Classifications

• • 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
  • 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
  • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • 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
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention is in the field of antiviral agents for the treatment of hepatitis virus infection.
• Hepatitis C virus (HCN) infection is the most common chronic blood borne infection in the United States. Although the numbers of new infections have declined, the burden of chronic infection is substantial, with Centers for Disease Control estimates of 3.9 million (1.8%) infected persons in the United States. Clironic liver disease is the tenth leading cause of death among adults in the United States, and accounts for approximately 25,000 deaths annually, or approximately 1% of all deaths. Studies indicate that 40% of chronic liver disease is HCN-related, resulting in an estimated 8,000-10,000 deaths each year. HCN-associated end- stage liver disease is the most frequent indication for liver transplantation among adults.
• Interferon-alpha (IF ⁇ - ⁇ ) treatment is the therapy of choice for treating hepatitis C virus infection.
• the serum half-life of IF ⁇ - ⁇ is in the range of 8 to 8.5 hours
• repeat doses of IF ⁇ - ⁇ are administered in order to maintain a higher serum level.
• an accepted dosage regimen is administration of IF ⁇ - ⁇ three times in week (TIW) for a period of 24 - 48 weeks. The peaks and troughs of drug levels associated with such repeat drug dosing has been assumed to result in the severe side effects of the interferons during therapy.
• Interferon alpha (IFN- ⁇ ) linked to a linear polyethylene glycol (PEG) molecule having an average molecular weight of about 30 kD, and compositions comprising the same, are provided.
• the invention further provides methods of treating a viral infection with the subject PEG-modified IFN- ⁇ .
• Figure 1 depicts the amino acid sequence of the consensus interferon IFN-alpha conl
• Figure 2 is a chromatogram depicting the size exclusion chromatographic profile of a pegylated consensus interferon alpha designated PEG-Alfacon.
• Figure 3 is a photograph depicting the results of SDS-PAGE electrophoretic (reducing and non-reducing) analyses of a pegylated consensus interferon alpha designated PEG- Alfacon.
• Figure 4 is a chromatogram depicting reversed phase HPLC chromatographic profiles of PEG-Alfacon, CIFN (Infergen) and a mixture of the two compounds.
• Figure 5 is a graph depicting the serum concentration-time profile of pegylated interferon alpha analogs (IM-001, IM-003, IM-005, and IM-006), Pegasys, and PEG-Intron administered by subcutaneous injection in rats.
• IM-001, IM-003, IM-005, and IM-006 pegylated interferon alpha analogs
• Pegasys Pegasys
• PEG-Intron administered by subcutaneous injection in rats.
• Figure 6 is a semilog plot depicting the serum concentration-time profile for PEG-
• Alfacon (preparation no. IM-006) administered by subcutaneous injection in rats.
• Figure 7 is a semilog plot depicting the pharmacokinetic profile of subcutaneously
• Figure 8 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,
• PEG-Intron, and Pegasys expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in A549 cell/EMCN assays.
• WHO World Health Organization
• Figure 8 depicts an analysis of the results with A549 cells.
• Figure 9 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,
• PEG-Intron, and Pegasys expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in HeLa cell/ECMN assays.
• WHO World Health Organization
• Figure 9 depicts an analysis of the results with HeLa cells.
• Figure 10 is a log plot depicting the antiviral activities of Infergen, PEG-Alfacon- 1,
• PEG-Intron, and Pegasys expressed as International Units of activity per milligram normalized to the reference standard of human leukocyte-derived interferon-alpha supplied by the World Health Organization (WHO), measured in ME 180 cell/ECMN assays.
• WHO World Health Organization
• Figure 10 depicts an analysis of the results with Me 180 cells.
• Figure 11 is a graph depicting mean PEG-alfacon serum pharmacokinetic profiles from six subjects/group.
• Figures 12A and B depict dose corrected C max (pg/mL; Figure 12A) and AUCo-i ast
• Figure 13 depicts mean serum pharmacokinetic profiles for 4-6 subjects and corresponding fitted curves using a 1 -compartment model.
• Figures 14A-14G depict simulated serum pharmacokinetic profiles for various dosing regimens. Each panel in Figures 14A-14G uses different datasets that are fitted to a 1- compartment model.
• Figure 14A depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 60 ⁇ g.
• Figure 14B depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 100 ⁇ g.
• Figure 14C depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 150 ⁇ g.
• Figure 14D depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 10 days with 200 ⁇ g.
• Figure 14E depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 100 ⁇ g.
• Figure 14F depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 150 ⁇ g.
• Figure 14G depicts simulated serum pharmacokinetic profiles for a dosing regimen with dosing every 7 days with 200 ⁇ g.
• Figure 15 depicts mean percent (%) change in serum 2',5'-oligoadenylate synthetase
• Figure 16 depicts measured and predicted percent (%) change in OAS serum values expressed as percent of baseline (pretreatment) values, from pharmacokinetic/pharmacodynamic (PK-PD) modeling of mean serum profiles using an Emax model with minimum response fixed at 0. FEATURES OF THE INVENTION
• the present invention features a monopegylated consensus interferon (CIFN) molecule comprised of a single CIFN polypeptide and a single polyethylene glycol (PEG) moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly linked through a stable covalent linkage to either the N-terminal residue in the CIFN polypeptide or a lysine residue in the CIFN polypeptide.
• CIFN monopegylated consensus interferon
• the PEG moiety is linked to either the alpha-amino group of the
• the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon- amino group of the lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide, thereby forming a hydrolytically stable linkage between the PEG moiety and the CIFN polypeptide.
• the PEG moiety is linked to the N-terminal residue in the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the alpha-amino group of the N-terminal residue of the CIFN polypeptide.
• the PEG moiety is linked to a lysine residue in the CIFN polypeptide. In other embodiments, the PEG moiety is linked to the epsilon-amino group of a lysine residue in the CIFN polypeptide. In further embodiments, the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide. In still further embodiments, the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon- amino group of the lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the PEG moiety is linked to a lysine chosen from lys 31 , lys 5 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the PEG moiety is linked to a lysine chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 1 5 of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the PEG moiety is linked to a lysine chosen from lys 121 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety is linked to the epsilon-amino group of a lysine chosen from lys 121 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon- amino group of the chosen lysine residue in the CIFN polypeptide.
• the invention contemplates embodiments of each such molecule where the CIFN polypeptide is chosen from interferon alpha-co , interferon alpha-con 2 , and interferon alpha-con 3 , the amino acid sequences of which CIFN polypeptides are disclosed in U.S. Pat. No. 4,695,623.
• the invention also features a composition
• a composition comprising a population of monopegylated consensus interferon (CIFN) molecules, where the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue in the CIFN polypeptide.
• CIFN monopegylated consensus interferon
• the linkage comprises an amide bond between the PEG moiety and either the alpha- amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached tlirough a covalent linlcage to either the N-terminal residue or a surface-exposed lysine residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between the PEG moiety and either the alpha- amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linlcage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N- terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linkage to either the N-terminal residue or a lysine residue chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon- amino group of the lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the population consists of one or more species of molecules, where each species is comprised of a single CIFN polypeptide and a single PEG moiety, where the PEG moiety is linear and about 30 kD in molecular weight and is directly or indirectly attached through a covalent linlcage to either the N-terminal residue or a lysine residue chosen from lys , lys , lys , and lys of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between a propionyl group of the PEG moiety and either the alpha-amino group of the N-terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega- propanoic acid activated ester of the PEG moiety and either the alpha-amino group of the N- terminal residue or the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to the N-terminal residue of the CIFN polypeptide.
• the PEG moiety in each such species in the population is linked to the alpha-amino group of the N-terminal residue in the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the alpha-amino group of the N-terminal residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the alpha-amino group of the N- terminal residue of the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue in the CIFN polypeptide.
• the PEG moiety in each such species in the population is linked to the epsilon-amino group of a lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the lysine group in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the lysine residue in the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a surface-exposed lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the surface-exposed lysine residue in the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety in each such species in the population is linked to the epsilon- amino group of a lysine chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linlcage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the linlcage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety in each such species in the population is linked to the epsilon-amino group of a lysine chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linlcage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha- methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the population consists of one or more species of monopegylated CIFN in which the PEG moiety is linked to a lysine residue chosen from lys , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety in each such species in the population is linked to the epsilon-amino group of a lysine chosen from lys 121 , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the linkage comprises an amide bond between the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide. In still further embodiments, in each such species in the population the linkage comprises an amide bond between a propionyl group of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the amide bond is formed by condensation of an alpha-methoxy, omega-propanoic acid activated ester of the PEG moiety and the epsilon-amino group of the chosen lysine residue in the CIFN polypeptide.
• the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide and a second monopegylated CIFN molecule comprising a PEG moiety linked to a lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, and a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, where each of the first, second and third CIFN polypeptides can be the same or different from any of the other CIFN polypeptides, and where the location of the linkage site in the second CI
• the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, and a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue in a fourth CIFN polypeptide, where each of the first,
• the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue in a fourth CIFN polypeptide, and a fifth monopeg
• the invention contemplates embodiments where the population consists of a first monopegylated CIFN molecule comprising a PEG moiety linked to the N-terminal residue of a first CIFN polypeptide, a second monopegylated CIFN molecule comprising a PEG moiety linked to a first lysine residue in a second CIFN polypeptide, a third monopegylated CIFN molecule comprising a PEG moiety linked to a second lysine residue in a third CIFN polypeptide, a fourth monopegylated CIFN molecule comprising a PEG moiety linked to a third lysine residue i a fourth CIFN polypeptide, a fifth monopeg
• the population can include at least one additional monopegylated CIFN molecule comprising a PEG moiety linked to a lysine residue of an additional CIFN polypeptide, where the CIFN polypeptide is the same or different as any of the first, second, third, fourth, fifth and sixth CIFN polypeptides, and where the location of the linlcage site in the additional CIFN polypeptide is not the same as the location of the linlcage site in any other CIFN polypeptide.
• each of the above-described populations of monopegylated CIFN molecules where each such species comprises a PEG moiety linked to a lysine residue of a CIFN polypeptide
• the invention contemplates embodiments characterized by a plurality of species of monopegylated, lysine-derivatized CIFN molecules, where each such species is characterized by a site of linkage that is not the same as the site of linlcage in any other species, in a manner analogous to the description of embodiments relating to populations including monopeyglated, N-terminally derivatized CIFN molecules above.
• the embodiments of the invention characterized by a plurality of species of monopegylated, lysine-derivatized CIFN molecules can be obtained by modifying the description of embodiments relating to populations including monopeyglated, N-terminally derivatized CIFN molecules above so as to remove the N-terminally derivatized species contained therein.
• the invention contemplates embodiments where the molecules in each such population comprise a CIFN polypeptide chosen from interferon alpha-co , interferon alpha- con 2 , and interferon alpha-con 3 .
• the invention further features a product that is produced by the process of reacting
• CIFN polypeptide with a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa) that is linear and about 30 kD in molecular weight where the reactants are initially present at a molar ratio of about 1 : 1 to about 1 :5 CIFN:mPEGspa, and where the reaction is conducted at a pH of about 7 to about 9, followed by recovery of the monopegylated CIFN product of the reaction.
• the reactants are initially present at a molar ratio of about 1 :3 CIFN:mPEGspa and the reaction is conducted at a pH of about 8.
• the reactants are initially present in a molar ratio of 1 :2 CIFN:mPEGspa and the reaction is conducted at a pH of about 8.0.
• the invention contemplates embodiments where the CIFN reactant is chosen from interferon alpha-con ! , interferon alpha- con , and interferon alpha-con 3 .
• the monopegylated CIFN molecule(s) or population(s) of the invention exhibit an antiviral activity that is at least 10-fold greater than that of PEGASYS® Peg- interferon-alfa2a, an approved marketed product to treat chronic hepatitis C.
• the monopegylated CIFN molecule(s) or population(s) of the invention exhibit an antiviral activity that is approximately the same as that of INFERGEN® Alfacon-1.
• the invention additionally features a pharmaceutical composition comprising any monopegylated CIFN molecule or population of such molecules described above and a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a viral disease in a patient.
• the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a hepatitis viral disease in a patient.
• the pharmaceutical composition comprises the monopegylated CIFN molecule or population of the invention in an amount that is therapeutically effective in the treatment of a hepatitis C viral (HCN) disease in a patient.
• HCN hepatitis C viral
• treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
• the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
• Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCN infection); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
• the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.
• pharmacokinetic profile refers to the profile of the curve that results from plotting serum concentration of IF ⁇ - ⁇ over time, following administration of IF ⁇ - ⁇ to a subject.
• Area under the curve refers to the integrated area under the curve generated by plotting serum concentration of IF ⁇ - ⁇ over time following administration of IF ⁇ - ⁇ .
• hepatitis virus infection refers to infection with one or more of hepatitis A
• hepatitis viral infection is of particular interest, particularly hepatitis C virus infection.
• the present invention provides IFN- ⁇ that is modified with linear polyethylene glycol
• the invention provides an IFN- ⁇ polypeptide that is linked to a single molecule of PEG (i.e., the IFN- ⁇ polypeptide is "monopegylated").
• the pharmacokinetic profile of the PEG-modified IFN- ⁇ of the invention is such that the PEG-modified IFN- ⁇ is effective for treating viral infections, particularly hepatitis virus infections.
• the invention further provides methods of treating a hepatitis virus infection. The methods generally involve administering an effective amount of a PEG-modified IFN- ⁇ of the invention to an individual having or susceptible to a viral hepatitis infection.
• the present invention provides IFN- ⁇ that is modified with a polyethylene glycol
• the invention provides an IFN- ⁇ polypeptide that is linked to a single molecule of PEG of less than about 40 lcDa, with a PEG molecule of about 30 kDa being of particular interest, more particularly a linear 30 kDa PEG molecule attached to IFN- ⁇ , with CIFN being of particular interest.
• IFN- ⁇ is linked to a single molecule of PEG of less than about 40 lcDa, with a PEG molecule of about 30 kDa being of particular interest, more particularly a linear 30 kDa PEG molecule attached to IFN- ⁇ , with CIFN being of particular interest.
• interferon-alpha refers to a family of related polypeptides that inhibit viral replication and cellular proliferation and modulate immune response.
• IFN- ⁇ includes IFN- ⁇ polypeptides that are naturally occurring; non-naturally- occurring IFN- ⁇ polypeptides; and analogs of naturally occurring or non-naturally occurring IFN- ⁇ that retain antiviral activity of a parent naturally-occurring or non-naturally occurring IFN- ⁇ .
• Suitable alpha interferons include, but are not limited to, naturally-occurring IFN- ⁇
• IFN- ⁇ 2a including, but not limited to, naturally occurring IFN- ⁇ 2a, IFN- ⁇ 2b
• recombinant interferon alpha-2b such as Intron®A interferon available from Schering Corporation, Kenilworth, N.J.
• recombinant interferon alpha-2a such as Roferon® interferon available from Hoffmann-La Roche, Nutley, N.
• interferon alpha-2C such as Berofor® alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.
• interferon alpha- nl a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon® interferon alpha-nl (INS) available from the Glaxo- Wellcome Ltd., London, Great Britain
• interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the Alferon® Tradename.
• IFN- ⁇ also encompasses consensus IFN- ⁇ .
• consensus IFN- ⁇ refers to a non-naturally-occurring polypeptide, which includes those amino acid residues that are common to all naturally-occurring human leukocyte IFN- ⁇ 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, provided that at any such position where there is no amino acid common to all subtypes, the polypeptide excludes any amino acid residue which is not present in at least one naturally-occurring subtype.
• a consensus interferon is a wholly synthetic Type I interferon developed by scanning several interferon-alpha non-allelic subtypes and assigning the most frequently observed amino acids in each position.
• Consensus IFN- ⁇ (also referred to as “CIFN” and “IFN-con” and “IFN-alpha con”) encompasses but is not limited to the amino acid sequences designated IFN-coni (sometimes referred to as “CIFN-alpha conl,” “IFN-alpha conl,” or “IFN-conl,” or “alphacon”), IFN-con 2 and IFN-con 3 which are disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471; and Infergen® (Amgen, Thousand Oaks, Calif).
• Consensus interferons are generally defined by determination of a consensus sequence of naturally occurring interferon alphas. PEG-modified CIFN, especially Infergen®, is of particular interest in some embodiments.
• IFN- ⁇ polypeptides can be produced by any known method. DNA sequences encoding
• IFN-con may be synthesized as described in the above-mentioned patents or other standard methods.
• IFN- ⁇ polypeptides are the products of expression of manufactured DNA sequences transformed or transfected into bacterial hosts, e.g., E. coli, or in eukaryotic host cells (e.g., yeast; mammalian cells, such as CHO cells; and the like).
• the IFN- ⁇ is "recombinant IFN- ⁇ .”
• the host cell is a bacterial host cell
• the IFN- ⁇ is modified to comprise an N-terminal methioiiine.
• IFN- ⁇ produced in E. coli is generally purified by procedures known to those skilled in the art and generally described in Klein et al. ((1988) J Chromatog. 454:205-215) for IFN-con L
• Bacterially produced IFN- ⁇ may comprise a mixture of isoforms with respect to the N- terminal amino acid residue.
• purified IFN-con may comprise a mixture of isoforms with respect to the N-terminal methionine status.
• an IFN-con comprises a mixture of N-terminal methionyl IFN-con, des- methionyl IFN-con with an unblocked N-terminus, and des-methionyl IFN-con with a blocked N-terminus.
• purified IFN-coni comprises a mixture of methionyl IFN-con ⁇ des-methionyl IFN-coni and des-methionyl IFN-coni with a blocked N- terminus.
• 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.
• IFN- ⁇ as described herein may comprise one or more modified amino acid residues, e.g., glycosylations, chemical modifications, and the like.
• PEG is coupled either directly (i.e., without a linking group), or via a linker (as described in detail below), to an amino group on the IFN- ⁇ polypeptide.
• the PEGylated IFN- ⁇ is PEGylated at or near the amino terminus (N-terminus) of the IFN- ⁇ polypeptide, e.g., the PEG moiety is conjugated to the IFN- ⁇ polypeptide at an amino acid residue from amino acid 1 through amino acid 4, or from amino acid 5 through about 10.
• the PEGylated IFN- ⁇ is PEGylated at an amino acid residues from about 10 to about 28.
• the PEGylated IFN- ⁇ is PEGylated at an amino acid residue from amino acids 100-114.
• the PEG molecule is linked to the NH 2 terminal amino acid residue of the CIFN polypeptide.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• the PEGylated IFN- ⁇ comprises CIFN PEGylated at the epsilon amino group of a lysine residue. See Figure 1 for the amino acid sequence of an exemplary IFN- ⁇ , showing the locations of the lysine residues.
• the PEG moiety is linked to a surface-exposed lysine (“lys”) residue.
• Whether a lysine is surface exposed can be determined using any known method. Generally, analysis of hydrophilicity (e.g., Kyte-Doolittle and Hoppe- Woods analysis) and/or predicted surface-forming regions (e.g., Emini surface-forming probability analysis) is carried out using appropriate computer programs, which are well known to those skilled in the art. Suitable computer programs include PeptideStructure, and the like.
• NMR investigations can identify the surface accessible residues by virtue of the chemical shift of the protons of a specific functional group in the spectrum and how they are affected by the inclusion of "shift reagents". In other cases, the inaccessibility or accessibility of residues to solvents or environment can be assessed by fluorescence. In yet other cases, the surface exposure of accessible lysines can be ascertained by the chemical reactivity to water soluble reagents e.g., Trinitrobenzene sulfonate or TNBS, and like measurements.
• water soluble reagents e.g.,
• the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 .
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 .
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 lcD
• the invention provides PEG-modified CIFN, where the PEG moiety is attached to a lysine residue chosen from lys 121 , lys 134 , lys 135 , and lys 165 .
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• the invention further provides a composition comprising a population of monopegylated IFN ⁇ molecules, where the population consists of one or more species of monopegylated IFN ⁇ molecules as described above.
• the PEG-modified IFN- ⁇ of the invention comprises a single PEG molecule per IFN- ⁇ polypeptide molecule.
• a subject composition comprises a population of modified IFN- ⁇ polypeptides, each with a single PEG molecule linked to a single amino acid residue of the polypeptide.
• the population comprises a mixture of a first IFN- ⁇ polypeptide linked to a PEG molecule at a first amino acid residue; and at least a second IFN- ⁇ polypeptide linked to a PEG molecule at a second amino acid residue, wherein the first and second IFN- ⁇ polypeptides are the same or different, and wherein the location of the first amino acid residue in the amino acid sequence of the first IFN- ⁇ polypeptide is not the same as the location of the second amino acid residue in the second IFN- ⁇ polypeptide.
• a subject composition comprises a population of PEG-modified IFN- ⁇ polypeptides, the population comprising an IFN- ⁇ polypeptide linked at its amino terminus to a linear PEG molecule; and an IFN- ⁇ polypeptide linked to a linear PEG molecule at a lysine residue.
• a given modified IFN- ⁇ species represents from about 0.5% to about 99.5% of the total population of monopegylated IFN ⁇ polypeptide molecules in a population, e.g, a given modified IFN- ⁇ species represents about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.5% of the total population of monopegylated IFN- ⁇ polypeptide molecules in a population.
• a subject composition comprises a population of monopegylated IFN- ⁇ polypeptides, which population comprises at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, IFN- ⁇ polypeptides linked to PEG at the same site, e.g., at the N-terminal amino acid.
• a subject composition comprises a population of monopegylated CIFN molecules, the population consisting of one or more species of molecules, where each species is a single CIFN polypeptide linked, directly or indirectly in a covalent linkage, to a single linear PEG moiety of about 30 kD in molecular weight, and where the linkage is to either a lysine residue in the CIFN polypeptide, or the N-terminal amino acid residue of the CIFN polypeptide.
• the amino acid residue to which the PEG is attached is in many embodiments the N- terminal amino acid residue.
• the PEG moiety is attached (directly or via a linker) to a surface-exposed lysine residue.
• the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys , lys , lys , lys , lys , lys , lys , lys , and lys of the CIFN polypeptide.
• the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys , lys , lys 134 , lys 135 , and lys 165 of the CIFN polypeptide.
• the PEG moiety is attached (directly or via a linker) to a lysine residue chosen from lys 121 , lys 13 , lys 135 , and lys 165 of the CIFN polypeptide.
• a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue of a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• a subject composition can further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to a lysine residue in the CIFN polypeptide, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated
• CIFN molecules consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first surface- exposed lysine residue of a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• a subject composition can further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to a surface- exposed lysine residue in the CIFN polypeptide, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated
• CIFN molecules consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• a subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys , lys , lys , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide.
• a subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 , where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated
• CIFN molecules consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• a subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide.
• a subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 , where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated
• CIFN molecules consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at the N-terminal amino acid residue of a first CIFN polypeptide, and a second monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue selected from one of lys 121 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different.
• a subject composition can further comprise a third monopegylated CIFN polypeptide species having a PEG moiety linked to a second lysine residue selected from one of lys 121 , lys 4 , lys 135 , and lys 1 5 in a third CIFN polypeptide, where the third CIFN polypeptide is the same or different from either of the first and second CIFN polypeptides, where the second lysine residue is located in a position in the amino acid sequence of the third CIFN polypeptide that is not the same as the position of the first lysine residue in the amino acid sequence of the second CIFN polypeptide.
• a subject composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 121 , lys 134 , lys 135 , and lys 165 , where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked to a first lysine residue in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the first lysine is located in a position in the amino acid sequence of the first CIFN polypeptide that is not the same as the position of the second lysine residue in the amino acid sequence of the second CIFN polypeptide.
• a subject composition may further comprise at least one additional monopegylated CIFN species having a PEG moiety linked to a lysine residue in the CIFN polypeptide, where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN
• the composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 31 , lys 50 , lys 71 , lys 84 , lys 121 , lys 122 , lys 134 , lys 5 , and lys 1 5 , where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linkage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the second lysine residue is located in a position in the amino acid sequence of in the second CIFN polypeptide that is not the same as the position of the first lys
• the composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 , where the location of the linlcage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a population of monopegylated CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked at a first lysine residue chosen from lys 121 , lys 134 , lys 135 , and lys 165 in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second lysine residue chosen from lys 121 , lys 134 , lys 135 , and lys 165 in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the second lysine residue is located in a position in the amino acid sequence of in the second CIFN polypeptide that is not the same as the position of the first lysine residue in the first CIFN polypeptide.
• the composition may further comprise at least one additional monopegylated CIFN polypeptide species having a PEG moiety linked to one of lys 121 , lys 134 , lys 135 , and lys 165 , where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 kD.
• a subject composition comprises a monopegylated population of CIFN molecules, consisting of a first monopegylated CIFN polypeptide species having a PEG moiety linked to a first surface-exposed lysine residue in a first CIFN polypeptide; and a second monopegylated CIFN polypeptide species having a PEG moiety linked at a second surface-exposed lysine residue in a second CIFN polypeptide, where the first and second CIFN polypeptides are the same or different, and where the first surface- exposed lysine is located in a position in the amino acid sequence of the first CIFN polypeptide that is not the same as the position of the second surface-exposed lysine residue in the amino acid sequence of the second CIFN polypeptide.
• a subject composition may further comprise at least one additional monopegylated CIFN species having a PEG moiety linked to a surface- exposed lysine residue in the CIFN polypeptide, where the location of the linkage site in each additional monopegylated CIFN polypeptide species is not the same as the location of the linlcage site in any other species.
• the PEG moiety is a linear PEG moiety having an average molecular weight of about 30 lcD.
• PEG is attached to IFN- ⁇ via a linking group.
• the linking group is any biocompatible linking group, where "biocompatible" indicates that the compound or group is essentially non-toxic and may be utilized in vivo without causing a significant adverse response in the subject, e.g., injury, sickness, disease, undesirable immune response, or death.
• PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thio ether bond or an amide bond.
• Suitable biocompatible linking groups include, but are not limited to, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group (including, for example, succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl butanoic acid (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazole group (including, for example, carbonyldimidazole (CDI)), a nitro phenyl group (including, for example, nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group,
• the PEG is a monomethoxyPEG molecule that reacts with primary amine groups on the IFN- ⁇ polypeptide.
• Methods of modifying polypeptides with monomethoxy PEG via reductive alkylation are known in the art. See, e.g., Chamow et al. (1994) Bioconj. Chem. 5:133-140.
• PEG is linked to IFN- ⁇ via an SPA linking group.
• SPA esters of PEG, and methods for making same, are described in U.S. Patent No. 5,672,662.
• SPA linkages provide for linkage to free amine groups on the IFN- ⁇ polypeptide.
• a PEG molecule is covalently attached via a linkage that comprises an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the IFN- ⁇ polypeptide.
• a linkage that comprises an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the IFN- ⁇ polypeptide.
• Such a bond can be formed, e.g., by condensation of an ⁇ -methoxy, omega propanoic acid activated ester of PEG (mPEGspa).
• monopegylated CIFN has a linear PEG moiety of about
• the covalent linkage is an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the CIFN polypeptide, where the surface-exposed lysine residue is chosen from lys 50 , lys 71 , lys 134 , lys 135 , and lys 165 , and the amide bond is formed by condensation of an ⁇ -methoxy, omega propanoic acid activated ester of PEG.
• monopegylated CIFN has a linear PEG moiety of about 30 kD attached via a covalent linlcage to the CIFN polypeptide, where the covalent linlcage is an amide bond between a propionyl group of the PEG moiety and the epsilon amino group of a surface-exposed lysine residue in the CIFN polypeptide, where the surface-exposed lysine residue is chosen from lys 121 , lys 134 , lys 135 , and lys 165 , and the amide bond is formed by condensation of an ⁇ -methoxy, omega propanoic acid activated ester of PEG.
• Polyethylene glycol is soluble in water at room temperature, and has the general formula R-O-(CH 2 -CH 2 O) n -R, where R is hydrogen or a protective group such as an allcyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.
• PEG has at least one hydroxyl group, e.g., a terminal hydroxyl group, which hydroxyl group is modified to generate a functional group that is reactive with an amino group, e.g., an epsilon amino group of a lysine residue, a free amino group at the N- terminus of a polypeptide, or any other amino group such as an amino group of asparagine, glutamine, arginine, or histidine.
• an amino group e.g., an epsilon amino group of a lysine residue, a free amino group at the N- terminus of a polypeptide, or any other amino group such as an amino group of asparagine, glutamine, arginine, or histidine.
• PEG is derivatized so that it is reactive with free carboxyl groups in the IFN- ⁇ polypeptide, e.g., the free carboxyl group at the carboxyl terminus of the IFN- ⁇ polypeptide.
• Suitable derivatives of PEG that are reactive with the free carboxyl group at the carboxyl-terminus of IFN- ⁇ include, but are not limited to PEG-amine, and hydrazine derivatives of PEG (e.g., PEG-NH-NH 2 ).
• PEG is derivatized such that it comprises a terminal thiocarboxylic acid group, -COSH, which selectively reacts with amino groups to generate amide derivatives.
• -SH a terminal thiocarboxylic acid group
• selectivity of certain amino groups over others is achieved.
• -SH exhibits sufficient leaving group ability in reaction with N-terminal amino group at appropriate pH conditions such that the ⁇ -amino groups in lysine residues are protonated and remain non-nucleophilic.
• reactions under suitable pH conditions may make some of the accessible lysine residues to react with selectivity.
• the PEG comprises a reactive ester such as an N-hydroxy succinimidate at the end of the PEG chain.
• a reactive ester such as an N-hydroxy succinimidate at the end of the PEG chain.
• Such an N-hydroxysuccinimidate-containing PEG molecule reacts with select amino groups at particular pH conditions such as neutral pH 6.5- 7.5.
• the N-terminal amino groups may be selectively modified under neutral pH conditions.
• accessible-NH 2 groups of lysine may also react.
• the PEG comprises a sufficiently reactive N-hydroxy succinimidyl ester at the end of the PEG chain by virtue of having a suitable spacer e.g., a propionyl group, between the end of the PEG chain and the ester such that the ester does not hydrolyze rapidly and reacts more selectively at particular pH conditions ranging from neutral to alkaline i.e., pH 7.0-9.0.
• a suitable spacer e.g., a propionyl group
• the ⁇ - amino group of certain lysines in the polypeptide chain may be selectively modified with a N-hydroxysuccinimdyl propionate ester- activated PEG.
• the specific process conditions used are selected to yield products of definite compositions and activities.
• the PEG can be conjugated directly to the IFN- ⁇ polypeptide, or through a linker.
• a linker is added to the IFN- ⁇ polypeptide, forming a linker-modified IFN- ⁇ polypeptide.
• Such linkers provide various functionalities, e.g., reactive groups such as sulfl ydryl, amino, or carboxyl groups to couple a PEG reagent to the linker-modified IFN- ⁇ polypeptide.
• the PEG conjugated to the IFN- ⁇ polypeptide is linear. In other embodiments, the PEG conjugated to the IFN- ⁇ polypeptide is branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, "star-PEG's” and multi-armed PEG's such as those described in Shearwater Polymers, Inc. catalog "Polyethylene Glycol Derivatives 1997-1998.” Star PEGs are described in the art including, e.g., in U.S. Patent No. 6,046,305.
• the PEG conjugated to the IFN- ⁇ polypeptide is linear.
• the PEG-modified IFN- ⁇ polypeptides of the invention comprise a single molecule of
• PEG having a molecular weight of less than 40 kDa.
• "30 kDa” PEG has an average molecular weight of 30 kDa.
• PEG having an average molecular weight in a range of from about 2 kDa to about 30 kDa is generally used.
• the molecular weight of the linear PEG molecule is in the range of from about 20 kD to about 40 lcD, from about 22 lcD to about 38 kD, from about 24 lcD to about 36 kD, from about 26 lcD to about 34 lcD, or from about 28 lcD to about 32 lcD.
• the PEG has a molecular weight of about 30 lcD and is linear.
• the molecular weight of PEG molecules is ascertained by gel filtration column chromatography with suitable molecular weight markers, or by MALDI-TOF mass spectrometry.
• PEG-modified IFN- ⁇ is ascertained by gel filtration column chromatography with suitable molecular weight markers, or by MALDI-TOF mass spectrometry.
• PEG-modified IFN- ⁇ of the invention has a molecular weight that is less than that of
• IFN- ⁇ 2a linked to a single molecule of branched 40 kDa PEG is referred to as Pegasys® (Reddy et al. ((2002) Adv. Drug Deliv. Rev. 54:571-586).
• the molecular weight of a subject PEG- modified IFN- ⁇ polypeptide is from about 5 kDa to about 20 kDa, from about 6 kDa to about 15 IcDa, from about 8 kDa to about 12 kDa, or from about 7 kDa to about 10 kDa less than the molecular weight of Pegasys®.
• a subject PEG-modified IFN- ⁇ polypeptide has a molecular weight that is about 8 kDa to about 12 kDa less than that of Pegasys®.
• Pegasys® can be readily determined using standard methods of determining the molecular weight of a protein. Such methods include, but are not limited to, high performance liquid chromatography (HPLC); reverse phase HPLC; size exclusion chromatography; sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC size exclusion chromatography (SEC); HPLC/SEC/laser light scattering; and matrix-assisted laser desorption ionization Stime- of-flight mass spectroscopy (MALDI-TOF MS).
• HPLC high performance liquid chromatography
• reverse phase HPLC size exclusion chromatography
• size exclusion chromatography sodium dodecyl sulfate polyacrylamide gel electrophoresis
• HPLC size exclusion chromatography SEC
• HPLC/SEC/laser light scattering HPLC/SEC/laser light scattering
• MALDI-TOF MS matrix-assisted laser desorption ionization Stime- of-flight mass
• the subject PEG- modified IFN- ⁇ and the Pegasys® are subjected to size exclusion HPLC under the same conditions.
• a PEG-modified IFN- ⁇ polypeptide of the invention has a molecular weight of less than about 60 IcDa, and generally is from about 40 kDa to about 55 kDa, or from about 45 kDa to about 50 IcDa.
• a subject PEG-modified IFN- ⁇ polypeptide has a molecular weight of about 50 IcDa, e.g., from about 48 kDa to about 52 kDa.
• the PEG moiety can be attached, directly or via a linker, to an amino acid residue at or near the N-terminus, or internally (e.g., at a surface-exposed lysine residue). Conjugation can be carried out in solution or in the solid phase.
• known methods for selectively obtaining an N-terminally chemically modified IFN- ⁇ are used.
• a method of protein modification by reductive all viation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein can be used.
• substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• the reaction is performed at pH which allows one to take advantage of the pK a differences between the ⁇ - amino groups of the lysine residues and that of the ⁇ -amino group of the N-terminal residue of the protein.
• PEGylated IFN- ⁇ is separated from unPEGylated IFN- ⁇ using any known method, including, but not limited to, ion exchange chromatography, size exclusion chromatography, and combinations thereof.
• ion exchange chromatography size exclusion chromatography
• the products are first separated by ion exchange chromatography to obtain material having a charge characteristic of monoPEGylated material (other multi- PEGylated material having the same apparent charge may be present), and then the monoPEGylated materials are separated using size exclusion chromatography.
• a modified IFN- ⁇ is prepared by reacting an IFN- ⁇ polypeptide with a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa).
• mPEGspa succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol)
• the reaction is carried out with IFN- ⁇ and mPEGspa in a molar ratio of from about 1 : 1 to about 1 :5.
• the reaction is carried out in a solution having a pH of from about 7 to about 9.
• CIFN is reacted with mPEGspa that is linear and has a molecular weight of about 30 kD, where the reaction is carried out with a CIFNmiPEGspa molar ratio of from about 1 : 1 to about 1:5, and where the pH of the reaction is from about 7 to about 9.
• the CIFN:mPEGspa ratio is about 1 :2 and the pH of the reaction is about 8.
• the invention provides a modified CIFN that is produced by the process of reacting CIFN and a succinimidyl ester of alpha-methoxy, omega- propionylpoly(ethylene glycol) (mPEGspa) that is linear and has a molecular weight of about 30 lcD, where the reaction is carried out with a CIFN:mPEGspa molar ratio of from about 1 : 1 to about 1:5, and where the pH of the reaction is from about 7 to about 9.
• mPEGspa succinimidyl ester of alpha-methoxy, omega- propionylpoly(ethylene glycol)
• the invention provides a modified CIFN that is produced by the process of reacting CIFN and a succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol) (mPEGspa) that is linear and has a molecular weight of about 30 kD, where the reaction is carried out with a CIFN:mPEGspa ratio of about 1:3, and at apH of about 8.
• mPEGspa succinimidyl ester of alpha-methoxy, omega-propionylpoly(ethylene glycol)
• a PEG-modified IFN- ⁇ polypeptide of the invention has a serum half-life (e.g., mean plasma residence time) of at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 17 hours, at least about 20 hours, or at least about 25 hours, or longer.
• serum half-life e.g., mean plasma residence time
• Serum clearance of the PEG-modified IFN- ⁇ is reduced compared to serum clearance of unmodified IFN- ⁇ , e.g., the serum clearance of PEG-modified IFN- ⁇ is at least about 25%, at least about 50%, at least about 75%, or at least about 90%) less than the serum clearance of unmodified IFN- ⁇ of the same amino acid sequence.
• AUC area under the curve
• a PEG-modified IFN- ⁇ polypeptide of the invention has substantially similar pharmacokinetic profile (e.g., as described by the AUC) as interferon- ⁇ 2a linked to a branched PEG molecule having a molecular weight of 40 kDa.
• pharmacokinetic profile e.g., as described by the AUC
• a once- weekly dose of 180 ⁇ g of a PEG-modified IFN- ⁇ of the invention for a period of 48 weeks produces the same pharmacokinetic profile as a once- weekly dose of 180 ⁇ g of Pegasys® for a period of 48 weeks.
• a PEG-modified IFN- ⁇ polypeptide of the invention exhibit antiviral activity that is at least 5-fold greater than that of PEGASYS® Peg-interferon- ⁇ 2a.
• a monopegylated IFN- ⁇ molecule(s) or population(s) of the invention exhibits antiviral activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg-interferon- ⁇ 2a.
• a subject monopegylated CIFN molecule(s) or population(s) exhibits antiviral activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg-interferon- ⁇ 2a.
• the basis for comparison of antiviral activities can be any known assay, including, but not limited to, the MDBK/VSV assay as described in Example 1.
• a PEG-modified IFN- ⁇ polypeptide of the invention retains at least about 5%, at least about 7%, at least about 10%, at least about 12%, at least about 15%), or at least about 20%, or more, of the antiviral activity of the parent (non-PEGylated) IFN- ⁇ polypeptide.
• a subject PEG-Alfacon- 1 molecule retains at least about 5%, at least about 7%, at least about 10%, at least about 12%, at least about 15%, or at least about 20%, or more, of the antiviral activity of INFERGEN® Alfacon-1.
• the basis for comparison of antiviral activities can be any known assay, including, but not limited to, the MDBK/VSV assay as described in Example 1.
• a PEG-modified IFN- ⁇ polypeptide of the invention exhibit anti-proliferative activity that is at least 5-fold greater than that of PEGASYS® Peg-interferon- ⁇ 2a.
• a monopegylated IFN- ⁇ molecule(s) or population(s) of the invention exhibits anti-proliferative activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg- interferon- ⁇ 2a.
• a subject monopegylated CIFN molecule(s) or population(s) exhibits anti-proliferative activity that is at least 5-fold greater, at least 10-fold greater, at least 15-fold greater, or at least 20-fold greater, than that of PEGASYS® Peg- interferon- ⁇ 2a.
• the basis for comparison of anti-proliferative activities can be any known assay, including, but not limited to, the Daudi cell assay as described in Example 1.
• compositions can be formulated using well-known reagents and methods.
• Compositions are provided in formulation with a pharmaceutically acceptable excipient(s).
• a pharmaceutically acceptable excipient A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.
• Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C.
• compositions such as vehicles, adjuvants, carriers or diluents
• pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
• a PEGylated IFN- ⁇ is formulated in an aqueous buffer.
• Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5mM to lOOmM.
• the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like.
• the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80.
• the formulations may further include a preservative.
• Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like.
• the formulation is stored at 2-8°C.
• Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.
• the active agents may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect.
• the agents can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
• administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc.
• the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
• the following methods and excipients are merely exemplary and are in no way limiting.
• the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
• conventional additives such as lactose, mannitol, corn starch or potato starch
• binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
• disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
• lubricants such as talc or magnesium stearate
• the agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
• the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
• the compounds of the present invention can be administered rectally via a suppository.
• the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
• Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
• unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
• unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
• the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
• Effective dosages of the subject PEG-modified IFN- ⁇ range from about 50 ⁇ g to about
• Effective dosages the subject PEG- modified IFN- ⁇ range from 0.5 ⁇ g/kg body weight to 3.5 ⁇ g/kg body weight per dose.
• the instant invention provides method of treating a hepatitis virus infection.
• the methods generally involve administering an effective amount of a PEG-modified IFN- ⁇ polypeptide of the invention to an individual.
• an "effective amount" of the subject PEG-modified IFN- ⁇ is an amount that is effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viral titer in the serum of the individual within a period of from about 12 hours to about 48 hours, from about 48 hours to about 3 days, from about 3 days to about 7 days, from about 7 days to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 4 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks after the beginning of the dosing regimen.
• Patients with chronic hepatitis C generally have circulating virus at levels of 10 5 -10 7 genome copies/ml.
• An effective amount of a subject PEG-modified IFN- ⁇ is an amount that is effective to reduce HCV titer down to about 5 x 10 4 to about 10 5 , to about 10 4 to about 5 x 10 4 , or to about 5 x 10 3 to about 10 4 genome copies per milliliter serum.
• an effective amount of the subject PEG-modified IFN- ⁇ is an amount that is effective to reduce HCV titer down to about 5 x 10 4 to about 10 5 , to about 10 4 to about 5 x 10 4 , or to about 5 x 10 3 to about 10 genome copies per milliliter serum within a period of from about 12 hours to about 48 hours, or from about 16 hours to about 24 hours after the beginning of the dosing regimen.
• an effective amount of a PEG-modified IFN- ⁇ of the invention is an amount that is effective to reduce viral titers to undetectable levels, e.g., to about 1000 to about 5000, to about 500 to about 1000, or to about 100 to about 500 genome copies/mL serum.
• an effective amount of a subject PEG-modified IFN- ⁇ polypeptide is an amount that is effective to reduce viral load to lower than 100 genome copies/mL serum.
• an effective amount of a PEG-modified IFN- ⁇ of the invention is an amount that is effective to achieve a sustained viral response, e.g., no detectable HCV RNA (e.g., less than about 500, less than about 400, less than about 200, or less than about 100 genome copies per milliliter serum) is found in the patient's serum for a period of at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or more preferably to at least about six months following cessation of therapy.
• no detectable HCV RNA e.g., less than about 500, less than about 400, less than about 200, or less than about 100 genome copies per milliliter serum
• a PEG-modified IFN- ⁇ of the invention provides for a serum concentration of PEG- modified IFN- ⁇ in the serum.
• the serum concentration of PEG-modified IFN- ⁇ of the invention is maintained for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• a subject PEG-modified IFN- ⁇ provides for a serum concentration of PEG-modified IFN- ⁇ that is at or near the maximum level that is tolerable by the patient.
• the serum concentration that is achieved is in a range of from about 10 to about 1000, from about 10 to about 500, from about 20 to about 250, from about 30 to about 100, or from about 50 to about 75 International Units (IU)/ml.
• the serum concentration is maintained for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• PEG-modified IFN- ⁇ of the invention is administered in an amount that is effective to achieve and maintain a serum concentration of the subject PEG- modified IFN- ⁇ that is from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100% of the maximum tolerated dose (MTD).
• MTD maximum tolerated dose
• a serum concentration of the subject PEG-modified IFN- ⁇ is achieved that is from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80%) to about 85%, from about 85%> to about 90%, from about 90% to about 95%), or from about 95% to about 100% ⁇ of the maximum tolerated dose (MTD).
• MTD maximum tolerated dose
• the achieved serum concentration can be maintained for a period of about 7 days to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• the administered dose of PEG-modified IFN- ⁇ is in a range of from about 50 ⁇ g to about 300 ⁇ g, e.g., from about 50 ⁇ g to about 70 ⁇ g, from about 70 ⁇ g to about 90 ⁇ g, from about 90 ⁇ g to about 100 ⁇ g, from about 100 ⁇ g to about 120 ⁇ g, from about 120 ⁇ g to about 150 ⁇ g, from about 150 ⁇ g to about 170 ⁇ g, from about 170 ⁇ g to about 200 ⁇ g, from about 200 ⁇ g to about 230 ⁇ g, from about 230 ⁇ g to about 270 ⁇ g, or from about 270 ⁇ g to about 300 ⁇ g-
• Amounts of PEG-modified IFN- ⁇ to be administered are expressed in micrograms, as described above. Alternatively, the doses are also expressed as Units or International Units (IU) of activity. Units or IU are measured in vitro as the ability of the interferon to inhibit the cytopathic effect of a suitable virus (e.g. endomyocarditis virus (EMC), vesicular stomatitis virus, Semliki forest virus) after infection of an appropriate cell line (e.g., the human lung carcinoma cell lines, A549; HEP2/C; and the like).
• EMC endomyocarditis virus
• vesicular stomatitis virus Semliki forest virus
• the antiviral activity is measured against a reference standard such as human interferon alpha supplied by WHO.
• the invention provides a method of treating a hepatitis virus infection, the method involving administering the subject PEG-modified IFN- ⁇ in an amount effective to reduce viral load.
• PEG-modified IFN- ⁇ of the invention is administered daily, twice a week, once a week, once every two weeks, or three, times a week for a period of from about 24 hours to about 48 hours, from about 2 days to about 4 days, from about 4 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• a PEG-modified IFN- ⁇ of the invention is administered once per week for a period of from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• the subject PEG-modified IFN- ⁇ is administered at a dosage of about 45 ⁇ g to about 270 ⁇ g, or about 180 ⁇ g, or about 120 ⁇ g per week subcutaneously for a period of from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.
• the weekly dosage may be delivered by a single bolus injection or by a pump- controlled continuous infusion system
• the subject PEG-modified IFN- ⁇ is administered in a combination therapy, e.g., another anti-viral agent or other therapeutic agent is administered: (1) substantially simultaneously and in a separate formulation; (2) substantially simultaneously and in the same formulation; or (3) in separate formulations, and at separate times.
• a combination therapy e.g., another anti-viral agent or other therapeutic agent is administered: (1) substantially simultaneously and in a separate formulation; (2) substantially simultaneously and in the same formulation; or (3) in separate formulations, and at separate times.
• Combination therapies are discussed in detail below. Combination therapies
• the methods provide for combination therapy comprising administering a composition of the invention and an additional therapeutic agent such as IFN- ⁇ and/or ribavirin.
• an additional therapeutic agent such as IFN- ⁇ and/or ribavirin.
• the additional therapeutic agent(s) is administered during the entire course of PEG-modified IFN- ⁇ treatment, and the beginning and end of the treatment periods coincide.
• the additional therapeutic agent(s) is administered for a period of time that is overlapping with that of the PEG-modified IFN- ⁇ treatment, e.g., treatment with the additional therapeutic agent(s) begins before the PEG-modified IFN- ⁇ treatment begins and ends before the PEG-modified IFN- ⁇ treatment ends; treatment with the additional therapeutic agent(s) begins after the PEG-modified IFN- ⁇ treatment begins and ends after the IFN- ⁇ treatment ends; treatment with the additional therapeutic agent(s) begins after the PEG-modified IFN- ⁇ treatment begins and ends before the PEG-modified IFN- ⁇ treatment ends; or treatment with the additional therapeutic agent(s) begins before the PEG-modified IFN- ⁇ treatment begins and ends after the PEG-modified IFN- ⁇ treatment ends.
• the additional therapeutic agent(s) is administered before the PEG-modified IFN- ⁇ treatment begins, and ends once PEG-modified IFN- ⁇ treatment begins, e.g., the additional therapeutic agent is used in a "priming" dosing regimen.
• Ribavirin and other antiviral agents are administered before the PEG-modified IFN- ⁇ treatment begins, and ends once PEG-modified IFN- ⁇ treatment begins, e.g., the additional therapeutic agent is used in a "priming" dosing regimen.
• Ribavirin is administered orally in dosages of about 400, about 800, or about 1200 mg per day.
• antiviral agents can be delivered in the treatment methods of the invention.
• compounds that inhibit inosine monophosphate dehydrogenase may have the potential to exert direct anti viral activity, and such compounds can be administered in combination with an IFN- ⁇ composition, as described herein.
• Drugs that are effective inhibitors of hepatitis C NS3 protease may be administered in combination with an IFN- ⁇ composition, as described herein.
• Hepatitis C NS3 protease inhibitors inhibit viral replication.
• Other agents such as inhibitors of HCV NS3 helicase are also attractive drugs for combinational therapy, and are contemplated for use in combination therapies described herein.
• Ribozymes such as HeptazymeTM and phosphorothioate oligonucleotides which are complementary to HCV protein sequences and which inhibit the expression of viral core proteins are also suitable for use in combination therapies described herein.
• suitable analogs of ribavirin including enantiomers and immune enhancers such as Zadaxin® are also suitable for use in combination therapies described herein. Determining effectiveness of treatment
• Whether a subject method is effective in treating a hepatitis virus infection, particularly an HCV infection can be determined by measuring viral load, or by measuring a parameter associated with HCV infection, including, but not limited to, liver fibrosis.
• Viral load can be measured by measuring the titer or level of virus in serum.
• PCR quantitative polymerase chain reaction
• bDNA branched DNA
• quantitative assays for measuring the viral load (titer) of HCV RNA have been developed.
• Many such assays are available commercially, including a quantitative reverse transcription PCR (RT-PCR) (Amplicor HCV MonitorTM, Roche Molecular Systems, New Jersey); and a branched DNA (deoxyribonucleic acid) signal amplification assay (QuantiplexTM HCV RNA Assay (bDNA), Chiron Corp., Emeryville, California). See, e.g., Gretch et al. (1995) Ann. Intern. Med. 123:321-329.
• liver fibrosis reduction is determined by analyzing a liver biopsy sample.
• An analysis of a liver biopsy comprises assessments of two major components: necro inflammation assessed by "grade” as a measure of the severity and ongoing disease activity, and the lesions of fibrosis and parenchymal or vascular remodeling as assessed by "stage” as being reflective of long-term disease progression. See, e.g., Brant (2000) Hepatol 31:241-246; and METAVIR (1994) Hepatology 20:15-20.
• a score is assigned.
• Serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method.
• Serum markers of liver fibrosis include, but are not limited to, hyaluronate, N-terminal procollagen III peptide, 7S domain of type IV collagen, C-terminal procollagen I peptide, and laminin.
• Additional biochemical markers of liver fibrosis include ⁇ - 2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.
• ALT serum alanine aminotransferase
• an effective amount of IFN ⁇ is an amount effective to reduce ALT levels to less than about 45 IU/ml serum.
• HAV, HBV, HCV, delta, etc. are suitable for treatment with the methods of the instant invention.
• Individuals who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti-HCV antibody in their serum.
• Such individuals include naive individuals (e.g., individuals not previously treated for HCV, particularly those who have not previously received IFN- ⁇ -based or ribavirin-based therapy) and individuals who have failed prior treatment for HCV ("treatment failure" patients).
• Treatment failure patients include non-responders (e.g., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, particularly a previous IFN- ⁇ monotherapy using a single form of IFN- ⁇ ); and relapsers (e.g., individuals who were previously treated for HCV (particularly a previous IFN- ⁇ monotherapy using a single form of IFN- ⁇ ), whose HCV titer decreased significantly, and subsequently increased).
• non-responders e.g., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, particularly a previous IFN- ⁇ monotherapy using a single form of IFN- ⁇
• relapsers e.g., individuals who were previously treated for HCV (particularly a previous IFN- ⁇ monotherapy using a single form of IFN- ⁇ ), whose HCV titer decreased significantly, and subsequently increased).
• individuals have an HCV titer of at least about
• HCV 10 5 at least about 5 x 10 5 , or at least about 10 , genome copies of HCV per milliliter of serum.
• the patient may be infected with any HCV genotype (genotype 1, including la and lb, 2, 3, 4, 6, 7, 8, 9, etc. and subtypes (e.g., 2a, 2b, 3a, etc.)), particularly a difficult to treat genotype such as HCV genotype 1 and particular HCV subtypes and quasispecies.
• HCV genotype genotype
• subtypes e.g., 2a, 2b, 3a, etc.
• HCV-positive individuals are HCV-positive individuals (as described above) who exhibit severe fibrosis or early cirrhosis (non-decompensated, Child' s-Pugh class A or less), or more advanced cirrhosis (decompensated, Child' s-Pugh class B or C) due to chronic HCV infection and who are viremic despite prior anti- viral treatment with IFN- ⁇ -based therapies or who cannot tolerate IFN- ⁇ -based therapies, or who have a contraindication to such therapies.
• HCV-positive individuals with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the methods of the present invention.
• individuals suitable for treatment with the methods of the instant invention are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation.
• individuals suitable for treatment with the methods of the instant invention include patients with milder degrees of fibrosis including those with early fibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoring systems; or stages 1, 2, or 3 in the Ishalc scoring system.)
• the HCV patient is selected according to certain disease parameters exhibited by the patient, such as the initial viral load, genotype of the HCV infection in the patient, antiviral treatment history of the patient, liver histology and/or stage of liver fibrosis in the patient.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having advanced or severe stage liver fibrosis as measured by a Knodell score of 3 or 4 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having advanced or severe stage liver fibrosis as measured by a Knodell score of 3 or 4 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN- ⁇ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and no or early stage liver fibrosis as measured by a Knodell score of 0, 1, or 2 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of greater than 2 million viral genome copies per ml of patient serum and no or early stage liver fibrosis as measured by a Knodell score of 0, 1, or 2 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 20 weeks to about 50 weeks, or about 24 weeks to about 48 weeks, or about 30 weeks to about 40 weeks, or up to about 20 weeks, or up to about 24 weeks, or up to about 30 weeks, or up to about 36 weeks, or up to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 20 weeks to about 24 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 1 infection and an initial viral load of less than or equal to 2 million viral genome copies per ml of patient serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 20 weeks to about 50 weeks, or about 24 weeks to about 48 weeks, or about 30 weeks to about 40 weeks, or up to about 20 weeks, or up to about 24 weeks, or up to about 30 weeks, or up to about 36 weeks, or up to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ and for a time period of about 20 weeks to about 24 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 2 or 3 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of at least about 24 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV genotype 4 infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG- modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks, or about 30 weeks to about one year, or about 36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or at least about 24 weeks, or at least about 30 weeks, or at least about 36 weeks, or at least about 40 weeks, or at least about 48 weeks, or at least about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 20 weeks to about 50 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient having an HCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of at least about 24 weeks and up to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and an initial viral load greater than 2 million HCV RNA genomes/ml of serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 1 HCV infection and an initial viral load less than or equal to 2 million HCV RNA genomes/ml of serum and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 2 or 3 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 12 weeks to about 24 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying an antiviral treatment naive patient having a genotype 4 HCV infection and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying patient who has an HCV infection and who failed and earlier course of antiviral treatment and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of IFN- ⁇ therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of IFN- ⁇ 2a or 2b therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of PEGASYS® peginterferon alfa-2a or PEG-INTRON® peginterferon alfa-2b therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who failed an earlier course of consensus interferon therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 60 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has a genotype 2 or 3 HCV infection and who relapsed after responding to an earlier course of IFN- ⁇ therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 24 weeks to about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has a genotype 1 or 4 HCV infection and who relapsed after responding to an earlier course of IFN- ⁇ therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 48 weeks.
• the invention provides a method for treatment of HCV infection comprising the steps of (1) identifying a patient who has an HCV infection and who did not respond to an earlier course of IFN- ⁇ therapy and then (2) administering to the patient a therapeutically effective amount of a subject PEG-modified IFN- ⁇ for a time period of about 48 weeks to about 60 weeks.
• the invention also contemplates co-administering to the patient a therapeutically effective amount of ribavirin for the duration of the desired course of PEG-modified IFN- ⁇ therapy.
• the subject method includes co- administering to the patient about 800 to about 1200 mg ribavirin orally per day, the daily dosage optionally being divided into two doses per day, for the desired course of PEG- modified IFN- ⁇ therapy.
• the subject method includes co- administering to the patient for the duration of the desired course of PEG-modified IFN- ⁇ therapy (a) 1000 mg ribavirin orally per day if the patient has a body weight less than 75 kg or (b) 1200 mg ribavirin orally per day if the patient has a body weight greater than or equal to 75 kg, where the daily dosage is optionally divided into two doses per day.
• KITS PEG-modified IFN- ⁇ therapy
• Kits with unit doses of a subject PEG-modified IFN- ⁇ e.g. in oral or injectable doses, are provided.
• kits in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating hepatitis.
• Preferred agents and unit doses are those described herein above.
• a subject kit includes a container comprising a solution comprising a unit dose of subject PEG-modified IFN- ⁇ and a pharmaceutically acceptable excipient; and instructions to administer a unit dose once per week.
• the instructions can be printed on a label affixed to the container, or can be a package insert that accompanies the container.
• CIFN (having an approximate one third of the protein as N-blocked variant, 3 mg/ml) in 20 mM sodium phosphate buffer containing 100 mM sodium chloride was treated with a two-fold molar excess of succinimido propionyl derivative of linear 30 kDa MPEG (mSPA 30K PEG) for one hour at 20°C.
• the crude mixture when analyzed by SEC, showed that the monopegylated products accounted for -52% of the mixture.
• the major fraction was purified on a cation exchange column to provide the monopegylated CIFN with a purity of -97% or greater.
• This material (3 OK MPEG-monopegylated CIFN) retained a significant amount of the antiviral activity of the parent CIFN.
• CIFN was N-terminally modified with 30 kD (linear) methoxypolyethylene glycol aldehyde to form N-terminal monopegylated CIFN (3 OK MPEG- ⁇ -amino-monopegylated CIFN) according to the method described in Example 3 of U.S. Pat. No. 5,985,265, except that 30 lcD (linear) methoxypolyethylene glycol aldehyde was substituted for 12 kD (linear) methoxypolyethylene glycol aldehyde.
• This material (3 OK MPEG- ⁇ -amino-monopegylated CIFN) retained a significant amount of the antiviral activity of the parent CIFN.
• N-terminal derivatization requires eight fold molar excess of methoxy PEG-propionaldehyde and sodium cyanoborohydride as reductant to effect the alkylation reaction at pH 4.0.
• CIFN was manufactured and purified by Amgen and the final product was received as drug substance with a Certificate of Analysis at a concentration of 0.2 mg/ml.
• Succinimido propionyl methoxy PEG Linear 30 lcD was obtained as either research grade or GMP material from Shearwater Corporation, Huntsville, AL.
• the reaction mixture was then diluted ten fold with 50mM sodium acetate, pH 4.5 and loaded on a Fractogel column at 2.0 mg total protein/ml of resin.
• the column was washed with equilibration buffer (50mM sodium acetate, pH 4.5) first and subsequently with 50mM acetate buffer containing lOOmM NaCl and 50mM acetate buffer containing 250 mM NaCl.
• the monopegylated product was then subjected to ultrafiltration diafiltration (UF/DF) and equilibrated with the formulation buffer (25mM sodium phosphate containing lOOmM NaCl, pH 7.0) and concentration adjusted to desired amounts for fill-finish activities.
• UF/DF ultrafiltration diafiltration
• CIFN monopegylated with 30 IcDa PEG are referred to below by product codes such as IM-002 or sometimes with an additional prefix, for example, IM-396-002.
• Preparation 1 (IM-002): Purified consensus ⁇ - interferon at a concentration of 3.02 mg/ml [Prepared by diluting the protein in solution A (25 mM sodium phosphate buffer containing 100 mM NaCl at pH 7.0) with sufficient amounts of 100 mM sodium phosphate buffer to provide a final pH of 6.5] was treated with SPA-linear 30 kD mPEG at a molar ratio of 5: 1 at a pH 6.5 for 5.5 hours at ambient temperature. The monopegylated product mixture assayed by SEC chromatography accounted for 47.0% of the crude mixture. This experiment was conducted close to physiological conditions. The reaction conditions and the product antiviral activities with the samples from the early bench scale materials are summarized in Tables 1 and 2. The preliminary antiproliferative activity obtained with the early small scale products is summarized in Table 3.
• Preparation 2 (IM-003): CIFN at a concentration of 2.73 mg/ml [prepared by diluting the protein in solution A ( 25 mM sodium phosphate buffer containing lOOmM NaCl at pH 7.0) with sufficient amounts of 50mM sodium phosphate dibasic solution to provide pH 7.4] was stirred with the peg reagent at a molar ratio of 1 :3 at ambient temperature for 3.5 hours and then at 2-8 °C for -15 hours. Product analysis showed that the monopegylated products accounted for -46% (Table 1) and the retention of antiviral activity was 14.5% (Table 2). This reaction was conducted at physiologic conditions and the product retains a significant amount of the parent IFN activity.
• Preparation 2 retained 60% of the antiproliferative activity of the CIFN molecule.
• Preparation 3 (IM-004): CIFN at a concentration of 3.0 mg/ml in 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0 was treated with a five fold molar excess of the PEG reagent at ambient temperature for 1.8 hours. This reaction also conducted at the physiologic pH provided a mixture of monopegylated compounds that accounted for a 41.1% yield in the crude mixture (Table 1) and the antiviral activities (Table 2) and antiproliferative activities (Table 3) of this material show that this compound also is comparable to others discussed above.
• Preparation 4 (IM-005): CIFN at a concentration of 2.57 mg/ml [ prepared by diluting the protein in 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH7.0 with sufficient amounts of 15mM sodium hydroxide to provide pH 8.0] was treated with a three fold molar excess of the PEG reagent at a pH of 8.0 ⁇ 0.2 at 2-8 °C for a period of approximately 15 hours Analysis of the product mixture showed that the yield of monopegylated compounds accounted for 47.0%) and the product retained significant amounts of the antiviral (13.8% of CIFN, see Table 2) and antiproliferative activities (150% of CIFN activity in the early experiments, see Table 3) of the parent molecule.
• the PEG-Alfacon 1 drug substance was produced by a slight modification of the conditions used above.
• CIFN was concentrated by a prior ultrafiltration/diafiltration step to ⁇ 3mg/mL protein concentration into the reaction buffer (25mM sodium phosphate, lOOmM NaCl, pH 8.0).
• the conjugation reaction used 2:1 molar ratio of the PEG reagent to protein at pH 8.0 and incubation at ambient temperature for one hour.
• the reaction was quenched by the addition of excess glycine followed by purification using cation exchange chromatography, anion exchange and formulation prior to fill-finish.
• the mono pegylated products (a mixure of regioisomers) were obtained in > 90%) purity after these steps. Purities of the different lots of products ranged from 93-97%.
• Fractogel column preequilibrated with acetate buffer at pH 4.5 The products were separated by washing with buffers differing in salt concentrations.
• the monopegylated isomers were isolated as a fraction and the product was subjected to ultrafiltration/diafiltration using 25mM sodium phosphate buffer containing lOOmM sodium chloride at pH 7.0. Final buffer exchange and dilution was carried out by an anion exchange over a Q Sepharose column preequilibrated with the formulation buffer.
• RP-HPLC Reversed Phase High Performance Liquid Chromatography
• SEC Size Exclusion Chromatography
• Bovine serum albumin was used as a standard along with molecular weight markers. The bands were visualized by colloidal blue staining method. The protein(s) were reduced prior to loading on the gel. Compared to CIFN standard, the product(s) ran as high molecular weight band, indicative of pegylation.
• RP-HPLC RP-HPLC
• a Phenomenex Synergi 4 ⁇ MAX-RP column was used for analysis.
• the product(s) were eluted with varying TFA-Acetonitrile combinations.
• the individual peaks were visualized by monitoring the DAD1 ultraviolet (uv) absorption at 214 nm and DAD2 wavelength of 280 nm.
• the major peak corresponded to monopegylated product and was used as the reference peak characteristic of the product. This method was used to characterize the preparations 1-6 described above. When the optimal pegylated product was identified for further scale-up manufacture, the HPLC analytical method was modified.
• Test material was injected on to a Zorbax 300 SB-C3 column ( 4.6 x 150 mm, Agilent Technologies, Palo Alto, CA). Two solvents (A: 0.1% TFA in water; B: 0.1% TFA in 80% acetonitrile) were used as mobile phases. The product was eluted using a gradient composed of the two mobile phases over 26 minutes. A flow rate of 1.0 mL/min was used and samples were detected by uv absorbance at 214 nm. PEG-Alfaconl eluted as a main peak with a retention time of 11.2 minutes (see Figure 4). SEC-HPLC
• CIFN was reductively alkylated with 30 lcD linear monomethoxypolyethylene glycol aldehyde, essentially as described in Example 3 of U.S. Pat. No. 5,985,265, except that a 30 lcD linear PEG reagent (a reactive aldehyde) was substituted for the 12 kD linear PEG reagent specified in Example 3.
• 3 OK MPEG- ⁇ -amino-monopegylated CIFN was recovered and purified from the alkylation reaction mixture as described in Example 3 of U.S. Pat. No. 5,985,265. This preparation (IM-001) is referred to as N-terminal PEG-CIFN below.
• MDBK Madin-Darby Bovine Kidney
• VSV Virus
• CIFN Peg-Alfacon or N-terminal PEG-CIFN
• Test represents the cell staining observed in the presence of test article
• VC represents the cell staining in the presence of viras but no test article
• CC represents the cell staining in the absence of virus.
• PEG-Alfacon and N-terminal PEG-CIFN exhibited a dose dependent antiviral activity against VSV viras. Similar antiviral activity has also been measured with human lung carcinoma (A549) cells infected with Endomyocardi
• N-terminal PEG-CIFN at predetermined concentrations for three days and cell proliferation was measured by the incorporation of tritiated thymidine. Percent inhibition was calculated by the following formula:
• Percent Inhibition [l-(test cpm/total cpm)] x 100 where test counts per minute (cpm) represents the value measured for the test compound at the concentrations indicated and total cpm represents the value measured for proliferation of cells in the absence of test compound.
• test counts per minute cpm
• total cpm represents the value measured for proliferation of cells in the absence of test compound.
• Dawley rats Pegylated interferons were administered by subcutaneous route (50 - 250 ⁇ g/kg) in rats. Serum concentrations were monitored over a period of one week. Cmax, AUC and the elimination t- 1/2 were determined using a pharmacokinetic analysis. A significant increase in half-life was seen with all the monopegylated CIFN mixtures. Consequently, PEG-Alfacon and N-terminal PEG-CIFN demonstrate a significantly increased plasma exposure.
• OAS Oligoadenylate synthetase
• PEG-Alfacon monopegylated product(s) as a mixture with purity of 97% and greater.
• the purity was ascertained by a SEC-HPLC and SDS-PAGE gel electrophoresis. Data obtained are shown in Figures 2 and 3.
• a reversed phase HPLC analysis ( Figure 4) showed that the purified product appeared as a single major peak with a retention time of 12 minutes.
• the average molecular mass of pegylated CIFN was measured using MALDI-TOF mass spectrometry. The observed mass of 52260 is consistent with one unit of nominal 30kd PEG (average MW of 32700 Da as measured by gel permeation chromatography (GPC) attached to one molecule of CIFN.
• Antiviral activity
• CIFN products any of preparation nos. IM-002, IM-003, IM-004, IM-005, IM-006 and IM- 007 and the N-terminal PEG-CIFN product (preparation no. IM-001) exhibited approximately 10%o of the antiviral activity of the parent molecule (CIFN).
• the derivative, IM-005 was chosen as the lead candidate for further development and designated as PEG-Alfacon.
• the antiviral activities measured depended on the actual cell lines used and infecting virus combinations.
• the PEG-Alfacon product and in the N-terminal PEG-CIFN product exhibited somewhat less than 10%) retention of CIFN antiviral activity.
• Roferon®interferon-alfa2a exhibits an antiviral activity that is 5-10 fold lower than that of Infergen® alfacon-1 when measured in the MDBK/VSV system [Ozes ON et al. J Interferon Res 12: 55-59 (1992)]. Extrapolating from the data in Table 2 above, it is evident that Pegasys®peg-interferon-alfa2a retains approximately 1.5-3.0 % of the antiviral activity of Roferon®interferon-alfa2a (compared to 0.3%> of the antiviral activity of Infergen® or Interferon Alfacon-1) when measured in the MDBK/VSV system.
• the pegylated consensus interferon molecules' percent retention of the parental interferon molecule's (Infergen®alfacon-l's) antiviral activity is approximately 10-fold greater than Pegasys®peg- interferon-alfa2a's percent retention of the parental interferon molecule's (Roferon®interferon- alfa2a's) antiviral activity. Similar comparisons were also made with a reference standard of PEG-Alfacon (pegylated CIFN or Infergen) and other commercial products such as PEG- Intron or PEGASYS (see Tables 4 and 5, below) in several cell lines using different infecting viruses.
• Antiproliferative activity was less affected by pegylation.
• the PEG-alfacon product exhibited approximately 40-150%) of the antiproliferative activity of the parent molecule (CIFN).
• the N-terminal PEG-CIFN product exhibited approximately 45% of the antiproliferative activity of the parent molecule (CIFN).
• the pegylated consensus interferon molecules exhibited approximately 5-15 fold higher antiproliferative activity than that exhibited by PEGASYS ®PEG-interferon-alfa2a in the Daudi cell-based antiproliferation assay.
• IM-001 denotes N-terminal PEG-CIFN and IM-003
• IM-005 and IM-006 denote pegylated CIFN preparations 2, 4 and 5, respectively.
• the semilogarithmic plot of the serum concentration-time profile for one of these analogs designated IM-006 (or IM-396-006) is shown in Figure 6.
• the pharmacokinetic profile of the pegylated consensus interferon molecules exhibited characteristics very similar to pegylated interferon ⁇ -2a (Pegasys®).
• Example 2 Antiviral activity characterization of PEG-modified CIFN and other PEGylated alpha-interferons
• HeLa or ME 180 cells were grown in DMEM or RPMI-1640 medium supplemented with 10%) heat-inactivated serum (calf or fetal as required) (Hyclone Laboratories, Inc., Logan, UT), L-glutamine (2 mM), streptomycin (100 ⁇ g/ml), and penicillin (100 u/ml). Cells were grown at 37°C in a 5% CO 2 humidified incubator. HeLa or ME 180 cells (2 x 104 cells per well in 96-well microtiter plates) were treated with IFN for 24 hours prior to the addition of the virus.
• heat-inactivated serum calf or fetal as required
• L-glutamine 2 mM
• streptomycin 100 ⁇ g/ml
• penicillin 100 u/ml
• VSV Vesicular Stomatitis Virus
• EMCV endomyocarditis virus
• PEG-Alfacon 1 retains a significant amount of the antiviral activity of the parent, Infergen molecule. In the head-to- head comparison, it is clear that PEG-Alfacon 1 retains comparable level of activity to PEG- Intron and significantly higher level of activity than Pegasys. However, in terms of the pharmacokinetic properties (Figure 5), PEG-Alfacon 1 does much better compared to PEG- Intron and the half-life for terminal elimination of the drag is comparable to Pegasys. Thus PEG-Alfacon is expected to have a superior in vivo activity based on a combination of antiviral activity and pharmacokinetic properties.
• Both sets of data indicate that PEG-alfacon- 1 (and other pegylated CIFN molecules) exhibits an antiviral activity that is at least 10-fold greater than that of Pegasys®peg-interferon- alfa2a.
• the MDBK assay data in Table 2 and the A549 and ME 180 data in Tables 4 and 5 indicate that PEG-Alfacon-1 retains approximately 10%) of the antiviral activity of the parental interferon molecule (CIFN) whereas Pegasys®peg-interferon-alfa2a retains approximately 1.0% of the parental interferon molecule's activity (i.e.
• Roferon®interferon- alfa2a's activity in the same assay systems based on the data presented here and published in the literature which has repeatedly shown a 5-10 fold greater activity of Infergen vs Roferon.
• PEG-Alfacon- l's percent retention of the parental interferon molecule's antiviral activity i.e. the antiviral activity of Infergen®alfacon-l
• Pegasys®peg-interferon-alfa2a's percent retention of the parental interferon molecule's antiviral activity i.e. the antiviral activity of Roferon®interferon-alfa2a.
• Example 3 Pharmacokinetic and pharmacodynamics analysis of PEG-alfaeon
• the following is a summary of the results of pharmacokinetic and pharmacodynamic data analysis that uses results from a single dose study of PEG-alfacon. Individual and mean PK profiles were examined and mean PD profiles of serum 2'5'-oligoadenylate synthetase (OAS) activity were examined. Additionally, PK profiles were used to simulate serum drug profiles predicted for multiple PEG-Alfacon doses given at 10 day intervals.
• the pharmacokinetic data were taken from the single dose, range finding clinical study of PEG-alfacon.
• Groups of 6 healthy male and female volunteers were given a single subcutaneous injection of PEG-alfacon in doses ranging from 15 to 210 ⁇ g in an escalating dose design. Samples from volunteers who received doses from 60 ⁇ g and higher were selected for this analysis. The final treatment group received a 210 ⁇ g dose that was identified in the escalation phase as the maximum tolerated dose. Baseline serum PEG-alfacon values from samples obtained 5 min before dosing were measurable in two cases (subjects 531 and 532, 120 ⁇ g dose group). Baseline values were subtracted from subsequent samples to obtain baseline corrected values for these two cases and data are shown with and without baseline correction.
• Results from noncompartmental pharmacokinetic analysis of serum drag concentrations are summarized in Table 6. Peak concentrations were achieved within 36 to 72 h after sc dosing and increased with dose from a mean of 665 pg/mL after a 60 ⁇ g dose to approximately 3600 pg/mL after 210 ⁇ g. Mean profiles are shown in Figure 11. Table 6 shows mean PK parameters calculated from individual profiles and PK parameters calculated from mean serum profiles. In general there was reasonable agreement between the methods although there was considerable variation between subjects within each group that could not be readily explained by sex, body mass index (BMI; kg/m 2 ) or thigh vs abdominal injection site variables. As an example, subject 424 had one quantifiable value following a 90 ⁇ g dose and other samples were below assay detection, while samples from subject 425 were all above 6000 pg/mL for PEG-alfacon following the same dose.
• BMI body mass index
• Table 6 compares the mean pharmacokinetic parameters for each dose group calculated using noncompartmental methods with corresponding parameters calculated using compartmental analysis of mean serum concentrations for each dose group. Data were insufficient to calculate parameters with noncompartmental methods for subjects 318, 423, 425, 529 and 747. Both methods resulted in calculated parameters that were in general agreement with values for AUC parameters (reflecting total drug exposure over time), and elimination half-life (t ⁇ / ) showing similar values with each method. Peak serum concentrations tended to be slightly lower for mean data compared to means of individual maximum concentrations. There was considerable variability within groups with %CV ranging from approximately 30 to 70%> across all parameters.
• Figure 14A simulates the serum profiles expected with a dosing regimen of 60 ⁇ g administered every 10 days using mean parameters calculated from each dose group. Baseline corrected values were used for subjects 531 and 532 to calculate mean parameters in the 120 ⁇ g dose group for all simulations. In general, there is good agreement among each simulation. Steady state is reached during the first dose interval and trough levels fall below the 300 pg/mL level that is the limit of assay quantification.
• Simulations based on mean data from the 60 ⁇ g dose group had peak concentrations that were approximately 50% of peak concentrations in simulations calculated from mean data from subjects given a 150 ⁇ g dose of PEG-alfacon. The difference is can be attributed to variability among treatment groups, as there is no apparent treatment group (dose) related change in simulation profiles. Additional simulations are shown in Figures 14B-G for 100, 150 and 200 ⁇ g doses administered every 10 and every 7 days. Profiles show the expected pattern of change with increasing dose. There is little effect from shortening the dosing interval to 7 days.
• Figure 16 shows the results of PK-PD modeling. Calculated EC 50 values, which reflect the concentration of drag resulting in 50% of maximum effect, varied greatly among doses. Similarly, the calculated Emax value had large differences with dose as shown in Table 8. Table 8

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