WO2013024158A1

WO2013024158A1 - Combinations of protein kinase inhibitors and interferons or of protein kinase inhibitors and direct acting antivirals for the treatment and the prevention of hcv infection

Info

Publication number: WO2013024158A1
Application number: PCT/EP2012/066109
Authority: WO
Inventors: Joachim Lupberger; Thomas Baumert
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2011-08-17
Filing date: 2012-08-17
Publication date: 2013-02-21

Abstract

The present invention provides combinations for use in the treatment or the prevention of HCV infection. In particular, combinations are provided that comprise at least one protein kinase inhibitor and at least one interferon or one direct acting antiviral, wherein the protein kinase inhibitor and interferon or the protein kinase inhibitor and direct acting antiviral act in a highly synergistic manner to inhibit HCV infection of susceptible cells. Also provided are pharmaceutical compositions and kits comprising such combinations and methods of using these compositions and kits for treating or preventing HCV infection.

Description

Combinations of Protein Kinase Inhibitors and Interferons or of Protein Kinase Inhibitors and Direct Acting Antivirals for the Treatment and the Prevention of HCV Infection

Related Patent Application

The present application claims priority to European Patent Application

No. EP 11 306 051.1 filed on August 17, 2011. The European patent application is incorporated herein by reference in its entirety.

Background of the Invention

Hepatitis C virus (HCV) is a major global health problem, with an estimated 150-200 million people infected worldwide, including at least 5 million in Europe (Pawlotsky, Trends Microbiol, 2004, 12: 96-102). According to the World Health Organization, 3 to 4 million new infections occur each year. The infection is often asymptomatic; however, the majority of HCV-infected individuals develop chronic infection (Hoof agle, Hepatology, 2002, 36: S21-S29; Lauer et al, N. Engl. J. Med., 2001, 345: 41-52; Seeff, Semin. Gastrointest., 1995, 6: 20-27). Chronic infection frequently results in serious liver disease, including fibrosis and steatosis (Chisari, Nature, 2005, 435: 930-932). About 20% of patients with chronic HCV infection develop liver cirrhosis, which progresses to hepatocellular carcinoma in 5% of the cases (Hoofnagle, Hepatology, 2002, 36: S21-S29; Blonski et al, Clin. Liver Dis., 2008, 12: 661-674; Jacobson et al, Clin. Gastroenterol. Hepatol, 2010, 8: 924-933; Castello et al., Clin. Immunol, 2010, 134: 237-250; McGivern et al., Oncogene, 2011, 30: 1969-1983).

Chronic HCV infection is the leading indication for liver transplantations (Seeff et al., Hepatology, 2002, 36: 1-2). Unfortunately, liver transplantation is not a cure for hepatitis C; viral recurrence being an invariable problem and the leading cause of graft loss (Brown, Nature, 2005, 436: 973-978; Watt et al, Am. J. Transplant, 2009, 9: 1707-1713). No vaccine protecting against HCV is yet available. Current therapies include administration of ribavirin and/or interferon-alpha (IFN-Cc), two non-specific anti-viral agents. Using a combination treatment of pegylated IFN-CC and ribavirin, persistent clearance is achieved in about 50% of patients with genotype 1 chronic hepatitis C. However, a large number of patients have contraindications to one of the components of the combination; cannot tolerate the treatment; do not respond to interferon therapy at all; or experience a relapse when administration is stopped. In addition to limited efficacy and substantial side effects such as neutropenia, haemo lytic anemia and severe depression, current antiviral therapies are also characterized by high cost. To improve efficacy of standard of care (SOC), a large number of direct acting antivirals (DAAs) targeting viral polyprotein processing and replication have been developed (Hofmann et al, Nat. Rev; Gastroenterol. Hepatol., 2011, 8: 257-264). These include small molecule compounds targeting HCV nonstructural proteins including the HCV protease, polymerase and NS5A protein. Although a marked improvement of antiviral response was observed when protease inhibitors were combined with SOC (Hofmann et al, Nat. Rev; Gastroenterol. Hepatol, 2011, 8: 257-264; Bacon et al, New Engl. J. Med., 2011, 364: 1207-1217; McHutchison et al, New Engl. J. Med., 2010, 362: 1292-1303; Poordad et al, New Engl. J. Med., 201 1, 364: 1195-1206; Hezode et al, New Engl. J. Med., 2009, 360: 1839-1850; Kwo et al, Lancet, 2010, 376: 705-716), toxicity of the individual compounds and rapid development of viral resistance in a substantial fraction of patients remain major challenges (Pawlotsky, Hepatology, 2011, 53: 1742-1751; Pereira et al, Nat. Rev. Gastroenterol. Hepatol., 2009, 6: 403-411; Sarrazin et al, Gastroenterol., 2010, 138: 447-462). New therapeutic approaches against HCV are therefore still needed.

HCV entry into target cells is a promising target for antiviral preventive and therapeutic strategies since it is essential for initiation, spread, and maintenance of infection (Timpe et al, Gut, 2008, 57: 1728-1737; Zeisel et al, Hepatology, 2008, 48: 299-307). Indeed, HCV initiates infection by attaching to molecules or receptors on the surface of hepatocytes. Current evidence suggests that HCV entry is a multistep process involving several host factors including heparan sulfate (Barth et al, J. Biol. Chem., 2003, 278: 41003-41012), the tetraspanin CD81 (Pileri et al, Science, 1998, 282: 938-941), the scavenger receptor class B type I (SR-BI) (Zeisel et al, Hepatology, 2007, 46: 1722-1731; Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Grove et al, J. Virol, 2007, 81 : 3162-3169; Kapadia et al, J. Virol, 2007, 81 : 374- 383; Scarselli et al, EMBO J., 2002, 21 : 5017-5025), Occludin (Ploss et al, Nature, 2009, 457: 882-886) and Claudin-1 (CLDN1), an integral membrane protein and a component of tight-junction strands (Evans et al, Nature, 2007, 446: 801-805). Identification of these (co)-entry factors or (co)-receptors for HCV has opened up new avenues for the development of therapeutic and prophylactic agents as drug candidates for the prevention and/or treatment of HCV infection. Indeed, proof-of- concept studies in cell culture and animal models have demonstrated that entry inhibitors are a promising novel class of antivirals for prevention and treatment of HCV infection (for review see Zeisel et al, J. Hepatol, 2011, 54: 566-576). Entry inhibitors in preclinical or early clinically development include HCV-receptor- and HCV-envelop-specific antibodies as well as small molecules (Zeisel et al, J. Hepatol, 2011, 54: 566-576; Catanese et al, J. Virol, 2007, 81 : 8063-8071; Fafi-Kremer et al, J. Exp. Med., 2010, 207: 2019-2031; Matsumura et al, Gastroenterology, 2009, 137: 673-681; Keck et al, J. Virol, 2005, 79: 13199-13208; Law et al, Nat. Med., 2008, 14: 25-27; Syder et al, J. Hepatol, 2011, 54: 48-55; Fofana et al, Gastroenterology, 2010, 139: 953-964, el-4).

Cross-neutralizing antibodies inhibiting HCV entry have been shown to be associated with control of HCV infection and prevention of HCV re-infection in cohorts with self- limited acute infection (Osburn et al, Gastroenterology, 2009, 138: 315-324; Pestka et al, Proc. Natl. Acad. Sci. USA, 2007, 104: 6025-630). For example, monoclonal antibodies raised against native human SR-BI have been shown to inhibit HCV E2 binding to SR-BI and to efficiently block HCVcc infection of hepatoma cells in a dose-dependent manner (Catanese et al, J. Virol, 2007, 81 : 8063- 8071 ; WO 2006/005465). European patent application No. EP 1 256 348 discloses substances, including antibodies, with antiviral effects that inhibit binding of HCV E2 and CD81. International patent application WO 2007/130646 describes in vitro and cell-based assays for identifying agents that interfere with HCV interactions with Claudin-1 thereby preventing HCV infection. The present Applicants have generated monoclonal antibodies that efficiently inhibit HCV infection by targeting host entry factor Claudin-1 (EP 08 305 597 and WO 2010/034812).

Recently, the present Applicants have demonstrated that blocking the activity of the newly discovered HCV entry co-factors, epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EphA2), using the approved kinase inhibitors, erlotinib and dasatinib respectively, broadly impaired infection by all major HCV genotypes and viral escape variants in vitro and in the human liver-chimeric Alb-uPA/SCID mouse model (Lupberger et al, Nature Medicine, 2011, 17: 589-595). Furthermore, the Applicants have shown that HCV entry is inhibited by antibodies directed against EGFR and EphA2.

Since the development of novel therapeutic approaches against HCV remains a high-priority goal, these studies are encouraging as they demonstrate that antibodies against receptor or co-receptors that affect HCV entry into susceptible cells may constitute an effective and safe alternative to current HCV therapies.

Summary of the Invention

The present invention relates to systems and improved strategies for the prevention and/or treatment of HCV infection and HCV-related diseases. More specifically, the present Applicants have demonstrated that interferon-alpha in combination with erlotinib or with dasatinib (which are both protein kinase inhibitors) act in a highly synergistic manner on the inhibition of HCV infection (see Example 1), suggesting that a combination of a protein kinase inhibitor and an interferon may be an effective antiviral approach to prevent primary HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients. Similarly, the Applicants have shown that synergy takes place for combinations of erlotinib or dasatinib with a direct acting antiviral (the protease inhibitors: telaprevir, boceprevir, danoprevir and TMC-435; the NS5A inhibitor, daclatasvir; and the polymerase inhibitors: mericitabine and GS7977) (see Example 5). Consequently, in one aspect, the present invention provides a combination of at least one protein kinase inhibitor and at least one interferon for use in the treatment or prevention of HCV infection.

In preferred embodiments, the at least one interferon is a human interferon. The human interferon may be a natural human interferon, a recombinant human interferon, a synthetic version of a human interferon, or derivatives thereof.

In certain preferred embodiments, the at least one interferon of an inventive combination is selected from the group consisting of interferon-alpha (IFN-Cc), pegylated IFN-CC, albumin-IFN-cc, interferon-beta (IFN-β), pegylated IFN-β, albumin- IFN-β, interferon-omega (IFN-CO), pegylated IFN-CO, albumin-IFN-CO, interferon- gamma (IFN-γ), pegylated IFN-γ, albumin-IFN-γ, interferon-lambda (IFN-λ), pegylated IFN-λ, albumin-IFN-λ, equivalents thereof, analogs thereof, derivatives thereof and any combination thereof. In certain preferred embodiments, the at least on interferon is interferon-alpha (IFN-Cc), pegylated IFN-CC, or albumin-IFN-CC.

In certain preferred embodiments, the at least one interferon of an inventive combination is interferon-alpha-a (IFN-Cc2a) or interferon-alpha-b (IFN-Cc2b). In a related aspect, the present invention provides a combination of at least one protein kinase inhibitor and at least one direct acting antiviral for use in the treatment or prevention of HCV infection.

In preferred embodiments, the at least one direct acting antiviral is a HCV protease inhibitor or a HCV polymerase inhibitor of an NS5A inhibitor. In certain embodiments, the at least one direct acting antiviral is selected from the group consisting of telaprevir, boceprevir, danoprevir, TMC-435, daclatasvir, mericitabine and GS7977.

In the combinations according to the present invention, the at least one protein kinase inhibitor is a tyrosine kinase inhibitor. In particular, the tyrosine kinase inhibitor may be a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR) or a tyrosine kinase inhibitor that acts on the ephrin type-A receptor 2 (EphA2).

Tyrosine kinase inhibitors that act on EGFR may be selected from the group consisting of erlotinib, gefitinib, vandetanib, lapatinib, neratinib, afatinib, equivalents thereof, and any combination thereof. Tyrorine kinase inhibitors that act on EphA2 may be dasatinib.

In certain embodiments, the tyrosine kinase inhibitor is an anti-receptor tyrosine kinase antibody. In other embodiments, the tyrosine kinase inhibitor is a small molecule. In a combination according to the present invention, the protein kinase inhibitor and interferon or the protein kinase inhibitor and the direct acting antiviral act in a highly synergistic manner to inhibit HCV infection. In certain embodiments, the interferon or the direct acting antiviral decreases the IC₅₀ for the inhibition of HCV infection by the protein kinase inhibitor by a factor of at least 10 fold or at least 25 fold, preferably at least 50 fold or at least 75 fold, more preferably at least 100 fold or 200 fold, and even more preferably more than 200 fold (e.g., more than 350 fold). In other embodiments, the protein kinase inhibitor decreases the IC₅₀ for the inhibition of HCV infection by the interferon or the direct acting antiviral by a factor of at least 10 fold or at least 25 fold, preferably at least 50 fold or at least 75 fold, more preferably at least 100 fold or 200 fold, and even more preferably more than 200 fold (e.g., more than 350 fold). The combination index (CI) of the at least one protein kinase inhibitor and at least one interferon or direct acting antiviral is lower than 1 , preferably lower than 0.90, more preferably lower than 0.80, and even more preferably lower than 0.50.

The combinations of the present invention can find application in a variety of prophylactic and therapeutic treatments. Thus, the combinations are provided for use in the prevention of HCV infection of a cell (e.g., a susceptible cell or a population of susceptible cells); for preventing or treating HCV infection or a HCV-related disease in a subject; for controlling chronic HCV infection; and for preventing HCV recurrence in a liver transplantation patient. HCV infection may be due to HCV of a genotype selected from the group consisting of genotype 1 , genotype 2, genotype 3, genotype 4, genotype 5, genotype 6, and genotype 7, or more specifically of a subtype selected from the group consisting of subtype la, subtype lb, subtype 2a, subtype 2b, subtype 2c, subtype 3a, subtype 4a-f, subtype 5a, and subtype 6a.

In a related aspect, the present invention provides a method of reducing the likelihood of a susceptible cell of becoming infected with HCV as a result of contact with HCV, which comprises contacting the susceptible cell with an effective amount of an inventive combination. Also provided is a method of reducing the likelihood of a subject's susceptible cells of becoming infected with HCV as a result of contact with HCV, which comprises administering to the subject an effective amount of an inventive combination or a pharmaceutical composition thereof. The present invention also provides a method of treating or preventing HCV infection or a HCV- associated disease (e.g., a liver disease or pathology) in a subject in need thereof, which comprises administering to the subject an effective amount of an inventive combination or a pharmaceutical composition thereof. The invention also provides a method for controlling chronic HCV infection in a subject in need thereof, which comprises administering to the subject an effective amount of an inventive combination or a pharmaceutical composition thereof. Also provided is a method of preventing HCV recurrence in a liver transplantation patient, which comprises administering to the patient an effective amount of an inventive combination or a pharmaceutical composition thereof. Administration of an inventive combination to a subject may be by any suitable route, including, for example, parenteral, aerosol, oral and topical routes. The inventive combination, or pharmaceutical composition thereof, may be administered alone or in combination with a therapeutic agent, such as an anti-viral agent.

In the context of the present invention, the HCV infection or HCV-related disease or HCV re-infection may be caused by a Hepatitis C virus that is resistant to a direct acting antiviral and/or that is transmitted by cell-cell transmission.

The inventive combinations may be administered per se or as pharmaceutical compositions. Accordingly, in another aspect, the present invention provides for the use of an inventive combination for the manufacture of medicaments, pharmaceutical compositions, or pharmaceutical kits for the treatment and/or prevention of HCV infection and HCV-associated diseases.

In a related aspect, the present invention provides a pharmaceutical composition comprising an effective amount of an inventive combination (i.e., at least one protein kinase inhibitor and at least one interferon as described herein or at least one protein kinase inhibitor and at least one direct acting antiviral) and at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is adapted for administration in combination with an additional therapeutic agent, such as an antiviral agent. In other embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antiviral agent. Antiviral agents suitable for use in methods and pharmaceutical compositions of the present invention include, but are not limited to, ribavirin, anti- HCV (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, entry inhibitors, micro-RNA antagonists, cytokines, therapeutic vaccines, NS5A antagonists, polymerase inhibitors, cyclophilin A antagonists, and any combination thereof. These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Drawing

Figure 1 is a set of graphs illustrating the synergistic effects of interferon-alpha and the protein kinase inhibitors, erlotinib and dasatinib, on the inhibition of HCVcc infection. Combination of EGFR inhibitor erlotinib with IFN-a potentiates the antiviral impact of IFN-a. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) IFN-a2a or (B) IFN-a2b and 0.1 μΜ erlotinib or dasatinib before incubation with HCVcc Luc-Jcl in the presence of both compounds. Dotted lines at CI values of 0.9 and 1.1 indicate the boundaries of an additive interaction. (C- D) Combination of IFN-a with 0.1 μΜ erlotinib resulted in a shift in the IC₅₀ of IFN- a2a and IFN-a2b up to lOOfold. Means ± SD from at least three independent experiments measured in triplicates. (E-F) Synergy was confirmed using three- dimensional analysis of the inhibitor-inhibitor combinations according to Prichard and Shipman. Surface amplitudes >20 % above the zero plane highlight inhibitor combinations acting synergistically. One representative experiment is shown.

Figure 2 is a set of graphs illustrating the synergistic effects of interferon-alpha and the protein kinase inhibitor, dasatinib, on the inhibition of HCVcc infection. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) IFN- a2a or (B) IFN-a2b and 0.1 μΜ dasatinib before incubation with HCVcc Luc-Jcl in the presence of both compounds. Combination of IFN-a with 0.1 μΜ dasatinib resulted in a shift in the IC₅₀ of IFN-a2a and IFN-a2b up to 10 fold. Means ± SD from at least three independent experiments measured in triplicates. (C-D) Synergy was confirmed using three-dimensional analysis of the inhibitor- inhibitor combinations according to Prichard and Shipman. Surface amplitudes >20 % above the zero plane highlight inhibitor combinations acting synergistically. One representative experiment is shown.

Figure 3 is a set of graphs showing the synergistic activity of combinations of protein kinase inhibitors and DAAs. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) a protease inhibitors: telaprevir, boceprevir, TMC- 435 or danoprevir, (B) a NS5A inhibitor: daclatasvir or (C) a polymerase inhibitor: mericitabine or GS-7977 and 0.1 μΜ PKIs (erlotinib or dasatinib) before incubation with HCVcc Luc-Jcl in the presence of both compounds. Dotted lines at combination values of 0.9 and 1.1 indicate the boundaries of an additive interaction. Means±SEM from at least three independent experiments performed in triplicate are shown. Figure 4 is a set of graphs showing the synergistic activity of combinations of the protein kinase inhibitor, erlotinib and the DAA, telaprevir. Combinations were performed as in Figure 3. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of protease inhibitor telaprevir and protein kinase inhibitors (PKIs) (erlotinib or dasatinib) before incubation with HCVcc Luc-Jcl in the presence of both compounds. HCVcc infection was analysed by quantification of luciferase reporter gene expression. (A) %HCV infection relative to cells infected in the absence of inhibitors is shown. Data are indicated as means±SEM from at least three independent experiments performed in triplicate are shown (HCV infection in the absence of inhibitors = 100%). (B) Synergy was confirmed using the method of Prichard and Shipman. One representative experiment is shown.

Figure 5 is a set of graphs showing the synergistic activity of combinations of the protein kinase inhibitor, erlotinib and the DAA, daclatasvir. Combinations were performed as in Figures 3 and 4. (A) %HCV infection relative to cells infected in the absence of inhibitors is shown. Data are indicated as means±SEM from at least three independent experiments performed in triplicate are shown (HCV infection in the absence of inhibitors = 100%). (B) Synergy was confirmed using the method of Prichard and Shipman. One representative experiment is shown.

Figure 6 is a set of graphs showing the synergistic activity of combinations of a protein kinase inhibitors, erlotinib or dasatinib and the DAA, GS-7977. Combinations were performed as in Figure 3. Combination of GS-7977 and (A) dasatinib or (B) erlotinib decreased the IC₅₀ of GS-7977 up to 210 fold. Means ± SEM from at least three independent experiments performed in triplicate are shown. (C) Synergy was confirmed using the method of Prichard and Shipman. One representative experiment is shown. Figure 7 is a set of graphs showing that protein kinase inhibitors inhibit cell-free entry of protease inhibitor-resistant variants without cross-resistance. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) telaprevir, (B) boceprevir, (C) erlotinib or respective isotype control reagents before incubation with HCVcc-Jcl-Luc containing the DAA-resistant mutations R155T and A156S, respectively, in the presence of each compound. HCV infection was analyzed 72 hours post-incubation by luciferase reporter gene expression in cell lysates as described in Example 7. Means ± SEM from at least three independent experiments performed in triplicate are shown.

Figure 8 is a set of graphs showing cell-cell transmission of protease inhibitor resistant variants and its inhibition by erlotinib. NS5A+ HCV producer cells (Pi) were transfected with HCV RNA encoding for HCV Luc-Jcl A156S (A-C) or Jcl L36M R155K (D-F). NS5A+ HCV producer cells and target GFP-expressing cells (T) were co-cultivated with an anti-E2 mAb to block cell-free transmission. Protease inhibitor- resistant HCV variant Luc-Jcl A156S (A-C) and Jcl L36M R155K (D-F) producer cells (Pi) cultured with uninfected target cells (T) were then incubated with erlotinib (B and E) or control IgG (A and D). HCV-infected target cells were quantified by flow cytometry. Cell-cell transmission of DAA-resistant variants, not affected by controls, is inhibited by erlotinib. Percentage of infected target cells is shown as histograms (C and F) and is represented as means ± SD from three experiments performed in triplicate.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

As used herein, the term "subject" refers to a human or another mammal {e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can be the host of Hepatitis C virus (HCV), but may or may not be infected with the virus, and/or may or may not suffer from a HCV-related disease. Non-human subjects may be transgenic or otherwise modified animals. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an individual". The term "individual" does not denote a particular age, and thus encompasses newborns, children, teenagers, and adults. As used herein, the term "HCV refers to any major HCV genotype, subtype, isolate and/or quasispecies. HCV genotypes include, but are not limited to, genotypes 1, 2, 3, 4, 5, 6 and 7; HCV subtypes include, but are not limited to, subtypes la, lb, 2a, 2b, 2c, 3a, 4a-f, 5a and 6a.

The terms "afflicted with HCV" or "infected with HCV" are used herein interchangeably. When used in reference to a subject, they refer to a subject that has at least one cell which is infected by HCV. The term "HCV infection" refers to the introduction of HCV genetic information into a target cell, such as by fusion of the target cell membrane with HCV or an HCV envelope glycoprotein-positive cell.

The terms "HCV-related disease" and "HCV-associated disease" are herein used interchangeably. They refer to any disease or disorder known or suspected to be associated with and/or caused, directly or indirectly, by HCV. HCV-related (or HCV- associated) diseases include, but are not limited to, a wide variety of liver diseases, such as subclinical carrier state of acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The term includes symptoms and side effects of any HCV infection, including latent, persistent and sub-clinical infections, whether or not the infection is clinically apparent.

The term "treatment is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition (e.g., HCV infection or HCV-related disease); (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; or (4) curing the disease or condition. A treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively or additionally, a treatment may be administered after initiation of the disease or condition, for a therapeutic action. A "pharmaceutical composition" is defined herein as comprising an effective amount of a combination of the invention, and at least one pharmaceutically acceptable carrier or excipient.

As used herein, the term "effective amount" refers to any amount of a compound, agent, antibody, composition, or combination that is sufficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject. For example, in certain embodiments of the present invention, the purpose(s) may be: to prevent HCV infection, to prevent the onset of a HCV-related disease, to slow down, alleviate or stop the progression, aggravation or deterioration of the symptoms of a HCV-related disease (e.g., chronic hepatitis C, cirrhosis, and the like); to bring about amelioration of the symptoms of the disease, or to cure the HCV- related disease. The term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not significantly toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion, media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example "Remington 's Pharmaceutical Sciences" ^', E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

The terms "susceptible cell" and "HCV-susceptible cell" are used interchangeably. They refer to any cell that may be infected with HCV. Susceptible cells include, but are not limited to, liver or hepatic cells, primary cells, hepatoma cells, CaCo2 cells, dendritic cells, placental cells, endometrial cells, lymph node cells, lymphoid cells (B and T cells), peripheral blood mononuclear cells, and monocytes/macrophages. The term "preventing, inhibiting or blocking HCV infection" when used in reference to an inventive combination means reducing the amount of HCV genetic information introduced into a susceptible cell or susceptible cell population as compared to the amount of HCV genetic information that would be introduced in the absence of the combination. The term "isolated?', as used herein in reference to a protein or polypeptide, means a protein or polypeptide, which by virtue of its origin or manipulation is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained. By "isolated", it is alternatively or additionally meant that the protein or polypeptide of interest is produced or synthesized by the hand of man.

The terms "protein", "polypeptide" , and "peptide" are used herein interchangeably, and refer to amino acid sequences of a variety of lengths, either in their neutral (uncharged) forms or as salts, and either unmodified or modified by glycosylation, side-chain oxidation, or phosphorylation. In certain embodiments, the amino acid sequence is a full-length native protein. In other embodiments, the amino acid sequence is a smaller fragment of the full-length protein. In still other embodiments, the amino acid sequence is modified by additional substituents attached to the amino acid side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains such as oxidation of sulfydryl groups. Thus, the term "protein" (or its equivalent terms) is intended to include the amino acid sequence of the full-length native protein, or a fragment thereof, subject to those modifications that do not significantly change its specific properties. In particular, the term "protein" encompasses protein isoforms, i.e., variants that are encoded by the same gene, but that differ in their pi or MW, or both. Such isoforms can differ in their amino acid sequence {e.g., as a result of allelic variation, alternative splicing or limited proteolysis), or in the alternative, may arise from differential post-translational modification {e.g., glycosylation, acylation, phosphorylation) .

The term "analog", as used herein in reference to a protein, refers to a polypeptide that possesses a similar or identical function as the protein but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the protein or a structure that is similar or identical to that of the protein. Preferably, in the context of the present invention, a protein analog has an amino acid sequence that is at least 30%, more preferably, at least 35%, 40%>, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of the protein. The term "fragment" or the term "portion", as used herein in reference to a protein, refers to a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues (preferably, at least about: 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 or more amino acid residues) of the amino acid sequence of a protein. The fragment of a protein may or may not possess a functional activity of the protein.

The term "biologically active", as used herein to characterize a protein variant, analog or fragment, refers to a molecule that shares sufficient amino acid sequence identity or homology with the protein to exhibit similar or identical properties to the protein. For example, in many embodiments of the present invention, a biologically active fragment of an inventive antibody is a fragment that retains the ability of the antibody to bind to a HCV receptor. The term "homologous" (or "homology"), as used herein, is synonymous with the term "identity" and refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, the respective molecules are then homologous at that position. The percentage of homology between two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum homology. Homologous amino acid sequences share identical or similar amino acid sequences. Similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in a reference sequence. "Conservative substitutions" of a residue in a reference sequence are substitutions that are physically or functionally similar to the corresponding reference residue, e.g. that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" as described by Dayhoff et al. ("Atlas of Protein Sequence and Structure", 1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).

The terms "approximately" and "about", as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention provides combinations and methods for the treatment and prevention of HCV infection. I - Combinations

A combination according to the invention comprises at least one protein kinase inhibitor and at least one interferon, or at least one protein kinase inhibitor and at least one direct acting antiviral, and is intended for use in the treatment or the prevention of HCV infection.

A. Protein Kinase Inhibitors

The combinations of the present invention comprise at least one protein kinase inhibitor. The term 'protein kinase inhibitor" refers to any molecule that specifically blocks the action of one or more protein kinases. Protein kinase inhibitors are subdivided by the amino acids whose phosphorylation is inhibited. Most kinases act on both serine and threonine, the tyrosine kinases act on tyrosine, and a number (dual- specificity) kinases act on all three. As used herein, the term "serine/threonine kinase inhibitor" refers to a molecule that specifically blocks the action of one or more serine and/or threonine kinases. As used herein, the term "tyrosine kinase inhibitor" refers to a molecule that specifically blocks the action of one or more tyrosine kinases.

As mentioned above, the present Applicants have shown that interferons and direct acting antivirals act in synergy with erlotinib or dasatinib to inhibit HCV infection. Both erlotinib and dasatinib are tyrosine kinase inhibitors. Therefore, in certain preferred embodiments, the at least one protein kinase inhibitor present in a combination according to the invention is a tyrosine kinase inhibitor.

Tyrosine kinase inhibitors are generally used in cancer therapy. Indeed, research indicates that mutations which make tyrosine kinases constantly active can be a contributing factor in the development of cancerous cells. So, when a tyrosine kinase inhibitor is administered, the cell communication and reproduction is reduced, and cancerous cell growth can be lowered to the point of stopping growth. However, the research team of the laboratory of the Applicants has recently demonstrated that blocking the activity of HCV entry cofactors, EGFR (epidermal growth factor receptor) and EphA2 (ephrin type-A receptor A), by the approved tyrosine kinase inhibitors, erlotinib and dasatinib, broadly impaired infection by all major HCV genotypes and viral escape variants in vitro and in the human liver-chimeric Alb- uPA/SCID mouse model (Lupberger et al, Nature Medicine, 2011, 17: 589-595). They showed that erlotinib and dasatinib interfere with CD81-CLDN1 co-receptor interactions and with glycoprotein-dependant viral fusion. These results suggest that tyrosine kinase inhibitors that act on these HCV entry cofactors may represent a promising class of novel antivirals that target the first step of the viral life cycle. Thus, in certain embodiments, the at least one protein kinase inhibitor present in a combination of the invention is a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR). The target protein EGFR is also sometimes referred to as Herl or ErbB-1. Examples of tyrosine kinase inhibitors that act on EGFR include, but are not limited to, erlotinib, gefitinib, vandetanib, and lapatinib. Thus, in certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is erlotinib. Erlotinib is marketed under the tradename TARCEVA^® by Genentech and OSI pharmaceuticals in the United States and by Roche elsewhere. Erlotinib binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the epidermal growth factor receptor. For the signal to be transmitted, two members of the EGFR family need to come together to form a homodimer. These then use the molecule of ATP to autophosphorylate each other, which causes a conformational change in their intracellular structure, exposing a further binding site for binding proteins that cause a signal cascade to the nucleus. By inhibiting the ATP, autophosphorylation is not possible and the signal is stopped. The FDA (U.S. Food and Drug Administration) has approved the use of erolotinib for the treatment of locally advanced or metastatic non-small cell lung cancer that has failed at least one prior chemotherapy regimen.

In certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is gefitinib. Gefitinib (tradename IRES S A^®) is marketed by AstraZeneca and Teva. In Europe, gefitinib is indicated in advanced non-small cell lung cancer in all lines of treatment for patients harboring EGFR mutations.

In certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is vandetanib. Vandetanib, also known as ZD6474 is being developed by AstraZeneca. It is an antagonist of the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR). In April 2011, Vandetanib became the first drug to be approved by the FDA for the treatment of late-stage (metastatic) medullatory thyroid cancer in adult patients who are ineligible for surgery.

In certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is lapatinib. Lapatinib, in the form of lapatinib ditosylate (tradenames TYKERB^® in the U.S. and TYVERB^® in Europe) is marketed by Glaxo SmithKline. Lapatinib is a dual tyrosine kinase inhibitor, which inhibits the tyrosine kinase activity associated with EGFR and HER2/neu (Human EGFR type 2). In February 2010, lapatinib received accelerated approval as front-line therapy in triple positive breast cancer. Other tyrosine kinase inhibitors that act on EGFR and that are suitable for use in the present invention include molecules that are currently under development for human use, including, but not limited to, neratinib (also known as HKI-272, being developed by Pfizer), which is under investigation for the treatment of breast cancer and other solid tumors; and afatinib (also known as BIBW 2992, being developed by Boehringer Ingelheim), which is a candidate drug against non-small cell lung carcinoma, both of which are dual inhibitors of EGFR and Her2.

Other examples of tyrosine kinase inhibitors that act on EGFR include anti- EGFR antibodies, such as Cetuximab, Panitumumab, Matuzumab, Zalutumumab, Nimotuzumab, Necitumumab, and the like. In other embodiments, the at least one protein kinase inhibitor present in a combination of the invention is a tyrosine kinase inhibitor that acts on the ephrin type- A receptor 2 (EphA2). Examples of such tyrosine kinase inhibitors include, for example, dasatinib.

Dasatinib (BMS-354825) is sold under the tradename SPRYCEL^® by Bristol- Myers Squibb. Dasatinib is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, but which also inhibits multiple Eph kinases, including EphA2. Dasatinib is approved for use in patients with chronic myelogenous leukemia (CML) after imatinib treatment, and Philadelphia chromosome-positive acute lymphoblastic leukemia. It is being evaluated for use in numerous other cancers, including advanced prostate cancer. Other examples of tyrosine kinase inhibitors that act on EphA2 include anti- EphA2 antibodies, such as those developed by Medlmmune Inc.

In other embodiments, the at least one protein kinase inhibitor present in a combination of the present invention is an antibody against a receptor tyrosine kinase (RTK) other than EGFR and EphA2. The receptor tyrosine kinases may belong to the insulin receptor family, PDGF receptor family, VEGF receptor family, HGF receptor family, Trk receptor family, AXL receptor family, LTK receptor family, TIE receptor family, ROR receptor family, DDR receptor family, RET receptor family, KLG receptor family, RYK receptor family, or MuSK receptor family. Examples of such anti-RTK monoclonal antibodies include, but are not limited to, anti-VEGF antibodies such as Bevacizumab and Ranibizumab; anti-Erb2 antibodies such as Trastuzumab; anti-HER2/neu antibodies such as Trastuzumab, Ertimaxomab, and Pertuzumab; anti-VEGFR2 antibodies such as Ramucirumab and Alacizumab pegol; anti-VEGF-A antibodies such as Ranibizumab and Bevacizumab; anti-PDGF-R antibodies such as Olaratumab; and anti-IGF-1 receptor antibodies such as Figitumumab; Robatumumal and Cixutumumab.

B. Interferons

Certain combinations of the present invention comprise at least one protein kinase inhibitor and at least one interferon. The terms "interferon", "IFN" and "interferon molecule" are used herein interchangeably. They refer to any interferon or interferon derivative {e.g., pegylated interferon) that can be used in the prevention or treatment of HCV infection and/or in the prevention or treatment of HCV-related diseases, in particular cirrhosis and liver cancer.

Interferons are a family of cytokines produced by eukaryotic cells in response to viral infection and other antigenic stimuli, which display broad-spectrum antiviral, antiproliferative and immunomodulatory effects. Recombinant forms of interferons have been widely applied in the treatment of various conditions and diseases, such as viral infections {e.g., HCV, HBV and HIV), inflammatory disorders and diseases (e.g., multiple sclerosis, arthritis, cystic fibrosis), and tumors {e.g., liver cancer, lymphomas, myelomas, etc...). Interferons are classified as Type I, Type II and Type III, depending on the cell receptor to which they bind. Type I interferons bind to a specific cell surface receptor complex known as the IFN-a receptor (IFNAR) that consists of two chains (IFNAR1 and IFNAR2). The type I interferons present in humans are interferon-alpha (IFN-a), interferon-beta (IFN-β) and interferon-omega (IFN-CO). Treatments based on the use of IFN-a or pegylated IFN-a remain the cornerstone of therapy for chronic HCV infection. A new form of IFN-a with an extended in vivo half- life, albumin- interferon or albinterferon (a recombinant formulation of IFN-a genetically fused to the human blood protein albumin), has been developed. Albumin-interferon has been shown to exhibit high antiviral activity and to offer safety/tolerability comparable to the current standard of care and fared well in phase III clinical trials in patients with chronic HCV infection (Zeuzem et al, Gastroenterology, 2010, 139: 1257-1266; Nelson et al, Gastroenterology, 2010, 139: 1267-1276). Interferon-β has also been demonstrated to display antiviral activity against HCV and to be useful in the treatment of HCV infection, alone or in combination with ribavirin (Fukutomi et al, J. Hepatology, 2001, 34: 100-107; Sang Hoon Ahn et al, Gut and Liver, 2009, 3: 20-25). Pegylated IFN-β is currently undergoing clinical testing in Japan for HCV patients who do not respond well to the conventional combination therapy of ribavirin and IFN-a (Toray Industries Inc., Press Release, Feb. 2009). Interferon-co also exhibits antiviral properties (Buckwold et al, Antiviral Res., 2007, 73: 118-125) and has been tested for anti-HCV effect using an implantable infusion pump for the continuous delivery and consistent dose of drug for 3 to 12 months (Rohloff et al, J. Diabetes Sci. Technol, 2008, 2: 461-467).

Type II interferons bind to the interferon-gamma receptor (IFNGR). The only type II interferon is interferon-gamma (IFN-γ). IFN-γ does have anti- viral and antitumor effects, however these effects are weaker when compared to INF-a.

Type III interferons signal through a receptor complex consisting of the interferon-lambda receptor (IFNLR1 or CRF2-12) and the interleukin 10 receptor 2 (IL10R2 or CRF2-4). In humans, type III interferons include three interferon lambda (IFN-λ) proteins referred to as IFN-λΙ, ΙΡΝ-λ2 and ΙΡΝ-λ3 also known as interleukin 29 (IL29), interleukin 28A (IL28A) and interleukin 28B (IL28B), respectively. It has recently been reported (European Association for the Study of Liver, 46^th Annual Meeting, April 2011) that pegylated IFN-λ showed virological responses superior to the standard of care, when tested in patients infected with HCV genotypes 1 or 4 and was better tolerated and safer.

Therefore, in certain embodiments, the at least one interferon molecule present in a combination according to the invention is selected from the group consisting of IFN-cc, IFN-β, IFN-co, IFN-γ, IFN-λ, analogs thereof and derivatives thereof. In certain preferred embodiments, the interferon present in the combination is selected from the group consisting of IFN-cc, analogs thereof and derivatives thereof. In other preferred embodiments, the interferon present in the combination is selected from the group consisting of IFN-CO, analogs thereof and derivatives thereof. In yet other preferred embodiments, the interferon present in the combination is selected from the group consisting of IFN-λ, analogs thereof and derivatives thereof.

As used herein, the terms "interferon", "IFN and "IFN molecule" more specifically refer to a peptide or protein having an amino acid substantially identical (e.g., et least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identical) to all or a portion of the sequence of an interferon (e.g., a human interferon), such as IFN-CC, IFN-β, IFN-CO, IFN-γ, and IFN-λ that are known in the art. Interferons suitable for use in the present invention include, but are not limited to, natural human interferons produced using human cells, recombinant human interferons produced from mammalian cells, E-co/z-produced recombinant human interferons, synthetic versions of human interferons and equivalents thereof. Other suitable interferons include consensus interferons which are a type of synthetic interferons having an amino acid sequence that is a rough average of the sequence of all the known human IFN subtypes (for example, all the known IFN-CC subtypes, or all the known IFN-β subtypes, or all the known IFN-co subtypes, or all the known IFN-γ subtypes, or all the known IFN-λ subtypes). In certain embodiments, interferons present in combinations according to the invention have been approved for human use. In other embodiments, interferons present in combinations according to the present are undergoing human clinical trials. The terms "interferon", "IFN", and "IFN molecule" also include interferon derivatives, i.e., molecules of interferon (as described above) that have been modified or transformed. A suitable transformation may be any modification that imparts a desirable property to the interferon molecule. Examples of desirable properties include, but are not limited to, prolongation of in vivo half-life, improvement of therapeutic efficacy, decrease of dosing frequency, increase of solubility/water solubility, increase of resistance against proteolysis, facilitation of controlled release, and the like. As mentioned above, pegylated interferons have been produced {e.g., pegylated IFN-a) and are currently used to treat hepatitis. Pegylated interferons exhibit longer half-lifes, which allows for less frequent administration of the drug. Pegylating an interferon molecule involves covalently binding the interferon to polyethylene glycol (PEG), an inert, non-toxic and biodegradable organic polymer. Therefore, in certain embodiments, the at least one interferon present in a combination according to the invention is a pegylated interferon. Interferons have also been produced as fusion proteins with human albumin {e.g., albumin-IFN-Cc). The albumin- fusion platform takes advantage of the long half-life of human albumin to provide a treatment that allows the dosing frequency of IFN to be reduced in individuals with chronic hepatitis C. Therefore, in certain embodiments, the at least one interferon present in a combination according to the invention is an albumin- interferon fusion protein.

The terms "alpha interferon", "interferon-alpha" , "interferon-a" and "IFN- " are used herein interchangeably and refer to the family of highly homologous species- specific proteins (i.e., glycoproteins) that are known in the art and inhibit viral replication and cellular proliferation, and modulate immune response. Typical IFN-a molecules suitable for use in the present invention include, but are not limited to, recombinant IFN-a-2b (such as INTRON-A^® interferon available from Schering Corporation); recombinant IFN-a-2a (such as ROFERON^® interferon available from Hoffman-La Roche); recombinant IFN-a-2C (such as BEROFOR^® alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc); IFN-a-nl, a purified blend of natural alpha interferons (such as SUMERIFERON^® available from Sumitomo, Japan or WELLFERON^® interferon alpha-nl (INS) available from Glaxo-Wellcome Ltd); IFN-a-n3, a mixture of natural alpha interferons (such as ALFERON^® made by Interferon Sciences); human leukocyte interferon-a obtained from the leukocyte fraction of human blood following induction with Sendai virus (such as MULTIFERON^®, available from Swedish Orphan Biovitrium, which contains several naturally occurring IFN-CC subtypes); a consensus IFN-CC (such as INFERGEN , interferon alfacon-1, available from Three Rivers Pharmaceuticals, LLC, and those described in U.S. Pat. Nos. 4,897,471); and equivalents thereof.

Other suitable interferon alpha molecules include IFN-CC derivatives, including, but not limited to, pegylated IFN-CC-2a (such as PEGASYS^® available from Hoffman- La Roche); pegylated IFN-CC-2b (such as PEGINTRON^® available from Schering Corporation); albumin IFN-CC-2b also known as albinterferon (such as ALBUFERON^® available from Human Genome Sciences), and equivalents thereof.

The terms "beta interferon", "interferon-beta" , "interferon-^)" and "ZFTV-β" are used herein interchangeably and refer to the family of highly homologous species- specific proteins (i.e., glycoproteins) that are known in the art and have the ability to induce resistance to viral antigens. Typical IFN-β molecules suitable for use in the present invention include, but are not limited to, recombinant ΙΡΝ-β-la (such as, REBIF^® available from Pfizer or AVONEX^® available from Biogen Idex), recombinant IFN-p-lb (such as BETAFERON^®/BETASERON^® available from Bayer HealthCare or EXTAVIA^®, the generic form of BETAFERON, available from Novartis, or ZIFERON^®, an interferon-β lb biosimilar, available from Zistdaru Danesh Ltd), IFN-β molecules described in U.S. Pat. Nos. 4,820,638 and 5,795,779) and, equivalents thereof. Other suitable interferon beta molecules include IFN-β derivatives, including, but not limited to, pegylated INF-β (such as TRK-560 being developed by Toray Industries, Inc.), pegylated ΙΡΝ-β-la (such as BUBO 17 being developed by Biogen Idee); pegylated ΙΡΝ-β-lb (such as NUlOO and NU400 being developed by Nuron Biotech); albumin- IFN-β fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "omega interferon", "interferon-omega" , "interferon-^ and "IFN- co" are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have the ability to inhibit viral replication and cellular proliferation and modulate immune response. Typical IFN-CO molecules suitable for use in the present invention include, but are not limited to, IFN-CO described in European patent No. EPO 170 204, ITCA being developed by Intarcia Therapeutics, Inc., and equivalents thereof.

Other suitable interferon omega molecules include IFN-CO derivatives, including, but not limited to, pegylated INF-CO that can be obtained using a method described in U.S. Pat. Nos. 5,612,460; 5,711,944; 5,951,974 or 5,951,974; albumin-IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "gamma interferon" , "interferon-gamma" , "interferon-¹^" and "IFN- γ" are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have the ability to induce resistance to certain viral antigens. Typical IFN-γ molecules suitable for use in the present invention include, but are not limited to, IFN-γ described in U.S. Pat. Nos. 4,727,138, 4,762,791, 4,845,196, 4,929,554, 5,574,137, and 5,690,925; interferon gamma lb (such as ACTIMMUNE^® available from InterMune, Inc.), and equivalents thereof. Other suitable interferon omega molecules include IFN-γ derivatives, including, but not limited to, pegylated INF-γ that can be obtained using a method described in U.S. Pat. Nos. 5,612,460; 5,711,944; 5,951,974 or 5,951,974 albumin-IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "lambda interferon", "interferon-lambda" , "interferon-λ" and "IFN-λ" are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have antiviral properties. Typical IFN-λ molecules suitable for use in the present invention include, but are not limited to, IFN-λΙ, ΙΡΝ-λ2 and ΙΡΝ-λ3 molecules described in international patent applications number WO02/086087, WO2004/037995 and WO/2005/023862 and equivalents thereof.

Other suitable interferon omega molecules include IFN-γ derivatives including, but not limited to, pegylated ΙΡΝ-λ-la (such as BMS-914143 being developed by Bristol-Myers Squibb), albumin- IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof. The terms "interferon" , "/FN", and "/FN molecule" also include interferon- like molecules, i.e., molecules that have functional and/or structural features exhibited by or similar to known interferons or interferon analogs, such as those described above.

C. Direct Acting Antivirals

Certain combinations of the present invention comprise at least one protein kinase inhibitor and at least one direct acting antiviral. The terms "direct acting antiviral", "direct acting antiviral agent", "DAA", "specifically targeted antiviral therapy for hepatitis C and "STAT-C are used herein interchangeably. They refer to molecules that interfere with specific steps of the lifecycle of HCV and are thus useful in the prevention or treatment of HCV infection.

A major effort of the pharmaceutical industry is being focused on the development of direct-acting antiviral agents. Direct acting antivirals have been shown to increase the efficacy of the standard of care in randomized clinical trials (for a review, see Hofmann et al, Nat. Rev. Gastroenterol. Hepatol.; 2011, 8: 257-264). However, along with these encouraging results, significant treatment-related adverse events including rash, gastrointestinal side-effects, and anemia as well as the emergence of HCV resistance have been reported (Poordad et al, New Engl. J. Med., 2011, 364: 1195-1206; Hezode et al, New Engl. J. Med., 2009, 360: 1839-1850; Pawlotsky et al, Hepatology, 2011, 53: 1742-1751). As mentioned above, the present Applicants have shown that a protein kinase inhibitor, in combination with a direct acting antiviral act in high synergy to inhibit HCV infection. The efficient inhibition observed for these combinations suggest that they may represent a new approach for interferon- or ribavirin-free treatment strategies. Furthermore, such combinations may overcome antiviral resistance as they result in a greater decrease of the viral load in shorter periods of time, thereby limiting the frequency of appearance of resistant variants.

A direct acting antiviral agent suitable for use in a combination of the present invention may exert its effects by any mechanism that interferes with one or more specific steps of the lifecycle of HCV. For example, telaprevir, boceprevir, danoprevir and TMC-435 used by the present Applicants are protease inhibitors, daclatasvir is a NS5A inhibitor; and mericitabine and GS7977 are polymerase inhibitors. Thus, in certain preferred embodiments, the at least one direct acting antiviral present in a combination according to the invention is a HCV protease inhibitor or a HCV polymerase inhibitor or an NS5A inhibitor.

Protease inhibitors suitable for use in the context of the present invention include NS3/4A protease inhibitors. Examples of NS3/4A protease inhibitors that can be present in a combination of the present invention include, but are not limited to, VX- 950 (also known as telaprevir), ITMN-191 (also known as danoprevir), boceprevir, BMS-650032, VX-985, BI 201335, and TMC-435. In certain preferred embodiments, the at least one direct acting antiviral present in a combination according to the invention is NS3/4A protease inhibitor telaprevir, boceprevir, danoprevir or TMC- 435.

Telaprevir (also known as VX-950), marketed under the tradename INCIVEK^®, was co-developed by Vertex Pharmaceuticals, Inc. and Johnson & Johnson. In May 2011, the FDA approved telaprevir for the treatment of patients with genotype 1 chronic hepatitis C. ITMN-191 (also known as R7227 or danoprevir) was being co- developed by Roche and InterMune Inc., but is now fully owned by Roche. Boceprevir (initially developed by Schering-Plough, and then by Merck and marketed under the tradename VICTRELIS^®) was approved by the FDA for the treatment of hepatitic C genotype 1 in May 2011. BMS-650032 is being developed by Bristol- Myers-Squibb. VX-985 is a NS3/4A protease inhibitor being developed by Vertex Pharmaceuticals, Inc. BI 201335 is being developed by Boehringer Ingelheim and is now in Phase III clinical trials in the United States. TMC-435, a NS3/4A protease inhibitor being developed by Medivir/Tibotec/ Johnson & Johnson, is also in Phase III clinical trials.

Other examples of NS3/4A protease inhibitors that can be present in a combination according to the invention include, but are not limited to, NS3/4A protease inhibitors that are currently in phase II clinical trials such as GS 9256 and GS 9451 (being developed by Gilead), MK-7009 (also known as vaniprevir, being developed by Merck), ACH-1625 (being developed by Achillion), and ABT-450 (being developed by Abbott/Enanta); NS3/4A protease inhibitors that are currently in phase I clinical trials such as BMS-791325 (being developed by Bristol-Myers Squibb), VX-985 and VX-500 (being developed by Vertex pharmaceuticals), and PHX1766 (being developed by Phenomix); and NS3/4A protease inhibitors that are currently in preclinical trials such as VX-813 (being developed by Vertex), AVL-181 and AVL-192 (being developed by Avila Therapeutics), and ACH-2684 (being developed by Achillion).

Polymerase inhibitors suitable for use in the context of the present invention include NS5B polymerase inhibitors. The NS5B, an R A-dependent R A polymerase (RdRp) enzyme, is a highly conserved structure across all hepatitis C genotypes. It is therefore, an ideal target for drug therapy. There are two classes of polymerase inhibitors: nucleoside/nucleotide analogues and non-nucleoside RdRp inhibitors. Nucleoside inhibitors target the catalytic sites of the enzyme and act as chain terminators. Non-nucleoside inhibitors are allosteric inhibitors. In certain embodiments, the at least one direct acting antiviral present in a combination according to the invention is the HCV NS5B polymerase inhibitor, mericitabine or GS-7977.

Mericitabine (also known as RG7128 or RO5024048), is a prodrug of PSI-6130, an oral cytidine nucleoside analogue. It is being developed by Roche and Pharmasset. Mericitabine has shown in vitro activity against all of the most common HCV genotypes.

GS-7977 (also known as PSI-7977) is being developed by Gilead Sciences. It is currently in Phase III clinical trials. It is being studied as a treatment to be used in combination with ribavirin. GS-78977 is a prodrug that is metabolized to the active antiviral agent 2'-deoxy-2'-a-fluoro-P-C-methyluridine-5 '-monophosphate.

Other examples of NS5B polymerase inhibitors that can be present in a combination according to the invention include, but are not limited to, nucleoside/nucleotide polymerase inhibitors that are currently in Phase II clinical trials such as IDX184 (being developed by Idenix); non-nucleoside polymerase inhibitors that are currently in Phase II clinical trials such as VX-222 (initially developed by ViroChem, now owned by Vertex); PF-868554 (being developed by Pfizer); ABT-072 and ABT-333 (being developed by Abbott), GS 9190 (being developed by Gilead) and ANA598 (also known as setrobuvir, being developed by Anadys); nucleoside/nucleotide polymerase inhibitors that are currently in Phase I clinical trials such as BI 207127 (being developed by Boehringer Ingelheim), MK- 0608 (being developed by Isis/Merck), TMC649128 (being developed by Medivir/Tibotec), RG7348 (being developed by Roche/Ligand (Metabasis)), PSI-938 (being developed by Pharmasset), and INX-189 (being developed by Inhibitex); and non-nucleoside polymerase inhibitors that are currently in Phase I clinical trials such as VCH-759 (initially developed by ViroChem Pharma, now owned by Vertex), IDX375 (being developed by Idenix), and A-837093 (being developed by Abbott).

NS5A inhibitors suitable for use in the context of the present invention include, in particular daclatasvir (also known as BMS-790052), which is developed by Bristol- Myers- Squibb.

D. Properties of the Combinations

A combination according to the present invention is such that (1) it is intended for use in the treatment or the prevention of HCV infection and (2) the at least one protein kinase inhibitor and at least one interferon or the least one protein kinase inhibitor and at least one direct acting antiviral act in a highly synergistic manner on the inhibition of HCV infection. In certain embodiments, the interferon or the direct acting antiviral decreases the

IC₅₀ for the inhibition of HCV infection by the protein kinase inhibitor by a factor of at least 10 fold or at least 25 fold, preferably at least 50 fold or at least 75 fold, more preferably at least 100 fold or 200 fold, and even more preferably more than 200 fold {e.g., more than 350 fold). In other words, in the presence of interferon or direct acting antiviral, the concentration of protein kinase inhibitor necessary to obtain a 50% inhibition of HCV entry is at least 10 times or at least 25 times, preferably at least 50 times or at least 75 times, more preferably at least 100 times or 200 times, and even more preferably more than 200 times {e.g., more than 350 times) lower than the concentration of protein kinase inhibitor that would be necessary to obtain the same HCV entry inhibition in the absence of interferon or direct acting antiviral.

In other embodiments, the protein kinase inhibitor decreases the IC₅₀ for the inhibition of HCV infection by the interferon or the direct acting antiviral by a factor of at least 10 fold or at least 25 fold, preferably at least 50 fold or at least 75 fold, more preferably at least 100 fold or 200 fold, and even more preferably more than 200 fold {e.g., more than 350 fold). In other words, in the presence of protein kinase inhibitor, the concentration of interferon or direct acting antiviral necessary to obtain a 50% inhibition of HCV entry is at least 10 times or at least 25 times, preferably at least 50 times or at least 75 times, more preferably at least 100 times or 200 times, and even more preferably more than 200 times (e.g., more than 350 times) lower than the concentration of interferon or direct acting antiviral that would be necessary to obtain the same HCV entry inhibition in the absence of protein kinase inhibitor. In certain embodiments, a combination of the present invention is characterized by a combination index (CI) that is lower than 1 (which is defined as a marked synergy). A combination of the present invention is preferably characterized by a CI lower than 1, preferably lower than 0.90, more preferably by a CI lower than 0.85, and even more preferably by a CI lower than 0.50. In other embodiments, a combination of the present invention is characterized by a surface amplitude > 20% as determined by the method of Prichard and Shipman (Prichard et al, Antiviral Res., 1990, 14: 181-205).

II - Treatment or Prevention of HCV infection and HCV-associated Diseases A. Indications

The combinations according of the present invention may be used in therapeutic and prophylactic methods to treat and/or prevent HCV infection, or to treat and/or prevent a liver disease or a pathological condition affecting HCV-susceptible cells, such as liver cells, lymphoid cells, or monocytes/macrophages.

Methods of treatment of the present invention may be accomplished using an inventive combination or a pharmaceutical composition comprising an inventive combination (see below). These methods generally comprise administration of an effective amount of at least one interferon (or at least one direct acting antiviral) and at least one protein kinase inhibitor, or a pharmaceutical composition thereof, to a subject in need thereof. The interferon (or direct acting antiviral) and protein kinase inhibitor may be administered concurrently (i.e., together or separately but at about the same time, e.g., within 5 minutes, 15 minutes or 30 minutes of each other), or alternatively, they may be administered sequentially (i.e., separately and at different times, e.g., different times of the same day or different times of the same week or different times of the same month, etc...). Administration may be performed using any of the methods known to one skilled in the art. In particular, the combination of protein kinase inhibitor and interferon or direct acting antiviral, or a composition thereof may be administered by various routes including, but not limited to, aerosol, parenteral, oral or topical route.

In general, the combination, or pharmaceutical composition thereof, will be administered in an effective amount, i.e. an amount that is sufficient to fulfill its intended purpose. The exact amount of the combination or pharmaceutical composition to be administered will vary from subject to subject, depending on the age, sex, weight and general health condition of the subject to be treated, the desired biological or medical response (e.g., prevention of HCV infection or treatment of HCV-associated liver disease), and the like. In many embodiments, an effective amount is one that inhibits or prevents HCV from entering into a subject's susceptible cells and/or infecting a subject's cells, so as to prevent HCV infection, treat or prevent liver disease or another HCV-associated pathology in the subject.

Combinations and pharmaceutical compositions of the present invention may be used in a variety of therapeutic or prophylactic methods. In particular, the present invention provides a method for treating or preventing a liver disease or pathology in a subject, which comprises administering to the subject an effective amount of at least one protein kinase inhibitor and at least one interferon or at least one protein kinase inhibitor and at least one direct acting antiviral (as defined above) (or pharmaceutical composition thereof) which inhibits HCV from entering or infecting the subject's cells, so as to treat or prevent the liver disease or pathology in the subject. The liver disease or pathology may be inflammation of the liver, liver fibrosis, cirrhosis, and/or hepatocellular carcinoma (i.e., liver cancer) associated with HCV infection.

The present invention also provides a method for treating or preventing a HCV- associated disease or condition (including a liver disease) in a subject, which comprises administering to the subject an effective amount of at least one interferon and at least one protein kinase inhibitor or at least one protein kinase inhibitor and at least one direct acting antiviral (as defined above) (or pharmaceutical composition thereof) which inhibits HCV from entering or infecting the subject's cells, so as to treat or prevent the HCV-associated disease or condition in the subject. In certain embodiments of the present invention, the combination (or pharmaceutical composition thereof) is administered to a subject diagnosed with acute hepatitis C. In other embodiments of the invention, the combination (or pharmaceutical composition thereof) is administered to a subject diagnosed with chronic hepatitis C.

Administration of an inventive combination, or pharmaceutical composition, according to such methods may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, symptoms of acute hepatitis C such as decreased appetite, fatigue, abdominal pain, jaundice, itching, and flu-like symptoms; symptoms of chronic hepatitis C such as fatigue, marked weight loss, flu-like symptoms, muscle pain, joint pain, intermittent low-grade fevers, itching, sleep disturbances, abdominal pain, appetite changes, nausea, diarrhea, dyspepsia, cognitive changes, depression, headaches, and mood swings; symptoms of cirrhosis such as ascites, bruising and bleeding tendency, bone pain, varices (especially in the stomach and esophagus), steatorrhea, jaundice and hepatic encephalopathy; and symptoms of extrahepatic manifestations associated with HCV such as thyroiditis, porphyria cutanea tarda, cryoglobulinemia, glomerulonephritis, sicca syndrome, thrombocytopenia, lichen planus, diabetes mellitus and B-cell lymphoproliferative disorders.

Alternatively or additionally, administration of a combination or pharmaceutical composition thereof according to such methods may slow down, reduce, stop or alleviate the progression of HCV infection or an HCV-associated disease, or reverse the progression to the point of eliminating the infection or disease. Administration of a combination or pharmaceutical composition of the present invention according to such methods may also result in reduction in the number of viral infections, reduction in the number of infectious viral particles, and/or reduction in the number of virally infected cells. The effects of a treatment according to the invention may be monitored using any of the assays known in the art for the diagnosis of HCV infection and/or liver disease. Such assays include, but are not limited to, serological blood tests, liver function tests to measure one or more of albumin, alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), and gamma glutamyl transpeptidase (GGT), and molecular nucleic acid tests using different techniques such as polymerase chain reaction (PCR), transcription mediated amplification (TMA), or branched DNA (bDNA). Combinations and compositions of the present invention may also be used in immunization therapies. Accordingly, the present invention provides a method of reducing the likelihood of susceptible cells of becoming infected with HCV as a result of contact with HCV. The method comprises contacting the susceptible cells with an effective amount of at least one interferon and at least one protein kinase inhibitor or at least one protein kinase inhibitor and at least one direct acting antiviral (as defined above) or a pharmaceutical composition thereof which inhibits HCV from entering or infecting the susceptible cells, so as to reduce the likelihood of the cells to become infected with HCV as a result of contact with HCV. The present invention also provides a method of reducing the likelihood of a subject's susceptible cells of becoming infected with HCV as a result of contact with HCV. In this method, contacting the susceptible cells with the combination or composition may be performed by administrating a combination or a pharmaceutical composition thereof to the subject. Reducing the likelihood of susceptible cells or of a subject of becoming infected with HCV means decreasing the probability of susceptible cells or a subject to become infected with HCV as a result of contact with HCV. The decrease may be of any significant amount, e.g., at least a 2-fold decrease, more than a 2-fold decrease, at least a 10-fold decrease, more than a 10-fold decrease, at least a 100-fold decrease, or more than a 100-fold decrease.

In certain embodiments, the subject is infected with HCV prior to administration of the inventive composition. In other embodiments, the subject is not infected with HCV prior to administration of the inventive composition. In yet other embodiments, the subject is not infected with, but has been exposed to, HCV. In certain embodiments, the subject may be infected with HIV or HBV.

For example, the methods of the present invention may be used to reduce the likelihood of a subject's susceptible cells of becoming infected with HCV as a result of liver transplant. As already mentioned above, when a diseased liver is removed from a HCV-infected patient, serum viral levels plummet. However, after receiving a healthy liver transplant, virus levels rebound and can surpass pre-transplant levels within a few days (Powers et al., Liver TranspL, 2006, 12: 207-216). Liver transplant patients may benefit from administration of a combination, or pharmaceutical composition, according the invention. Administration may be performed prior to liver transplant, during liver transplant, and/or following liver transplant.

Other subjects that may benefit from administration of a combination of interferon and protein kinase inhibitor according to the present invention include, but are not limited to, babies born to HCV-infected mothers, in particular if the mother is also HIV-positive; health-care workers who have been in contact with HCV- contaminated blood or blood contaminated medical instruments; drug users who have been exposed to HCV by sharing equipments for injecting or otherwise administering drugs; and people who have been exposed to HCV through tattooing, ear/body piercing and acupuncture with poor infection control procedures.

Other subjects that may benefit from administration of a combination according to the invention include, but are not limited to, subjects that exhibit one or more factors that are known to increase the rate of HCV disease progression. Such factors include, in particular, age, gender (males generally exhibit more rapid disease progression than females), alcohol consumption, HIV co-infection (associated with a markedly increased rate of disease progression), and fatty liver.

Still other subjects that may benefit from administration of a combination according to the invention include patients with HCV infections that are resistant to the standard of care or to other combinations of antivirals - antiviral resistance being a major challenge in HCV prevention and treatment (Pawlotsky et al., Hepatology, 2011, 53: 1742-1751).

In certain embodiments, the HCV infection or HCV-related disease to be treated by a combination according to the invention is caused by a Hepatitis C virus that is resistant to a direct acting antiviral. The recent development of direct acting antiviral molecules, together with clinical studies showing that these drugs may lead to the selection of resistant viruses if administered alone or in combination therapy, has raised concerns that resistance may undermine DAA-based therapy (Pawlotsky, Hepatology, 2011, 53: 1742-1751; Schaefer et al, Gastroenterology, 2012, 142: 1340- 1350 el341). However, the present Applicants have shown that a protein kinase inhibitor efficiently prevents dissemination of DAA-resistant HCV variants resulting in more rapid and sustained virus elimination. The Applicants have also shown that a combination of an anti-HCV entry agent and of a direct acting antiviral prevents DAA resistance allowing suppression of viral infection below the detection limit in a sustained manner.

In certain embodiments, a combination of an interferon and a protein kinase inhibitor or of at least one protein kinase inhibitor and at least one direct acting antiviral or a pharmaceutical composition thereof is administered alone according to a method of treatment of the present invention. In other embodiments, a combination of an interferon and a protein kinase inhibitor or of at least one protein kinase inhibitor and at least one direct acting antiviral or a pharmaceutical composition thereof is administered in combination with at least one additional therapeutic agent. The combination or pharmaceutical composition may be administered prior to administration of the therapeutic agent, concurrently with the therapeutic agent, and/or following administration of the therapeutic agent.

Therapeutic agents that may be administered in combination with an inventive combination or pharmaceutical composition may be selected among a large variety of biologically active compounds that are known to have a beneficial effect in the treatment or prevention of HCV infection, or a HCV-associated disease or condition. Such agents include, in particular, antiviral agents including, but not limited to, ribavirin, anti-HCV (monoclonal or polyclonal) antibodies, R A polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, micro-RNA antagonists, cytokines, therapeutic vaccines, NS5A antagonists, polymerase inhibitors, and any combination thereof.

B. Administration

An inventive combination (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used to administer combinations of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. An inventive combination or composition may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, mucosa, rectal and intestinal mucosa, etc). Administration can be systemic or local. Parenteral administration may be preferentially directed to the patient's liver, such as by catheterization to hepatic arteries or into a bile duct. As will be appreciated by those of ordinary skill in the art, in embodiments where the interferon (or direct acting antiviral) and protein kinase inhibitor are administered sequentially (i.e., at different times or separately but at substantially the same time), the interferon (or direct acting antiviral) and protein kinase inhibitor may be administered by the same route (e.g., intravenously) or by different routes (e.g., orally and intravenously). Similarly, in embodiments where an inventive combination is administered along with an additional therapeutic agent, the combination and therapeutic agent may be administered by the same route or different routes.

C. Dosage

Administration of an inventive combination (or a composition thereof) of the present invention will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the therapeutic effect desired, the severity of the HCV- related condition to be treated if already present, the presence of any infection, the age, sex, weight, and general health condition of the patient as well as upon the potency, bioavailability, and in vivo half-life of the interferon and protein kinase inhibitor used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively or additionally, the dosage to be administered can be determined from studies using animal models (e.g., chimpanzee or mice). Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using the inventive combination of an interferon and a protein kinase inhibitor, further information will emerge regarding the appropriate dosage levels and duration of treatment. A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of an inventive combination, or composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in a time release form). Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week; two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

In general, the amount of combination administered will preferably be in the range of about 1 ng/kg to about 100 mg/kg body weight of the subject, for example, between about 100 ng/kg and about 50 mg/kg body weight of the subject; or between about 1 g/kg and about 10 mg/kg body weight of the subject, or between about 100 M-g/kg and about 1 mg/kg body weight of the subject.

Ill - Pharmaceutical Compositions

As mentioned above, a combination of the invention may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of at least one interferon and at least one protein kinase inhibitor, or at least one protein kinase inhibitor and at least one direct acting antiviral as described herein and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents.

The combinations and pharmaceutical compositions thereof may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and/or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient. The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "unit dosage form", as used herein, refers to a physically discrete unit of an interferon or of a protein kinase inhibitor or of both an interferon and a protein kinase inhibitor for the patient to be treated. The expression "unit dosage form" also refers to a physically discrete unit of a direct acting antiviral or of a protein kinase inhibitor or of both a direct acting antiviral and a protein kinas inhibitor for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

A. Formulation

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

In order to prolong the effect of an active ingredient (i.e., a combination according to the invention), it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the active principles, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils {e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, an inventive combination may be mixed with at least one inert, physiologically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannital, and silicic acid; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition such that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally, in a delaying manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

In certain embodiments, it may be desirable to administer an inventive composition locally to an area in need of treatment (e.g., the liver). This may be achieved, for example, and not by way of limitation, by local infusion during surgery (e.g., liver transplant), topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or other implant.

For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient by either passive or active release mechanisms. Transdermal administrations include all administration across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient (i.e., a combination of an interferon and a protein kinase inhibitor) and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil- in- water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used. When a pharmaceutical composition of the present invention is used as

"vaccine" to prevent HCV-susceptible cells from becoming infected with HCV, the pharmaceutical composition may further comprise vaccine carriers known in the art such as, for example, thyroglobulin, albumin, tetanus toxoid, and polyamino acids such as polymers of D-lysine and D-glutamate. The vaccine may also include any of a variety of well known adjuvants such as, for example, incomplete Freund's adjuvant, alum, aluminium phosphate, aluminium hydroxide, monophosphoryl lipid A (MPL, GlaxoSmithKline), a saponin, CpG oligonucleotides, montanide, vitamin A and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005, L-121 and combinations thereof.

Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations for the delivery of antibodies can be found, for example, in "Remington 's Pharmaceutical Sciences^'", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA.

B. Additional Biologically Active Agents

In certain embodiments, an inventive combination is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, vaccine adjuvants and therapeutic agents such as anti-viral agents (as described above), antiinflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof.

In such pharmaceutical compositions, an interferon (or a direct acting antiviral), a protein kinase inhibitor and additional therapeutic agent(s) may be combined in one or more preparations for simultaneous, separate or sequential administration of the different components. More specifically, an inventive composition may be formulated in such a way that the interferon (or the direct acting antiviral), protein kinase inhibitor and therapeutic agent(s) can be administered together or independently from one another. For example, an interferon (or a direct acting antiviral), a protein kinase inhibitor and a therapeutic agent can be formulated together in a single composition. Alternatively, they may be maintained {e.g., in different compositions and/or containers) and administered separately.

C. Pharmaceutical Packs of Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of a combination of the present invention. Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s) (e.g., one or more anti- viral agents, as described above). Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein. An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data are actually obtained.

Example 1: Inhibition of HCVcc Infection

Using an Inventive Combination of a Protein Kinase Inhibitor and an Interferon

Materials and Methods

Cell Lines. Cultures of Huh7.5.1 cells which have previously been described (Zhong et αί, Proc. Natl. Acad. Sci. USA, 2005, 102: 9294-2929), were used in this study. Protein Kinase Inhibitors. Erlotinib, dasatinib and Sorafmib were obtained from IC Laboratoires.

Interferon-alpha. Interferon-alpha-2a was obtained from Roche; Interferon- alpha-2b was obtained from Merck. HCVcc Production and Infection. Cell-culture derived HCVcc (Luc-Jcl) were generated as previously described (Koutsoudakis et al, J. Virol, 2006, 80: 5308- 5320; Zeisel et al, Hepatology, 2007, 46: 1722-1731). For infection experiments, Huh7.5.1 cells were incubated with HCVcc infected as described previously (Zeisel et al., World J. Gastroenterol, 2007, 13: 4824-4830; Fofana et al., Gastroenterology, 2010, 139: 953-964; Lupberger et al. , Nature Medicine, 2011, 17: 589-595).

Combination Experiments. The protein kinase inhibitors and interferon-alpha were tested individually and in combination. Huh7.5.1 cells were pre-incubated with erlotinib or dasatinib and with IFN-CC for 1 hour. The Huh7.5.1 cells were then incubated with HCVcc Luc-Jcl in the presence of the compounds. HCVcc infection was analyzed two days later by luciferase reporter gene expression as previously described (Krieger et al., Hepatology, 2010, 54: 1144-1157; Fofana et al., Gastroenterology, 2010, 139: 953-964; Koutsoudakis et al, J. Virol, 2006, 80: 5308- 5320). The Combination Index (CI) was calculated as described (Zhao et al., Clin. Cancer Res., 2004, 10: 7994-8004). A CI of less than 0.9 indicates synergy; a CI equal to 0.9-1.1 indicates additivity; and a CI of more than 1.1 indicates antagonism (Zhao et al, Clin. Cancer Res., 2004, 10: 7994-8004; Zhu et al, J. Infect. Dis., 2012, 205: 656-662).

Synergy was also assessed using the method of Prichard and Shipman applied as described (Zhao et al, Clin. Cancer Res., 2004, 10: 7994-8004; Prichard et al, Antiviral Res., 1990, 14: 181-205). Surface amplitudes > 20% above the zero plane indicate a synergistic effect, while surface amplitudes < 20% below the zero plane indicate antagonism (Prichard et al, Antiviral Res., 1990, 14: 181-205). The validity of the assay and methods were confirmed by comparative analyses of combinations showing a non-synergistic effect or an antagonistic effect. Statistical Analysis. Results are expressed as means ± standard deviation (SD).

Statistical analyses were performed using Student's t test with a P value of <0.05 being considered statistically significant. Results and Discussion

The results obtained are presented on Figures 1 and 2 and in Table 1 below.

Table 1. IC₅₀ and CI for the combinations studied on HCVcc infection.

IC50 IC50 (Ul/ml) for

Compound 1 Compound 2 CI

(Ul/ml) combination

erlotinib 0.0035±0.004 0.21 ±0.09

IFN-a2a 0.3±0.16

dasatinib 0.07±0.008 0.48±0.09 erlotinib 0.002±0.003 0.21 ±0.04

IFN-a2b 0.2±0.05

dasatinib 0.015±0.005 0.33±0.02

As can be concluded from Figure 1 and Table 1 , IFN-a combined with clinical kinase inhibitors targeting HCV entry co-factors EGFR or EphA2 inhibit HCV infection in a synergistic manner. The present Applicants have previously identified EGFR and EphA2 as co-factors for HCV entry; and demonstrated that the clinical kinase inhibitors of EGFR (erlotinib) and of EphA2 (dasatinib) inhibit infection and viral spread at IC₅₀ 0.45-0.53 μΜ (Lupberger et al, Nature Medicine, 2011, 17: 589- 595). To investigate the effect of receptor tyrosine kinase (RTK) inhibition on the antiviral activity of IFN-a, they incubated Huh7.5.1 cells with IFN-a2a or IFN-a2b and a sub-IC₅o concentration of erlotinib which exerts only minimal effects on infection with recombinant HCV (HCVcc). They observed an enhanced and synergistic antiviral effect on HCVcc infection of IFN-a2a and IFN-a2b when combined with 0.1 μΜ erlotinib (CIs of 0.21±0.09 and 0.21±0.04, see Figure 1A-B). In contrast, an antagonistic effect was observed when IFN-a2a and IFN-a2b were combined with a different RTK inhibitor sorafenib (CIs of 1.31±0.03 and 1.32±0.05, respectively; see Figure 1A-1B), demonstrating that the observed synergies are specific for the compounds. The addition of a very low dose of erlotinib to IFN-a2a or IFN-a2b decreased their IC₅₀ up to 10-fold (from 0.3±0.16 to 0.0035±0.004 IU/ml for IFN-a2a; and from 0.2±0.05 to 0.002±0.003 IU/ml for IFN-a2b; Fig.l C-D). Furthermore, three-dimensional modeling according to the method of Prichard and Shipman was used to validate synergy. Surface amplitudes >20 % above the zero plane confirmed synergistic effects of the indicated combination of IFN-a with erlotinib (see Figure 1D-E). Similar results were obtained when IFN-a2a and IFN- a2b were combined with dasatinib (see Figure 1A-B and Figure 2). These data suggest that erlotinib and dasatinib enhance the antiviral activity of IFN-a in a highly synergistic manner.

These results demonstrate that protein kinase inhibitors in combination with interferons could provide a valuable alternative to currently available HCV combination therapy.

Example 2: Further Characterization in in vitro Models for HCV Infection

Following screening of combinations including other protein kinase inhibitors and other interferons using different HCV genotypes and model systems, combinations according to the invention will be further characterized by comparative analysis of neutralization in state-of-the-art in vitro models (Krieger et al., Hepatology, 2010, 51 : 1144-1157; Fofana et al, 2010, 139: 953-964).

Example 3: Characterization in an in vivo Model for HCV Infection

As a first step to evaluate the combinations according to the present invention, and establish the essential parameters for protection and treatment of HCV infection, the human liver-chimeric SCID/Alb-uPA mouse model will be used in a preclinical study. This model is a well characterized preclinical model for the in vivo assessment of antivirals. Pharmacokinetic and toxicity of selected combinations in uPA/SCID mice will be examined as previously described (Law et al, Nat. Med., 2008, 14: 25- 27; Vanwolleghem et al, Hepatology, 2008, 47: 1846-1855). Briefly, transplanted SCID/Alb-uPA mice will be infected with HCV-infected human serum intravenously and the effect of combinations of the invention on viral load will be assessed. Treatment outcome will be evaluated clinically (toxicity), virologically (viral load), and morphologically (histopathology of transplanted hepatocytes and other tissues) as described recently (Vanwolleghem et al, Gastroenterology, 2007, 133: 1144-1155). The safety profile will be further assessed in non-human primates.

Example 4: Phase I/IIa Clinical Trials

Following completion of the studies in the uPA-SCID mouse model as well as toxicity studies in non-human primates, clinical phase I/IIa trials will be initiated in HCV infected humans resistant or not eligible to standard of care using a longstanding collaboration of Inserm U748 -University of Strasbourg with the Strasbourg Center for Clinical Investigation (CIC) at Strasbourg. Two study designs are required to assess safety and efficacy for prevention and treatment of HCV infection:

Prevention of HCV infection in subjects undergoing liver transplantation. The combinations will be evaluated for their ability to prevent the universal re-infection of the liver graft following liver transplantation by achieving a reduction in viral load (as measured quantitatively by HCV RT-PCR) post-transplant by > 1 log 10 from the baseline value

Treatment of HCV infection in subjects chronically infected patients. The combinations will be evaluated for their ability to achieve reduction in viral load by > 1 logio from the baseline value.

Example 5: Inhibition of HCVcc Infection using an Inventive Combination of a Protein Kinase Inhibitor and a Direct Acting Antiviral

Materials and Methods

Cell Lines; Protein Kinase Inhibitors; HCVcc Production and Infection; Combination Experiments; and Statistical Analysis. See Example 1

Direct Acting Antiviral. Protease inhibitors (telaprevir, boceprevir, danoprevir and TMC-435), NS5A inhibitor (daclatasvir) and polymerase inhibitors (mericitabine and GS-7977 (formally known as PSI-7977)) were synthesized by Acme Bioscience, Inc. Results and Discussion

The results obtained are presented on Figures 3-6 and in Table 2 below.

A major effort of the pharmaceutical industry and current clinical research is the further improvement of IFN-based therapies using DAAs and the development of IFN-free combinations based on the combination of DAAs with or without ribavirin. Addressing these future concepts, the present Applicants have studied the combined antiviral effect of the protein kinase inhibitors, erlotinib and dasatinib, with the clinically licensed protease inhibitors - such as telaprevir and boceprevir - as well as second-generation protease inhibitors in late stage development - such as TMC-435 and danoprevir in the HCVcc model system. Table 2. IC₅₀ and CI for the combinations studied on HCVcc infection, protein kinase inhibitors: erlotinib, 0.5±0.06 μΜ; dasatinib, 0.4±0.3 μΜ.

IC50 IC50 (|JM or nM¹)

Compound 1 Compound 2 CI

(μΜ or nM¹) for combination

erlotinib 0.001 ±0.002 0.19±0.02 telaprevir 0.15±0.06 ^■

dasatinib 0.02±0.03 0.40±0.22 erlotinib 0.008±0.004 0.24±0.03 boceprevir 0.14±0.02 ^■

dasatinib 0.001 ±0.0005 0.26±0.002 erlotinib 0.0005±0.002 0.57±0.1

TMC-435 0.013±0.001 ^■

dasatinib 0.0015±0.0002 0.37±0.02 erlotinib 0.001 ±0.0005 0.30±0.06 danoprevir 0.006±0.003 ^■

dasatinib 0.0016±0.0007 0.45±0.009 erlotinib 0.0009±0.0004 0.25±0.03 daclatasvir 0.012±0.003 ^■

dasatinib 0.002±0.0008 0.40±0.07 erlotinib 0.003±0.006 0.21 ±0.05 mericitabine 0.12±0.03 ^■

dasatinib 0.035±0.01 0.56±0.09 erlotinib 0.0001 ±0.00002 0.19±0.002

GS-7977 0.021 ±0.002 ^■

dasatinib 0.00029±0.0001 0.26±0.007

Telaprevir, boceprevir, danoprevir, TMC-435, mercitabine, PSI-7977: μΜ ; daclatasvir : nM

Combination of clinically licensed protease inhibitors telaprevir or boceprevir with a sub-IC₅o concentration of protein kinase inhibitors - which exerts only minimal inhibitory effect on HCV infection - resulted in CIs of 0.19 to 0.56, reflective of synergy (Figure 3A and Table 2), the most effective combination being the combination of telaprevir and erlotinib. In contrast, combination of two protease inhibitors (telaprevir and boceprevir) resulted in an additive activity confirming the validity of the assay (Figure 3 A).

Second-generation protease inhibitors have beend demonstrated to have a higher genetic barrier for resistance. However, single amino acid substitutions are able to confer drug resistance in vivo. Importanly, it has been demonstrated that several telaprevir- and boceprevir-resistance mutations confer cross-resistance to these second-generation protease inhibitors (Sarrazin et al, J. Hepatol, 2012, 56(1): S88- 100). Combination of a second-generation protease inhibitor, TMC-435 or danoprevir, and a protein kinase inhibitor (CIs of 0.3 to 0.57 - see Figure 3A and Table 2), demonstrating the relevance of adding an entry- inhibitor as a concept to improve antiviral efficacy. A number of novel DAAs have reached early- to late stage clinical development, including NS5A inhibitors and polymerase inhibitors. The first NS5A inhibitor, daclatasvir (Gao et al., Nature, 2010, 465: 96-100), has been shown to have potent antiviral activity against HCV genotype 1 in monotherapy. However, its genetic barrier to resistance is low and resistant variants developed rapidly without imposing a loss of in vivo viral fitness (Gao et al, Nature, 2010, 465: 96-100). Combination of daclatasvir with erlotinib or dasatinib also resulted in a synergistic activity with CIs of 0.25 to 0.40 (see Figure 3C and Table 2). The most effective combinations was the combination of daclatasvir and erlotinib decreasing its IC₅₀ up to 60 fold (Figure 5A- B).

Finally, the Applicants investigated synergy between protein kinase inhibitors and the polymerase inhibitor GS-7977. GS-7977 is currently in clinical development and has been suggested as having the potential to become the cornerstone of an efficacious, all-oral combination regiment for many patients with chronic hepatitis C infection (Zeisel et al, Front Biosci., 2009, 14: 3274-3285; Zeisel et al, J. Hepatol, 2011, 54: 566-576). Thus, the Applicants investigated whether protein kinase inhibitors potentiate the antiviral activity of GS-7977. Combination of GS-7977 with erlotinib and dasatinib resulted in a synergistic activity (CIs of 0.19 and 0.26, respectively; Figure 3D and Table 2), decreasing the IC50 of GS-7977 up to 210 fold from 0.021 μΜ to 0.0001 μΜ and 0.00029 μΜ, respectively (Figure 6A-C). These clearly demonstrate the potential of combining GS7977 with protein kinase inhibitors to improve its antiviral activity.

Similar results were obtained for the combination of another polymerase inhibitor, mericitabine (Gane et al, Lancet, 2010, 376: 1467-1475) and protein kinase inhibitors (CIs of 0.21 to 0.56; see Figure 3D and Table 2).

To further confirm the synergistic effect over a broad range of concentrations of both compounds, the Applicants performed combinations testing a full checker-board of compounds dose-response curves by using the method of Prichard and Shipman (Prichard et al, Antiviral Res., 1990, 14: 181-205). Noteworthy, in particular low doses of both compounds resulted in an antiviral activity above the expected value (Figure 4B, Figure 5B, Figure 6C). Noteworthy, none of all DAA-protein kinase inhibit combinations tested in the present study resulted in detectable toxicity in primary human hepatocytes (Table 3).

Table 3. Absence of toxicity of combinations of DAAs and protein kinase inhibitors in primary human hepatocytes (PHH). Cytotoxic effects on PHH using the highest concentrations of each compound used in combination (IFN-CC, 10 IU/ml; DAAs, 10 μΜ; and PKIs, 10 μΜ) were assessed by analyzing the ability to metabolize MTT. Anti-Fas antibody (10 μg/ml) was used as a positive control of toxicity. Toxicity analyses of the most efficient combinations are shown. Data are presented as relative cell viability compared to PHH cultured in the absence of compounds or solvent (=100%). Means ± SD from one representative experiment performed in triplicate is shown.

Relative cell

Compound 1 Concentration Compound 2 Concentration

viability (%) telaprevir 10 μΜ erlotinib 10 μΜ 93±5

GS-7977 10 μΜ dasatinib 10 μΜ 103±8

GS-7977 10 μΜ erlotinib 10 μΜ 101 ±4 anti-Fas 10 Mg/ml 16±2

The results obtained suggest an opportunity to reduce the doses of both compounds - a key requirement for improvement of future antiviral treatment. Taken together, the present Applicants have demonstrated that the addition of a sub-IC₅o concentration of protein kinase inhibitor is sufficient to markedly decrease the IC50 concentration of the different DAAs currently evaluated in IFN-free regimens, without displaying any toxic effects in vitro. These data demonstrate the proof-of-concept that entry inhibitors and DAAs are highly synergistic and define novel antiviral combinations for further preclinical and clinical development in IFN-free regimens.

Example 6: IFN-cc Signal Transducer STAT3 is Relevant for HCV Infection

To investigate the molecular mechanisms of the synergistic effects, the present Applicants first investigated the role of IFN signal transducers in HCV entry and infection using functional silencing of gene expression by STAT-specific siRNAs. STAT3 is an important regulator in the STAT1 mediated IFN-a response (Ho et al., J. Biol. Chem., 2006, 281 : 14111-14118) and has been described as an interaction partner of EGFR in proliferative cells (Lo et al, Br. J. Cancer, 2006, 94: 184-188). The impact of STAT3 on HCV entry and infection was studied using RNAi in HCV permissive Huh7.5.1 cells. Silencing of STAT3 expression in Huh7.5.1 cells was found to decrease entry of HCV pseudoparticles (HCVpp) that display the HCV glycoproteins as well as infection of cell-culture derived HCV (HCVcc) (data not shown). Furthermore, STAT3 inhibitor Cpdl88 (Xu et al, PLoS One, 2009, 4:e4783) was found to impair HCVcc infection without any cell toxicity (data not shown). Taken together, these data suggest that STAT3 is relevant for HCV infection, and most likely plays a mechanistic role for the observed synergy between protein kinase inhibitors and interferon-alpha. Since STAT3 is a co-factor for HCV infection, it is also an antiviral target, using compounds that inhibit STAT3 function or expression.

Example 7: Erlotinib Prevents Antiviral Resistance by Blocking Cell-Cell

Transmission of DAA-Resistant Variants

Materials and Methods

Production of Recombinant Viruses Containing Resistance Mutations. The drug-resistant mutations were introduced into the Jcl -Luc plasmid (Koutsoudakis et al, J. Virol, 2006, 80: 5308-5320; Pietschmann et al, Proc. Natl. Acad. Sci. USA, 2006, 103: 7408-7413) using in vitro site directed mutagenesis (Quickchange XL, Stratagene) as previously described (Zhu et al, J. Infect. Dis., 2012, 205: 656-662). Nucleotide changes were made in the HCV Luc- Jcl construct to generate the A156S or L36M or R155K amino acid substitutions in the NS3 protein. A one-step polymerase chain reaction (PCR) mutagenesis was performed using mutation primers. The introduction of mutations into Jcl constructs was confirmed by DNA sequence analysis.

Analysis of HCV Cell-Cell Transmission. Cell-cell transmission of HCV was assessed as previously described (Witteveldt et al, J. Gen. Virol, 20069, 90: 48-58). Briefly, producer Huh7.5.1 cells were electroporated with HCV Jcl RNA and cultured with naive target Huh7.5-GFP cells in the presence of 10 μΜ erlotinib or DMSO solvent/rat IgG control. An HCV anti-E2-neutralizing antibody (Witteveldt et al. , J. Gen. Virol, 20069, 90: 48-58) (25 μg/mL) was added to block cell-free transmission (Witteveldt et al, J. Gen. Virol, 20069, 90: 48-58). After 24 hours of coculture, cells were fixed with paraformaldehyde, stained with an NS5A-specific antibody (0.1 μg/mL) (Virostat) and analyzed by flow cytometer. Cell-cell transmission was defined as percentage HCV infection of Huh7.5-GFP+ target cells in the presence of an HCV E2-specific antibody (Witteveldt et al, J. Gen. Virol, 20069, 90: 48-58).

Results and Discussion

Infection of DAA-resistant viruses is sensitive to Protein Kinase Inhibitors. Clinically approved protease inhibitors telaprevir and boceprevir have a low genetic barrier for resistance in several amino acid substitutions conferring resistance without imposing a large viral fitness cost. First, to assess whether entry inhibitors inhibit protease-inhibitor resistant variants, the present Applicants introduced two well characterized mutations at positions 155 and 156 in the backbone of the HCVcc-Luc genome, known to confer resistance in vivo (Sarrazin et al, J. Hepatol, 2012, 56 Suppl. 1, S88-100). As shown on Figure 7A and B, introduction of mutations R155S and A156S into HCVcc-Jcl increased the IC₅₀ of telaprevir and boceprevir up to 10- fold, respectively. In contrast, no differences were observed in the inhibition for both wild-type and protease-resistant viruses by erlotinib (Fig. 7C). These data indicate that erlotinib efficiently inhibits DAAs-resistant variants without exhibiting cross- resistance.

DAA-resistant variants are efficiently transmitted by cell-cell transmission.

Cell-cell transmission is considered more rapid and efficient than cell-free spread because it obviates rate-limiting early steps in the virus life cycle, such as virion attachment (Timpe et al., Hepatology, 2008, 47: 17-24). The cell-cell transmission of DAA-resistant variants may thus accelerate viral spread, leading to viral breakthrough and treatment failure. First, the Applicants investigated whether DAA variants are efficiently transmitted by cell-cell spread. To address this question they used a well- established cell-cell transmission assay (Lupberger et al., Nature Medicine, 2011, 17: 589-595) and recombinant protease inhibitor-resistant virus HCVcc Jcl containing either the single mutation at position 156 (A156S) or the double mutations at positions 155 and 36 (R155K, L36M). As shown on Figure 8A and D, protease resistant- viruses very efficiently spread through cell-cell transmission indicating that this mode of transmission is relevant for spread of DAA-resistant variants in the infected liver. Cell-cell transmission of DAA resistant variant is inhibited by erlotinib.

Since EGFR has been suggested as co-factor for cell-cell transmission (Brimacombe et al., J. Virol, 2011, 85: 596-605), the present Applicants next investigated whether cell-cell transmission of DAA-resistant variants can be inhibited by erlotinib. Erlotinib efficiently inhibited cell-cell transmission of protease-resistant viruses (Figure 8B and E). These data demonstrate that erlotinib efficiently inhibits cell-cell transmission of protease inhibitor-resistant viruses and thus provides a previously undiscovered opportunity to inhibit the dissemination of protease inhibitor-resistant viruses throughout the liver.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

Claims

Claims claimed is:

A combination comprising at least one protein kinase inhibitor and at least one interferon or at least one direct acting antiviral for use in the treatment or the prevention of HCV infection, wherein the at least one protein kinase inhibitor and at least one interferon or at least one direct acting antiviral act in synergy to inhibit HCV infection.

The combination according to claim 1 , wherein the at least one protein kinase inhibitor is a tyrosine kinase inhibitor.

The combination according to claim 2, wherein the at least one protein kinase inhibitor is a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR) or on the ephrin type-A receptor 2 EphA2

The combination according to claim 3, wherein the at least one protein kinase inhibitor is selected from the group consisting of erlotinib, gefitinib, vandetanib, lapatinib, neratinib, afatinib, dasatinib, equivalents thereof, and any combination thereof.

The combination according to claim 2, wherein the tyrosine kinase inhibitor is an anti-receptor tyrosine kinase antibody.

The combination according to any one of claims 1 to 4, wherein the at least one interferon is a human interferon.

The combination according to claim 6, wherein the at least one interferon is a human interferon selected from the group consisting of interferon-alpha (IFN- cc), pegylated IFN-cc, albumin-IFN-cc, interferon-beta (IFN-β), pegylated IFN- β, albumin-IFN-β, interferon-omega (IFN-CO), pegylated IFN-CO, albumin-IFN- co, interferon-gamma (IFN-γ), pegylated IFN-γ, albumin-IFN-γ, interferon- lambda (IFN-λ), pegylated IFN-λ, albumin- IFN-λ, equivalents thereof, and combinations thereof.

The combination according to claim 7, wherein the at least one interferon is human interferon-cc2a or human interferon- a 2b. The combination according to any one of claims 1 to 4, wherein the at least one direct acting antiviral is selected from the group consisting of protease inhibitors, NS5A inhibitors, and polymerase inhibitors.

The combination according to claim 9, wherein the at least one direct acting antiviral is selected from the group consisting of telaprevir, boceprevir, danoprevir, TMC-435, daclatasvir, mericitabine and GS7977.

The combination according to any one of claims 1 to 10, wherein the combination index (CI) of the combination is lower than 1, preferably lower than 0.90, more preferably lower than 0.80, and even more preferably lower than 0.60.

The combination according to any one of claims 1 to 11, wherein the combination is used for the treatment of HCV infection or a HCV-related disease in a subject, or for the control of chronic HCV infection in a subject.

The combination according to any one of claims 1 to 11, wherein the combination is used for preventing HCV re-infection and recurrence in a liver transplantation patient.

The combination according to claim 12 or claim 13, wherein the HCV infection or HCV-related disease or HCV re-infection is caused by a Hepatitis C virus that is resistant to a direct acting antiviral and/or that is transmitted by cell-cell transmission.

A pharmaceutical composition comprising a combination according to any one of claims 1 to 14 and at least one pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to claim 15 further comprising at least one anti-viral agent.

The pharmaceutical composition according to claim 16, wherein the anti- viral agent is selected from the group consisting of ribavirin, anti-hepatitis C virus monoclonal antibodies, anti-hepatitis C virus polyclonal antibodies, R A polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, micro-RNA antagonists, cytokines, therapeutic vaccines, NS5A antagonists, polymerase inhibitors, cyclophilin A antagonists, and any combination thereof.

A kit comprising at least one protein kinase inhibitor and at least one interferon or at least one protein kinase inhibitor and at least one direct acting antiviral for simultaneous or sequential use in the treatment or prevention of HCV infection, wherein the at least on protein kinase inhibitor and at least one interferon or the at least one protein kinase inhibitor and at least one direct acting antiviral act in synergy to inhibit HCV infection.

The kit according to claim 18, wherein the at least one protein kinase inhibitor is as defined in any one of claims 2 to 5 and the at least one interferon is as defined in any one of claims 6 to 8 and the at least one direct acting antiviral is as defined in any one of claims 9-10.

The kit according to claim 18 or claim 19, wherein the HCV infection is caused by a Hepatitis C virus that is resistant to a direct acting antiviral and/or that is transmitted by cell-cell transmission.