EP0804248A2 - Interferent chimiquement modifie - Google Patents

Interferent chimiquement modifie

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Publication number
EP0804248A2
EP0804248A2 EP96903420A EP96903420A EP0804248A2 EP 0804248 A2 EP0804248 A2 EP 0804248A2 EP 96903420 A EP96903420 A EP 96903420A EP 96903420 A EP96903420 A EP 96903420A EP 0804248 A2 EP0804248 A2 EP 0804248A2
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EP
European Patent Office
Prior art keywords
ifn
con
interferon
chemically modified
glycosyl
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EP96903420A
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German (de)
English (en)
Inventor
David Collins
Jennifer Lin-Chun Liu
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Amgen Inc
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Amgen Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the modification of consensus interferon and, more particularly, to neoglycosylated analog compositions of recombinant consensus interferon, wherein glycosyl ligands are conjugated to the recombinant consensus interferon.
  • Interferon ⁇ are a subclass of cytokines that exhibit both antiviral and antiproliterative activity.
  • human interferons are grouped into three classes: interferon-alpha (leukocyte), interferon-beta (fibroblast) and interferon-gamma (immune) .
  • interferon-alpha leukocyte
  • fibroblast fibroblast
  • interferon-gamma immunoglobulin-gamma
  • At least fourteen alpha interferons (grouped into subtypes A through H) having distinct amino acid sequences have been identified by isolating and sequencing DNA encoding these polypeptides.
  • Alpha interferons have received considerable attention as potential therapeutic agents due to their antiviral and antitumor growth inhibitior..
  • Alpha-interferon is currently approved in the United States and other countries for the treatment of hairy cell leukemia, venereal warts, Kaposi's Sarcoma (a cancer commonly afflicting patients suffering from Acquired Immune Deficiency Syndrome (AIDS) ) , and chronic hepatitis C virus (HCV) infection.
  • Two variants of alpha interferon have received approval for therapeutic use: Interferon alfa-2a, marketed under the trade name Roferon®-A, and Interferon alfa-2b, marketed under the trade name INTRON® A.
  • alpha-interferon is being used or evaluated alone or in conjunction with chemotherapeutic agents in a variety of other cellular proliferation disorders, including chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancers (basal cell carcinoma and malignant melanoma) , renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma.
  • Alpha-interferon may be effective in combination with other chemotherapy agents for the treatment of solid tumors that arise from lung, colorectal and breast cancer (see Rosenberg et al.
  • Alpha-interferons are known to affect a variety of cellular functions, including DNA replication and RNA and protein synthesis, in both normal and abnormal cells. Thus, cytotoxic effects of interferon are not restricted to tumor or virus infected cells but are also manifested in normal, healthy cells as well. As a result, undesirable side effects arise during interferon therapy, particularly when high doses are required. Administration of interferon can lead to myelosuppression resulting in reduced red blood cell, white blood cell and platelet levels. Higher doses of interferon commonly give rise to flu-like symptoms (e.g., fever, fatigue, headaches and chills), gastrointestinal disorders (e.g., anorexia, nausea and diarrhea), dizziness and coughing.
  • flu-like symptoms e.g., fever, fatigue, headaches and chills
  • gastrointestinal disorders e.g., anorexia, nausea and diarrhea
  • Natural human interferon-beta is a glycoprotein with an apparent 22-23 kDa molecular weight and an antiviral specific activity of 2 - 5 X 10 8 international units (IU)/mg protein.
  • Human IFN- ⁇ has been clinically applied to the treatment of glioma, melanoma, viral dermatropic diseases and hepatitis; see Interferon, Principles and Medical Applications, 1st Edition (1992, Univ. of Texas Medical Branch of Galveston) pages 107-116.
  • U.S. Patents Nos. 4,695,623 and 4,897,471 disclose novel interferon polypeptides having amino acid sequences which include common or predominant amino acids found at each position among naturally-occurring alpha interferon subtype polypeptides, and are referred to as consensus interferons (IFN-con) .
  • the specific IFN-con amino acid sequences disclosed are designated IFN-coni, IFN-con 2 , and IFN-con 3 .
  • the preparation of manufactured genes encoding IFN-con and the expression of said genes in E. coli are also disclosed.
  • IFN-con ⁇ produced in £. coli is described in Klein et al. ( J. Chromatog. 454, 205-215 (1988)). IFN-coni purified in this manner is reported to have a specific activity of 3 x 109 units/mg. protein as measured in the cytopathic effect inhibition assay using the T98G human cell line (Fish et al. J. Interferon Res . , 97-114 (1989)).
  • U.S. Patent No. 5,372,808 discloses methods of treatment of diseases using consensus interferon. It is shown that IFN-con, when used in the treatment of diseases susceptible to treatment by alpha interferons, does not cause the same degree of side effects in patients as do the alpha interferons.
  • the present invention encompasses neoglycosylated analogs of recombinant consensus interferon.
  • the invention is based on the discovery that multiple lactose ligands can be conjugated to the IFN-con to give neoglycosylated forms of IFN-con
  • lacIFN-con (lacIFN-con) .
  • the lactose ligands are conjugated through the ⁇ -NH of lysine and the N-terminal amine of the IFN-con.
  • lacIFN-con is highly targeted to the liver.
  • IFN-con is a polypeptide having the amino acid sequence of IFN-coni, IFN-con 2 , or IFN-con 3 , and the conjugate will have between 1 and 10 glycosyl residues per IFN-con molecule.
  • IFN-con has the amino acid sequence of IFN-coni, and the neoglycosylated IFN-con will have between 4 and 7 lactose residues for each IFN-con molecule.
  • the invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of lacIFN-con along with suitable diluents, adjuvants, carriers, preservatives and/or solublizers. These compositions may provide therapeutic benefit in the treatment of those conditions currently susceptible to treatment with IFN-con.
  • the present invention also encompasses a method of delivering an interferon directly to the liver of a patient, comprising administering to the patient a therapeutically effective amount of a chemically modified interferon, wherein the chemically modified interferon is comprised of an interferon conjugated to at least one glycosyl ligand, wherein said ligand comprises a terminal galactose, and wherein said interferon is selected form the group consisting of IFN- ⁇ , IFN- ⁇ , and IFN-con.
  • Figure 1 is a schematic diagram showing the preparation of lacIFN-con from IFN-con and lactose through reductive amination.
  • Figure 3 is a graphical representation of the biodistribution of radiolabeled IFN-con in rats. The % injected dose was determined at indicated time intervals.
  • Figure 4 is a graphical representation of the biodistribution of radiolabeled lacIFN-con in rats. The % injected dose was determined at indicated time intervals.
  • Figure 6 is a graphical representation of 2-5A synthetase activity measured in hamster serum after intravenous administration of IFN-con (- ⁇ -), lacIFN-con
  • Figure 7 is a graphical representation of the survival rate of untreated EMCV-infected hamsters (-0-), EMCV-infected hamsters preinjected with IFN-con (- ⁇ -), or EMCV-infected hamsters pretreated with lacIFN-con (-0-) . The animals were monitored twice daily for 27 days.
  • consensus human leukocyte interferon means a nonnaturally-occurring polypeptide, which predominantly includes those amino acid residues that are common to all naturally-occurring human leukocyte interferon subtype sequences and which includes, at one or more of those positions where there is no amino acid common to all subtypes, an amino acid which predominantly occurs at that position and in no event includes any amino acid residue which is not extant in that position in at least one naturally- occurring subtype.
  • IFN-con encompasses but is not limited to the amino acid sequences designated IFN-coni, IFN-con 2 and IFN-con 3 which are disclosed in commonly owned U.S. Patents 4,695,623 and 4,897,471, the entire disclosures of which are hereby incorporated by reference. DNA sequences encoding IFN-con may be synthesized as described in the above-mentioned patents or other standard methods.
  • IFN-con polypeptides are preferably the products of expression of manufactured DNA sequences transformed or transfected into bacterial hosts, especially E. coli . That is, IFN-con is preferably recombinant IFN-con. Recombinant IFN-con is preferably produced in E. coli and is purified by procedures known to those skilled in the art, as generally described in Klein et al. , supra (1988) for IFN-coni.
  • Purified IFN-con may comprise a mixture of isoforms, e.g., purified IFN-coni may comprise a mixture of methionyl IFN-coni, des-methionyl IFN-coni an des-methionyl IFN-coni with a blocked N-terminus (Klein et al. , supra (1990) ) .
  • IFN-con may comprise a specific, isolated isoform. Isoforms of IFN-con are separated from each other by techniques such as isoelectric focusing which are known to those skilled in the art .
  • the chemically modified IFN-con of the present invention will be comprised of at least one terminal galactose-containing glycosyl ligand per protein.
  • Such glycosyl ligands will be selected from the group consisting of galactose, terminal glycosyl oligosaccharides, asialoglycopeptides, and asialoglycoproteins.
  • the glycosyl ligand is a monosaccharide, it will generally be galactose.
  • the oligosaccharides of this invention are generally those having at least two sugar moieties, wherein, in each case, galactose residue(s) will be attached to the terminal end(s) of the oligosaccharide.
  • Some representative oligosaccharides useful for attachment to IFN-con are lactose, N-acetyllactosamine and galactan.
  • the neoglycosylated IFN-con analog will contain four to seven ligands and the glycosyl ligand will be an oligosaccharide.
  • the neoglycosylated IFN-con analog will contain four to seven ligands and the glycosyl ligand will be lactose.
  • the multiple ligands are preferably identical, but mixtures of ligands are also encompassed by this invention.
  • the neoglycosylated IFN-con analogs of the present invention can be obtained by a number of conventional methods. Such methods have been very well summarized; see Kataoka and Tavassoli, Jour, of Histochem . and Cytochemistry, 3_2: 1091-1098 (1984); Chipowsky and Lee, Carbohy. Research , 3_1: 339-346 (1973) and references cited therein.
  • the neoglycosylated IFN-con analogs are obtained by a method which comprises conjugating multiple glycosyl ligands to the protein backbone through active amino acid residue(s) such as lysine, cysteine, serine, threonine, or asparagine.
  • active amino acid residue(s) such as lysine, cysteine, serine, threonine, or asparagine.
  • IFN-con analogs are obtained by a method which comprises conjugating multiple glycosyl ligands through the ⁇ -NH 2 of lysine and/or the N-terminal a ine of the IFN-con.
  • This chemical modification is achieved by reacting glycosyl ligands with IFN-con under certain reaction conditions, preferably non-denaturing conditions, in sufficient amounts such that the aminc groups are accessible for the reductive aminaticn.
  • the glycosyl ligand is lactose and the reactions are carried out at pH 7.0, require the addition of a reducing agent, e.g.
  • the invention also encompasses pharmaceutical compositions which comprise a therapeutically effective amount of lacIFN-con in a mixture with a pharmaceutically acceptable carrier, diluent, preservative, solublizer, adjuvants and/or emulsifier; see Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • Substantially homogenous as used herein means that the only neoglycosylated IFN-con analogs observed are those having four to seven ligands attached per protein.
  • the preparation may contain unreacted (i.e., lacking glycosyl ligands) protein.
  • the chemically modified IFN-con is at least 90% one product (as in the working example below) and most preferably, the chemically modified IFN-con is >98% one product.
  • the chemically modified IFN-con contemplated by the present invention are those analogs which are active in a biological assay such as that described ir. Example 1.
  • the analogs will have activity of at lease 50% compared to that of native IFN-con.
  • the analogs will have activity of at least 60%. Those skilled in the art will be able to readily evaluate such analogs and determine whether they demonstrate activity in such assays.
  • compositions of the present invention can be used in the same manner as that described previously for IFN-con and it is contemplated that the compositions will be used for treating those conditions treatable ith a consensus interferon.
  • exemplary conditions include, but are not limited to, cell proliferation disorders and viral infections.
  • Viral conditions treatable by IFN-con include, but are not limited to, hepatitis A, hepatitis C, other non-A, non-B hepatitis, hepatitis B, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adenovirus, rhino virus, HTLV I, HTLV II, and human rotavirus.
  • IFN-con is also effective in treating cell proliferation disorders frequently associated with cancer. Such disorders include, but are not limited to, hairy cell leukemia and Kaposi's Sarcoma.
  • the conditions to be treated are hepatic disorders.
  • IFN-con may be used alone or in combination with one or more factors that stimulate myeloid cell proliferation or differentiation, such as granulocyte colony stimulating factor (G-CSF) , granulocyte/ macrophage colony stimulating factor (GM-CSF) , interleukin-1 (IL-1), interleukin-3 (IL-3), interleukin-6 (IL-6) , erythropoietin, stem cell factor (SCF) , and megakaryocyte growth and development factor (MGDF) .
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte/ macrophage colony stimulating factor
  • IL-1 interleukin-1
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • SCF stem cell factor
  • MGDF megakaryocyte growth and development factor
  • IFN-con modified with ligands containing terminal galactose residues will be selectively bound to asialoglycoprotein-binding receptors on the hepatocyte cell surface, it is further contemplated that the modified IFN-con will be particularly useful in the treatment of hepatic disorders such as HCV.
  • the present invention also contemplates a method of delivering an interferon directly to the liver of a patient, comprising administering to the patient a therapeutically effective amount of a chemically modified interferons of the present invention.
  • Interferons contemplated for use in such methods include IFN- ⁇ , IFN- ⁇ , and IFN-con.
  • IFN- ⁇ and IFN- ⁇ contain lysine residues which allow for chemical modification by glycosyl ligands in a similar manner as that described for IFN-con in the detailed examples.
  • the amount of the chemically modified interferon that will be effective in the treatment of a particular disorder will depend on the nature of the disorder, and other factors, and can be determined by standard clinical techniques or based on dosage amounts or regimens already established for interferon.
  • This example demonstrates chemically modified human recombinant consensus interferon. More specifically, this example demonstrates a method of preparing a homogeneous preparation of neoglycosylated IFN-coni, and characterization of the preparation.
  • IFN-coni as described in Figure 2 of U.S. Patent No. 4,695,623, which is incorporated by reference in its entirety, was used for preparation of the neoglycosylated human recombinant consensus interferon.
  • the IFN-coni was produced by expression of exogenous DNA in bacteria, and contained a methionyl residue at the N-terminus.
  • LacIFN-con was prepared by conjugating multiple lactose ligands through the ⁇ -NH 2 of lysine and the N-terminal amine of the IFN-con as depicted in
  • Figure 1 In general, the conjugation reaction can be described as follows: ⁇ -D-Lactose (Sigma, St. Louis, MO) was dissolved in phosphate buffer saline (PBS) containing IFN-coni at various lactose: IFN-con molar ratios. The reaction was stirred at room temperature for various lengths of time, and sodium cyanoborohydride (NaBH 3 CN) (Sigma, St. Louis, MO) was added to the reaction mixture twice a day. The progression of the reactions was monitored on 16% SDS-PAGE. The reaction is then stopped by the passing the mixture through a phosphate buffer saline (PBS) containing IFN-coni at various lactose: IFN-con molar ratios. The reaction was stirred at room temperature for various lengths of time, and sodium cyanoborohydride (NaBH 3 CN) (Sigma, St. Louis, MO) was added to the reaction mixture twice a day. The progression of the reactions was monitored on 16%
  • the proteins were further purified on FPLC with a Pharmacia HiLoad Superdex 75pg 26/60 column (Piscataway, NJ) eluted with PBS at 1.2 ml/min flow rate and monitored for absorption at 280 nm. Fractions of 2 ml were collected and the protein fractions pooled.
  • the purified lacIFN-con preparations were analyzed on gel filtration HPLC with a Pharmacia Superose 12 H/R 10/30 column (Piscataway, NJ) or a Phenomenex BioSep
  • ⁇ EC-2000 column (Torrance, CA) at 0.5 ml/min eluted with PBS.
  • the derivatized proteins were characterized using 16% SDS-PAGE gels (Novex, San Diego, CA) and pH 3-10 isolectric focusing gels (Novex, San Diegeo, CA) and mass spectroscopy.
  • the reaction condition was optimized by studying various factors such as the reaction pH, reaction time, and reagent concentra ion.
  • the pH effect was studied by conducting reaction in pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 PBS at room temperature for 3 days. The progression of the reactions was monitored on 16% SDS-PAGE.
  • the degree of derivatization was greater at higher pH as the amino groups were less protonated and more accessible for the nucleophilic attack to the aldose form of lactose.
  • Based on results from gel filtration HPLC no more than 10% of the derivatized proteins were found as dimer or aggregate at all reaction conditions except for the one conducted at pH 5.5, since the pi for IFN-con is 5.7.
  • the amount of reducing agent added to the reactions was found to directly affect the reaction progression and the degree of protein denaturation.
  • Sodium cyanoborohydride was dissolved in water into a 10M solution just prior to addition to the reaction in order to preserve protein stability.
  • As high as 20,000 molar excess reducing agent was added twice a day during the reaction to facilitate the reaction progression while minimum amount of aggregation was detected under this treatment.
  • the degree of glycosylation, and in vi tro bioactivity of the derivatized proteins were examined.
  • the lacIFN-con used for this analysis was prepared as follows: one gram of ⁇ -D-Lactose was dissolved in 5 ml
  • IFN-con was determined by mass spectroscopy using Kra os Kompact MALDI-TOF mass spectrometer. The number of lactose attached to IFN-con was calculated by subtracting 19,516 from the analog molecular weight, and then dividing that number by 326 (lactose - oxygen) . The in vi tro bioactivity was determined by measurement of the inhibition of viral replication in a cultured cell line.
  • HeLa cells were plated into 96-well plates at 15,000 cells/well and incubated for twenty four hours at 37°C under 5% carbon dioxide in base medium (Dulbecco's modified Eagles medium (DMEM) , containing 100 units/ml of penicillin, 100 mg/ml of streptomycin, 2 mM L-glutamine, 1% by weight of non- essential amino acids, 0.1% by weight of gentamicin sulfate and 1% HEPES buffer) , with 10% FBS.
  • IFN-con and lacIFN-con was prepared at multiple dilutions in base medium and 0.2% FBS. One hundred microliters of each standard and appropriately diluted IFN-con and lacIFN- con were added to each well.
  • the medium was aspirated and replaced with 100 microliters of the challenge virus, i.e., Encephalomyocarditis virus (EMCV) , at a dilution equal to 100-1000 tissue culture infected dose (TCID) units in DMEM with 1% FBS.
  • EMCV Encephalomyocarditis virus
  • TCID tissue culture infected dose
  • the plates were further incubated for twenty two hours, the medium removed, and the cells were fixed with 200 microliters of anhydrous methyl alcohol for five minutes. The fixative is removed and the cells are stained for thirty minutes in 0.5% Gentian dye, then rinsed free of dye and air-dried for one to two hours. The dye was eluted with two hundred microliter of ethylene glycol monoethyl ether and shaken for thirty minutes.
  • the absorbance of each well at 650 nm was determined in a Vmax Kinetic Microplate Reader, model 88026 (Molecular Devices) .
  • the results for the standard were graphed as the log concentration of IFN-con versus the percentage of dye uptake,- and the bioactivity of the IFN-con and lacIFN-con determined.
  • This example relates to biodistribution studies using IFN-con and lacIFN-con as prepared in
  • Example 1 The tissue-targeting ability of the lactose conjugated IFN-con was determined by studying the total body distribution of the 125 I-labeled protein in hamsters and in rats.
  • mice Male Syrian-Golden hamsters (85-130 g) were obtained from Charles River Laboratories and maintained on a 12/12 light dark cycle in a controlled environment of 21°C. A diet of Purina Chow and water was available ad libitum. The animals were caged in groups of two and five, and were allowed one week to acclimate to the facility after arrival. Three hamsters (85-130 g) per group were given the 125 I-labeled proteins at a dose of 2 mg/kg (2 x 10 7 cpm/hamster) intravenously through the penile vein. At 3, 15, 30, 60 and 120 minutes, the animals were sacrificed and the radioactivity associated with the organs was determined. Percent injected dose per gram of organ weight was measured at various time points. The data is summarized in Table 2. Table 2
  • Radioactivity in one lobe of liver was measured and calculated for whole organ.
  • mice Male Sprague-Dawley rats (250-300 g) were obtained from Charles River Laboratories and maintained on a 12/12 light dark cycle and in a controlled environment of 21°C. A diet of Purina Chow and water was available ad libitum. The animals were caged in groups of two and five. Three rats (250-300 g) per group were given the 125 I-labeled proteins at a dose of 12 ⁇ g/kg (2 x 106 cpm/rat) intravenously through the penile vein. At 5, 60 and 360 minutes, the animals were sacrificed and the whole organs were removed to determine the total associated radioactivity. Percent injected dose per gram of organ weight was measured at various time points. The data is summarized in Table 3.
  • kidney 4 18.81(2.11) 2.42(1.91) 0.68(0.02)
  • Radioactivity in one lobe of liver was measured and calculated for whole organ.
  • the lacIFN-con rapidly accumulated in the liver after 3-5 minutes, and the amount of native IFN-con accumulated in the liver was less than one third as compared to lacIFN- con (see also Figures 3 and 4) . Furthermore, less than 1% dose was accumulated in the spleen, duodenum, brain and heart, and the amount of lacIFN-con found in the serum was 40% the amount of native IFN-con. Therefore, despite the fact that the in vi tro bioactivity of lacIFN-con is lower (see Example 1), the lacIFN-con is still advantageous in that it is targeted specifically to the liver and may provide improved treatment of certain hepatic disorders.
  • the in vi tro assay in Example 1 used EMCV as a marker and EMCV is not a liver virus.
  • This example relates to pharmacokinetic analysis in hamsters using IFN-con and lacIFN-con as prepared in Example 1.
  • IFN-con and lacIFN-con was administered intravenously through the penile vein to male Syrian Golden hamsters (85-130 g) at a dose of 300 ⁇ g/kg.
  • Serum samples were obtained using serum separation tubes (Becton Dickinson) .
  • the serum samples were analyzed with a sandwich-type ELISA assay.
  • the primary, immobilizing antibody polyclonal rabbit derived anti-CIFN antibody
  • the secondary antibody mouse-derived monoclonal anti-IFN IgGl
  • Three different batches of rabbit polyclonal anti-IFN antibody were screened.
  • the detection antibody was goat-derived anti-mouse IgGl conjugated with horse radish peroxidase (Boehringer Mannheim) , and the color development from the treatment of 3, 3 ' , 5, 5 ' -tetramethylethylenedia ine (TMB) peroxidase substrate (Boehringer Mannheim) was used to determine the concentration of CIFN and lac-CIFN in the serum samples.
  • TMB trimethylethylenedia ine
  • the sensitivity of this assay has a linear correlation for consensus interferon concentration between 0.156 to 10 ng/mL.
  • the initial distribution half life (alpha-T ⁇ /2 ) for both proteins v;as -11 minutes and the terminal clearance half life (beta- T 1/2 ) for IFN-con and lacIFN-con was 1.3 and 1.0 hr respectively.
  • the AUC for IFN-con and lacIFN-con was 1817 and 691 ng-hr/mL, and Vss was 198 and 434 mL/kg respectively.
  • the in vivo bioactivity of IFN-con and lacIFN-con was determined in hamsters by measuring the plasma level of 2' , 5'-oligoadenylate synthetase (2-5A synthetase) .
  • 2-5A synthetase is an enzyme produced as a direct response of interferon binding to its receptor and is believed to be the first step in the mechanism of antiviral activity; see Interferon, Principles and Medical Applications, 1st Edition (1992, Univ. of Texas Medical Branch of Galveston) pages 225-237.
  • Three male Syrian Golden hamsters (85-130 g) per group were intravenously administered through the penile vein with 300 ⁇ g/kg of IFN-con and lacIFN-con.
  • the vehicle groups were dosed with 100 ⁇ L of PBS.
  • the animals were sacrificed at 6, 12, 24, 48, 72 and 96 hr and blood samples were obtained by cardiac puncture.
  • the 2-5A synthetase activity in the serum was assayed using the 2-5A RIA kit (Eiken, Kitaku, Japan) using the method described by Sawai et al . , Biomed. Res . , 9.:59-66 (1988) .
  • the 2-5A synthetase produced in hamster serum after intravenous administration of IFN-con and lacIFN- con were compared (see Figure 6) .
  • Minimum amounts of 2-5A synthetase activity was found in the hamster vehicle groups and the time zero group, indicating the injection procedure induced very low background level of enzyme production of hamsters.
  • the enzyme activity was found elevated in a biphasic manner. The first peak was found at 24 hour after the injection and the enzyme activity was found to be seven times higher than that of the vehicle group. The enzyme activity was decreased down to base-line level at 48 hours, and elevated again at 72 hours to four times higher than that of the vehicle group. At 96 hours, the enzyme activity was lowered down to base-line level.
  • Encephalomyocarditis virus (EMCV) and then comparing the survival rates.

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Abstract

La présente invention se rapporte à un interférent de consensus chimiquement modifié possédant au moins un ligand glycosyle, ce ou ces ligands glycosyles étant conjugués à l'interférent de consensus. Des compositions pharmaceutiques contenant cet analogue et des procédés de traitement utilisant les compositions de l'invention sont également décrits.
EP96903420A 1995-01-13 1996-01-11 Interferent chimiquement modifie Withdrawn EP0804248A2 (fr)

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EP2042196B1 (fr) * 2001-10-10 2016-07-13 ratiopharm GmbH Remodelage et glycoconjugation de facteur stimulant les colonies de granulocytes (G-CSF)
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
WO2006127896A2 (fr) 2005-05-25 2006-11-30 Neose Technologies, Inc. Facteur ix glycopegyle
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
TW201245229A (en) 2003-10-14 2012-11-16 Hoffmann La Roche Macrocyclic carboxylic acids and acylsulfonamides as inhibitors of HCV replication
US20060040856A1 (en) 2003-12-03 2006-02-23 Neose Technologies, Inc. Glycopegylated factor IX
EP3061461A1 (fr) 2004-10-29 2016-08-31 ratiopharm GmbH Remodelage et glycopegylation du facteur de croissance des fibroblastes (fgf)
JP5249028B2 (ja) 2005-07-25 2013-07-31 インターミューン・インコーポレーテッド Ｃ型肝炎ウイルス複製の新規大環状阻害剤
GEP20104956B (en) 2005-10-11 2010-04-12 Array Biopharma Inc Compounds for inhibiting hepatitis c viral replication and use thereof
AR078117A1 (es) 2006-06-20 2011-10-19 Protech Pharma S A Una muteina recombinante del interferon alfa humano glicosilado, un gen que codifica para dicha muteina, un metodo de produccion de dicho gen, un metodo para obtener una celula eucariota productora de dicha muteina, un metodo para producir dicha muteina, un procedimiento para purificar dicha muteina
DK2257311T3 (da) 2008-02-27 2014-06-30 Novo Nordisk As Konjugerede Faktor VIII-Molekyler
US20110027229A1 (en) 2009-07-31 2011-02-03 Medtronic, Inc. Continuous subcutaneous administration of interferon-alpha to hepatitis c infected patients
CN102711871A (zh) 2010-06-16 2012-10-03 麦德托尼克公司 用于稳定药物递送装置中的药物的阻尼系统

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BR9607599A (pt) 1998-06-09
IL116730A0 (en) 1996-05-14
AU4751896A (en) 1996-07-31
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JPH10513440A (ja) 1998-12-22
ZA96206B (en) 1996-08-19

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