agents, preservatives, etc.
  • 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.
  • 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.
  • BHA butylated hydroxy anisole
  • preservatives useful in a pharmaceutical composition of the invention are parabens and benzalkonium chloride.
  • humectants useful in a pharmaceutical composition of the invention are glycerin, propylene glycol, sorbitol and urea.
  • chelating agents useful in a pharmaceutical composition of the invention are sodium EDTA, citric acid and phosphoric acid.
  • 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”.
  • 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 ⁇ or an interferon ⁇ .
  • Interferon ⁇ and/or ⁇ 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 ⁇ 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.
  • 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.
  • bacteria including filamentous bacteria, chlamydia, and rickettsias
  • mycoplasmata chlamydia
  • spirochetes protozoans
  • fungi filaria and other nematodes
  • trematodes and cestodes cestodes.
  • 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.
  • inflammatory diseases are degenerative processes of unclear etiology such as, e.g., arthritis, Alzheimer's disease, rheumatic disorders, Crohn's disease and ulcerative colitis.
  • 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.
  • B cell or T cell or combined B cell and T cell deficiencies
  • 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.
  • 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.
  • heparin-binding proteins 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 ⁇ ; proteins involved in lipid metabolism such as, e.g., lipoprotein lipase, hepatic triglyceride lipase, apolipoprotein B, apolipoprotein E; serine protease inhibitors such as,
  • interferons a and ⁇ 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.
  • 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.
  • 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.
  • Figure 1 shows the affinity of interferon ⁇ 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 ⁇ (see Example 2)
  • Figure 3 shows how a mixture of interferon ⁇ 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.
  • 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 ⁇ 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.
  • 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.
  • An adduct is determined to be stable if no interferon ⁇ is detected in the supernatant fraction and interferon a is found in the sediment fraction.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Utilisation de composés d'octasulfate de saccharose dans la préparation d'interferons. L'addition d'octasulfate de saccharose à des solutions contenant des interférons entraîne la précipitation de ces derniers sous forme d'adduits sensiblement insolubles dans l'eau qui gardent l'activité des interférons, séparant et/ou purifiant ainsi lesdits interférons. Les adduits sont très stables, et comme les octasulfates de saccharose ne se métabolisent pas in vivo, les adduits peuvent être utilisés directement comme compositions pharmaceutiques, p.ex. pour le traitement de maladies infectieuses ou inflammatoires ou de troubles à caractère néoplasique ou prolifératif.
PCT/DK1994/000196 1993-05-18 1994-05-18 Procede de preparation d'interferons Ceased WO1994028020A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67941/94A AU6794194A (en) 1993-05-18 1994-05-18 A method for the preparation of interferons

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6285293A 1993-05-18 1993-05-18
US08/062,852 1993-05-18

Publications (1)

Publication Number Publication Date
WO1994028020A1 true WO1994028020A1 (fr) 1994-12-08

Family

ID=22045262

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1994/000196 Ceased WO1994028020A1 (fr) 1993-05-18 1994-05-18 Procede de preparation d'interferons

Country Status (2)

Country Link
AU (1) AU6794194A (fr)
WO (1) WO1994028020A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011090A1 (fr) * 1995-09-19 1997-03-27 Weisz Paul B Inhibition de la croissance tumorale
EP0770390A1 (fr) * 1995-11-01 1997-05-02 Dott Research Laboratory Compositons administrables par voie intranasale contenants des peptides physiologiquement actifs
US5843920A (en) * 1996-09-16 1998-12-01 Biocell Technology, Llc Anionic saccharides for extraction of anti-angiogenic protein from cartilage
WO2008152423A3 (fr) * 2007-06-11 2009-02-19 Diosamine Dev Corp Nouvelle utilisation médicale

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420049A1 (fr) * 1989-09-28 1991-04-03 F. Hoffmann-La Roche Ag Interféron leucocytaire stabilisé
EP0457223A1 (fr) * 1990-05-15 1991-11-21 Children's Medical Center Corporation Composition stable du facteur de croissance fibroblastique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420049A1 (fr) * 1989-09-28 1991-04-03 F. Hoffmann-La Roche Ag Interféron leucocytaire stabilisé
EP0457223A1 (fr) * 1990-05-15 1991-11-21 Children's Medical Center Corporation Composition stable du facteur de croissance fibroblastique

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011090A1 (fr) * 1995-09-19 1997-03-27 Weisz Paul B Inhibition de la croissance tumorale
EP0770390A1 (fr) * 1995-11-01 1997-05-02 Dott Research Laboratory Compositons administrables par voie intranasale contenants des peptides physiologiquement actifs
US5908824A (en) * 1995-11-01 1999-06-01 Dott Research Laboratory Nasally administrable compositions containing physiologically active peptides
US5843920A (en) * 1996-09-16 1998-12-01 Biocell Technology, Llc Anionic saccharides for extraction of anti-angiogenic protein from cartilage
WO2008152423A3 (fr) * 2007-06-11 2009-02-19 Diosamine Dev Corp Nouvelle utilisation médicale

Also Published As

Publication number Publication date
AU6794194A (en) 1994-12-20

Similar Documents

Publication Publication Date Title
US5202311A (en) Stabilized fgf composition
EP0230023B1 (fr) Compositions pharmaceutiques pour l'amélioration de la guérison des blessures
EP3103459B1 (fr) Acides hyaluroniques sulfatés en tant qu'agents antiviraux
HU228478B1 (en) Erythropoietin conjugates with polyethylenglycol
US5356874A (en) Angiogenic factor isolated from live yeast cell derivatives and its use in treating wounds or burns in mammals
KR100273389B1 (ko) 조직노화의 예방과 치료에의 올리고당류의 사용
JP2003089647A (ja) 関節性疾患治療剤
EP0495116A1 (fr) Agent anti-vih
CA1270764A (fr) Substance ayant une activite anti-infectieuse
KR100442758B1 (ko) 조직인자응고계억제제함유혈관신생저해제
WO1994028020A1 (fr) Procede de preparation d'interferons
US6653294B2 (en) Use of chitinous materials for inhibiting cellular nitric oxide production
JP5025045B2 (ja) 多硫酸化多糖類によるｔｉｍｐ産生の亢進
JP4317350B2 (ja) 顆粒球マクロファージコロニー刺激因子分泌促進剤
CN114948974A (zh) 肝素五糖化合物在制备脓毒症药物中的应用
US7015202B2 (en) Use of glycyrrhizin and its derivatives as MCP-1 production inhibitors
JP2550452B2 (ja) グリコサミノグリカンとアミノ酸エステルとの塩、その製造法およびそれを含有する経口投与用薬学的製剤
JP3455783B2 (ja) 血管内膜肥厚抑制剤
EP1127574A1 (fr) L'utilisation des produits contenant du chitin pour inhiber la production de l'oxyde nitrique
JPH04282322A (ja) 生物活性ペプチド製剤
WO2007111535A1 (fr) Composition immunomodulatrice
JP4633232B2 (ja) 成長因子誘導剤
WO2002083155A1 (fr) Utilisation de polysaccharides bacteriens sulfates pour l'inhibition de l'angiogenese
US5190750A (en) Methods for the treatment of demyelinating disease, uveitis, or graft-versus-host disease using tnf
AU1335599A (en) Use of glycosaminoglycans for producing pharmaceutical preparations for treating diabetes-associated diseases of the eye

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CN CZ CZ DE DE DK DK FI FI GE HU JP KG KP KR KZ LK LV MD MG MN MW NO NZ PL RO RU SD SI SK SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT.BUL.27/94 UNDER INID (54)"TITLE",REPLACE THE EXISTING TEXT BY "A METHOD FOR THE PREPARATION OF INTERFERONS BY USING SUCROSE OCTASULFATE COMPOUNDS"

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA