WO2004078194A1

WO2004078194A1 - Interferon drug therapy for the treatment of viral diseases and liver fibrosis

Info

Publication number: WO2004078194A1
Application number: PCT/US2003/006687
Authority: WO
Inventors: Lawrence M. Blatt; Henry H. Hsu
Original assignee: Intermune, Inc.
Priority date: 2003-02-28
Filing date: 2003-02-28
Publication date: 2004-09-16
Also published as: CA2516776A1; JP2006522008A; WO2004078193A1; AU2003287145A1; CN1764475A; AU2003225670A1

Abstract

The present invention provides methods of reducing liver fibrosis; methods of increasing liver function in an individual suffering from liver fibrosis; methods of reducing the incidence of complications associated with HCV and cirrhosis of the liver; methods of reducing viral load in a patient suffering from HCV infection; methods of treating an HCV infection; methods of treating West Nile infection; and methods of treating alphaviral infection. The methods generally involve administering a therapeutically effective amount of IFN-a; and IFN-y; concurrently.

Description

INTERFERON DRUG THERAPY FOR THE TREATMENT OF VlRAL DISEASES AND LlVER

FIBROSIS

FIELD OF THE INVENTION This invention is in the field of flaviviral infection, particularly West Nile viral infection and hepatitis C viral infection, and in the field of liver fibrosis.

BACKGROUND OF THE INVENTION

The United States is currently experiencing an increase in the number of West Nile viral infections. West Nile virus is a member ofthe alpha-like Flaviviridae, as is hepatitis C virus. Most alpha-like viruses, including hepatitis C virus and poliovirus, are highly sensitive to type I interferon treatment. It appears that West Nile virus will become endemic in the United States because it has an avian reservoir and is transmitted by mosquitoes. West Nile virus can cause a harsh, self-limiting fever, body aches, brain swelling, coma, paralysis, and death. Although it is generally accepted that West Nile viral disease results in death in only one out of 40,000 cases, the death rate in the U.S. appears to be higher. In particular, 5 deaths were reported recently in the State of Louisiana. It is possible that the U.S. strain is more virulent or that the U.S. population is genetically predisposed to more severe clinical courses. In middle eastern countries, West Nile virus has been endemic for centuries, which may have allowed natural selection to create populations with resistance to the virus. There is no effective treatment for the disease.

Hepatitis C virus (HCV) 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. Chronic 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.

Antiviral therapy of chronic hepatitis C has evolved rapidly over the last decade, with significant improvements seen in the efficacy of treatment. Nevertheless, even with combination therapy using pegylated IFN-α plus ribavirin, 40% to 50% of patients fail therapy, i.e., are nonresponders or relapsers. These patients currently have no effective therapeutic alternative. In particular, patients who have advanced fibrosis or cirrhosis on liver biopsy are at significant risk of developing complications of advanced liver disease, including ascites, jaundice, variceal bleeding, encephalopathy, and progressive liver failure, as well as a markedly increased risk of hepatocellular carcinoma. The high prevalence of chronic HCN infection has important public health implications for the future burden of chronic liver disease in the United States. Data derived from the National Health and Nutrition Examination Survey (NHANES III) indicate that a large increase in the rate of new HCV infections occurred from the late 1960s to the early 1980s, particularly among persons between 20 to 40 years of age. It is estimated that the number of persons with long-standing HCN infection of 20 years or longer could more than quadruple from 1990 to 2015, from 750,000 to over 3 million. The proportional increase in persons infected for 30 or 40 years would be even greater. Since the risk of HCN-related chronic liver disease is related to the duration of infection, with the risk of cirrhosis progressively increasing for persons infected for longer than 20 years, this will result in a substantial increase in cirrhosis-related morbidity and mortality among patients infected between the years of 1965-1985.

Fibrosis occurs as a result of a chronic toxic insult to the liver, such as chronic hepatitis C virus (HCN) infection, autoimmune injury, and chronic exposure to toxins such as alcohol. Chronic toxic insult leads to repeated cycles of hepatocyte injury and repair accompanied by chronic inflammation. Over a variable period of time, abnormal extracellular matrix progressively accumulates as a consequence ofthe host's wound repair response. Left unchecked, this leads to increasing deposition of fibrous material until liver architecture becomes distorted and the liver's regenerative ability is compromised. The progressive accumulation of scar tissue within the liver finally results in the histopathologic picture of cirrhosis, defined as the formation of fibrous septae throughout the liver with the formation of micronodules.

There is a need in the art for improved methods for treating flaviviral infections, e.g. West Nile viral infection and hepatitis C viral infection, and for treating liver fibrosis. The present invention addresses this need.

Literature

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SUMMARY OF THE INVENTION The present invention provides methods of treating alphavirus infection; methods of treating hepatitis C virus (HCN) infection; methods of treating West Nile virus infection; methods of reducing liver fibrosis; methods of increasing liver function in an individual suffering from liver fibrosis; methods of reducing the incidence of complications associated with HCN and cirrhosis ofthe liver; and methods of reducing viral load, or reducing the time to viral clearance, or reducing morbidity or mortality in the clinical outcomes, in patients suffering from viral infection. The methods generally involve administering a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN- γ for the treatment of viral infection or liver fibrosis.

FEATURES OFTHE INVENTION The invention features a method of treating alphaviral infection, generally involving administering to an individual IFN-γ and a Type I or Type III interferon receptor agonist concurrently, in an amount effective to ameliorate the clinical course ofthe disease. The invention also features a method of treating alphavirus infection by administering to an individual IFN-γ and a Type I or Type III interferon receptor agonist in a synergistically effective amount to ameliorate the clinical course ofthe disease.

The invention features a method of treating West Nile viral infection, generally involving administering to an individual a Type I or Type III interferon receptor agonist, IFN-γ, or IFN-γ and a Type I or Type III interferon receptor agonist concurrently, in an amount effective to reduce the time to viral clearance or to reduce morbidity or mortality in clinical outcomes. The invention also features a method of treating West Nile viral infection by administering to an individual IFN-γ and a Type I or Type III interferon receptor agonist in a synergistically effective amount to reduce the time to viral clearance or to reduce morbidity or mortality in clinical outcomes.

The invention features a method of treating hepatitis C virus (HCN) infection, generally involving administering to an individual IFΝ-γ and a Type I or Type III interferon receptor agonist concurrently, in an amount effective to achieve a sustained viral response. The invention also features a method of treating HCV infection by administering to an individual IFΝ-γ and a Type I or Type III interferon receptor agonist in a synergistically effective amount to achieve a sustained viral response. The invention features a method of reducing liver fibrosis in an individual, generally involving administering a Type I or Type III interferon receptor agonist and IFΝ-γ concurrently, in an amount effective to reduce liver fibrosis. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or Type III interferon receptor agonist and IFΝ-γ along with an amount of pirfenidone or a pirfenidone analog effective to enhance the anti-fibrotic effect or the reduction of liver fibrosis achieved by the a Type I or Type III interferon receptor agonist and/or IFΝ-γ therapy. The invention also features a method of reducing liver fibrosis in an individual by administering a Type I or Type III interferon receptor agonist and IFΝ-γ in a synergistically effective amount to reduce liver fibrosis, optionally including co-administering to the patient an amount of pirfenidone or a pirfenidone analog effective to enhance the anti-fibrotic effect or the reduction of liver fibrosis achieved by the a Type I or Type III interferon receptor agonist and/or IFN-γ therapy. In some embodiments, the degree of liver fibrosis is determined by pre-treatment and post-treatment staging of a liver biopsy, wherein the stage of liver fibrosis, as measured by a standardized scoring system, is reduced by at least one unit when comparing pre-treatment with post-treatment liver biopsies.

The invention features a method of increasing liver function in an individual suffering from liver fibrosis, generally involving administering a Type I or Type III interferon receptor agonist and IFN-γ concurrently, in an amount effective to increase a liver function. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or Type III interferon receptor agonist and IFN-γ along with an amount of pirfenidone or a pirfenidone analog effective to enhance the anti-fibrotic effect or the increase in liver function achieved by the a Type I or Type III interferon receptor agonist and/or IFN-γ therapy. The invention also features a method of increasing liver function in an individual suffering from liver fibrosis by administering a Type I or Type III interferon receptor agonist and IFN-γ in a synergistically effective amount to increase a liver function, optionally including co-administering to the patient an amount of pirfenidone or a pirfenidone analog effective to enhance the anti-fibrotic effect or the increase in liver function achieved by the Type I or Type III interferon receptor agonist and/or IFN-γ therapy. Liver function may be indicated by measuring a parameter selected from the group consisting of serum transaminase level, prothrombin time, serum bilirubin level, blood platelet count, serum albumin level, improvement in portal wedge pressure, reduction in degree of ascites, reduction in a level of encephalopathy, and reduction in a degree of internal varices. The invention features a method of reducing the incidence of a complication of cirrhosis ofthe liver. The methods generally involve administering a Type I or Type III interferon receptor agonist and IFN-γ concurrently, in an amount effective to reduce the incidence of a complication of cirrhosis ofthe liver. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or Type III interferon receptor agonist and IFN-γ along with an amount of pirfenidone or a pirfenidone analog effective to enhance the anti-fibrotic effect or the reduction ofthe incidence of a complication of cirrhosis ofthe liver achieved by the Type I or Type III interferon receptor agonist and/or IFN-γ therapy. The invention also features a method of reducing the incidence of a complication of cirrhosis ofthe liver by administering a Type I or Type III interferon receptor agonist and IFN-γ in a synergistically effective amount to reduce the incidence of a complication of cirrhosis ofthe liver, optionally including co-administering to the patient an amount of pirfenidone or a pirfenidone analog effective to enhance the anti- fibrotic effect or the reduction ofthe incidence of a complication of cirrhosis ofthe liver achieved by the a Type I or Type III interferon receptor agonist and/or IFN-γ therapy.

Examples of complications of cirrhosis ofthe liver are portal hypertension, progressive liver insufficiency, and hepatocellular carcinoma.

In carrying out the methods of combination therapy for alphaviral infection, hepatitis C viral infection, West Nile viral infection and/or liver fibrosis in an individual as described above, a Type I or Type III interferon receptor agonist and IFN-γ are administered to the individual. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in the same formulation. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in separate formulations. When administered in separate formulations, a Type I or Type III interferon receptor agonist and IFN-γ can be administered substantially simultaneously, or can be administered within about 24 hours of one another. In many embodiments, a Type I or Type III interferon receptor agonist and IFN-γ are administered subcutaneously in multiple doses. Optionally, the Type I or Type III interferon receptor agonist and/or IFN-γ is administered to the individual by a controlled drug delivery device. Optionally, the Type I or Type III interferon receptor agonist and/or IFN-γ is administered to the individual substantially continuously or continuously by a controlled drug delivery device. Optionally, the controlled drug delivery device is an implantable infusion pump and the infusion pump delivers the a Type I or Type III interferon receptor agonist and/or IFN-γ to the individual by subcutaneous infusion.

In some embodiments utilizing combination therapy, IFN-γ is administered during the entire course of a Type I or Type III interferon receptor agonist treatment. In other embodiments, IFN-γ is administered for a period of time that is overlapping with that ofthe Type I or Type III interferon receptor agonist treatment, e.g., the IFN-γ treatment can begin before the Type I or Type III interferon receptor agonist treatment begins and end before the Type I or Type III interferon receptor agonist treatment ends; the IFN-γ treatment can begin after the Type I or Type III interferon receptor agonist treatment begins and end after the IFN-γ treatment ends; the IFN-γ treatment can begin after the Type I or Type III interferon receptor agonist treatment begins and end before the Type I or Type III interferon receptor agonist treatment ends; or the IFN-γ treatment can begin before the IFN-α treatment begins and end after the Type I or Type III interferon receptor agonist treatment ends. In embodiments utilizing co-administration of pirfenidone or a pirfenidone analog, the duration of therapy with pirfenidone or a pirfenidone analog can be coincident with the duration of therapy with a Type I or Type III interferon receptor agonist and/or IFN-γ. In other embodiments, the course of therapy with pirfenidone or a pirfenidone analog can overlap with the course of therapy with a Type I or Type III interferon receptor agonist and/or IFN-γ, e.g., the pirfenidone or pirfenidone analog treatment can begin before the treatment with a Type I or Type III interferon receptor agonist and/or IFN-γ begins and end before treatment with a Type I or Type III interferon receptor agonist and/or IFN-γ ends; the pirfenidone treatment can being after the treatment with IFN-α and/or IFN-γ begins and end before the treatment with a Type I or Type III interferon receptor agonist and/or IFN-γ ends; or the pirfenidone treatment can begin before the treatment with a Type I or Type III interferon receptor agonist and/or IFN-γ begins and end after treatment with a Type I or Type III interferon receptor agonist and/or IFN-γ ends.

In other embodiments, ribavirin is co-administered with a Type I or Tjφe III interferon receptor agonist and IFN-γ. In still other embodiments, ribavirin is co- administered with a Type I or Type III interferon receptor agonist, IFN-γ and pirfenidone (or a specific pirfenidone analog).

In many embodiments, any ofthe above-described methods involve administering IFN-α and IFN-γ. In some of these embodiments, the methods involve co-administering ribavirin, IFN-α, and IFN-γ. In other embodiments, the methods involve co-administering IFN-α, IFN-γ, and pirfenidone or a pirfenidone analog.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a graph depicting HCV replicon assay results after treatment of infected cells with various doses of Actimmune, Infergen, a combination of 2.5 ng/mL Actimmune and 0.2 ng/mL Infergen, or a combination of 25 ng/mL Actimmune and 0.2 ng/mL Infergen.

DEFINITIONS

As used herein, the term "hepatic fibrosis," used interchangeably herein with "liver fibrosis," refers to the growth of scar tissue in the liver that can occur in the context of a chronic hepatitis infection.

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. As used herein, the term "alphavirus," and its grammatical variants, refers to a group of viruses characterized by (i) an RNA genome (ii) viral replication in the cytoplasm of host cells and (iii) no DNA phase occurs in the viral replication cycle.

As used herein, the term "liver function" refers to a normal function ofthe liver, including, but not limited to, a synthetic function, including, but not limited to, synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5'-nucleosidase, γ- glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; a hemodynamic function, including splanchnic and portal hemodynamics; and the like.

The term "sustained viral response" (SVR; also referred to as a "sustained response" or a "durable response"), as used herein, refers to the response of an individual to a treatment regimen for HCV infection, in terms of serum HCV titer. Generally, a "sustained viral response" refers to no detectable HCV RNA (e.g., less than about 500, less than about 200, or less than about 100 genome copies per milliliter serum) 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 at least about six months following cessation of treatment.

"Treatment failure patients" as used herein generally refers to HCV-infected patients who failed to respond to previous therapy for HCV (referred to as "non-responders") or who initially responded to previous therapy, but in whom the therapeutic response was not maintained (referred to as "relapsers"). The previous therapy generally can include treatment with IFN-α monotherapy or IFN-α combination therapy, where the combination therapy may include administration of IFN-α and an antiviral agent such as ribavirin.

As used herein, the terms "treatment," "treating," and the like, refer 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," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression ofthe disease.

The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

A "specific pirfenidone analog," and all grammatical variants thereof, refers to, and is limited to, each and every pirfenidone analog shown in Table 1.

As used herein, the term "a Type I interferon receptor agonist" refers to any naturally occurring or non-naturally occurring ligand of human Type I interferon receptor, which binds to and causes signal transduction via the receptor. Type I interferon receptor agonists include interferons, including naturally-occurring interferons, modified interferons, synthetic interferons, pegylated interferons, fusion proteins comprising an interferon and a heterologous protein, shuffled interferons; antibody specific for an interferon receptor; non- peptide chemical agonists; and the like. As used herein, the term "a Type III interferon receptor agonist" refers to any naturally occurring or non-naturally occurring ligand of humanIL-28 receptor α ("IL-28R"), the amino acid sequence of which is described by Sheppard, et al., infra., that binds to and causes signal transduction via the receptor.

The term "dosing event" as used herein refers to administration of an antiviral agent to a patient in need thereof, which event may encompass one or more releases of an antiviral agent from a drug dispensing device. Thus, the term "dosing event," as used herein, includes, but is not limited to, installation of a continuous delivery device (e.g., a pump or other controlled release injectible system); and a single subcutaneous injection followed by installation of a continuous delivery system. "Continuous delivery" as used herein {e.g., in the context of "continuous delivery of a substance to a tissue") is meant to refer to movement of drug to a delivery site, e.g., into a tissue in a fashion that provides for delivery of a desired amount of substance into the tissue over a selected period of time, where about the same quantity of drug is received by the patient each minute during the selected period of time. "Controlled release" as used herein {e.g., in the context of "controlled drug release") is meant to encompass release of substance {e.g., a Type I or Type III interferon receptor agonist, e.g., IFN-α) at a selected or otherwise controllable rate, interval, and/or amount, which is not substantially influenced by the environment of use. "Controlled release" thus encompasses, but is not necessarily limited to, substantially continuous delivery, and patterned delivery {e.g., intermittent delivery over a period of time that is interrupted by regular or irregular time intervals).

"Patterned" or "temporal" as used in the context of drug delivery is meant delivery of drug in a pattern, generally a substantially regular pattern, over a pre-selected period of time {e.g. , other than a period associated with, for example a bolus injection). "Patterned" or "temporal" drug delivery is meant to encompass delivery of drug at an increasing, decreasing, substantially constant, or pulsatile, rate or range of rates {e.g., amount of drug per unit time, or volume of drug formulation for a unit time), and further encompasses delivery that is continuous or substantially continuous, or chronic. The term "controlled drug delivery device" is meant to encompass any device wherein the release (e.g., rate, timing of release) of a drug or other desired substance contained therein is controlled by or determined by the device itself and not substantially influenced by the environment of use, or releasing at a rate that is reproducible within the environment of use. By "substantially continuous" as used in, for example, the context of "substantially continuous infusion" or "substantially continuous delivery" is meant to refer to delivery of drug in a manner that is substantially uninterrupted for a pre-selected period of drug delivery, where the quantity of drug received by the patient during any 8 hour interval in the pre-selected period never falls to zero. Furthermore, "substantially continuous" drug delivery can also encompass delivery of drug at a substantially constant, pre-selected rate or range of rates {e.g., amount of drug per unit time, or volume of drug formulation for a unit time) that is substantially uninterrupted for a pre-selected period of drug delivery.

By "substantially steady state" as used in the context of a biological parameter that may vary as a function of time, it is meant that the biological parameter exhibits a substantially constant value over a time course, such that the area under the curve defined by the value ofthe biological parameter as a function of time for any 8 hour period during the time course (AUC₈ι_ιr) is no more than about 20% above or about 20% below, and preferably no more than about 15% above or about 15% below, and more preferably no more than about 10% above or about 10% below, the average area under the curve ofthe biological parameter over an 8 hour period during the time course (AUC₈hr average)- The AUC₈h_r average is defined as the quotient (q) ofthe area under the curve ofthe biological parameter over the entirety ofthe time course (AUCt_otai) divided by the number of 8 hour intervals in the time course (t_totaiι/3days), i.e., q = (AUCtotai)/ (ttotaiι/3days)- For example, in the context of a serum concentration of a drug, the serum concentration ofthe drug is maintained at a substantially steady state during a time course when the area under the curve of serum concentration of the drug over time for any 8 hour period during the time course (AUC₈h_r) is no more than about 20% above or about 20% below the average area under the curve of serum concentration ofthe drug over an 8 hour period in the time course (AUC₈ι_{ιr av}erage), i.e., the AUCs_hr is no more than 20% above or 20% below the AUC₈h_r a_verage for the serum concentration ofthe drug over the time course.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth ofthe unit ofthe lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both ofthe limits, ranges excluding either both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing ofthe present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date ofthe present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating alphaviral infections, including methods of treating West Nile viral infection and methods of treating HCV infection, and methods of treating liver fibrosis, including reducing clinical liver fibrosis, reducing the likelihood that liver fibrosis will occur, and reducing a parameter associated with liver fibrosis. The methods generally involve administering an effective amount of a Type I or Type III interferon receptor agonist and IFN-γ, to an individual in need thereof. Of particular interest in many embodiments is treatment of humans.

Liver fibrosis is a precursor to the complications associated with liver cirrhosis, such as portal hypertension, progressive liver insufficiency, and hepatocellular carcinoma. A reduction in liver fibrosis thus reduces the incidence of such future complications.

Accordingly, the present invention further provides methods of reducing the likelihood that an individual will develop complications associated with cirrhosis ofthe liver.

Some methods ofthe invention generally involve administering a therapeutically effective amount of a Type I or Type III interferon receptor agonist or IFN-γ for the treatment of West Nile viral infection. As used herein, a "therapeutically effective amount" of a Type I or Type III interferon receptor agonist or IFN-γ is an amount of a Type I or Type III interferon receptor agonist or IFN-γ that is effective in treating a West Nile viral infection.

Other methods ofthe invention generally involve administering a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ. As used herein, a "therapeutically effective amount" of a Type I or Type III interferon receptor agonist and IFN-γ is an amount of a Type I or Type III interferon receptor agonist and IFN-γ that is effective in treating an alphaviral infection, or in treating a West Nile viral infection, or in treating an HCV infection, or in achieving a sustained viral response in an HCV infection, or in reducing liver fibrosis, or in reducing the likelihood that an individual will develop liver fibrosis, or in reducing a parameter associated with liver fibrosis, or in reducing a disorder associated with cirrhosis ofthe liver.

In certain embodiments, the methods involve administering a synergistically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ. As used herein, a "synergistically effective amount" of a Type I or Type III interferon receptor agonist and IFN-γ used in combination therapy for a particular disease, and any grammatical equivalent thereof, is an amount of a Type I or Type III interferon receptor agonist and an amount of IFN-γ that in combination have a greater effect on the disease than would be expected for a merely additive effect of an identical amount of a Type I or Type III interferon receptor agonist used in monotherapy for the disease and an identical amount of IFN-γ used in monotherapy for the disease.

For example, in certain embodiments, the methods involve administering a synergistically effective amount of IFN-α and IFN-γ. As used herein, a "synergistically effective amount" of IFN-α and IFN-γ used in combination therapy for a particular disease, and any grammatical equivalent thereof, is an amount of IFN-α and an amount of IFN-γ that in combination have a greater effect on the disease than would be expected for a merely additive effect of an identical amount of IFN-α used in monotherapy for the disease and an identical amount of IFN-γ used in monotherapy for the disease. In some embodiments ofthe invention, a selected amount of a Type I or Type III interferon receptor agonist and a selected amount of IFN-γ are effective when used in combination therapy for a disease, but the selected amount of a Type I or Type III interferon receptor agonist and/or the selected amount of IFN-γ is ineffective when used in monotherapy for the disease. Thus, the invention encompasses (1) regimens in which a selected amount of IFN-γ enhances the therapeutic benefit of a selected amount of a Type I or Type III interferon receptor agonist when used in combination therapy for a disease, where the selected amount of IFN-γ provides no therapeutic benefit when used in monotherapy for the disease (2) regimens in which a selected amount of a Type I or Type III interferon receptor agonist enhances the therapeutic benefit of a selected amount of IFN-γ when used in combination therapy for a disease, where the selected amount of a Type I or Type III interferon receptor agonist provides no therapeutic benefit when used in monotherapy for the disease and (3) regimens in which a selected amount of a Type I or Type III interferon receptor agonist and a selected amount of IFN-γ provide a therapeutic benefit when used in combination therapy for a disease, where each ofthe selected amounts ofthe Type I or Type III interferon receptor agonist and IFN-γ, respectively, provides no therapeutic benefit when used in monotherapy for the disease. As used herein, a "synergistically effective amount" of a Type I or Type III interferon receptor agonist and IFN-γ, and its grammatical equivalents, shall be understood to include any regimen encompassed by any of (l)-(3) above. Alphavirus

The present invention provides methods for treating alphaviral infection. The methods generally involve administering a Type I or Type III interferon receptor agonist and IFN-γ to an individual in an amount that is effective to ameliorate the clinical course ofthe disease.

Whether a subject method is effective in treating an alphaviral infection can be determined by a reduction in number or length of hospital stays, a reduction in time to viral clearance, a reduction of morbidity or mortality in clinical outcomes, or other indicator of disease response. In general, an effective amount of a Type I or Type III interferon receptor agonist and

IFN-γ is an amount that is effective to (i) reduce the time to viral clearance, (ii) reduce morbidity or mortality in the clinical course ofthe disease or (iii) reduce viral load in the patient. West Nile Virus

The present invention provides methods for treating West Nile viral infection. The methods generally involve administering a Type I or Type III interferon receptor agonist, or IFN-γ, or a Type I or Type III interferon receptor agonist and IFN-γ, to an individual in an amount that is effective to reduce the time to viral clearance in the individual, and/or to ameliorate the clinical course ofthe disease.

Whether a subject method is effective in treating West Nile viral infections can be determined by a reduction in number or length of hospital stays, a reduction in time to viral clearance, a reduction of morbidity or mortality in clinical outcomes, or other indicator of disease response.

In general, an effective amount of a Type I or Type III interferon receptor agonist, an effective amount of IFN-γ, or an effective amount of a Type I or Type III interferon receptor agonist and IFN-γ, is an amount that is effective to reduce the time to viral clearance, or an amount that is effective to reduce morbidity or mortality in the clinical course ofthe disease. Hepatitis C Virus

The present invention provides methods for treating HCV. The methods generally involve administering a Type I or Type III interferon receptor agonist and IFN-γ, to an individual in an amount that is effective to decrease viral load in the individual, and to achieve a sustained viral response.

Whether a subject method is effective in treating 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, elevations in serum transaminase levels, and necroinflammatory activity in the liver. Indicators of liver fibrosis are discussed in detail below.

Viral load can be measured by measuring the titer or level of virus in serum. These methods include, but are not limited to, a quantitative polymerase chain reaction (PCR) and a branched DNA (bDNA) test. 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 Monitor™, Roche Molecular Systems, New Jersey); and a branched DNA (deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNA Assay (bDNA), Chiron Corp., Emeryville, California). See, e.g., Gretch et al. (1995) Ann. Intern. Med. 123:321-329.

In general, an effective amount of IFNα and IFNγ is an amount that is effective to reduce viral load to undetectable levels, e.g., to less than about 5000, less than about 1000, less than about 500, or less than about 200 genome copies/mL serum. In some embodiments, an effective amount of a Type I or Type III interferon receptor agonist and/or IFN-γ is an amount that is effective to reduce viral load to less than 100 genome copies/mL serum. In many embodiments, the methods ofthe invention achieve a sustained viral response, e.g., the viral load is reduced to undetectable levels 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 at least about six months following cessation of treatment.

As noted above, whether a subject method is effective in treating an HCV infection can be determined by measuring a parameter associated with HCV infection, such as liver fibrosis. Methods of determining the extent of liver fibrosis are discussed in detail below. In some embodiments, the level of a serum marker of liver fibrosis indicates the degree of liver fibrosis.

As one non-limiting example, levels of serum alanine aminotransferase (ALT) are measured, using standard assays. In general, an ALT level of less than about 45 international units is considered normal. In some embodiments, an effective amount of IFNα and IFNγ is an amount effective to reduce ALT levels to less than about 45 U/ml serum. A therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is an amount that is effective to reduce a serum level of a marker of liver fibrosis by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the level ofthe marker in an untreated individual, or to a placebo-treated individual. Methods of measuring serum markers include immunological- based methods, e.g., enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and the like, using antibody specific for a given serum marker. Fibrosis

Whether treatment with a Type I or Type III interferon receptor agonist and IFN-γ is effective in reducing liver fibrosis is determined by any of a number of well-established techniques for measuring liver fibrosis and liver function. Liver fibrosis reduction is determined by analyzing a liver biopsy sample. An analysis of a liver biopsy comprises assessments of two major components: necroinfiammation assessed by "grade" as a measure ofthe 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., Brunt (2000) Hepatol 31:241-246; and METAVIR (1994) Hepatology 20: 15-20. Based on analysis ofthe liver biopsy, a score is assigned. A number of standardized scoring systems exist which provide a quantitative assessment of the degree and severity of fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.

The METAVIR scoring system is based on an analysis of various features of a liver biopsy, including fibrosis (portal fibrosis, centrilobular fibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis, acidophilic retraction, and ballooning degeneration); inflammation (portal tract inflammation, portal lymphoid aggregates, and distribution of portal inflammation); bile duct changes; and the Knodell index (scores of periportal necrosis, lobular necrosis, portal inflammation, fibrosis, and overall disease activity). The definitions of each stage in the METAVIR system are as follows: score: 0, no fibrosis; score: 1, stellate enlargement of portal tract but without septa formation; score: 2, enlargement of portal tract with rare septa formation; score: 3, numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index, classifies specimens based on scores in four categories of histologic features: I. Periportal and/or bridging necrosis; II. Intralobular degeneration and focal necrosis; III. Portal inflammation; and IV. Fibrosis. In the Knodell staging system, scores are as follows: score: 0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion); score: 2, moderate fibrosis; score: 3, severe fibrosis (bridging fibrosis); and score: 4, cirrhosis. The higher the score, the more severe the liver tissue damage. Knodell (1981) Hepatol. 1 :431. In the Scheuer scoring system scores are as follows: score: 0, no fibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2, periportal or portal-portal septa, but intact architecture; score: 3, fibrosis with architectural distortion, but no obvious cirrhosis; score: 4, probable or definite cirrhosis. Scheuer (1991) J Hepatol. 13:372. The Ishak scoring system is described in Ishak (1995) J. Hepatol. 22:696-699. Stage

0, No fibrosis; Stage 1, Fibrous expansion of some portal areas, with or without short fibrous septa; stage 2, Fibrous expansion of most portal areas, with or without short fibrous septa; stage 3, Fibrous expansion of most portal areas with occasional portal to portal (P-P) bridging; stage 4, Fibrous expansion of portal areas with marked bridging (P-P) as well as portal-central (P-C); stage 5, Marked bridging (P-P and/or P-C) with occasional nodules (incomplete cirrhosis); stage 6, Cirrhosis, probable or definite.The benefit of anti-fibrotic therapy can also be measured and assessed by using the Child-Pugh scoring system which comprises a multicomponent point system based upon abnormalities in serum bilirubin level, serum albumin level, prothrombin time, the presence and severity of ascites, and the presence and severity of encephalopathy. Based upon the presence and severity of abnormality of these parameters, patients may be placed in one of three categories of increasing severity of clinical disease: A, B, or C.

In some embodiments, a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is an amount of a Type I or Type III interferon receptor agonist and IFN-γ that effects a change of one unit or more in the fibrosis stage based on pre- and post-therapy liver biopsies. In particular embodiments, a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ reduces liver fibrosis by at least one unit in the METAVIR, the Knodell, the Scheuer, the Ludwig, or the Ishak scoring system. Secondary, or indirect, indices of liver function can also be used to evaluate the efficacy of Type I or Type III interferon receptor agonist and IFN-γ treatment. Morphometric computerized semi-automated assessment ofthe quantitative degree of liver fibrosis based upon specific staining of collagen and/or serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Secondary indices of liver function include, but are not limited to, serum transaminase levels, prothrombin time, bilirubin, platelet count, portal pressure, albumin level, and assessment of the Child-Pugh score. An effective amount of IFN-α and IFN-γ is an amount that is effective to increase an index of liver function by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the index of liver function in an untreated individual, or to a placebo-treated individual. Those skilled in the art can readily measure such indices of liver function, using standard assay methods, many of which are commercially available, and are used routinely in clinical settings.

Serum markers of liver fibrosis can also be measured as an indication ofthe 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 α-2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.

A therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is an amount that is effective to reduce a serum level of a marker of liver fibrosis by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the level ofthe marker in an untreated individual, or to a placebo-treated individual. Those skilled in the art can readily measure such serum markers of liver fibrosis, using standard assay methods, many of which are commercially available, and are used routinely in clinical settings. Methods of measuring serum markers include immunological-based methods, e.g., enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and the like, using antibody specific for a given serum marker.

Quantitative tests of functional liver reserve can also be used to assess the efficacy of treatment with a Type I or Type III interferon receptor agonist and IFN-γ. These include: indocyanine green clearance (ICG), galactose elimination capacity (GEC), aminopyrine breath test (ABT), antipyrine clearance, monoethylglycine-xylidide (MEG-X) clearance, and caffeine clearance.

As used herein, a "complication associated with cirrhosis ofthe liver" refers to a disorder that is a sequellae of decompensated liver disease, i.e., or occurs subsequently to and as a result of development of liver fibrosis, and includes, but it not limited to, development of ascites, variceal bleeding, portal hypertension, jaundice, progressive liver insufficiency, encephalopathy, hepatocellular carcinoma, liver failure requiring liver transplantation, and liver-related mortality. A therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is an amount that is effective in reducing the incidence (e.g., the likelihood that an individual will develop) of a disorder associated with cirrhosis ofthe liver by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to an untreated individual, or to a placebo-treated individual.

Whether treatment with a Type I or Type III interferon receptor agonist and IFN-γ is effective in reducing the incidence of a disorder associated with cirrhosis ofthe liver can readily be determined by those skilled in the art.

Reduction in liver fibrosis increases liver function. Thus, the invention provides methods for increasing liver function, generally involving administering a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ. Liver functions include, but are not limited to, synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5'-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; a hemodynamic function, including splanchnic and portal hemodynamics; and the like.

Whether a liver function is increased is readily ascertainable by those skilled in the art, using well-established tests of liver function. Thus, synthesis of markers of liver function such as albumin, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin, and the like, can be assessed by measuring the level of these markers in the serum, using standard immunological and enzymatic assays. Splanchnic circulation and portal hemodynamics can be measured by portal wedge pressure and/or resistance using standard methods. Metabolic functions can be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normal range can be determined by measuring the levels of such proteins, using standard immunological and enzymatic assays. Those skilled in the art know the normal ranges for such serum proteins. The following are non-limiting examples. The normal level of alanine transaminase is about 45 U per milliliter of serum. The normal range of aspartate transaminase is from about 5 to about 40 units per liter of serum. Bilirubin is measured using standard assays. Normal bilirubin levels are usually less than about 1.2 mg/dL. Serum albumin levels are measured using standard assays. Normal levels of serum albumin are in the range of from about 35 to about 55 g/L. Prolongation of prothrombin time is measured using standard assays. Normal prothrombin time is less than about 4 seconds longer than control. A therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is one that is effective to increase liver function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more. For example, a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is an amount effective to reduce an elevated level of a serum marker of liver function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, or to reduce the level ofthe serum marker of liver function to within a normal range. A therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ is also an amount effective to increase a reduced level of a serum marker of liver function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, or to increase the level ofthe serum marker of liver function to within a normal range. Type I interferon receptor agonists In any ofthe above-described methods, in some embodiments a Type I interferon receptor agonist is administered. Type I interferon receptor agonists include an IFN-α; an IFN-β; an IFN-tau; an IFN-ω; antibody agonists specific for a Type I interferon receptor; and any other agonist of Type I interferon receptor, including non-polypeptide agonists. Interferon-Alpha Any known IFN-α can be used in the instant invention. The term "interferon-alpha" as used herein refers to a family of related polypeptides that inhibit viral replication and cellular proliferation and modulate immune response. The term 'TFN-a" includes naturally occurring IFN-α; synthetic IFN-α; derivatized IFN-α (e.g., PEGylated IFN-α, glycosylated IFN-α, and the like); and analogs of naturally occurring or synthetic IFN-α; essentially any IFN-α that has antiviral properties, as described for naturally occurring IFN-α.

Suitable alpha interferons include, but are not limited to, naturally-occurring IFN-α (including, but not limited to, naturally occurring IFN-α2a, IFN-α2b); 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. J.; recombinant 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; and 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.

The term "IFN-α" also encompasses consensus IFN-α. Consensus IFN-α (also referred to as "CIFN" and "IFN-con" and "consensus interferon") encompasses but is not limited to the amino acid sequences designated IFN-con_l3 IFN-con₂ and IFN-con₃ which are disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471; and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (e.g., Infergen®, InterMune, Inc., Brisbane, Calif). IFN-con_t is the consensus interferon agent in the Infergen® alfacon-1 product. The Infergen® consensus interferon product is referred to herein by its brand name (Infergen®) or by its generic name (interferon alfacon-1). DNA sequences encoding IFN-con may be synthesized as described in the aforementioned patents or other standard methods. Use of CIFN is of particular interest.

Also suitable for use in the present invention are fusion polypeptides comprising an IFN-α and a heterologous polypeptide. Suitable IFN-α fusion polypeptides include, but are not limited to, Albuferon-alpha™ (a fusion product of human albumin and IFN-α; Human Genome Sciences; see, e.g., Osborn et al. (2002) J. Pharmacol Exp. Therap. 303:540-548). Also suitable for use in the present invention are gene-shuffled forms of IFN-α. See., e.g., Masci et al. (2003) Curr. Oncol Rep. 5:108-113. PEGylated Interferon-Alpha The term "IFN-α" also encompasses derivatives of IFN-α that are derivatized (e.g., are chemically modified) to alter certain properties such as serum half-life. As such, the term "IFN-α" includes glycosylated IFN-α; IFN-α derivatized with polyethylene glycol ("PEGylated IFN-α"); and the like. PEGylated IFN-α, and methods for making same, is discussed in, e.g., U.S. Patent Nos. 5,382,657; 5,981,709; and 5,951,974. PEGylated IFN-α encompasses conjugates of PEG and any ofthe above-described IFN-α molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, N. J.), interferon alpha 2b (Intron, Schering-Plough, Madison, N. J.), interferon alpha- 2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen®, InterMune, Inc., Brisbane, Calif).

Any ofthe above-mentioned IFN-α polypeptides can be modified with one or more polyethylene glycol moieties, i.e., PEGylated. The PEG molecule of a PEGylated IFN-α polypeptide is conjugated to one or more amino acid side chains ofthe IFN-α polypeptide. In some embodiments, the PEGylated IFN-α contains a PEG moiety on only one amino acid. In other embodiments, the PEGylated IFN-α contains a PEG moiety on two or more amino acids, e.g., the IFN-α contains a PEG moiety attached to two, three, four, five, six, seven, eight, nine, or ten different amino acid residues. IFN-α may be coupled directly to PEG (i.e., without a linking group) through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.

In some embodiments, the PEGylated IFN-α is PEGylated at or near the amino terminus (N-terminus) ofthe IFN-α polypeptide, e.g., the PEG moiety is conjugated to the IFN-α polypeptide at one or more amino acid residues from amino acid 1 through amino acid 4, or from amino acid 5 through about 10.

In other embodiments, the PEGylated IFN-α is PEGylated at one or more amino acid residues from about 10 to about 28.

In other embodiments, the PEGylated IFN-α is PEGylated at or near the carboxyl terminus (C-terminus) ofthe IFN-α polypeptide, e.g., at one or more residues from amino acids 156-166, or from amino acids 150 to 155.

In other embodiments, the PEGylated IFN-α is PEGylated at one or more amino acid residues at one or more residues from amino acids 100-114.

The polyethylene glycol derivatization of amino acid residues at or near the receptor- binding and/or active site domains ofthe IFN-α protein can disrupt the functioning of these domains. In certain embodiments ofthe invention, amino acids at which PEGylation is to be avoided include amino acid residues from amino acid 30 to amino acid 40; and amino acid residues from amino acid 113 to amino acid 149.

In some embodiments, PEG is attached to IFN-α via a linking group. The linking group is any biocompatible linking group, where "biocompatible" indicates that the compound or group is non-toxic and may be utilized in vitro or in vivo without causing injury, sickness, disease, or death. PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thiol 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 carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazole group (including, for example, carbonyldimidazole (GDI)), 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, a tyrosine group, a cysteine group, a histidine group or a primary amine. Methods for attaching a PEG to an IFN-α polypeptide are known in the art, and any known method can be used. See, for example, by Park et al, Anticancer Res., 1:373-376 (1981); Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992); and U.S. PatentNo. 5,985,265.

Pegylated IFN-α, and methods for making same, is discussed in, e.g., U.S. Patent Nos. 5,382,657; 5,981,709; 5,985,265; and 5,951,974. Pegylated IFN-α encompasses conjugates of PEG and any ofthe above-described IFN-α molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon, Hoffman LaRoche, Nutley,

N.J.), where PEGylated Roferon is known as Pegasys (Hoffman LaRoche); interferon alpha 2b (Intron, Schering-Plough, Madison, N. J.), where PEGylated Intron is known as PEG- Intron (Schering-Plough); interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon (CIFN) as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen®, InterMune, Inc., Brisbane, Calif), where PEGylated Infergen is referred to as PEG-Infergen. Polyethylene glycol

Polyethylene glycol suitable for conjugation to an IFN-α polypeptide is soluble in water at room temperature, and has the general formula R(O-CH₂-CH₂)_nO-R, where R is hydrogen or a protective group such as an alkyl 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.

In many embodiments, 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.

In other embodiments, PEG is derivatized so that it is reactive with free carboxyl groups in the IFN-α polypeptide, e.g., the free carboxyl group at the carboxyl terminus ofthe IFN-α polypeptide. Suitable derivatives of PEG that are reactive with the free carboxyl group at the carboxyl-terminus of IFN-α include, but are not limited to PEG-amine, and hydrazine derivatives of PEG (e.g.,.PEG-NH-NH₂).

In other embodiments, PEG is derivatized such that it comprises a terminal thiocarboxylic acid group, -COSH, which selectively reacts with amino groups to generate amide derivatives. Because ofthe reactive nature ofthe thio acid, selectivity of certain amino groups over others is achieved. For example, -SH exhibits sufficient leaving group ability in reaction with N-terminal amino group at appropriate pH conditions such that the ε- amino groups in lysine residues are protonated and remain non-nucleophilic. On the other hand, reactions under suitable pH conditions may make some ofthe accessible lysine residues to react with selectivity.

In other embodiments, the PEG comprises a reactive ester such as an N-hydroxy succinimidate at the end ofthe PEG chain. Such an N-hydroxysuccinimidate-containing PEG molecule reacts with select amino groups at particular pH conditions such as neutral 6.5-7.5. For example, the N-terminal amino groups may be selectively modified under neutral pH conditions. However, if the reactivity ofthe reagent were extreme, accessible- NH groups of lysine may also react.

The PEG can be conjugated directly to the IFN-α polypeptide, or through a linker. In some embodiments, a linker is added to the IFN-α polypeptide, forming a linker-modified IFN-α polypeptide. Such linkers provide various functionalities, e.g., reactive groups such sulfhydryl, amino, or carboxyl groups to couple a PEG reagent to the linker-modified IFN-α

In some embodiments, the PEG conjugated to the IFN-α polypeptide is linear. In other embodiments, the PEG conjugated to the IFN-α 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. PatentNo. 6,046,305.

PEG having a molecular weight in a range of from about 2 kDa to about 100 kDa, is generally used, where the term "about," in the context of PEG, indicates that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight. For example, PEG suitable for conjugation to IFN-α has a molecular weight of from about 2 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 40 kDa, from about 40 kDa to about 50 kDa, from about 50 kDa to about 60 kDa, from about 60 kDa to about 70 kDa, from about 70 kDa to about 80 kDa, from about 80 kDa to about 90 kDa, or from about 90 kDa to about 100 kDa. Preparing PEG-IFN-α conjugates As discussed above, the PEG moiety can be attached, directly or via a linker, to an amino acid residue at or near the N-terminus, internally, or at or near the C-terminus ofthe IFN-α polypeptide. Conjugation can be carried out in solution or in the solid phase.

N-terminal linkage

Methods for attaching a PEG moiety to an amino acid residue at or near the N- terminus of an IFN-α polypeptide are known in the art. See, e.g., U.S. PatentNo. 5,985,265.

In some embodiments, known methods for selectively obtaining an N-terminally chemically modified IFN-α are used. For example, a method of protein modification by reductive alkylation 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. Under the appropriate reaction conditions, 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 ofthe pK_a differences between the ε-amino groups ofthe lysine residues and that ofthe α-amino group ofthe N- terminal residue ofthe protein. By such selective derivatization attachment of a PEG moiety to the IFN-α is controlled: the conjugation with the polymer takes place predominantly at the N-terminus ofthe IFN-α and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs.

C-terminal linkage

N-terminal-specific coupling procedures such as described in U.S. PatentNo. 5,985,265 provide predominantly monoPEGylated products. However, the purification procedures aimed at removing the excess reagents and minor multiply PEGylated products remove the N-terminal blocked polypeptides. In terms of therapy, such processes lead to significant increases in manufacturing costs. For example, examination ofthe structure of the well-characterized Infergen® Alfacon-1 CIFN polypeptide amino acid sequence reveals that the clipping is approximate 5% at the carboxyl terminus and thus there is only one major C-terminal sequence. Thus, in some embodiments, N-terminally PEGylated IFN-α is not used; instead, the IFN-α polypeptide is C-terminally PEGylated.

An effective synthetic as well as therapeutic approach to obtain mono PEGylated Infergen product is therefore envisioned as follows: A PEG reagent that is selective for the C-terminal can be prepared with or without spacers. For example, polyethylene glycol modified as methyl ether at one end and having an amino function at the other end may be used as the starting material.

Preparing or obtaining a water-soluble carbodiimide as the condensing agent can be carried out. Coupling IFN-α (e.g., Infergen® Alfacon-1 CIFN or consensus interferon) with a water-soluble carbodiimide as the condensing reagent is generally carried out in aqueous medium with a suitable buffer system at an optimal pH to effect the amide linkage. A high molecular weight PEG can be added to the protein covalently to increase the molecular weight. The reagents selected will depend on process optimization studies. A non-limiting example of a suitable reagent is ED AC or l-ethyl-3- (3-dimethylaminopropyl) carbodiimide. The water solubility of ED AC allows for direct addition to a reaction without the need for prior organic solvent dissolution. Excess reagent and the isourea formed as the by-product ofthe cross-linking reaction are both water-soluble and may easily be removed by dialysis or gel filtration. A concentrated solution of ED AC in water is prepared to facilitate the addition of a small molar amount to the reaction. The stock solution is prepared and used immediately in view ofthe water labile nature ofthe reagent. Most ofthe synthetic protocols in literature suggest the optimal reaction medium to be in pH range between 4.7 and 6.0. However the condensation reactions do proceed without significant losses in yields up to pH 7.5. Water may be used as solvent. In view ofthe contemplated use of Infergen, preferably the medium will be 2-(N-mo holino)ethane sulfonic acid buffer pre-titrated to pH between 4.7 and 6.0. However, 0.1M phosphate in the pH 7-7.5 may also be used in view ofthe fact that the product is in the same buffer. The ratios of PEG amine to the IFN-α molecule is optimized such that the C-terminal carboxyl residue(s) are selectively PEGylated to yield monoPEGylated derivative(s).

Even though the use of PEG amine has been mentioned above by name or structure, such derivatives are meant to be exemplary only, and other groups such as hydrazine derivatives as in PEG-NH-NH₂ which will also condense with the carboxyl group ofthe IFN-α protein, can also be used. In addition to aqueous phase, the reactions can also be conducted on solid phase. Polyethylene glycol can be selected from list of compounds of molecular weight ranging from 300-40000. The choice ofthe various polyethylene glycols will also be dictated by the coupling efficiency and the biological performance ofthe purified derivative in vitro and in vivo i.e., circulation times, anti viral activities etc. Additionally, suitable spacers can be added to the C-terminal ofthe protein. The spacers may have reactive groups such as SH, NH₂ or COOH to couple with appropriate PEG reagent to provide the high molecular weight IFN-α derivatives. A combined solid/solution phase methodology can be devised for the preparation of C-terminal pegylated interferons. For example, the C-terminus of IFN-α is extended on a solid phase using a Gly- Gly-Cys-NH₂ spacer and then monopegylated in solution using activated dithiopyridyl-PEG reagent of appropriate molecular weights. Since the coupling at the C-terminus is independent ofthe blocking at the N-terminus, the envisioned processes and products will be beneficial with respect to cost (a third ofthe protein is not wasted as in N-terminal PEGylation methods) and contribute to the economy ofthe therapy to treat chronic hepatitis C infections, liver fibrosis etc.

There may be a more reactive carboxyl group of amino acid residues elsewhere in the molecule to react with the PEG reagent and lead to monoPEGylation at that site or lead to multiple PEGylations in addition to the -COOH group at the C-terminus ofthe IFN-α. It is envisioned that these reactions will be minimal at best owing to the steric freedom at the C- terminal end ofthe molecule and the steric hindrance imposed by the carbodiimides and the PEG reagents such as in branched chain molecules. It is therefore the preferred mode of PEG modification for Infergen and similar such proteins, native or expressed in a host system, which may have blocked N-termini to varying degrees to improve efficiencies and maintain higher in vivo biological activity.

Another method of achieving C-terminal PEGylation is as follows. Selectivity of C- terminal PEGylation is achieved with a steiϊcally hindered reagent which excludes reactions at carboxyl residues either buried in the helices or internally in IFN-α. For example, one such reagent could be a branched chain PEG ~40kd in molecular weight and this agent could be synthesized as follows:

OH₃C-(CH₂CH₂O)n-CH₂CH₂NH₂ + Glutamic Acid i.e., HOCO-CH₂CH₂CH(NH2)- COOH is condensed with a suitable agent e.g., dicyclohexyl carbodiimide or water-soluble EDC to provide the branched chain PEG agent OH₃C-(CH₂CH₂O)_n- CH₂CH₂NHCOCH(NH₂)CH₂OCH₃-(CH₂CH₂O)_n-CH₂CH NHCOCH₂. o

I' H₃C-θ- (CH₂CH₂θ)„-CH₂CH₂NH - CO

H₃C-θ-(CH₂CH₂θ)„-CH₂CH₂NH₂ + HO C-CH₂CH₂CH-COOH EDAC

^ CHNH₂

I CHNH₂ I I ¹ t (CH₂)₂

I

H₃C-θ-(CH₂CH₂θ)„-CH₂CH₂NH - CO This reagent can be used in excess to couple the amino group with the free and flexible carboxyl group of IFN-α to form the peptide bond.

If desired, PEGylated IFN-α is separated from unPEGylated IFN-α using any known method, including, but not limited to, ion exchange chromatography, size exclusion chromatography, and combinations thereof. For example, where the PEG-IFN-α conjugate is a monoPEGylated IFN-α, 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. IFN-β

The term interferon-beta ("IFN-β") includes IFN-β polypeptides that are naturally occurring; non-naturally-occurring IFN-β polypeptides; and analogs of naturally occurring or non-naturally occurring IFN-β that retain antiviral activity of a parent naturally-occurring or non-naturally occurring IFN-β.

Any of a variety of beta interferons can be delivered by the continuous delivery method ofthe present invention. Suitable beta interferons include, but are not limited to, naturally-occurring IFN-β; IFN-β la, e.g., Avonex® (Biogen, Inc.), and Rebif® (Serono, SA); IFN-βlb (Betaseron®; Berlex); and the like.

The IFN-β formulation may comprise an N-blocked species, wherein the N-terminal amino acid is acylated with an acyl group, such as a formyl group, an acetyl group, a malonyl group, and the like. Also suitable for use is a consensus IFN-β. IFN-β polypeptides can be produced by any known method. DNA sequences encoding IFN-β may be synthesized using standard methods. In many embodiments, IFN-β 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). In these embodiments, the IFN-β is "recombinant IFN-β." Where the host cell is a bacterial host cell, the IFN-β is modified to comprise an N-terminal methionine.

It is to be understood that IFN-β as described herein may comprise one or more modified amino acid residues, e.g., glycosylations, chemical modifications, and the like. IFN-tau

The term interferon-tau includes IFN-tau polypeptides that are naturally occurring; non-naturally-occurring IFN-tau polypeptides; and analogs of naturally occurring or non- naturally occurring IFN-tau that retain antiviral activity of a parent naturally-occurring or non-naturally occurring IFN-tau. Suitable tau interferons include, but are not limited to, naturally-occurring IFN-tau;

Tauferon® (Pepgen Corp.); and the like.

IFN-tau may comprise an amino acid sequence as set forth in any one of GenBank AccessionNos. P15696; P56828; P56832; P56829; P56831; Q29429; Q28595; Q28594; S08072; Q08071; Q08070; Q08053; P56830; P28169; P28172; and P28171. The sequence of any known IFN-tau polypeptide may be altered in various ways known in the art to generate targeted changes in sequence. A variant polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence changes may be substitutions, insertions or deletions. Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary amino acid sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation; changes in amino acid sequence that introduce or remove a glycosylation site; changes in amino acid sequence that make the protein susceptible to PEGylation; and the like. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine. The IFN-tau formulation may comprise an N-blocked species, wherein the N- terminal amino acid is acylated with an acyl group, such as a formyl group, an acetyl group, a malonyl group, and the like. Also suitable for use is a consensus IFN-tau.

IFN-tau polypeptides can be produced by any known method. DNA sequences encoding IFN-tau may be synthesized using standard methods. In many embodiments, IFN- tau 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). In these embodiments, the IFN- tau is "recombinant IFN-tau." Where the host cell is a bacterial host cell, the IFN-tau is modified to comprise an N-terminal methionine.

It is to be understood that IFN-tau as described herein may comprise one or more modified amino acid residues, e.g., glycosylations, chemical modifications, and the like. IFN-ω

The term interferon-omega ("IFN-ω") includes IFN-ω polypeptides that are naturally occurring; non-naturally-occurring IFN-ω polypeptides; and analogs of naturally occurring or non-naturally occurring IFN-ω that retain antiviral activity of a parent naturally-occurring or non-naturally occurring IFN-ω.

Any known omega interferon can be delivered by the continuous delivery method of the present invention. Suitable IFN-ω include, but are not limited to, naturally-occurring IFN-ω; recombinant IFN-ω, e.g., Biomed 510 (BioMedicines); and the like.

IFN-ω may comprise an amino acid sequence as set forth in GenBank Accession No. NP_002168; or AAA70091. The sequence of any known IFN-ω polypeptide may be altered in various ways known in the art to generate targeted changes in sequence. A variant polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence changes may be substitutions, insertions or deletions. Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine). Modifications of interest that may or may not alter the primary amino acid sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation; changes in amino acid sequence that introduce or remove a glycosylation site; changes in amino acid sequence that make the protein susceptible to PEGylation; and the like. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

The IFN-ω formulation may comprise an N-blocked species, wherein the N-terminal amino acid is acylated with an-acyl group, such as a formyl group, an acetyl group, a malonyl group, and the like. Also suitable for use is a consensus IFN-ω.

IFN-ω polypeptides can be produced by any known method. DNA sequences encoding IFN-ω may be synthesized using standard methods. In many embodiments, IFN-ω 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). In these embodiments, the IFN-ω is "recombinant IFN-ω." Where the host cell is a bacterial host cell, the IFN-ω is modified to comprise an N-terminal methionine.

It is to be understood that IFN-ω as described herein may comprise one or more modified amino acid residues, e.g., glycosylations, chemical modifications, and the like. Type III interferon receptor agonists

In any ofthe above-described methods or apparatus, the interferon receptor agonist is in some embodiments an agonist of a Type III interferon receptor (e.g., "a Type III interferon agonist"). Type III interferon agonists include an IL-28b polypeptide; and IL-28a polypeptide; and IL-29 polypeptide; antibody specific for a Type III interferon receptor; and any other agonist of Type III interferon receptor, including non-polypeptide agonists.

IL-28A, IL-28B, and IL-29 (referred to herein collectively as "Type III interferons" or "Type III IFNs") are described in Sheppard et al. (2003) Nature 4:63-68. Each polypeptide binds a heterodimeric receptor consisting of IL-10 receptor β chain and an IL-28 receptor α. Sheppard et al. (2003), supra. The amino acid sequences of IL-28 A, IL-28B, and IL-29 are found under GenBank Accession Nos. NP_742150, NP_742151, and NP_742152, respectively. The amino acid sequence of a Type III IFN polypeptide may be altered in various ways known in the art to generate targeted changes in sequence. A variant polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence changes may be substitutions, insertions or deletions. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids. Specific amino acid substitutions of interest include conservative and non- conservative changes. Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary amino acid sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation; changes in amino acid sequence that introduce or remove a glycosylation site; changes in amino acid sequence that make the protein susceptible to PEGylation; and the like. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

Included in the subject invention are polypeptides that have been modified using ordinary chemical techniques so as to improve their resistance to proteolytic degradation, to optimize solubility properties, or to render them more suitable as a therapeutic agent. For examples, the backbone ofthe peptide may be cyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789). Analogs may be used that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. The protein may be pegylated to enhance stability. The polypeptides may be fused to albumin. The polypeptides may be prepared by in vitro synthesis, using conventional methods as known in the art, by recombinant methods, or may be isolated from cells induced or naturally producing the protein. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the polypeptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. Interferon-Gamma

The nucleic acid sequences encoding IFN-γ polypeptides may be accessed from public databases, e.g., Genbank, journal publications, etc. While various mammalian IFN-γ polypeptides are of interest, for the treatment of human disease, generally the human protein will be used. Human IFN-γ coding sequence may be found in Genbank, accession numbers XI 3274; V00543; and NM_000619. The corresponding genomic sequence may be found in Genbank, accession numbers J00219; M37265; and V00536. See, for example. Gray et al. (1982) Nature 295:501 (Genbank XI 3274); and Rinderknecht et al {1984) J.B.C. 259:6790. IFN-γlb (Actimmune®; human interferon) is a single-chain polypeptide of 140 amino acids. It is made recombinantly in E.coli and is unglycosylated. Rinderknecht et al. (1984) J Biol. Chem. 259:6790-6797.

The IFN-γ to be used in the methods ofthe present invention may be any of natural IFN-γs, recombinant IFN-γs and the derivatives thereof so far as they have an IFN-γ activity, particularly human IFN-γ activity. Human IFN-γ exhibits the antiviral and anti-proliferative properties characteristic of the interferons, as well as a number of other immunomodulatory activities, as is known in the art. Although IFN-γ is based on the sequences as provided above, the production ofthe protein and proteolytic processing can result in processing variants thereof. The unprocessed sequence provided by Gray et al., supra, consists of 166 amino acids (aa). Although the recombinant IFN-γ produced in E. coli was originally believed to be 146 amino acids, (commencing at amino acid 20) it was subsequently found that native human IFN-γ is cleaved after residue 23, to produce a 143 aa protein, or 144 aa if the terminal methionine is present, as required for expression in bacteria. During purification, the mature protein can additionally be cleaved at the C terminus after reside 162 (referring to the Gray et al. sequence), resulting in a protein of 139 amino acids, or 140 amino acids if the initial methionine is present, e.g. if required for bacterial expression. The N-terminal methionine is an artifact encoded by the mRNA translational "start" signal AUG that, in the particular case of E. coli expression is not processed away. In other microbial systems or eukaryotic expression systems, methionine may be removed.

For use in the subject methods, any ofthe native IFN-γ peptides, modifications and variants thereof, or a combination of one or more peptides may be used. IFN-γ peptides of interest include fragments, and can be variously truncated at the carboxyl terminus relative to the full sequence. Such fragments continue to exhibit the characteristic properties of human gamma interferon, so long as amino acids 24 to about 149 (numbering from the residues of the unprocessed polypeptide) are present. Extraneous sequences can be substituted for the amino acid sequence following amino acid 155 without loss of activity. See, for example, U.S. Patent No. 5,690,925. Native IFN-γ moieties include molecules variously extending from amino acid residues 24-150; 24-151, 24-152; 24- 153, 24-155; and 24-157. Any of these variants, and other variants known in the art and having IFN-γ activity, may be used in the present methods.

The sequence ofthe IFN-γ polypeptide may be altered in various ways known in the art to generate targeted changes in sequence. A variant polypeptide will usually be substantially similar to the sequences provided herein, i.e., will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence changes may be substitutions, insertions or deletions. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids. Specific amino acid substitutions of interest include conservative and non- conservative changes. Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary amino acid sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation; changes in amino acid sequence that introduce or remove a glycosylation site; changes in amino acid sequence that make the protein susceptible to PEGylation; and the like. In one embodiment, the invention contemplates the use of IFN-γ variants with one or more non-naturally occurring glycosylation and/or pegylation sites that are engineered to provide glycosyl- and/or PEG-derivatized polypeptides with reduced serum clearance, such as the IFN- γ polypeptide variants described in International Patent Publication No. WO 01/36001. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Included in the subject invention are polypeptides that have been modified using ordinary chemical techniques so as to improve their resistance to proteolytic degradation, to optimize solubility properties, or to render them more suitable as a therapeutic agent. For examples, the backbone ofthe peptide may be cyclized to enhance stability (see Friedler et al. (2000) J Biol. Chem. 275:23783-23789). Analogs may be used that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The protein may be pegylated to enhance stability. The polypeptides may be prepared by in vitro synthesis, using conventional methods as known in the art, by recombinant methods, or may be isolated from cells induced or naturally producing the protein. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the polypeptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared ofthe expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight ofthe desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation ofthe product and its purification. Usually, the percentages will be based upon total protein.

PIRFENIDONE AND ANALOGS THEREOF

Pirfenidone (5-methyl-l-phenyl-2-(lH)-pyridone) and pirfenidone analogs are disclosed for the treatment of fibrotic conditions. A "fibrotic condition" is one that is amenable to treatment by administration of a compound having anti-fibrotic activity.

Pirfenidone

Pirfenidone analogs

Descriptions for Substituents Ri, R₂, X

Ri: carbocyclic (saturated and unsaturated), heterocyclic (saturated or unsaturated), alkyls (saturated and unsaturated). Examples include phenyl, benzyl, pyrimidyl, naphthyl, indolyl, pyrrolyl, furyl, thienyl, imidazolyl, cyclohexyl, piperidyl, pyrrolidyl, morpholinyl, cyclohexenyl, butadienyl, and the like.

Ri can further include substitutions on the carbocyclic or heterocyclic moieties with substituents such as halogen, nitro, amino, hydroxyl, alkoxy, carboxyl, cyano, thio, alkyl, aryl, heteroalkyl, heteroaryl and combinations thereof, for example, 4-nitrophenyl, 3- chlorophenyl, 2,5-dinitrophenyl, 4-methoxyphenyl, 5-methyl-pyrrolyl, 2, 5- dichlorocyclohexyl, guanidinyl-cyclohexenyl and the like.

R₂: alkyl, carbocylic, aryl, heterocyclic. Examples include: methyl, ethyl, propyl, isopropyl, phenyl, 4-nitrophenyl, thienyl and the like.

X: may be any number (from 1 to 3) of substituents on the carbocyclic or heterocyclic ring. The substituents can be the same or different. Substituents can include hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, halo, nitro, carboxyl, hydroxyl, cyano, amino, thio, alkylamino, haloaryl and the like.

The substituents may be optionally further substituted with 1-3 substituents from the group consisting of alkyl, aryl, nitro, alkoxy, hydroxyl and halo groups. Examples include: methyl, 2,3-dimethyl, phenyl, p-tolyl, 4-chlorophenyl, 4-nitrophenyl, 2,5-dichlorophenyl, furyl, thienyl and the like. Specific Examples include:

Table 1

U.S. Pat. Nos. 3,974,281; 3,839,346; 4,042,699; 4,052,509; 5,310,562; 5,518,729; 5,716,632; and 6,090,822 describe methods for the synthesis and formulation of pirfenidone and pirfenidone analogs in pharmaceutical compositions suitable for use in the methods of the present invention. DOSAGES, FORMULATIONS, AND ROUTES OF ADMINISTRATION

A Type I or Type III interferon receptor agonist and IFN-γ are administered to individuals in a formulation with a pharmaceutically acceptable excipient(s). 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. Ansel et al, eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, 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. In the subject methods, the active agents may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect. Thus, the agents can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents ofthe 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.

As such, administration ofthe agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, subcutaneous, intramuscular, transdermal, intratracheal,etc, administration. In some embodiments, two different routes of administration are used. For example, in some embodiments, IFN-α is administered subcutaneously, and pirfenidone or pirfenidone analog is administered orally.

Subcutaneous administration of a Type I or Type III interferon receptor agonist is accomplished using standard methods and devices, e.g., needle and syringe, a subcutaneous injection port delivery system, and the like. See, e.g., U.S. Patent Nos. 3,547,119;

4,755,173; 4,531,937; 4,311,137; and 6,017,328. A combination of a subcutaneous injection port and a device for administration of IFN-α to a patient through the port is referred to herein as "a subcutaneous injection port delivery system." In some embodiments, subcutaneous administration is achieved by a combination of devices, e.g., bolus delivery by needle and syringe, followed by delivery using a continuous delivery system.

In some embodiments, a Type I or Type III interferon receptor agonist is delivered by a continuous delivery system. The term "continuous delivery system" is used interchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable for use with the present invention. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, the present methods of drug delivery can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. Typically, the agent (e.g., a Type I or Type III interferon receptor agonist, e.g., IFN-α) is in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are generally preferred because of convenience in implantation and removal ofthe drug delivery device.

Drug release devices suitable for use in the invention may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system {e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material {e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.

Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present invention. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, the present methods of drug delivery can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are particularly preferred due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396)). Exemplary osmotically-driven devices suitable for use in the invention include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396 and the like.

In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted infra, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.

In some embodiments, a Type I or Type III interferon receptor agonist is delivered using an implantable drug delivery system, e.g., a system that is programmable to provide for administration of a Type I or Type III interferon receptor agonist. Exemplary programmable, implantable systems include implantable infusion pumps. Exemplary implantable infusion pumps, or devices useful in connection with such pumps, are described in, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplary device that can be adapted for the present invention is the Synchromed infusion pump (Medtronic). In pharmaceutical dosage forms, 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. For oral preparations, 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.

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.

Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds ofthe 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 ofthe composition containing one or more inhibitors. Similarly, 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. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds ofthe 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 ofthe present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

In some embodiments, at least one dose of IFN-γ is administered concurrently with at least one dose of a Type I or Type III interferon receptor agonist. As used herein, the term "concurrently" indicates that IFN-α and the Type I or Type III interferon receptor agonist are administered separately and are administered within about 5 seconds to about 15 seconds, within about 15 seconds to about 30 seconds, within about 30 seconds to about 60 seconds, within about 1 minute to about 5 minutes, within about 5 minutes to about 15 minutes, within about 15 minutes to about 30 minutes, within about 30 minutes to about 60 minutes, within about 1 hour to about 2 hours, within about 2 hours to about 6 hours, within about 6 hours to about 12 hours, within about 12 hours to about 24 hours, or within about 24 hours to about 48 hours of one another.

In some embodiments, IFN-γ is administered during the entire course of Type I or Type III interferon receptor agonist treatment. In other embodiments, IFN-γ is administered for a period of time that is overlapping with that ofthe Type I or Type III interferon receptor agonist treatment, e.g., the IFN-γ treatment can begin before the Type I or Type III interferon receptor agonist treatment begins and end before the Type I or Type III interferon receptor agonist treatment ends; the IFN-γ treatment can begin after the Type I or Type III interferon receptor agonist treatment begins and end after the IFN-γ treatment ends; the IFN- γ treatment can begin after the Type I or Type III interferon receptor agonist treatment begins and end before the Type I or Type III interferon receptor agonist treatment ends; or the IFN-γ treatment can begin before the Type I or Type III interferon receptor agonist treatment begins and end after the Type I or Type III interferon receptor agonist treatment ends. In connection with each ofthe methods described herein, the invention provides embodiments in which the Type I or Type III interferon receptor agonist and/or IFN-γ is administered to the patient by a controlled drug delivery device. In some embodiments, the Type I or Type III interferon receptor agonist and/or IFN-γ is delivered to the patient substantially continuously or continuously by the controlled drug delivery device. Optionally, an implantable infusion pump is used to deliver the Type I or Type III interferon receptor agonist and/or IFN-γ to the patient substantially continuously or continuously by subcutaneous infusion.

In other embodiments, the Type I or Type III interferon receptor agonist and/or IFN-γ is administered to the patient so as to achieve and maintain a desired average daily serum concentration ofthe Type I or Type III interferon receptor agonist and/or IFN-γ at a substantially steady state for the duration ofthe Type I or Type III interferon receptor agonist and/or IFN-γ therapy. Optionally, an implantable infusion pump is used to deliver the Type I or Type III interferon receptor agonist and/or IFN-γ to the patient by subcutaneous infusion so as to achieve and maintain a desired average daily serum concentration ofthe Type I or Type III interferon receptor agonist and/or IFN-γ at a substantially steady state for the duration ofthe Type I or Type III interferon receptor agonist and/or IFN-γ therapy.

Effective dosages of IFN-γ can range from about 0.5 μg/m to about 500 μg/m , usually from about 1.5 μg/m² to 200 μg/m², depending on the size ofthe patient. This activity is based on 10⁶ international units (U) per 50 μg of protein. IFN-γ can be administered daily, every other day, three times a week, or substantially continuously or continuously.

In specific embodiments of interest, IFN-γ is administered to an individual in a unit dosage form of from about 25 μg to about 500 μg, from about 50 μg to about 400 μg, or from about 100 μg to about 300 μg. In particular embodiments of interest, the dose is about 200 μg IFN-γ. In many embodiments of interest, IFN-γ lb is administered.

Where the dosage is 200 μg IFN-γ per dose, the amount of IFN-γ per body weight (assuming a range of body weights of from about 45 kg to about 135 kg) is in the range of from about 4.4 μg IFN-γ per kg body weight to about 1.48 μg IFN-γ per kg body weight.

The body surface area of subject individuals generally ranges from about 1.33 m to about 2.50 m . Thus, in many embodiments, an IFN-γ dosage ranges from about 150 μg/m to about 20 μg/m². For example, an IFN-γ dosage ranges from about 20 μg/m² to about 30 μg/m , from about 30 μg/m to about 40 μg/m , from about 40 μg/m to about 50 μg/m , from about 50 μg/m² to about 60 μg/m², from about 60 μg/m² to about 70 μg/m², from about

9 9 9 9 9

70 μg/m to about 80 μg/m , from about 80 μg/m to about 90 μg/m , from about 90 μg/m to about 100 μg/m², from about 100 μg/m² to about 110 μg/m², from about 110 μg/m² to about 120 μg/m², from about 120 μg/m² to about 130 μg/m², from about 130 μg/m² to about 140 μg/m², or from about 140 μg/m² to about 150 μg/m². In some embodiments, the dosage

9 9 groups range from about 25 μg/m to about 100 μg/m . In other embodiments, the dosage groups range from about 25 μg/m² to about 50 μg/m².

A Type I or a Type III interferon receptor agonist can be administered daily, every other day, once a week, three times a week, every other week, three times per month, once monthly, substantially continuously or continuously. In some embodiments, the Type I interferon receptor agonist is an IFN-α. Effective dosages of an IFN-α can range from about 1 μg to about 30 μg, from about 3 μg to about 27 μg, from about 1 MU to about 20 MU, from about 3 MU to about 10 MU, from about 90 μg to about 180 μg, or from about 18 μg to about 90 μg.

Effective dosages of Infergen® consensus IFN-α include about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg of drug per dose. Effective dosages of IFN-α2a and IFN-α2b can range from 3 million Units (MU) to 10 MU per dose. Effective dosages of PEGylated IFN-α2a can contain an amount of about 90 μg to 180 μg, or about 135 μg, of drug per dose. Effective dosages of PEGylated IFN-α2b can contain an amount of about 0.5 μg to 1.5 μg of drug per kg of body weight per dose. Effective dosages of PEGylated consensus interferon (PEG-CIFN) can contain an amount of about 18 μg to about 90 μg, or from about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG-CIFN. IFN-α can be administered daily, every other day, once a week, tliree times a week, every other week, three times per month, once monthly, substantially continuously or continuously.

In some embodiments, a Type I or a Type III interferon receptor agonist is administered in a first dosing regimen, followed by a second dosing regimen. The first dosing regimen of Type I or a Type III interferon receptor agonist (also referred to as "the induction regimen ") generally involves administration of a higher dosage ofthe Type I or Type III interferon receptor agonist. For example, in the case of Infergen® consensus IFN-α (CIFN), the first dosing regimen comprises administering CIFN at about 9 μg, about 15 μg, about 18 μg, or about 27 μg. The first dosing regimen can encompass a single dosing event, or at least two or more dosing events. The first dosing regimen ofthe Type I or Type III interferon receptor agonist can be administered daily, every other day, three times a week, every other week, three times per month, once monthly, substantially continuously or continuously.

The first dosing regimen ofthe Type I or Type III interferon receptor agonist is administered for a first period of time, which time period can be at least about 4 weeks, at least about 8 weeks, or at least about 12 weeks. The second dosing regimen ofthe Type I or Type III interferon receptor agonist (also referred to as "the maintenance dose") generally involves administration of a lower amount ofthe Type I or Type III interferon receptor agonist. For example, in the case of CIFN, the second dosing regimen comprises administering CIFN at least about 3 μg, at least about 9 μg, at least about 15 μg, or at least about 18 μg. The second dosing regimen can encompass a single dosing event, or at least two or more dosing events.

The second dosing regimen ofthe Type I or Type III interferon receptor agonist can be administered daily, every other day, three times a week, every other week, tliree times per month, once monthly, substantially continuously or continuously.

In some embodiments, where an "induction'V'maintenance" dosing regimen of a Type I or a Type III interferon receptor agonist is administered, a "priming" dose of IFN-γ is included. In these embodiments, IFN-γ is administered for a period of time from about 1 day to about 14 days, from about 2 days to about 10 days, or from about 3 days to about 7 days, before the beginning of treatment with the Type I or Type III interferon receptor agonist. This period of time is referred to as the "priming" phase. In some of these embodiments, IFN-γ treatment is continued throughout the entire period of treatment with the Type I or Type III interferon receptor agonist. In other embodiments, IFN-γ treatment is discontinued before the end of treatment with the Type I or Type III interferon receptor agonist. In these embodiments, the total time of treatment with IFN-γ (including the "priming" phase) is from about 2 days to about 30 days, from about 4 days to about 25 days, from about 8 days to about 20 days, from about 10 days to about 18 days, or from about 12 days to about 16 days. In still other embodiments, IFN-γ treatment is discontinued once Type I or a Type III interferon receptor agonist treatment begins.

In other embodiments, the Type I or Type III interferon receptor agonist is administered in single dosing regimen. For example, in the case of CIFN, the dose of CIFN is generally in a range of from about 3 μg to about 15 μg, or from about 9 μg to about 15 μg. The dose of Type I or a Type III interferon receptor agonist is generally administered daily, every other day, three times a week, every other week, tliree times per month, once monthly, or substantially continuously. The dose ofthe Type I or Type III interferon receptor agonist is administered for a period of time, which period can be, for example, from at least about 24 weeks to at least about 48 weeks, or longer.

In some embodiments, where a single dosing regimen of a Type I or a Type III interferon receptor agonist is administered, a "priming" dose of IFN-γ is included. In these embodiments, IFN-γ is administered for a period of time from about 1 day to about 14 days, from about 2 days to about 10 days, or from about 3 days to about 7 days, before the beginning of treatment with the Type I or Type III interferon receptor agonist. This period of time is referred to as the "priming" phase. In some of these embodiments, IFN-γ treatment is continued throughout the entire period of treatment with the Type I or Type III interferon receptor agonist. In other embodiments, IFN-γ treatment is discontinued before the end of treatment with IFN-α. In these embodiments, the total time of treatment with IFN-γ (including the "priming" phase) is from about 2 days to about 30 days, from about 4 days to about 25 days, from about 8 days to about 20 days, from about 10 days to about 18 days, or from about 12 days to about 16 days. In still other embodiments, IFN-γ treatment is discontinued once Type I or a Type III interferon receptor agonist treatment begins. Those of skill in the art will readily appreciate that dose levels can vary as a function ofthe specific compound, the severity ofthe symptoms and the susceptibility ofthe subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. Those of skill in the art will readily appreciate that dose levels can vary as a function ofthe specific compounds, the severity of the symptoms and the susceptibility ofthe subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in the same formulation, and are administered simultaneously. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered separately, e.g., in separate formulations. In some of these embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered separately, and are administered simultaneously. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered separately and are administered within about 5 seconds to about 15 seconds, within about 15 seconds to about 30 seconds, within about 30 seconds to about 60 seconds, within about 1 minute to about 5 minutes, within about 5 minutes to about 15 minutes, within about 15 minutes to about 30 minutes, within about 30 minutes to about 60 minutes, within about 1 hour to about 2 hours, within about 2 hours to about 6 hours, within about 6 hours to about 12 hours, within about 12 hours to about 24 hours, or within about 24 hours to about 48 hours of one another.

Multiple doses of Type I or a Type III interferon receptor agonist and IFN-γ can be admimstered, e.g., the Type I or Type III interferon receptor agonist and IFN-γ can be administered once per month, twice per month, three times per month, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, or daily, over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more. In particular embodiments of interest, Type I or a Type III interferon receptor agonist and IFN-γ is admimstered three times per week over a period of about 48 weeks. In some embodiments, where IFN-α and IFN-γ are administered in combination therapy, the IFN-α and IFN-γ are co-formulated in a single liquid formulation that is contained in a single reservoir, for use in a drug delivery device. Thus, the present invention provides a pharmaceutical formulation comprising a liquid formulation comprising a single dose of IFN-α and a single dose of IFN-γ. Thus, the present invention provides a drug reservoir or other container containing IFN-α and IFN-γ co-formulated in a liquid, wherein both IFN-α and IFN-γ are present in the formulation in an amount suitable for one dose each. Dosage amounts are described herein. The reservoir can be provided in any of a variety of forms, including, but not limited to, a cartridge, a syringe, a reservoir of a continuous delivery device, and the like. The invention further provides a drug delivery device comprising (e.g., pre-loaded with) a reservoir containing a liquid formulation that comprises a single dose of IFN-α and a single dose of IFN-γ. Exemplary, non-limiting drug delivery devices include injection devices, such as pen injectors, needle/syringe devices, continuous delivery devices, and the like. Any ofthe dosage amounts, including synergistically effective amounts, described herein can be used in the pharmaceutical formulation, in the reservoir, or in the drug delivery device.

In other embodiments, where IFN-α and IFN-γ are administered in combination therapy, the IFN-α and IFN-γ are in pharmaceutical formulations contained in separate reservoirs in the same drug delivery device. The invention further provides a drug delivery device that is pre-loaded with separate reservoirs, one reservoir containing a liquid formulation comprising a single dose of IFN-α, and a second reservoir containing a liquid formulation comprising a single dose of IFN-γ. Any ofthe dosage amounts, including synergistically effective amounts, described herein can be used in the pharmaceutical formulation, in the reservoir, or in the drug delivery device. In some embodiments, in a treatment method described herein, the interferon receptor agonist is an IFN-α, and the subject method comprises co-administering to the patient an effective amount of IFN-γ for the duration ofthe IFN-α therapy. In one embodiment, the IFN-γ is administered to the patient by bolus injection. In another embodiment, the IFN-α and IFN-γ are administered to the patient by a drug delivery device. Optionally, the device is used to deliver the IFN-α to the patient by substantially continuous or continuous administration and used to deliver the IFN-γ to the patient by bolus administration tiw, biw, qod, or qd. Optionally, the device is used to deliver the IFN-α and IFN-γ to the patient in the same manner and pattern of administration, such as substantially continuous or continuous administration. Optionally, the IFN-α and IFN-γ are contained in separate reservoirs in the drug delivery device. Optionally, the IFN-α and IFN-γ are co- formulated in a single liquid formulation that is contained in a single reservoir in the drug delivery device.

Where the agent is a polypeptide, polynucleotide (e.g., a polynucleotide encoding a Type I or a Type III interferon receptor agonist or IFN-γ), it may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al (1992) Anal Biochem. 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992) Nature 356: 152-154), where gold microprojectiles are coated with the therapeutic DNA, then bombarded into skin cells. Of particular interest in these embodiments is use of a liver-specific promoter to drive transcription of an operably linked IFN-α and IFN-γ coding sequences preferentially in liver cells. Additional therapeutic agents

In some embodiments, the method further includes administration of pirfenidone or a pirfenidone analog. Pirfenidone or a pirfenidone analog can be administered once per month, twice per month, three times per month, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, daily, or in divided daily doses ranging from once daily to 5 times daily over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

Effective dosages of pirfenidone or a specific pirfenidone analog include a weight- based dosage in the range from about 5 mg/kg/day to about 125 mg/kg/day, or a fixed dosage of about 400 mg to about 3600 mg per day, or about 800 mg to about 2400 mg per day, or about 1000 mg to about 1800 mg per day, or about 1200 mg to about 1600 mg per day, administered orally. Other doses and formulations of pirfenidone and specific pirfenidone analogs suitable for use in the treatment of fibrotic diseases are described in U.S. Pat. Nos., 5,310,562; 5,518,729; 5,716,632; and 6,090,822.

In some embodiments, pirfenidone or a pirfenidone analog is administered tliroughout the entire course of Type I or a Type III interferon receptor agonist and/or IFN-γ treatment. In other embodiments, pirfenidone or a pirfenidone analog is administered less than the entire course of Type I or a Type III interferon receptor agonist and/or IFN-γ treatment, e.g., only during the first phase of Type I or a Type III interferon receptor agonist and/or IFN-γ treatment, only during the second phase of Type I or a Type III interferon receptor agonist and/or IFN-γ treatment, or some other portion ofthe Type I or Type III interferon receptor agonist and/or IFN-γ treatment regimen.

In some embodiments, the method further includes administration of ribavirin. Ribavirin, l-β-D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif, is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771. The invention also contemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No. 6,277,830). The ribavirin may be administered orally in capsule or tablet form, or in the same or different administration form and in the same or different route as the Type I or Type III interferon receptor agonist. Of course, other types of administration of both medicaments, as they become available are contemplated, such as by nasal spray, transdermally, intravenously, by suppository, by sustained release dosage form, etc. Any form of administration will work so long as the proper dosages are delivered without destroying the active ingredient. Ribavirin is generally administered in an amount ranging from about 30 mg to about

60 mg, from about 60 mg to about 125 mg, from about 125 mg to about 200 mg, from about 200 mg to about 300 gm, from about 300 mg to about 400 mg, from about 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, or from about 700 to about 900 mg per day, or about 10 mg/kg body weight per day. In some embodiments, ribavirin is administered throughout the entire course ofthe

Type I or Type III interferon receptor agonist and/or IFN-γ treatment. In other embodiments, ribavirin is administered less than the entire course ofthe Type I or Type III interferon receptor agonist and/or IFN-γ treatment, e.g., only during the first phase ofthe Type I or Type III interferon receptor agonist and/or IFN-γ treatment, only during the second phase ofthe Type I or Type III interferon receptor agonist and/or IFN-γ treatment, or some other portion ofthe Type I or Type III interferon receptor agonist and/or IFN-γ treatment regimen. METHODS OF TREATMENT

1. TREATMENT OF ALPHAVIRAL INFECTION The present invention provides methods of treating alphaviral infection by administering a therapeutically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ to an individual in need thereof. Individuals who are to be treated according to the methods ofthe invention include individuals who have been clinically diagnosed with an alphaviral infection, as well as individuals who exhibit one or more ofthe signs and the symptoms of clinical infection but have not yet been diagnosed with an alphaviral infection.

In carrying out the methods of combination therapy for alphaviral infection in an individual as described above, a Type I or Type III interferon receptor agonist and IFN-γ are administered to the individual. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in the same formulation. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in separate formulations. When administered in separate formulations, the Type I or Type III interferon receptor agonist and IFN-γ can be administered substantially simultaneously, or can be administered within about 24 hours of one another. In many embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered subcutaneously in multiple doses.

Effective dosages of IFN-γ can range from about 25 μg/dose to about 300 μg/dose, from about 10 μg/dose to about 100 μg/dose, or from about 100 μg/dose to about 1000 μg/dose.

A Type I or a Type III interferon receptor agonist can be administered daily, every other day, once a week, tliree times a week, every other week, three times per month, once monthly, substantially continuously or continuously.

In some embodiments, the Type I interferon receptor agonist is an IFN-α. Effective dosages of an IFN-α can range from about 1 μg to about 30 μg, from about 3 μg to about 27 μg, from about 1 MU to about 20 MU, from about 3 MU to about 10 MU, from about 90 μg to about 180 μg, or from about 18 μg to about 90 μg.

Effective dosages of Infergen® consensus IFN-α can contain an amount of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg of drug per dose. Effective dosages of IFN-α2a and IFN-α2b can contain an amount of about 3 million Units (MU) to about 10 MU of drug per dose. Effective dosages of PEGASYS®PEGylated IFN-α2a can contain an amount of about 90 μg to about 180 μg, or about 135 μg, of drug per dose. Effective dosages of PEG-INTRON® PEGylated IFN-α2b can contain an amount of about 0.5 μg to about 1.5 μg of drug per kg of body weight per dose. Effective dosages of PEGylated consensus interferon (PEG-CIFN) can contain an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG- CIFN.

In many embodiments, the Type I or Type III interferon receptor agonist and/or IFN- γ is administered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time. Dosage regimens can include tid, bid, qd, qod, biw, tiw, qw, qow, three times per month, or monthly administrations.

In some embodiments, the invention provides methods using a synergistically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ in the treatment of alphaviral infection in a patient. In some embodiments, the invention provides methods using a synergistically effective amount of an IFN-α and IFN-γ in the treatment of alphaviral infection in a patient. In one embodiment, the invention provides a method using a synergistically effective amount of a consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient.

In general, a synergistically effective amount of a consensus interferon (CIFN) and IFN-γ suitable for use in the methods ofthe invention is provided by a dosage ratio of 1 μg CIFN : 10 μg IFN-γ, where both CIFN and IFN-γ are unPEGylated and unglycosylated species.

In one embodiment, the invention provides a method using a synergistically effective amount of INFERGEN® consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 30 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 9 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 50 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN® consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 9 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 90 μg to about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN® consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 30 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 200 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 4 μg to about 60 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drug per week in divided doses admimstered subcutaneously qd, qod, tiw, biw, , or substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 18 μg to about 24 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week in divided doses administered subcutaneously qd, qod, tiw, biw, or substantially continuously or continuously, for the desired treatment duration.

In general, a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ suitable for use in the methods ofthe invention is provided by a dosage ratio of 1 million Units (MU) IFN-α 2a or 2b or 2c : 30 μg IFN-γ, where both IFN-α 2a or 2b or 2c and IFN-γ are unPEGylated and unglycosylated species.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 1 MU to about 20 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 30 μg to about 600 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 3 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 10 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEGAS YS® containing an amount of about 90 μg to about 360 μg, of drag per dose of PEGASYS®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg, of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEGASYS® containing an amount of about 180 μg of drug per dose of PEGASYS®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg, of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON®PEGylated IFN-α2b and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEG- INTRON® containing an amount of about 0.75 μg to about 3.0 μg of drug per kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON® PEGylated IFN-α2b and IFN-γ in the treatment of alphaviral infection in a patient comprising administering to the patient a dosage of PEG- INTRON® containing an amount of about 1.5 μg of drug per kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week admimstered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration. The invention also provides methods for the treatment of alphaviral infection in which ribavirin therapy is added to any ofthe Type I or Type III interferon receptor agonist and IFN-γ combination therapies described above. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of 800 mg to 1200 mg ribavirin orally qd for the specified duration of therapy. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of 1000 mg ribavirin orally qd for the specified duration of therapy. In additional embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of about 10 mg of ribavirin/kg body weight orally qd for the specified duration of therapy. The daily ribavirin dosage can be administered in one dose per day or in divided doses, including one, two, three or four doses, per day.

2. TREATMENT OF WEST NILE VIRAL INFECTION

The present invention provides methods of treating West Nile viral infection by administering a therapeutically effective amount of Type I or Type III interferon receptor agonist and/or IFN-γ to an individual in need thereof. Individuals who are to be treated according to the methods ofthe invention include individuals who have been clinically diagnosed with West Nile viral infection, as well as individuals who exhibit one or more of the signs and the symptoms of clinical infection but have not yet been diagnosed with West Nile viral infection. In carrying out the methods of monotherapy for West Nile viral infection described above, a Type I or Type III interferon receptor agonist or IFN-γ is administered to the individual in need of such treatment. Effective dosages of IFN-γ can range from about 25 μg/dose to about 300 μg/dose, from about 10 μg/dose to about 100 μg/dose, or from about 100 μg/dose to about 1000 μg/dose.

A Type I or a Type III interferon receptor agonist can be administered daily, every other day, once a week, three times a week, every other week, three times per month, once monthly, substantially continuously or continuously.

Effective dosages of Infergen® consensus IFN-α containing an amount of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg, of drag per dose. Effective dosages of IFN-α2a and IFN-α2b can contain an amount of about 3 million Units (M) to about 10 MU, of drug per dose. Effective dosages of PEGAS YS®PEGylated IFN-α2a can contain an amount of about 90 μg to 180 μg, or about 135 μg, of drug per dose. Effective dosages of PEG-INTRON® PEGylated IFN-α2b can contain an amount of about 0.5 μg to about 1.5 μg of drug per kg of body weight per dose. Effective dosages of PEGylated consensus interferon (PEG-CIFN) can contain an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG- CIFN. In many embodiments, IFN-α or IFN-γ is administered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time. Dosage regimens can include tid, bid, qd, qod, biw, tiw, qw, qow, three times per month, or monthly administrations. In some embodiments, the dosages are administered subcutaneously.

In carrying out the methods of combination therapy for West Nile viral infection in an individual as described above, a Type I or Type III interferon receptor agonist and IFN-γ are administered to the individual. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in the same formulation. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in separate formulations. When administered in separate formulations, the Type I or Type III interferon receptor agonist and IFN-γ can be administered substantially simultaneously, or can be administered within about 24 hours of one another. In many embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered subcutaneously in multiple doses.

Effective dosages of Infergen® consensus IFN-α can contain an amount of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg, of drug per dose. Effective dosages of IFN-α2a and IFN-α2b can contain an amount of about 3 million Units (MU) to about 10 MU of drug per dose. Effective dosages of PEGAS YS®PEGylated IFN-α2a can contain an amount of about 90 μg to about 180 μg, or about 135 μg, of drug per dose. Effective dosages of PEG-INTRON®PEGylated IFN-α2b can contain an amount of about 0.5 μg to about 1.5 μg of drug per kg of body weight per dose. Effective dosages of PEGylated consensus interferon (PEG-CIFN) can contain an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG-CIFN. In many embodiments, IFN-α and/or IFN-γ is admimstered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time. Dosage regimens can include tid, bid, qd, qod, biw, tiw, qw, qow, three times per month, or monthly administrations.

In some embodiments, the invention provides methods using a synergistically effective amount of a Type I or Type III interferon receptor agonist and IFN-γ in the treatment of West Nile viral infection in a patient. In some embodiments, the invention provides a method using a synergistically effective amount of an IFN-α and IFN-γ in the treatment of West Nile viral infection in a patient. In one embodiment, the invention provides a method using a synergistically effective amount of a consensus IFN-α and IFN-γ in the treatment of West Nile viral infection in a patient. In general, a synergistically effective amount of a consensus interferon (CIFN) and

IFN-γ suitable for use in the methods ofthe invention is provided by a dosage ratio of 1 μg CIFN : 10 μg IFN-γ, where both CIFN and IFN-γ are unPEGylated and unglycosylated species. In one embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of West Nile viral (WNV) infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 30 μg, of drag per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 9 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 50 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 9 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 90 μg to about 100 μg of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 30 μg of drag per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 200 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 4 μg to about 60 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drug per week in divided doses administered subcutaneously qd, qod, tiw, or biw, or substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 18 μg to about 24 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week in divided doses administered subcutaneously qd, qod, tiw, biw, or substantially continuously or continuously, for the desired treatment duration. In general, a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ suitable for use in the methods ofthe invention is provided by a dosage ratio of 1 million Units (MU) IFN-α 2a or 2b or 2c : 30 μg IFN-γ, where both IFN-α 2a or 2b or 2c and IFN-γ are unPEGylated and unglycosylated species. In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 1 MU to about 20 MU of drag per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 30 μg to about 600 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 3 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 100 μg of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 10 MU of drag per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEGASYS® containing an amount of about 90 μg to about 360 μg, of drag per dose of PEGASYS®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg, of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEGASYS® containing an amount of about 180 μg of drug per dose of PEGASYS®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg, of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON®PEGylated IFN-α2b and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEG- INTRON® containing an amount of about 0.75 μg to about 3.0 μg of drug per kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON® PEGylated IFN-α2b and IFN-γ in the treatment of WNV infection in a patient comprising administering to the patient a dosage of PEG- INTRON® containing an amount of about 1.5 μg of drag per kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration. The invention also provides methods for the treatment of West Nile viral infection in which ribavirin therapy is added to any ofthe Type I or Type III interferon receptor agonist and IFN-γ combination therapies described above. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of 800 mg to 1200 mg ribavirin orally qd for the specified duration of therapy. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of 1000 mg ribavirin orally qd for the specified duration of therapy. In additional embodiments, the Type I or Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of about 10 mg of ribavirin kg body weight orally qd for the specified duration of therapy. The daily ribavirin dosage can be administered in one dose per day or in divided doses, including one, two, three or four doses, per day.

3. TREATMENT OF HEPATITIS C VIRAL INFECTION

The present invention provides methods of treating HCV infection by administering a combination of a Type I or Type III interferon receptor agonist and IFN-γ in a therapeutically effective amount to an individual in need thereof. Individuals who are to be treated according to the methods ofthe invention include individuals who have been clinically diagnosed as infected with HCV. Individuals who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti-HCV antibody in their serum.

Individuals who are clinically diagnosed as infected with HCV include naϊve individuals (e.g., individuals not previously treated for HCV) 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); and relapsers (e.g., individuals who were previously treated for HCV, whose HCV titer decreased, and subsequently increased). In particular embodiments of interest, individuals have an HCV titer of at least about 10⁵, at least about 5 x 10⁵, or at least about 10⁶, or at least about 2 x 10⁶, 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, 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.

Also of interest 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-α-based therapies or who cannot tolerate IFN-α-based therapies, or who have a contraindication to such therapies. In particular embodiments of interest, HCV-positive individuals with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the methods ofthe present invention. In other embodiments, individuals suitable for treatment with the methods ofthe instant invention are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation. In still other embodiments, individuals suitable for treatment with the methods ofthe 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 Ishak scoring system.).

In carrying out the methods of combination therapy for hepatitis C viral infection in an individual as described above, a Type I or Type III interferon receptor agonist and IFN-γ are administered to the individual. In some embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in the same formulation. In other embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered in separate formulations. When administered in separate formulations, the Type I or Type III interferon receptor agonist and IFN-γ can be administered substantially simultaneously, or can be admimstered within about 24 hours of one another. In many embodiments, the Type I or Type III interferon receptor agonist and IFN-γ are administered subcutaneously in multiple doses.

Effective dosages of Infergen® consensus IFN-α can contain an amount of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg, of drug per dose. Effective dosages of IFN-α2a and IFN-α2b can contain an amount of about 3 million Units (MU) to about 10 MU of drag per dose. Effective dosages of PEGASYS ©PEGylated IFN-α2a can contain an amount of about 90 μg to about 180 μg, or about 135 μg, of drug per dose.

Effective dosages of PEG-INTRON® PEGylated IFN-α2b can contain an amount of about 0.5 μg to about 1.5 μg of drug per kg body weight per dose. Effective dosages of PEGylated consensus interferon (PEG-CIFN) can contain an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG- CIFN. In many embodiments, IFN-α and/or IFN-γ is administered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time. Dosage regimens can include tid, bid, qd, qod, biw, tiw, qw, qow, three times per month, or monthly administrations.

In certain embodiments, the specific regimen of drug therapy used in treatment ofthe HCV patient is selected according to certain disease parameters exhibited by the patient, such as the initial viral load, genotype ofthe HCV infection in the patient, liver histology and/or stage of liver fibrosis in the patient. In one embodiment, 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 IFN-α and IFN-γ 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.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks. In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks. In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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. In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of about 40 weeks to about 50 weeks, or about 48 weeks.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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. In another embodiment, 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 Type I or a Type III interferon receptor agonists and IFN-γ for a time period of about 20 weeks to about 24 weeks.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of about 24 weeks to about 48 weeks.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of about 20 weeks to about 24 weeks.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of at least about 24 weeks. In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ 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.

In another embodiment, 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 IFN-α and IFN-γ for a time period of about 20 weeks to about 50 weeks.

In another embodiment, 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 Type I or a Type III interferon receptor agonist and IFN-γ for a time period of at least about 24 weeks and up to about 48 weeks. In practicing the methods ofthe invention that are specific for the HCV genotype and/or initial viral load ofthe patient described above, the clinician will administer a Type I or a Type III interferon receptor agonist and IFN-γ to the patient at a sufficient dosage to achieve a sustained viral response to the course of therapy. In one embodiment, such methods provide for treating the patient with an IFN-α regimen of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg Infergen® consensus IFN-α administered subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, or monthly, and an IFN-γ regimen of 25 μg to 300 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg Infergen® consensus IFN-α administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, and an IFN-γ regimen of 100 μg to 200 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy. In another embodiment, such methods provide for treating the patient with an IFN-α regimen of about 3 million Units (MU) to about 10 MU IFN-α2a or IFN-α2b administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, and an IFN-γ regimen of 25 μg to 300 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of about 3 million UnitsMU to about 10 MU IFN-α2a or IFN-α2b administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, and an IFN-γ regimen of 100 μg to 200 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of PEGAS YS®PEGylated IFN-α2a providing an amount of about 90 μg to about 180 μg, or about 135 μg, of drug per dose of PEGAS YS®PEGylated IFN-α2a administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 25 μg to 300 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of PEGAS YS®PEGylated IFN-α2a providing an amount of about 90 μg to about 180 μg, or about 135 μg, of drag per dose of PEGAS YS®PEGylated IFN-α2a administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 100 μg to 200 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of about 0.5 μg PEG-INTRON®PEGylated IFN-α2b/kg body weight to about 1.5 μg PEG-INTRON®/kg body weight administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 25 μg to 300 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of about 0.5 μg PEG-INTRON®PEGylated IFN-α2b/kg body weight to about 1.5 μg PEG-INTRON®/kg body weight administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 100 μg to 200 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, or monthly, for the specified duration of therapy. In another embodiment, such methods provide for treating the patient with an IFN-α regimen of PEGylated consensus interferon (PEG-CIFN) providing an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG-CIFN administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 25 μg to 300 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy.

In another embodiment, such methods provide for treating the patient with an IFN-α regimen of PEGylated consensus interferon (PEG-CIFN) providing an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG-CIFN administered subcutaneously qw, qow, three times per month, or monthly, and an IFN-γ regimen of 100 μg to 200 μg IFN-γ administered subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, or monthly, for the specified duration of therapy. In some embodiments, the invention provides methods using a synergistically effective amount of a Type I or a Type III interferon receptor agonist and IFN-γ in the treatment of hepatitis C viral infection in a patient. In some embodiments, the invention provides methods using a synergistically effective amount of an IFN-α and IFN-γ in the treatment of hepatitis C viral infection in a patient. In one embodiment, the invention provides a method using a synergistically effective amount of a consensus IFN-α and IFN-γ in the treatment of hepatitis C viral infection in a patient.

In one embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of hepatitis C viral (HCV) infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 30 μg, of drag per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg to about 9 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, tliree times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 1 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 10 μg to about 50 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 9 μg of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 90 μg to about 100 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of INFERGEN®consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of INFERGEN® containing an amount of about 30 μg of drag per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 200 μg to about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 4 μg to about 60 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drug per week in divided doses administered subcutaneously qd, qod, tiw, or biw, or substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGylated consensus IFN-α and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEGylated consensus IFN-α (PEG-CIFN) containing an amount of about 18 μg to about 24 μg of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, tliree times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week in divided doses administered subcutaneously qd, qod, tiw, biw, or substantially continuously or continuously, for the desired treatment duration.

In general, a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ suitable for use in the methods ofthe invention is provided by a dosage ratio of 1 million Units (MU) IFN-α 2a or 2b or 2c : 30 μg IFN-γ, where both IFN-α 2a or 2b or 2c and IFN-γ are unPEGylated and unglycosylated species. In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 1 MU to about 20 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 30 μg to about 600 μg of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 3 MU of drag per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 100 μg of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of IFN-α 2a or 2b or 2c and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of IFN-α2a containing an amount of about 10 MU of drag per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, in combination with a dosage of IFN-γ containing an amount of about 300 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantially continuously or continuously, for the desired treatment duration. In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEGASYS® containing an amount of about 90 μg to about 360 μg, of drug per dose of PEGASYS®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1 ,000 μg, of drug per week admimstered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEGAS YS®PEGylated IFN-α2a and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEGASYS® containing an amount of about 180 μg of drug per dose of PEGASYS®, subcutaneously qw, qow, tliree times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg, of drug per week admimstered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON®PEGylated IFN-α2b and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEG-INTRON® containing an amount of about 0.75 μg to about 3.0 μg of drug per kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg of drag per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration.

In another embodiment, the invention provides a method using a synergistically effective amount of PEG-INTRON®PEGylated IFN-α2b and IFN-γ in the treatment of HCV infection in a patient comprising administering to the patient a dosage of PEG-INTRON® containing an amount of about 1.5 μg of drug per kilogram of body weight per dose of PEG- INTRON®, subcutaneously qw, qow, three times per month, or monthly, in combination with a total weekly dosage of IFN-γ containing an amount of about 100 μg to about 300 μg of drug per week administered in divided doses subcutaneously qd, qod, tiw, or biw, or administered substantially continuously or continuously, for the desired treatment duration. The invention also provides methods for the treatment of HCV infection in which ribavirin therapy is added to any ofthe Type I or a Type III interferon receptor agonist and IFN-γ combination therapies described above. In some embodiments, the Type I or a Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of 800 mg to 1200 mg ribavirin orally qd for the specified duration of therapy. In other embodiments, the Type I or a Type III interferon receptor agonist and IFN- γ combination therapy is modified to include a ribavirin regimen of 1000 mg ribavirin orally qd for the specified duration of therapy. In additional embodiments, the Type I or a Type III interferon receptor agonist and IFN-γ combination therapy is modified to include a ribavirin regimen of about 10 mg of ribavirin/kg body weight orally qd for the specified duration of therapy. The daily ribavirin dosage can be administered in one dose per day or in divided doses, including one, two, three or four doses, per day. 3. TREATMENT OF LIVER FIBROSIS

Individuals with liver fibrosis who are suitable for treatment according to the methods ofthe invention include individuals who have been clinically diagnosed with liver fibrosis, as well as individuals who have not yet developed clinical liver fibrosis but who are considered at risk of developing liver fibrosis. Such individuals include, but are not limited to, individuals who are infected with HCV; individuals who are infected with HBV; individuals who are infected with Schistosoma mansoni; individuals who have been exposed to chemical agents known to result in liver fibrosis; individuals who have been diagnosed with Wilson's disease; individuals diagnosed with hemochromatosis; and individuals with alcoholic liver disease; individuals with non-alcoholic steatohepatitis; individuals with autoimmune hepatitis; individuals with primary sclerosing cholangitis, primary biliary cirrhosis, or alpha- 1-antitrysin deficiency.

In one aspect, the invention provides a method of treating liver fibrosis in a patient comprising administering to the patient an amount of a Type I or a Type III interferon receptor agonist and IFN-γ effective to reduce liver fibrosis. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or a Type III interferon receptor agonist and IFN-γ along with an amount of pirfenidone or a specific pirfenidone analog effective to enhance the anti-fibrotic effect or the reduction of liver fibrosis achieved by the Type I or a Type III interferon receptor agonist and IFN-γ therapy. In another aspect, the invention provides a method of increasing liver function in a patient suffering from liver fibrosis, comprising administering to the patient an amount of a Type I or a Type III interferon receptor agonist and IFN-γ effective to increase liver function. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or a Type III interferon receptor agonist and IFN-γ along with an amount of pirfenidone or a specific pirfenidone analog effective to enhance the anti- fibrotic effect or the increase in liver function achieved by the Type I or a Type III interferon receptor agonist and IFN-γ therapy.

In another aspect, the invention provides a method of reducing the incidence of a complication of cirrhosis ofthe liver in a patient suffering from liver fibrosis, comprising administering to the patient an amount of a Type I or a Type III interferon receptor agonist and IFN-γ effective to reduce the incidence of a complication of cirrhosis ofthe liver. Optionally, the method ofthe invention provides for administering to the patient the combination of a Type I or a Type III interferon receptor agonist and IFN-γ along with an amount of pirfenidone or a specific pirfenidone analog effective to enhance the anti-fibrotic effect or the reduction ofthe incidence of a complication of cirrhosis ofthe liver achieved by the Type I or a Type III interferon receptor agonist and IFN-γ therapy.

A Type I or a Type III interferon receptor agonist can be administered daily, every other day, once a week, tliree times a week, every other week, three times per month, once monthly, substantially continuously or continuously. In some embodiments, the Type I interferon receptor agonist is an IFN-α. Effective dosages of an IFN-α can range from about 1 μg to about 30 μg, from about 3 μg to about 27 μg, from about 1 MU to about 20 MU, from about 3 MU to about 10 MU, from about 90 μg to about 180 μg, or from about 18 μg to about 90 μg. In one embodiment, the methods of the invention for the treatment of liver fibrosis described above can be carried out by administering to the patient a dosage of IFN-γ containing an amount of about 25 μg to about 300 μg, or about 100 μg to about 200 μg, of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, and a dosage of INFERGEN®consensus IFN-α containing an amount of about 3 μg, about 9 μg, about 15 μg, about 18 μg, or about 27 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously. The anti-fibrotic effect or other therapeutic benefit of such regimens can be enhanced by co-administering to the patient a weight-based dosage of pirfenidone or a specific pirfenidone analog in the range of about 5 mg/kg of body weight to about 125 mg/kg of body weight, or a fixed dosage of pirfenidone or a specific pirfenidone analog in the range of about 400 mg to about 3600 mg, or about 800 mg to about 2400 mg, or about 1000 mg to about 1800 mg, or about 1200 mg to about 1600 mg, orally qd for the desired duration of IFN-α and IFN-γ therapy. In another embodiment, the methods ofthe invention for treatment of liver fibrosis described above can be carried out by administering to the patient a dosage of IFN-γ containing an amount of about 25 μg to about 300 μg, or about 100 μg to about 200 μg, of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, and a dosage of IFN- α2a or IFN-α2b containing an amount of about 3 million Units (MU) to about 10 MU of drug per dose of IFN-α2a or IFN-α2b, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously. The anti-fibrotic effect or other therapeutic benefit of such regimens can be enhanced by co- administering to the patient a weight-based dosage of pirfenidone or a specific pirfenidone analog in the range of about 5 mg/kg of body weight to about 125 mg/kg of body weight, or a fixed dosage of pirfenidone or a specific pirfenidone analog in the range of about 400 mg to about 3600 mg, or about 800 mg to about 2400 mg, or about 1000 mg to about 1800 mg, or about 1200 mg to about 1600 mg, orally qd for the desired duration of IFN-α and IFN-γ therapy. In another embodiment, the methods ofthe invention for treatment of liver fibrosis described above can be carried out by administering to the patient a dosage of IFN-γ containing an amount of about 25 μg to about 300 μg, or about 100 μg to about 200 μg, of drag per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, and a dosage of

PEGAS YS®PEGylated IFN-α2a containing an amount of about 90 μg to about 180 μg, or about 135 μg, of drag per dose of PEGASYS®, subcutaneously qw qow, tliree times per month, or monthly. The anti-fibrotic effect or other therapeutic benefit of such regimens can be enhanced by co-administering to the patient a weight-based dosage of pirfenidone or a specific pirfenidone analog in the range of about 5 mg/kg of body weight to about 125 mg/kg of body weight, or a fixed dosage of pirfenidone or a specific pirfenidone analog in the range of about 400 mg to about 3600 mg, or about 800 mg to about 2400 mg, or about 1000 mg to about 1800 mg, or about 1200 mg to about 1600 mg, orally qd for the desired duration of IFN-α and IFN-γ therapy. In another embodiment, the methods of the invention for treatment of liver fibrosis described above can be carried out by administering to the patient a dosage of IFN-γ containing an amount of about 25 μg to about 300 μg, or about 100 μg to about 200 μg, of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, and a dosage of PEG- INTRON® PEGylated IFN-α2b containing an amount of about 0.5 μg to about 1.5 μg of drug per kg body weight per dose of PEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly. The anti-fibrotic effect or other therapeutic benefit of such regimens can be enhanced by co-administering to the patient a weight-based dosage of pirfenidone or a specific pirfenidone analog in the range of about 5 mg/kg of body weight to about 125 mg/kg of body weight, or a fixed dosage of pirfenidone or a specific pirfenidone analog in the range of about 400 mg to about 3600 mg, or about 800 mg to about 2400 mg, or about 1000 mg to about 1800 mg, or about 1200 mg to about 1600 mg, orally qd for the desired duration of IFN-α and IFN-γ therapy.

In another embodiment, the methods ofthe invention for treatment of liver fibrosis described above can be carried out by administering to the patient a dosage of IFN-γ containing an amount of about 25 μg to about 300 μg, or about 100 μg to about 200 μg, of drug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, once monthly, or per day substantially continuously or continuously, and a dosage of PEGylated consensus interferon (PEG-CIFN) containing an amount of about 18 μg to about 90 μg, or about 27 μg to about 60 μg, or about 45 μg, of CIFN amino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, three times per month, or monthly.

The anti-fibrotic effect or other therapeutic benefit of such regimens can be enhanced by co-administering to the patient a weight-based dosage of pirfenidone or a specific pirfenidone analog in the range of about 5 mg/kg of body weight to about 125 mg/kg of body weight, or a fixed dosage of pirfenidone or a specific pirfenidone analog in the range of about 400 mg to about 3600 mg, or about 800 mg to about 2400 mg, or about 1000 mg to about 1800 mg, or about 1200 mg to about 1600 mg, orally qd for the desired duration of Type I or a Type III interferon receptor agonist and IFN-γ therapy. In many embodiments, a Type I or a Type III interferon receptor agonist and/or IFN- γ is administered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time. In embodiments utilizing co-administration of pirfenidone or a specific pirfenidone analog, the duration of therapy with pirfenidone or a specific pirfenidone analog can be coincident with the duration of therapy with Type I or a Type III interferon receptor agonist and/or IFN-γ.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Example 1: Antiviral Activity Characterization of CIFN and IFN-γ MATERIALS AND METHODS

HeLa 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 μg/ml), and penicillin (100 u/ml). Cells were grown at 37° C in a 5% CO humidified incubator. HeLa cells (2 x 10⁴ cells per well in 96-well microtiter plates) were treated with IFN for 24 hours prior to the addition ofthe virus. Virus at a MOI of 0.1 was added and the plates incubated for an additional 48 hours. At this time, the cell monolayer was stained with 0.5% crystal violet in 20% (vol/vol) methanol. All cytopathic effects (CPE) inhibition assays were done in duplicate. One unit of IFN was defined as that amount of IFN that inhibited CPE by 50%. The activity of all IFNs was measured against a NIH standard human IFN-α (Namalwa/Sendai) (Ga23-901-532).

Results

The data generated in the assays are shown in Table 2 below. Table 2: Activity of Infergen, Intron A. and Actimmune against West Nile Virus

*The value used here is from separate toxicity studies, as dilutions high enough to demonstrate the toxic effect were not performed on this plate.

EC50 = Effective concentration. Measures antiviral effect

IC50 = Inhibitory concentration. Drug only, no virus. Measurement of toxicity of the drug.

SI = selectivity index. IC50 / EC50. High SI is a standard indicator of a drug worth testing further.

The results ofthe HeLa/VSV assay indicate that Infergen and IFN-γ exhibit highly synergistic antiviral growth inhibition activity against West Nile virus. Given the role of interferons in immune systems that occur in nature, it is likely that the antiviral properties of interferons are not specific for or limited to particular viruses. It is believed that Infergen (and other interferon-α's) and IFN-γ will exhibit synergistic antiviral activity against hepatitis C virus and other alphaviruses.

A combination of INFERGEN® and Actimune were found to provide synergistic effects in an HCV replicon assay, where the results are depicted in Figure 1. The assay used is as described in Lohmann et al. (1999) Science 285:110-113.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope ofthe invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope ofthe present invention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method of reducing liver fibrosis in an individual, comprising administering IFN-α and IFN-γ in an amount effective to reduce liver fibrosis.

2. A method of treating a hepatitis C virus infection in an individual, comprising administering IFN-α and IFN-γ in an amount effective to achieve a sustained viral response.

3. The method of claim 1 , wherein a degree of liver fibrosis is determined by staging, wherein the stage of liver fibrosis, as measured by a standardized scoring system, is reduced by at least one unit.

4. The method according to claim 1 or claim 2, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein the IFN-α and IFN-γ are administered simultaneously.

5. The method according to claim 1 or claim 2, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein the IFN-α and IFN-γ are administered within about 24 hours of one another.

6. The method according to claim 1 or claim 2, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein the IFN-α and IFN-γ are administered in multiple doses.

7. A method of increasing liver function in an individual suffering from liver fibrosis, comprising administering IFN-α and IFN-γ in an amount effective to increase a liver function.

8. The method according to claim 7, wherein the liver function is indicated by measuring a parameter selected from the group consisting of serum transaminase level, prothrombin time, serum bilirubin level, blood platelet count, viral load and serum albumin level.

9. The method according to claim 7, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is admimstered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein IFN-α and IFN-γ are administered in serial doses.

10. A method of reducing the incidence of a complication of cirrhosis of the liver, comprising administering to an individual suffering from liver fibrosis a combination of

IFN-α and IFN-γ in an amount effective to reduce the incidence of a complication of cirrhosis ofthe liver.

11. The method according to claim 10, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein IFN-α and IFN-γ are administered in multiple doses.

12. A method of treating hepatitis C virus (HCV) in an individual, comprising administering a therapeutically effective amount of IFN-α and IFN-γ.

13. The method of claim 12, wherein the level of alanine aminotransferase is reduced to below about 45 international units per milliliter serum.

14. The method of claim 12, wherein the viral load ofthe individual is reduced to below about 500 HCV genome copies per milliliter serum.

15. The method according to claim 12, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein IFN-α and IFN-γ are administered simultaneously.

16. The method according to claim 12, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein IFN-α and IFN-γ are administered separately.

17. The method according to claim 12, wherein IFN-γ is administered subcutaneously in an amount of from about 25 μg to about 300 μg per dose, wherein IFN-α is administered subcutaneously in an amount of from about 3 μg to about 27 μg, and wherein IFN-α and IFN-γ are administered in serial doses.

18. A method of decreasing viral load in an individual suffering from hepatitis C virus, comprising administering IFN-α and IFN-γ in an amount effective to decrease viral load.

19. A method of treating hepatitis C virus in an individual, comprising the steps of: (a) identifying an individual suffering from a genotype 1 hepatitis C viral (HCV) infection and having an initial viral load of greater than 2 million HCV genome copies per milliliter of serum; and (b) administering to the individual an effective amount of IFN-α and IFN-γ for a period of about 48 weeks.

20. The method of claim 19, wherein the identification ofthe individual in step (a) further requires the individual to have no or early stage liver fibrosis as measured by a

Knodell score of 0, 1 or 2.

21. A method of treating hepatitis C virus in an individual, comprising the steps of: (a) identifying an individual suffering from a genotype 1 hepatitis C viral (HCV) infection and having an initial viral load of less than or equal to 2 million HCV genome copies per milliliter of serum; and (b) administering to the individual an effective amount of IFN-α and IFN-γ for a period of about 24 to 48 weeks.

22. A method of treating hepatitis C virus in an individual, comprising the steps of: (a) identifying an individual suffering from a genotype 2 or 3 hepatitis C viral (HCV) infection; and (b) administering to the individual an effective amount of IFN-α and IFN-γ for a period of about 24 to 48 weeks.

23. The method of any of claims 12-22, where the IFN-α is a consensus interferon.

24. The method of any of claims 12-23, further comprising co-administering to the individual an effective amount of ribavirin for the duration of IFN-α and IFN-γ therapy.

25. The method of any of claims 12-24, wherein the individual receives a synergistic amount of IFN-α and IFN-γ.

26. The method of any of claims 12-25, wherein the IFN-α is a PEGylated IFN-α.

27. The method of any of claims 12-26, wherein the IFN-α is administered subcutaneously by pump-controlled continuous infusion.

28. A method of treating an alphaviral infection in an individual, comprising administering to the individual an effective amount of IFN-α and IFN-γ.

29. The method of claim 28, wherein the individual receives a synergistic amount of IFN-α and IFN-γ.

30. The method of claim 28 or 29, further comprising co-administering to the individual an effective amount of ribavirin for the duration ofthe IFN-α and IFN-γ therapy.

31. The method of any of claims 28-30, wherein the IFN-α is a consensus interferon.

32. The method of any of claims 28-31, wherein the IFN-α is a PEGylated IFN-α.

33. A method of treating West Nile viral infection in an individual, comprising administering to the individual an effective amount of a consensus interferon (CIFN).

34. A method of treating West Nile viral infection in an individual, comprising administering to the individual an effective amount of IFN-γ.

35. A method of treating West Nile viral infection in an individual, comprising administering to the individual an effective amount of IFN-α and IFN-γ.

36. The method of claim 35, wherein the individual receives a synergistic amount of IFN-α and IFN-γ.

37. The method of claim 35 or 36, wherein the IFN-α is a consensus interferon.

38. The method of any of claims 35-37, wherein the IFN-α is a PEGylated IFN-α.

39. The method of any of claims 33-38, comprising further co-administering to the individual an effective amount of ribavirin for the duration ofthe IFN-α or IFN-γ therapy.

40. The method of any of claims 1-11, wherein the individual receives a synergistic amount of IFN-α and IFN-γ.

41. The method of claim 40, wherein the IFN-α is a consensus interferon.

42. The method of claim 40 or 41 , wherein the IFN-α is a PEGylated IFN-α.

43. The method of any of claims 1 and 40-42, further comprising co- administering to the individual an effective amount of pirfenidone or a specific pirfenidone analog for the duration ofthe IFN-α and IFN-γ therapy.