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  • A61K38/19—Cytokines; Lymphokines; Interferons
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  • A61K38/19—Cytokines; Lymphokines; Interferons
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  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5431—IL-11
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  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the use of a cytokine in combination with an interferon for the preparation of compositions for the treatment of viral diseases.
• the interferon system is the first line of defense against viral infections in mammals.
• Type I interferons (which includes several subtypes of IFN-alpha as well as IFN-beta) are molecules with antiviral activity that are induced in mammalian cells in response to viral infections.
• the action of IFN-alpha is mediated by interaction with a multisubunit of surface cell receptor, which consists of two receptor subunits, 1 IFN-alpha receptor (IFNAR1) and IFNAR2. Only one form of IFNARl chain has been identified; and three variants of the subunit.
• IFNAR2 have been recognized: one full length, IFNAR2C, and two truncated isoforms IFNAR2b and IFNAR2a.
• the IFNAR2c variant is involved in ligand binding and signal transduction, while the two truncated forms IFNAR2b and IFNAR2a - which do not have intracellular domains - inhibit the IFN-alpha signal, by competition with IFNAR2c by binding to IFN-alpha .
• the IFN-alpha signaling cascade starts when IFN-alpha binds to the receptor.
• the binding of IFN-alpha to the receptor leads to the activation of tyrosine kinases associated with IFNAR (Janus kinase 1 (Jakl) and tyrosine kinase 2 (Tyk2)), which phosphorylate both IFNARl and IFNAR2 subunits.
• Phosphorylated IFNAR provides a binding site for the activator of the transcription factor 2 and signal transducer that contains a domain of homology 2 with Src (STAT2), when it is phosphorylated by Tyk2 or Jakl.
• the other STATs including STAT1, STAT3 and STAT5, are consequently recruited to the recipient for phosphorylation and activation.
• the STAT1 and STAT2 activated monomers are then released back to the cytosol, where they form heterodimers and bind to interferon / Protein p48 regulation factor 9, to form an active complex of transcription factors known as interferon stimulated gene factor 3 (ISGF3 ).
• ISGF3 interferon stimulated gene factor 3
• the complex translocates to the nucleus and binds to the IFN stimulation response element (ISRE) to initiate transcription of the target genes, including some antiviral and immunoregulatory proteins.
• ISRE IFN stimulation response element
• IFN-alpha also induces the formation of other STAT complexes, including STAT1 / STAT1, STAT1 / STAT3 and STAT3 / STAT3, which bind to the activated ⁇ sequence in the promoter regions of sensitive genes.
• IFN-alpha activates STATl, STAT2, STAT3 and STAT5, followed by the induction of a wide variety of antiviral and proapoptotic genes that can contribute to the antitumor and antiviral activity of IFN-alpha in human livers.
• HBV hepatitis B
• HCV hepatitis C
• HBV causes chronic infection mainly in cases of vertical transmission and immunosuppressed individuals.
• HCV infection is notable for the tendency to develop chronicity in most cases, indicating that this virus has developed particularly effective mechanisms to evade the interferon system.
• Patients with chronic HCV infection as well as patients with chronic hepatitis B fail to respond to interferon therapy. In chronic hepatitis B, the sustained antiviral response occurs in less than 40% of cases [1].
• the response of the virus-infected cell to interferon-alpha depends on several determining factors, including those related to the virus and those specific to the host.
• Various HCV gene products have been shown to modulate the Host response to IFM therapy and influence the severity of viral disease, particularly liver disease.
• N5A non-structural proteins
• E2 structural proteins
• PKR one of the key molecules involved in the development of an antiviral state in response to IFN [3, 4]. This could block PKR resulting in an inhibition of IFN activity in HCV infected cells.
• IFN-alpha-induced STAT1 signal is affected in both HCV transgenic mice and in liver biopsies of patients with chronic HCV [5,6].
• the STAT1 activation is not affected in these cells when they were incubated with the IFN-gamma proinflammatory molecule, thus indicating that the blocking of the STAT1 activation produced by HCV is specific for the signaling cascade of Type I IFN, and that does not affect the signaling pathway of type II IFN.
• cytokines that activate the Jak-STAT signaling pathway, in particular, members of the IL-6 family, comprising IL-6, IL-Il, leukemia inhibitor factor (LIF), oncostatin (OSM ), cardiotrophin-1 (CT-I), ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) [7].
• cytokines bind to the plasma membrane receptor complexes that comprise the common signal transduction receptor chain gpl30 [7]. Signal transduction involves the activation of members of the Jak tyrosine kinase family, leading to the activation of STATl and STAT3 transcription factors. These cytokines potentially activate STAT3 and to a lesser extent STATl through their common gpl30 receptor subunit. However, although IL-6 has been shown to induce some antiviral effects [8], the antiviral activity of this cytokine is much lower than that of interferon-alpha.
• the present invention relates to the use of an interleukin of the IL-6 family, preferably, cardiotrophin-1 (CT-I) or oncostatin M (OSM):
• CT-I cardiotrophin-1
• OSM oncostatin M
• interferon alfa IFN-alpha
• interleukin of the IL-6 family preferably CT-I or OSM
• IFN-alpha as an improved antiviral therapy in any form of viral infection, and in particular in hepatitis C virus infection ( HCV), in which the preferred combination of CT-1-IFN-alpha or OSM-IFN-alpha has been found to be especially potent in inhibiting HCV replication.
• HCV hepatitis C virus infection
• interferon-alpha associated with a cytokine of the IL-6 family in particular, CT-I or oncostatin M, is capable of overcoming the blockade of the interferon-alpha signaling cascade (and consequently , the attenuation of the effect of interferon-alpha that occurs when the virus, preferably HCV, replicates in the infected cell).
• the present invention relates first to the use of at least one cytokine of the IL-6 family - gpl30 family - or a DNA sequence that encodes it, in the preparation of a pharmaceutical composition for administration combined with at least one interferon -alpha or a DNA sequence that encodes it, in the treatment of viral diseases, said cytokine being selected from cardiotrophin-1, IL-Il, the leukemia inhibiting factor, oncostatin M, ciliary neurotrophic factor, cytokine similar to cardiotrophin, and combinations thereof; and even more preferably, said cytokine is cardiotrophin-1 or oncostatin M.
• the ⁇ itoquine of the "IL-6 family" refers to: the complete native form of said cytokine; any active fraction of said cytokine, that is, any partial polypeptide sequence of said cytokine that maintains the physiological effects of the complete cytokine claimed in the present invention; and any polypeptide derivative of said cytokine, that is, any polypeptide sequence having a homology with said native cytokine greater than 80%, more preferably greater than 90% and more preferably even greater than 95%, and that it maintains the physiological effects of the complete cytokine claimed in the present invention.
• the cytokine of the IL-6 family can come from both the native form, and any form of recombinant cytokine-, from any polynucleotide form encoding the complete cytokine , the active fraction or the polypeptide derivative.
• complete or an active fraction, or recombinant cytokine one or more amino acids may have been suppressed, substituted or added to the protein in any of the aforementioned ways, provided it is maintained the activity provided in the present invention.
• the interferon-alpha of the invention is any type of interferon-alpha.
• said interferon-alpha is selected from interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-5, consensus interferon, purified interferon-alpha, pegylated interferon-alpha and combinations thereof, -
• Another particular embodiment of interferon-alpha is selected from pegylated interferon-alpha-2b, pegylated interferon-alpha-2a, pegylated interferon-alpha-5 and combinations thereof.
• the combined use of an interferon-alpha and a cytokine of the interleukin-6 family is directed to the treatment of a preferably viral disease.
• viral diseases that can be treated by the combined use of interferon and a cytokine of the interleukin-6 family, we can cite, without the use being limited to them, diseases caused by the encephalomyocarditis virus, hepatitis B and C, HIV, cutaneous viral infections (chickenpox, herpes zoster, measles), respiratory viral infections, viral diseases of the central nervous system, hepatic viral diseases, viral diseases of the salivary glands, infectious mononucleosis and genital warts.
• the viral disease is hepatitis C.
• the cytokine - or cytokines - of the IL-6 family and interferon-alpha can be administered separately being present in different pharmaceutical compositions; or they can be administered together, being present in the same pharmaceutical composition.
• a further object of the present invention is therefore a pharmaceutical composition comprising a pharmaceutically acceptable amount of at least one cytokine of the IL-6 family - gpl30 family -, or a DNA sequence encoding it, and a pharmaceutically acceptable amount. of at least one interferon-alpha, or a DNA sequence that encodes it.
• the IL family cytokine -6 is selected from IL-6, IL-11, leukemia inhibiting factor, M oncostatin, cardiotrophin-1, ciliary neurotrophic factor, cardiotrophin-like cytokine and combinations thereof, preferably between IL- 6, IL-Il, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, ciliary neurotrophic factor, cardiotrophin-like cytokine and combinations thereof; and even more preferably, said cytokine of the IL-6 family is cardiotrophin-1 or oncostatin M.
• interferon-alpha is any type of interferon-alpha.
• interferon-alpha has been selected from interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-5, consensus interferon, purified interferon-alpha, pegylated interferon-alpha and combinations thereof.
• the interferon-alpha is selected from pegylated interferon-alpha-2b, pegylated interferon-alpha-2a, pegylated interferon-alpha-5 and combinations thereof.
• the DNA sequence encoding the cytokine of the IL-6 family is incorporated into an expression vector, for example , a plasmid or a viral vector, preferably operably linked to a control sequence that regulates the expression of cytokine or interferon-alpha.
• an expression vector for example , a plasmid or a viral vector, preferably operably linked to a control sequence that regulates the expression of cytokine or interferon-alpha.
• the construction of said expression vector with the DNA sequence can be carried out by conventional methods of recombinant technology collected in manuals such as "Molecular Cloning: a Laboratory manual" by J. Sambrook, D. W. Russel Eds. 2001, 3rd ed. CoId Spring Harbor, New York.
• These embodiments of the pharmaceutical composition are of interest for gene transfer therapies (gene therapy).
• composition of the invention may further comprise, at the least one pharmaceutically compatible carrier with the cytokine IL-6 family, or with the DNA sequence encoding it , and is pharmaceutically compatible with the interferon-alpha or the sequence DNA that encodes it.
• the cytokine of the IL-6 family - or the DNA sequence that the it encodes - and the interferon-alpha - or the DNA sequence that encodes it - can be vehiculized in two vehicular agents.
• compositions of the invention are, without these being taken as a limitation thereof, any solid composition (eg, tablets, capsules, granules, etc.) or liquid
• said pharmaceutical composition may be in a pharmaceutical form for oral administration, either in solid or liquid form.
• pharmaceutical forms of oral administration include tablets, capsules, granules, solutions, suspensions, etc., and may contain conventional excipients, such as binders, diluents, disintegrants, lubricants, humectants, etc., and may be prepared. by conventional methods.
• the pharmaceutical composition may also be adapted for parenteral administration, in the form of, for example, sterile lyophilized solutions, suspensions or products, in the appropriate dosage form; in this case, said pharmaceutical composition will include suitable excipients, such as buffers, surfactants, etc.
• the excipients will be chosen based on the pharmaceutical form of administration selected.
• a review of the different pharmaceutical forms of drug administration, for these and other possible alternative routes, and their preparation can be found for example in the book “Pharmaceutical Technology", by JL ViIa Jato, 1997 VoIs I and II, Ed. Synthesis , Madrid; or in “Handbook of pharmaceutical manufacturing formulations ", by SK Niazi, 2004 VoIs I to VI, CRC Press, Boca Raton.
• the pharmaceutical composition is for parenteral administration, preferably subcutaneously, intravenously, intramuscularly or intraperitoneally.
• the interferon-alpha is in pegylated form.
• Some examples for the preparation of compositions with pegylated forms of interferon-alpha can be found in US5,762,923 and
• Intron pegylated interferon-alpha-2b from Schering Corporation (Kenilworth, N. J. USA) and PEGASYS
• both the IL-6 family cytokine and interferon-alpha will preferably be in a pharmaceutically acceptable or substantially pure form, that is, they will have a pharmaceutically acceptable level of purity excluding pharmaceutically acceptable excipients and not including Material considered toxic at normal dosage levels.
• the purity levels for the IL-6 family cytokine and interferon-alpha are preferably greater than 50%, more preferably, greater than 70%, more preferably, greater than 90%. In a preferred embodiment, they are greater than 95%.
• the therapeutically effective amount of the IL-6 family cytokine and the interferon-alpha to be administered will depend, among other factors, on the individual to be treated, on the severity of the disease suffered by said individual, on the way of chosen administration, etc.
• IL-6 family cytokine and interferon-alpha can be administered, one or more times a day, for example, 1, 2, 3 or 4 times a day.
• the typical total daily amount of cardiotrophin-1 is comprised between 1 ⁇ g / kg and 10 mg / kg body weight; and the typical total daily amount of interferon-alpha-2a is between 1.5 and 10 MUI daily or between 40 and 300 micrograms per week of pegylated interferon-alpha.
• the dosage level will be higher in the first weeks of treatment, reducing the dose in later stages.
• the administration regime can be daily, 3 times per week, or also weekly.
• cardiotrophin-1 and interferon-alpha can be administered according to different administration regimens (for example, different route of administration or different frequency).
• the present object of the present invention is a pharmaceutical kit for the treatment of a viral disease comprising at least:
• a first component comprising at least one cytokine of the IL-6 family -gpl30 family- (either complete, an active fraction or a polypeptide derivative, as defined above) or a DNA sequence encoding said cytokine;
• the kit according to the invention comprises preferably a cytokine of the IL-6 family selected from TL-G 1 IL-Il, the leukemia inhibitor factor, oncostatin M, cardiotrophin-1, ciliary neurotrophic factor, cardiotrophin-like cytokine and combinations thereof , preferably selected from IL-Il, the leukemia inhibitor factor, oncostatin M, cardiotrophin-1, ciliary neurotrophic factor, cardiotrophin-like cytokine and combinations thereof; and even more preferably the cytokine of the IL-6 family is cardiotrophin-1 or oncostatin M.
• the kit according to the invention comprises an interferon-alpha of any type, preferably one selected from interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-5, consensus interferon, purified interferon-alpha, interferon-alpha Pegylated and combinations thereof.
• the interferon-alpha is selected from pegylated interferon-alpha-2b, pegylated interferon-alpha-2a, pegylated interferon-alpha-5 and combinations thereof.
• the DNA sequence encoding the IL-6 family cytokine (either complete, an active fraction, or a polypeptide derivative), or the interferon-alpha of the kit, is incorporated into an expression vector.
• the first component and the second component may further comprise at least one pharmaceutically acceptable excipient and compatible with the IL-6 family cytokine - or a DNA sequence encoding it - and with the interferon- alpha- or a DNA sequence that encodes it -.
• the kit defined above may comprise the first and second components in separate pharmaceutical compositions; or the first and second components may be present in the kit in the same pharmaceutical composition.
• Said kit may further comprise a third component comprising one or more pharmaceutically compatible excipients with the IL-6 family cytokine - or a DNA sequence that encodes it - and with interferon-alpha - or a DNA sequence that encodes it. -.
• Said third component may further comprise one or more pharmaceutically compatible carrier agents with the IL-6 family cytokine - or a DNA sequence that encodes it - and with interferon-alpha - or a DNA sequence that encodes it.
• the present invention has as a further object a method for the treatment of a viral disease comprising administering in combination a therapeutically effective amount of at least one cytokine of the IL-6 family - gpl30 family - (either complete, an active fraction or a polypeptide derivative, as defined above), or a DNA sequence that encodes it, and a therapeutically effective amount of at least one interferon-alpha, or a DNA sequence that encodes it.
• the DNA sequence encoding the IL-6 family cytokine or the interferon-alpha of the method is incorporated into an expression vector.
• the viral disease can be caused by the encephalomyocarditis virus, hepatitis B and C, HIV, cutaneous viral infections (chickenpox, shingles, measles), respiratory viral infections, viral diseases of the central nervous system, diseases hepatic virals, viral diseases of the salivary glands, infectious mononucleosis and warts genitals.
• the viral disease is hepatitis C.
• the cytokine of the IL-6 family is selected from IL-6, IL-Il, the leukemia inhibitor factor, oncostatin M, cardiotrophin-1, ciliary neurotrophic factor, cytokine similar to cardiotrophin and combinations thereof, preferably between IL-11, the leukemia inhibitor factor, oncostatin M, cardiotrophin-1, ciliary neurotrophic factor, cardiotrophin-like cytokine and combinations thereof, and more still preferred, the cytokine of the IL-6 family is cardiotrophin-1 or oncostatin M.
• interferon-alpha is any type of interferon-alpha.
• interferon-alpha-2a interferon-alpha-2b, interferon-alpha-5, consensus interferon, purified interferon-alpha, pegylated interferon-alpha and combinations thereof;
• interferon-alpha is selected from pegylated interferon-alpha-2b, pegylated interferon-alpha-2a, pegylated interferon-alpha-5 and combinations thereof.
• it can comprise the combined and simultaneous administration of the cytokine of the interleukin family, preferably, cardiotrophin-1, and interferon-alpha.
• the cytokine of the IL-6 family (either complete, an active fraction or a polypeptide derivative) and interferon-alpha may be present in the same pharmaceutical composition that is administered to the patient; or the cytokine of the IL-6 family and interferon-alpha can be administered in separate pharmaceutical compositions.
• the present invention shows that when liver cells comprising a complete HCV replicon with interferon-alpha and an interleukin of the IL-6 family are stimulated, in particular, cardiotrophin-1 and oncostatin M.
• ISGs interferon-sensitive genes
• 2'-5'-oligoadenylate synthase (2'-5'OAS) 2'-5'-oligoadenylate synthase
• STAT1 and STAT3 higher levels of STAT1 and STAT3 than when cells are incubated with cytokine only.
• FIG. 1 shows an analysis of the phosphorylation of STAT1, STAT2 and STAT3.
• Huh7 cells Huh7 and Huh7 cells containing a complete genomic HCV replicon (Core-3 ') were treated for 15, 60 and 120 minutes with 50 IU / mi IFN-alpha-2 (IFKT ⁇ ) or with 20 ng / ml of cardiotrophin-1 (CT1), or with a combination of IFN-alpha-2 and CT-I, and the amount of phosphorylated STAT1, STAT2 and STAT3 present in cell extracts was analyzed by Western-blot.
• IFKT ⁇ 50 IU / mi IFN-alpha-2
• CT1 cardiotrophin-1
• Figure 2 shows a quantitative mRNA analysis by real-time RT-PCR of STATl, STAT3 and 2'-
• Figure 3 shows a quantitative analysis, using real-time RT-PCR, of HCV RNA present in Huh7 cells containing a complete HCV replicon, treated for 3 days with IFN-alpha-2 or IFN-alpha-5 (5 or 50 IU / mi) and 20 ng / ml of CT-I or OSM or IL6.
• Figure 4 shows the comparative antiviral effect of the combination IFN-alpha-2 or IFN-alpha-5 (5U / mL) plus CT-I or OSM or IL-6 (20ng / mL), using real-time RT-PCR of HCV RNA present in Huh7 cells containing a complete HCV replicon, treated for 3 days with said cytokines.
• FIG. 5 shows the percentage of Huh7 cells protected against infection with the encephalomyocarditis virus.
• Huh7 cells were pretreated for 24 hours with 5 ng / ml or 50 ng / ml of CT-I and different amounts of IFN-alpha-2, and were infected with 10 5 PFU of the encephalomyocarditis virus (EMCV), and after 24 hours, the cells were stained with violet crystal dye to measure the amount of viable cells.
• EMCV encephalomyocarditis virus
• Huh7 cells a hepatoma cell line
• HCV infection determines not only a defective activation of STAT1, but also a reduction in the levels of STAT3 and STAT2 proteins.
• the studies shown in Figure 1 show the short-term effects of incubation with either IFN-alpha-2 or CT-I, or with the combination of the two.
• incubation for 72 hours shows that the combined treatment of CT-I and IFN-alpha-2, or OSM and IFN-alpha-2 resulted in increased expression STAT3, thus counteracting the effect of HCV on infected cells (see figure 2).
• Huh7 cell lines carrying the full length HCV replicon Huh.7 cells expressing the full length HCV replicon were established as described [9]. Summing up, pI 389 / Core-3 '/5.1 was linearized with Seal
• RNA synthesis using T7 RNA polymerase 20 ⁇ g of synthesized RNA was used to electroporate 10 7 Huh7 cells and, at 24 hours, 500 ⁇ g / ml of G418 (Gibco, USA) was added. Twice a week, the culture medium supplemented with G418 was replaced and, at 4 weeks after transfection, mixed G418 resistant colonies were collected and used for further analysis. Western transfer analysis. Huh7 cells were expressed that expressed or did not express the full length HCV replicon at 200,000 / well in 6-well plates in D-MEM (Gibco) with 10% FCS (Gibco).
• the membranes were subjected to extensive washing in TBS-T and specific protein bands were visualized using the "Western Lightning chemiluminescence reagent plus” chemiluminescence detection system (Perkin Elmer, USA), according to the manufacturer's instructions. Subsequently, the membranes were autoradiographed and the bands were quantified by densitometric analysis using the Molecular Analyst / PC program (Bio-Rad Laboratories).
• Antibodies Anti-phospho-STATl tyr701 and anti-phospho-STAT3 tyr705 antibodies and the anti- rabbit IgG antibody bound to HRP were purchased from CeIl Signaling Technology
• the anti-STAT3, anti-phospho-STATl ser727 , anti- STAT2 and anti-phospho-STAT2 tyr689 antibodies were obtained from
• ISGs 2'-5'OAS, STAT1 and STAT3
• IFN-alpha-2 5 or 50 IU / mi
• CT-I 20 ng / ml
• OSM 20 ng / ml
• IFN-alpha-2 + CT-I combination or the IFN-alpha-2 + OSM combination see Fig. 2A-2F.
• RNA samples Two micrograms of total RNA were treated with DNase (Gibco-BRL, UK) before reverse transcription with M-MLV Reverse Transcriptase (Gibco BRL) in the presence of RNaseOUT (Gibco-BRL).
• STAT, 2'-5'OAS and ⁇ -actin expression was measured by real-time PCR using an ICycler and the SYBR Green Supermix IQ (Bio-Rad Laboratories, CA). 2 ⁇ l aliquots of the cDNA pool were used for each PCR, which contained specific sense and antisense primers for each gene (Table 1) in a final volume of 20 ⁇ l. To determine the specificity of the PCR products obtained, their dissociation temperature was analyzed.
• Huh7 cells bearing the HCV replicon were incubated with IFN-alpha (IFN-alpha-2 or IFN-alpha-5, at 5 or 50 Ul / ml), alone or in combination with cytokines of the IL-6 family ( IL-6, CT-I, OSM; at 20 ng / ml); or with the cytokines of the IL-6 family alone.
• IFN-alpha IFN-alpha-2 or IFN-alpha-5, at 5 or 50 Ul / ml
• IFN-alpha-2 as of IFN-alpha-5, when these cytokines were used at low (5 Ul / ml) or high doses (50 Ul / ml)
• FIG. 3A IL-6 weaker potentiated the antiviral effect of both IFN-alpha-2 and IFN-alpha-5 ( Figure 3C).
• the combination therapy IFN-alpha-2 or IFN-alpha-5 plus CT-I or OSM increases by a factor of approximately 5 and 10, respectively, the antiviral effect of IFN-alpha, and the combined therapy of IFN- alpha-2 or IFN-alpha-5 plus IL-6 increases by a factor of approximately 2.
• Figure 4 comparatively depicts the potentiating effect of IL-6, CT-I and OSM (20ng / mL) on the antiviral action of IFN-alpha-2 (5U / mL) ( Figure 4A) or IFN-alpha-5 ( Figure 4B).
• the greater antiviral enhancing effect of the IFN-alpha / CT-1 and IFN-alpha / OSM combinations compared to the IFN-alpha / lL-6 combination is clearly observed.
• HCV RNA analysis by quantitative real-time PCR Huh7 cells expressing the full length HCV replicon at 100,000 / well were seeded in 6-well plates in D-MEM with 10% FCS. 50 or 5 Ul / ml of IFN-alpha-2 or IFN-alpha-5 alone was added, or in combination with 20 ng / ml of CT-I or OSM or IL6. The cell culture was maintained for three days. The culture medium supplemented with the previous cytokines was replaced daily.
• RNA of Huh7 cells transfected with the full length HCV replicon was obtained following the "Ultraspec RNA isolation system" protocol (Biotech, USA), which is based on the method described by Chomczynski and Sacchi [10].
• Two micrograms of total RNA were treated with DNase (Gibco-BRL) before reverse transcription with M-MLV Reverse Transcriptase (Gibco-BRL) in the presence of RNaseOUT (Gibco-BRL).
• the expression of HCV RNA and ⁇ -actin mRNA was measured by quantitative real-time PCR using an ICycler and the SYBR Green Supermix IQ (Bio-Rad Laboratories).
• Huh7 cells were incubated with decreasing doses of IFN-alpha-2 from 250 to 1 UT / ml in the presence or absence of a low (5 ng / ml) or high (50 ng / ml) dose of CT-I and infected 24 hours later with EMCV. It is observed that CT-I alone (at low or high dose) had little cytoprotective effect on EMCV-infected cells, since the addition of this cytokine at very low doses of IFN-alpha-2 did not improve cell viability.
• Cytoprotective assay of IFN-alpha-2 and CT-I against EMCV in Huh7 cells The cytoprotective activity of IFN-alpha-2 and CT-I was determined by measuring the ability of these cytokines to protect Huh7 cells against the cytopathic effect of the encephalomyocarditis virus. The test was performed on a 96-well microtiter plate. First, 2xlO 4 Huh7 cells were plated per well in 150 ⁇ l of medium containing serial dilutions of IFN-alpha-2 alone (250 to 1 Ul / ml) or these serial dilutions of IFN-alpha-2 plus 50 or 5 ng / ml of CT-1 and incubated for 24 hours.
• Antiviral studies were performed combining fixed concentrations of IFN-alpha-2 and IFN-alpha-5 (5 or 50 Ul / ml) plus or minus IL-6, OSM and CT-I (20ng / mL) in Huh7 cells transfected with full length HCV replicon.
• Mathematical analysis of the type of interaction established between IFN-alpha and the cytokines of the IL-6 family was performed by multivariate analysis according to the method described by T. C Chou (11).
• I dl / Dl + d2 / D2
• di, d2 are the concentrations of the inhibitors in the combination
• Dl, D2 the concentrations of the inhibitors 1 and 2 which separately exert the same inhibition as the combination.
• Huh7 cells expressing the full length HCV replicon were plated at 20,000 cells / well in 24-well plates in D-MEM with 10% FCS. Different types of treatments were carried out; 50 or 5 UT / ml of IFN-alpha-2 or IFN alpha-5 plus or minus 20 ng / ml of CT-I, OSM or IL-6 (R&D Systems, UK) were added. The cell culture was maintained for three days. The culture medium supplemented with the previous cytokines was replaced daily.
• RNA was obtained from Huh7 cells transfected with the full length HCV replicon using (the Nucleic Acid Purification Lysis Solution Kit (Applied BioSystems, Foster City, CA) and the ABI PRISM 6100 Nucleic Acid PrepStation semi-automatic system (Applied BioSystems). Two micrograms of total RNA were treated with DNase (Gibco-BRL) before reverse transcription with M-MLV Reverse Transcriptase (Gibco-BRL) in the presence of
• HCV RNA and ⁇ -actin mRNA were measured by quantitative real-time PCR using an ICycler and the SYBR Green Supermix IQ (Bio-Rad Laboratories). 2 ⁇ l aliquots of the cDNA pool were used for each PCR, which contained sense and antisense primers specific for the 5 'untranslated region of HCV or for the ⁇ -actin gene (Table 1) in a final volume of 20 ⁇ l . To determine the specificity of the PCR products, their dissociation temperature was analyzed. The results were normalized according to the quantification of ⁇ -actin in the same sample. The amount of HCV RNA was expressed by the formula 2 ct l actin) - ct (HCV) s j_ endo ct the point at which the fluorescence rises appreciably above the background fluorescence.
• Tripodi M La Monica N, Heim MH. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology 2003/124: 1465-1475. 6. Duong FH, Filipowicz M, Tripodi M, La Monica N, Heim MH Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A. Gastroenterology 2004/126: 263-277.

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