WO1996021468A2

WO1996021468A2 - Chemically modified interferon

Info

Publication number: WO1996021468A2
Application number: PCT/US1996/000323
Authority: WO
Inventors: David Collins; Jennifer Lin-Chun Liu
Original assignee: Amgen Inc.
Priority date: 1995-01-13
Filing date: 1996-01-11
Publication date: 1996-07-18
Also published as: JPH10513440A; EP0804248A2; MX9705119A; BR9607599A; IL116730A0; ZA96206B; WO1996021468A3; KR19980701814A; AU4751896A; CA2209557A1

Abstract

The present invention relates to chemically modified consensus interferon having one or more glycosyl ligand(s) conjugated to consensus interferon. Pharmaceutical compositions containing such analog, and methods of treatment using the compositions of the invention are also provided.

Description

CHEMICALLY MODIFIED INTERFERON

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.

BACKGROUND OF THE INVENTION

Interferonε are a subclass of cytokines that exhibit both antiviral and antiproliterative activity. On the basis of biochemical and immunological properties, human interferons are grouped into three classes: interferon-alpha (leukocyte), interferon-beta (fibroblast) and interferon-gamma (immune) . 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.

In addition to the labeled indications, 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. "Principles and Applications of Biologic Therapy" in Cancer : Principles and Practices of Oncology, 3rd ed. , Devita et al. , eds. pp. 301-547 (1989), Balmer DICP, Ann Pharmac other 2A, 761-768 (1990)) .

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.

Natural human interferon-beta is a glycoprotein with an apparent 22-23 kDa molecular weight and an antiviral specific activity of 2 - 5 X 10⁸ international units (IU)/mg protein. Human IFN-βhas 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-con₂, and IFN-con₃. The preparation of manufactured genes encoding IFN-con and the expression of said genes in E. coli are also disclosed.

A purification of IFN-conχ 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. It was further shown that 3 to 5 times higher doses of IFN-con can be used, leading to enhanced therapeutic benefit, with substantially no corresponding increase in the frequency or severity of undesirable side effects. Because the interferon dosage form that will achieve the greatest therapeutic effect, with reduced adverse side effects, remains unclear, the need exists to develop improved forms of the interferons for therapeutic use. The development of interferon forms which would allow for high dosage without the undesirable side effects associated with interferon therapy would be of great benefit. It is therefore an object of the present invention to provide improved forms of interferon comprising conjugates of galactose residues to interferon, for use in treatment of conditions that are susceptible to interferon treatment. It is a further object of the present invention to provide compositions which allow for selective delivery of interferon to the liver, thereby increasing the opportunity of interferon binding to hepatocyte cell surface receptors, and diminishing the undesired binding of interferon to other organs during the antiviral treatment of such disease states as chronic HCV.

SUMMARY OF THE INVENTION

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) . Preferably, the lactose ligands are conjugated through the ε-NH of lysine and the N-terminal amine of the IFN-con. As compared to native consensus interferon, lacIFN-con is highly targeted to the liver. Preferably, IFN-con is a polypeptide having the amino acid sequence of IFN-coni, IFN-con₂, or IFN-con₃, and the conjugate will have between 1 and 10 glycosyl residues per IFN-con molecule. Most preferably, 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-α, IFN-β, and IFN-con.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the preparation of lacIFN-con from IFN-con and lactose through reductive amination.

Figure 2 is a graphical representation of the percentage of injected radiolabeled IFN-con (-□-) and lacIFN-con (-0-) accumulated in the liver of hamsters at indicated time intervals. The bars represent standard deviation (n=3) .

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 5 is a graphical representation of hamster serum pharmacokinetics of IFN-con (-Q-) and lacIFN-con (-O- ) after intravenous administration (300 μg/kg ) . Serum interferon concentration is plotted versus time. The bars represent standard deviation (n=3) .

Figure 6 is a graphical representation of 2-5A synthetase activity measured in hamster serum after intravenous administration of IFN-con (-□-), lacIFN-con

(-0-), and phosphate buffer vehicle group (-Δ-) at various time points. The bars represent standard deviation (n=3) .

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.

DETAILED DESCRIPTION OF THE INVENTION

As employed herein, consensus human leukocyte interferon (IFN-con) 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-con₂ and IFN-con₃ 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) ) . Alternatively, 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. When 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. Preferably, the neoglycosylated IFN-con analog will contain four to seven ligands and the glycosyl ligand will be an oligosaccharide. Most preferably, 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. Preferably, 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. Most preferably, the neoglycosylated

IFN-con analogs are obtained by a method which comprises conjugating multiple glycosyl ligands through the ε-NH₂ 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. In a preferred embodiment, the glycosyl ligand is lactose and the reactions are carried out at pH 7.0, require the addition of a reducing agent, e.g. sodium cyanoborohydride, in large molar excess, and are controlled such that the analog obtained contains four to seven lactose ligands per protein. The means used to control such reactions are readily determined by those skilled in the art. Because the methods of this invention provide a substantially homogeneous modified IFN-con preparation, 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. Preferably, 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. Preferably, 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.

In general, the 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. Preferably, 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) . It is contemplated by the present invention that treatment with chemically modified IFN-con will provide improved efficacy with substantially reduced or eliminated side effects as compared to treatment with alpha interferon. The reduction or elimination of side effects is expected to be demonstrated regardless of the condition being treated. More particularly, because 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.

Because the chemically modified interferons of the present invention are directed to the liver, 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-α, IFN-β, and IFN-con. IFN-α and IFN-β 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.

The following examples will illustrate in more detail the various aspects of the present invention.

EXAMPLE 1

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.

A. Preparation of Consensus Interferon

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. B. Preparation of LacIFN-con

LacIFN-con was prepared by conjugating multiple lactose ligands through the ε-NH₂ 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: β-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 (NaBH₃CN) (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

Sephadex G-25 column (Sigma, St. Louis, MO) eluted with PBS to remove unreacted lactose and NaBH₃CN.

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

ΞEC-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.

C. Characterization of LacIFN-con

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 β-D-Lactose was dissolved in 5 ml

PBS containing 12.5 mg IFN-coni. The reaction was stirred at room temperature for four days and 50 mg of sodium cyanoborohydride (NaBH₃CN) was added to the reaction mixture twice a day. The molecular weight of lactose-modified

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. After further incubation for 19-23 hours, 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. 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.

The results of the above analysis on four separate preparations is shown in Table I. Potency was compared using IFN-con activity = 1. Table 1

Preparat on jL of. ,ς Potencv

IFN-con 0 1 lacIFN-con #1 4 .55 lacIFN-con #2 5 .60 lacIFN-con #3 6 .62 lacIFN-con #4 7 .55

The Table I data indicates that the optimum reaction condition gave a lacIFN-con with an average of five-six lactose residues attached to IFN-con. These lacIFN-con analogs had an average in vi tro bioactivity of 6.4 X 10⁸ U/mg as compared to 11 X 10⁸ U/mg for IFN- con.

EXAMPLE 2

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 ¹²⁵I-labeled protein in hamsters and in rats.

A. Iodination of IFN-con and lacIFN-con

The iodination procedure was slightly modified from that described in Fraker and Speck, Biophys . Res .

Coπun. , £_: 849-857 (1978) . In a 12 x 75 mm borocilicate test tube was weighed 3 mg of IODO-GEN (Pierce) which was then dissolved in 100 μL CHCI₃ and dried under a gentle stream of N₂ to form a thin film in the tube. (¹²⁵I) sodium iodine (12 μL, 1.2 mCi) (New England

Nuclear) and 600 μg protein in PBS were added to the tube and the reaction was incubated for 10 min on ice. The reaction was transferred to a 1.5 mL eppendorf tube containing 20 μL of parahydroxybenzoate (110 nmol) and incubated for 10 min on ice. The iodinated protein was purified with PD-10 columns (Pharmacia) pre-equilibrated stepwisely with 25 mL of PBS containing 1% BSA and 0.05% Triton X100, and 25 mL of PBS. Fractions of 0.5 mL were collected and counted for radioactivity. The fractions containing the iodinated proteins were pooled and the contamination due to unbound 125- _was determined by precipitation with a 6% final solution of trichloric acid.

B. Biodistribution analysis in hamsters

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 ¹²⁵I-labeled proteins at a dose of 2 mg/kg (2 x 10⁷ 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

Biodistribution of IFN-con and lacIFN-con in hamsters¹

Tissue 3 mm 15 mm 30 mm 60 mm 120 min blood² 1.84(1.18) 4.23(0.52) 2.63(0.25) 1.30(0.13) 1.14(0.33) 4.94(0.8) 2.37(0.34 1.99(0.2) 1.15(0.0) 0.90(0.2) liver^ 1.58(0.76) 1.42(0.18) 0.95(0.11) 0.75(0.51) 0.33 (0.06) 4.71(0.4) 4.18(0.2) 2.04 (0.1) 0.72(0.0) 0.48(0.0) kidney* 25.80(9.6) 67.54(11) 23.08(2.5) 13.28(0.8) 5.09(0.8) 20.9(3.6) 36.8(3.9) 23.2(5.2) 11.1(0.2) 5.28(0.2) spleen 0.58(0.18) 0.44(0.18) 0.29(0.11) 0.28(0.01) 0.14(0.03) 0.36 ( .05) 0.62 ( .04) 0.19 ( .02) 0.26 ( .08) 0.18 ( .02) lung 0.21(0.02) 0.24(0.01) 0.26(0.00) 0.16(0.05) 0.38(0.16) 0.19 ( .01) 0.20 ( .11) 0.20 ( .02) 0.08 ( .01) 0.06 ( .01) duodenum-¹ 0.66(0.02) 0.59(0.05) 0.70(0.00) 1.04(0.13) 0.65(0.11) 0.36 ( .04) 0.49 ( .02) 0.78 ( .09) 0.48 ( .13) 0.69 ( .37) brain 2.05(0.84) 1.11(0.06) 0.77(0.09) 0.76(0.04) 0.65(0.06) 0.75 ( .11) 0.34 ( .01) 0.57 ( .03) 0.25 ( .00) 0.38 ( .28) heart 1.75(0.29) 0.97(0.14) 0.99(0.19) 0.42(0.03) 0.47 '2.02)

1.29 ( .34) 0 68 ( .15) 0 61 ( .02) 0 43 ( .05) 0.27 ( .05)

¹ The numbers for IFN-con are on top and the numbers for lacIFN- con are on the bottom in bold. Number in parentheses are S . 2 (n = 3) . 2 Calculated from collected blood and assuming that 7.3 % of :..e total body weight is blood.

3 Radioactivity in one lobe of liver was measured and calculated for whole organ.

⁴ Radioactivity m one kidney was measured and doubled for bo r. kidney.

⁵ Radioactivity in 10 cm of duodenum was measured. As depicted in Table 2 and in Figure 2, intravenous injection gave an instantaneous distribution of the drugs in the systemic circulation, and the lacIFN-con rapidly accumulated in the liver after 3-5 minutes. The amount of native IFN-con accumulated in the liver was less than one third as compared to lacIFN- con. Furthermore, the accumulation remained higher than that of native IFN-con for at least 30 minutes.

C. Biodistribution analysis in rats

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 ¹²⁵I-labeled proteins at a dose of 12 μg/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.

a e

Biodistribution of IFN-con and lacIFN-con in rats¹

Tissue 5 min 60 min 360 min blood² 29.92(1.61) 6.67(0 .21) 2.69(0.05)

5.96(0.16) 4.56(0.27) 2.29 (0.13) liver³ 11.01(0.45) 3.30(0.20) 1.46(0.06)

40.14(1.73) 4.85(0.37) 1.64(0.12) kidney⁴ 18.81(2.11) 2.42(1.91) 0.68(0.02)

6.69(0.33) 1.50(0.34) 0.37 (0.02) spleen 0.66(0.03) 0.18(0.02) 0.06(0.01)

0.15(0.02) 0.11(0.02) 0.05(0.01) lung 0.70(0.05) 0.32(0.04) 0.10(0.00)

0.21(0.03) 0.45(0.05) 0.84(0.01) duodenum⁵ 0.31(0.01) 0.43(0.01) 0.11(0.01)

0.13(0.06) 0.22 (0.02) 0.18(0.04)

¹ The numbers for IFN-con are on top and the numbers for lacIFN- con are on the bottom in bold. Number in parentheses are S.D (n = 3) . ² Calculated from collected blood and assuming that 7.3 % of the total body weight is blood.

³ Radioactivity in one lobe of liver was measured and calculated for whole organ.

⁴ Radioactivity in one kidney was measured and doubled for both kidney.

⁵ Radioactivity in 10 cm of duodenum was measured.

Again, as with the hamster study, 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.

EXAMPLE 3

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 μg/kg. At 0.05, 0.17, 0.25, 0.5, 1, 2, 4, 6 and

8 hr, groups of three animals were sacrificed and blood samples (100 μL) were collected by cardiac puncture.

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) , and the secondary antibody (mouse-derived monoclonal anti-IFN IgGl), were generated at Amgen Inc. 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. The sensitivity of this assay has a linear correlation for consensus interferon concentration between 0.156 to 10 ng/mL.

Both native and lactose conjugated IFN-con were able to bind to anti-IFN antibody but with different degrees of affinity. LacIFN-con has a 25% binding affinity compared to IFN-con to all of the polyclonal antibodies, and a linear correlation was obtained for the protein concentration between 0.156 - 2.0 ng/mL. IFN-con and lacIFN-con showed a similar biphasic clearance mechanism and no significant difference in the Tχ_/2 was found (see Figure 5). When given the same dose, lacIFN-con concentration in hamster serum was quickly dropped to about 50 % that of IFN-ccn 3 min after the injection and then maintained about the same ratio throughout the 8 hr period. The initial distribution half life (alpha-Tι_/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 area under the curve (AUC) and the apparent volume of distribution (Vss) for IFN-con and lacIFN-con, however, were very different. The AUC for IFN-con and lacIFN-con was 1817 and 691 ng-hr/mL, and Vss was 198 and 434 mL/kg respectively.

EXAMPLE 4

In this example 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 μg/kg of IFN-con and lacIFN-con. The vehicle groups were dosed with 100 μL 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. When treated with IFN- con, 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.

For lacIFN-con, the enzyme activity was found greatly increased at 48 hours and lasted to 96 hours. The elevation of the 2-5A synthetase activity was ten times higher than that of the vehicle group at 48 hours. These results indicate a different enzyme production pattern for IFN-con and lacIFN-con, suggesting that lacIFN-con might stimulates very different antiviral activity as compared to IFN-con, and possibly related to the different biodistribution profiles of the two proteins. EXAMPLE 5

In this example, the in vivo antiviral activity of IFN-con and lacIFN-con were studied by challenging the hamsters receiving the proteins with

Encephalomyocarditis virus (EMCV) and then comparing the survival rates.

Ten male Syrian-Golden hamsters (85-130 g) per group were injected intraperitoneally with IFN-con and lacIFN-con at dose of 10 μg/kg. Six hours later, each hamster was then injected intraperitoneally with 100 μL (2E+4 pfu/mL) of EMCV. The animals were monitored twice daily, and the end point was the sign of hind quarter paralysis. Hamsters which survived through 27 days were considered protected from the virus. When the interferon administered hamsters were challenged by EMCV, IFN-con protected 86% and lacIFN-con protected 57% of the animals as observed through 27 days after the challenge (see Figure 7). The control group which did not receive interferon all died on or before the fourth day after the EMCV challenge.

Claims

WHAT IS CLAIMED IS:

1. Chemically modified IFN-con comprised of a IFN-con conjugated to at least one glycosyl ligand, wherein said ligand comprises a terminal galactose.

2. A chemically modified IFN-con of claim 1 wherein said IFN-con is selected from the group consisting of IFN-coni, IFN-con2, and IFN-con3.

3. A chemically modified IFN-con of claim 2 wherein said IFN-con is IFN-coni.

4. A chemically modified IFN-con of claim 1 wherein said glycosyl ligand is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, glycopeptides, and glycoproteins.

5. A chemically modified IFN-con according to claim 4 wherein said glycosyl ligand is an oligosaccharide.

6. A chemically modified IFN-con according to claim 5 wherein said oligosaccharide has at least two sugar residues, and comprises a terminal galactose residue.

7. A chemically modified IFN-con according to claim 5 wherein said oligosaccharide is lactose.

8. A chemically modified IFN-con according to claim 1 wherein four to seven glycosyl ligands are conjugated to said IFN-con.

9. A chemically modified IFN-con according to claim 1 wherein said glycosyl ligand(s) is conjugated to the protein backbone through active amino acid residue(s) such as lysine, cysteine, serine, threonine, or asparagine.

10. A chemically modified IFN-con according to claim 1 wherein said glycosyl ligand(s) is connected to said IFN-con through the ε-NH₂ of lysine(s) and the N-terminal amine of said IFN-con directly.

11. A pharmaceutical composition comprising a substantially homogenous preparation of neoglycosylated IFN-con, said neoglycosylated IFN-con consisting of four to seven glycosyl ligands connected to a IFN-con and a pharmaceutically acceptable diluent, adjuvant or carrier.

12. A substantially homogeneous preparation of neoglycosylated IFN-con.

13. A method for treating a patient having a condition treatable by IFN-con which comprises administering to said patient a therapeutically effective amount of a chemically modified IFN-con of Claim 1.

14. A method according to Claim 13, wherein said condition is a cell proliferation disorder or a viral disease.

15. A method according to Claim 14, wherein said viral disease is a hepatic disorder.

16. A method according to Claim 15, wherein said hepatic disorder is hepatitis A, hepatitis B, hepatitis C or hepatitis Delta.

17. A method according to Claim 16, wherein said hepatic disorder is hepatitis C.

18. A method of delivering an interferon preferentially to the liver of a patient, comprising administering to said patient a chemically modified interferon, wherein said 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-α, IFN-β, and

IFN-con.

19. A method according to Claim 18, wherein said interferon is IFN-α.

20. A method according tc Claim 18, wherein said interferon is IFN-β.

21. A method according to Claim 18, wherein said interferon is IFN-con.

22. A method according to claim 18, wherein a therapeutically effective amount of said chemically modified interferon is administered to said patient in order to treat a hepatic disorder.

23. A method according to claim 22, wherein said hepatic disorder is hepatitis C.