JP2007262067A - Composition for hepatitis c treatment, agent for hepatitis c treatment, and kit for hepatitis c treatment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for hepatitis C treatment having as an active ingredient an anti-HCV substance obtained by a screening method using HCV full-length genome replicon cells which express a reporter gene product. <P>SOLUTION: The composition for hepatitis C treatment contains pitavastatin as an active ingredient. This is based on the finding that an anti-HCV effect can be recognized in statins as HMG Co-A reductase inhibitors known as hyperlipemia remedies by using the screening method. Alternatively, the composition comprises pitavastatin, interferon and/or cyclosporine A as active ingredients. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composition for treating hepatitis C, an agent for treating hepatitis C, and a therapeutic agent for hepatitis C, comprising as an active ingredient a substance having an anti-HCV action obtained by a screening method using HCV full-length genome replicating cells expressing a reporter gene product. The present invention relates to a hepatitis B treatment kit.

  Hepatitis C virus (hereinafter referred to as “HCV”) is an RNA virus belonging to the Flaviviridae family that was discovered and identified in 1989 as a causative virus for non-A non-B hepatitis. Since HCV is a virus that establishes persistent infection, hepatitis caused by HCV infection (hepatitis C) shifts to chronic hepatitis at a high rate. Since then, it has been revealed that cirrhosis and eventually hepatocellular carcinoma develop in the course of more than 20 years.

  It is estimated that there are about 2 million HCV infected people in Japan and about 200 million people worldwide. As can be seen from the situation of HCV infection by fibrinogen preparations, which has become a recent social problem, there are many so-called asymptomatic carriers who are not aware of being infected with HCV. Currently, there are about 35,000 victims of hepatocellular carcinoma annually in Japan, 80% of which are due to HCV infection. In addition, about 20,000 people are sacrificed annually in the previous stage of cirrhosis. Therefore, it can be said that HCV is a virus that causes serious infections.

  Currently, interferon (hereinafter referred to as “IFN”) is the only therapeutic agent that exhibits an antiviral effect on chronic hepatitis C alone. In Japan, IFN therapy for chronic hepatitis C was started in 1992, and the therapeutic effect has been improved by trial and error of the administration method of IFN. However, since about half of patients cannot be cured, the limitations of IFN therapy have become apparent.

  Many genotypes exist in HCV, but in Japan, type 1b is about 70%, type 2a is about 20%, type 2b is about 10%, and other genotypes are rarely found. It is known that the effect on IFN treatment differs depending on the genotype, and that the IFN treatment effect is high in the 2a and 2b types (high efficiency is 50% or more), whereas the IFN treatment effect is low in the 1b type. In addition, it is known that IFN is less effective in patients with a large amount of virus than in patients with a small amount of virus. Extremely low at ~ 8%. These patient groups are called “interferon resistance” or “interferon intractable”.

  The combination therapy of ribavirin (which alone has no effect on hepatitis C alone) and IFN, which has been known to have a wide range of antiviral activity with nucleic acid structural analogues in recent years, with the aim of increasing marked efficiency ( (Approved for insurance in December 2001) and therapy with peg IFN that increases the stability of IFN in the blood by binding polyethylene glycol to IFN and decreases renal excretion (approved for use in December 2003) ) Is to be performed.

  The year after the discovery of HCV, the entire structure of the RNA genome of HCV was elucidated, and it was revealed that 10 types of HCV proteins were produced from single-stranded positive-stranded RNA of about 9,600 bases (see FIG. 1). ). Virus particles are produced from the core and envelope of the first half, and NS proteins essential for replication of the HCV genome are produced from the nonstructural (NS) region of the second half.

  Although the HCV research has greatly progressed as a result of elucidation of the genome structure, the development of neutralizing antibodies, vaccines, and specific anti-HCV drugs is still difficult. The biggest reason is the lack of an experimental system for artificially growing HCV. Since the discovery of HCV, an attempt was made to develop an artificial growth system using many cultured cells and animals, but no practical one was obtained. Moreover, the chimpanzee is the only model animal for HCV infection, and no alternative animal has been found yet. Using chimpanzees for drug screening is impractical from a rarity and economic point of view.

  In 1999, the HCV replicon system appeared as a new experimental system that cleared the above problems to some extent (see Non-Patent Document 1). In this system, NS3 to NS5B regions essential for HCV genome replication and the HCV subgenome consisting of both ends of the genome are replicated in the cell, and the number of copies of HCV subgenome per cell is several thousand. To reach. Thereafter, subgenomic HCV replicon cells derived from several different HCV strains have been established (see, for example, Non-Patent Document 2). In addition, in the above replicon cells, a cell into which a subgenomic HCV replicon incorporating a reporter gene has been introduced has been developed for the purpose of easily monitoring the replication level of the subgenomic HCV replicon (Non-patent Documents 3 to 3). 5).

  Currently, in the development of a therapeutic agent for hepatitis C, since pharmacological tests using a large number of model animals (chimpanzees) cannot be carried out, it is essential to evaluate the efficacy using the subgenomic HCV replicon system.

  However, the subgenomic HCV replicon system has a problem that the influence of HCV structural proteins cannot be evaluated. It is conventionally known that a core protein, which is one of HCV structural proteins, affects host cellular factors. Therefore, the replicon system is not sufficient for attempts to see what actually occurs in human liver infected with HCV. In addition, it is expected that an agent that suppresses virus replication screened using a subgenomic HCV replicon system may not actually sufficiently suppress HCV replication.

  Accordingly, in order to solve the problems of the subgenomic HCV replicon system, a replication system for HCV full-length genomic RNA has been developed. To date, three types of HCV strains (N strain, Con-1 strain and H77 strain) have been developed. Establishment of cells capable of replicating the full-length genome (full-length HCV RNA replication system) has been reported (see Non-Patent Documents 6 to 8).

  Statins are HMG Co-A reductase inhibitors that are widely used clinically as therapeutic agents for hyperlipidemia and therapeutic agents for hypercholesterolemia. Recently, the antiviral action of statins has been reported, and there are several reports on the anti-HCV action.

  In Patent Document 1, the replication inhibitory effect of 5 types of atorvastatin, lovastatin, simvastatin, cerivastatin and pravastatin was examined using a subgenomic HCV replicon system, and antistatic HCV action was observed in 4 types of statins other than pravastatin. It is described. However, since it was not evaluated using the full-length HCV RNA replication system, the influence of structural proteins could not be evaluated. In addition, the use of other antiviral agents (IFN, ribavirin) and statins is mentioned, but no empirical data is given.

  Patent Document 2 and Non-Patent Document 9 describe the results of examining the anti-HCV action of lovastatin. In these reports, the full-length HCV RNA replication system is used, but the subgenomic HCV replicon system is used for the evaluation of RNA level, and the expression suppression of HCV protein is evaluated in the full-length HCV RNA replication system. Only.

Cyclosporine A is widely used clinically as an immunosuppressant. It has already been reported that cyclosporin A has an anti-HCV action, and its action mechanism has also been clarified (see Non-Patent Documents 10 to 13).
US Patent Application Publication No. 2005/0085528 US Patent Application Publication No. 2005/0085529 V. Lohmann, F. Korner, J. Koch, U. Herian, L. Theilmann, R. Bartenschlager, Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line, Science 285, 110-113 (1999) N.Kato, K.Sugiyama, K.Namba, H.Dansako, T.Nakamura, M.Takami, K.Naka, A.Nozaki, K.Shimotohno, Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in in vitro, Biochem.Biophys.Res.Commun. 306, 756-766 (2003) EMMurray, JAGrobler, EJMarkel, MFPagnoni, G. Paonessa, AJSimon, OAFlores, Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells, J. Virol. 77, 2928-2935 (2003) M.Yi, F.Bodola, SMLemon, Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein, Virology 304, 197-210 (2002) T.Yokota, N.Sakamoto, N.Enomoto, Y.Tanabe, M.Miyagishi, S.Maekawa, L.Yi, M.Kurosaki, K.Taira, M.Watanabe, H.Mizusawa, Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs, EMBO Rep. 4, 602-608 (2003) KJBlight, JAMcKeating, J. Marcocotrigiano, CMRice, Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture, J. Virol. 77, 3181-3190 (2003) M.Ikeda, M.Yi, K.Li, SMLemon, Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells, J. Virol. 76 , 2997-3006 (2002) T. Pietschmann, V. Lohmann, A. Kaul, N. Krieger, G. Rinck, G. Rutter, D. Strand, R. Bartenschlager, Persistent and transient replication of full-length hepatitis C virus genomes in cell culture, J. Virol. 76, 4008-4021 (2002) Jin Ye et al. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation.Proc Natl Acad Sci US A. Dec 23; 100 (26): 15865-70 (2003). Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 38: 1282-1288 (2003) Nakagawa M, Sakamoto N, Enomoto N, Tanabe Y, Kanazawa N, Koyama T, Kurosaki M, Maekawa S, Yamashiro T, Chen CH, Itsui Y, Kakinuma S, Watanabe M. Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem. Biophys. Res. Commun. 313; 42-47 (2004) Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol. Cell 19: 111-122 (2005) Nakagawa M, Sakamoto N, Tanabe Y, Koyama T, Itsui Y, Takeda Y, Chen CH, Kakinuma S, Oooka S, Maekawa S, Enomoto N, Watanabe M. Suppression of hepatitis C virus replication by cyclosporin A is mediated by blockade of cyclophilins. Gastroenterology 129: 1031-1041 (2005)

  It is considered that the problem of the subgenomic HCV replicon system has been solved by the establishment of a full-length HCV RNA replication system (HCV full-length genome replication cell). However, when monitoring the HCV replication level in already established HCV full-length genome-replicating cells, it is necessary to carry out complicated RT-PCR method or Northern blotting method, which requires a large amount of anti-HCV drug. It is difficult to use for screening. It is also difficult to accurately monitor the replication level with high sensitivity. Therefore, it is necessary to develop a full-length HCV RNA replication system incorporating a reporter gene so that the level of HCV replication can be monitored easily and accurately.

  However, as can be seen from the fact that there are very few HCV full-length genome replicating cells already established, it is not easy to establish HCV full-length genome replicating cells. The reason is that RNA with a region encoding a drug resistance marker gene added to the full-length genomic RNA of HCV exceeds 11 kb, and it is very difficult to introduce such a long RNA into a host cell and stably replicate it. It is. Therefore, it is very difficult to introduce RNA that is longer by adding a reporter gene sequence (about 12 kb) into a host cell and stably replicate it, and an HCV full-length genome replicating cell incorporating a reporter gene. Has not yet been established.

  On the other hand, since the current hepatitis C treatment method mainly composed of IFN cannot cure about 50% of patients, development of a drug that is more effective in combination with IFN or an alternative to IFN is eagerly desired. .

  The present invention has been made in view of the above problems, and its purpose is to realize a full-length HCV full-length genome replicating cell in which a full-length HCV genome into which a reporter gene has been incorporated is introduced and expressing a reporter gene product, An object of the present invention is to provide a screening method for anti-HCV drugs using cells, and further to provide a composition for treating hepatitis C, which comprises a substance having an anti-HCV action obtained by using the screening method as an active ingredient.

  The present inventors have intensively studied to solve the above-mentioned problems, and have used HCV full-length genome-replicating cells that have already been established by the present inventors as parent cells for introducing a full-length HCV genome incorporating a reporter gene. A so-called healing cell in which HCV RNA was completely eliminated by treatment with IFN-α was found to be suitable, and an HCV full-length genome replicating cell incorporating a reporter gene was established. The present inventors have confirmed that the expression level of the reporter gene product and the replication level of HCV RNA correlate very well. Furthermore, the present inventors screened a statin that has already been marketed as an HMG Co-A inhibitor using the cells, and as a result, found that it has an action of suppressing HCV genome replication and completed the present invention. .

  That is, the cell according to the present invention is a cell that replicates the full-length genome of hepatitis C virus (HCV) and expresses a reporter gene product, and includes a reporter gene sequence, a selectable marker gene sequence, and an HCV full-length genome sequence. It is characterized by the introduction of RNA.

  The RNA introduced into the cell according to the present invention preferably further contains an exogenous internal ribosomal entry site (IRES) sequence.

  The cell according to the present invention is a HCV full-length genome replicating cell into which RNA containing a selectable marker gene sequence and an HCV full-length genome sequence is introduced as a parent cell into which the RNA is inserted, in a medium containing a drug having an antiviral effect. It is preferable to use a healing cell obtained by culturing. The drug having antiviral action is preferably interferon. Furthermore, it is preferable that the said healing cell is an Oc cell (FERM P-20517) derived from human hepatoma cell line HuH-7.

  In the cell according to the present invention, it is more preferable that the HCV open reading frame sequence of RNA introduced into the parent cell contains a mutation with at least one amino acid substitution. More preferably, the mutation occurs in the NS3 region.

  In the cell that replicates the full-length genome of HCV according to the present invention, RNA introduced into the parent cell is HCV IRES sequence, reporter gene sequence, selectable marker gene sequence, foreign IRES sequence, HCV open reading from the 5 ′ end. It is preferable that the frame (ORF) sequence and the HCV 3 ′ untranslated sequence are linked in this order.

  The screening method according to the present invention is a method for screening a substance having an anti-HCV action, the step of contacting a test substance with the cell according to the present invention; the step of measuring the level of a reporter gene product; and the reporter A step of comparing the level of the gene product with the level when the test substance is not contacted.

  The screening kit according to the present invention is a kit for screening a substance having an anti-HCV action, and is characterized by comprising cells that replicate the full-length genome of HCV according to the present invention.

  The composition for treating hepatitis C according to the present invention is characterized in that a substance having an anti-HCV action obtained by the screening method according to the present invention is used as an active ingredient.

  The composition for the treatment of hepatitis C according to the present invention comprises (1) fluvastatin or a derivative thereof, (2) atorvastatin or a derivative thereof, (3) simvastatin or a derivative thereof, (4) lovastatin or a derivative thereof, (5) It is preferable to use pitavastatin or a derivative thereof as an active ingredient.

  The composition for treating hepatitis C according to the present invention is preferably applied to patients with hepatitis C to which interferon is administered.

  The composition for the treatment of hepatitis C according to the present invention is preferably applied to patients with hepatitis C administered with interferon and cyclosporin A.

  The therapeutic agent for hepatitis C according to the present invention comprises at least one selected from the group consisting of fluvastatin or a derivative thereof, atorvastatin or a derivative thereof, simvastatin or a derivative thereof, lovastatin or a derivative thereof and pitavastatin or a derivative thereof, and interferon It is characterized by combining.

  The therapeutic agent for hepatitis C according to the present invention preferably further comprises a combination of cyclosporin A in addition to the above.

  A kit for treating hepatitis C according to the present invention comprises the composition for treating hepatitis C according to the present invention.

  The kit for treating hepatitis C according to the present invention preferably further comprises a composition for treating hepatitis C containing interferon as an active ingredient, and further comprises a composition for treating hepatitis C containing cyclosporin A as an active ingredient. It is more preferable.

  A cell that replicates the full-length genome of HCV according to the present invention can replicate the full-length genome of HCV and expresses a reporter gene product. Since the amount of replicated genomic RNA and the amount of reporter gene product correlate very well, the use of this cell produces an effect that the replication level of full-length HCV genomic RNA can be monitored easily and accurately.

  The screening method according to the present invention uses cells that replicate the full-length genome of HCV according to the present invention, and has the effect of enabling high-throughput screening of substances having an anti-HCV action.

  If the composition for the treatment of hepatitis C according to the present invention is used, it is possible to achieve an improvement in marked efficiency and a reduction in side effects in the conventional method for treating hepatitis C.

  By using the kit for treating hepatitis C according to the present invention, the combined use of statin and IFN, or the combined use of statin, cyclosporin A and IFN, has an anti-HCV effect that significantly exceeds the anti-HCV effect obtained by the combined use of IFN and ribavirin. There is an effect that it can be realized. Moreover, even if the dose of IFN is reduced, it is possible to obtain an effect equal to or greater than the combined effect of IFN and ribavirin, and side effects due to IFN can be reduced.

(1) A cell that replicates the full-length genome of HCV and expresses a reporter gene product The present invention provides a cell that replicates the full-length genome of HCV and expresses a reporter gene product. The cell according to the present invention is a cell into which RNA comprising a reporter gene sequence, a selectable marker gene sequence and an HCV full-length genome sequence is introduced, as long as it replicates the full-length genome of HCV and expresses a reporter gene product. . In the cells according to the present invention, the expression level of the reporter gene product and the replication amount of HCV RNA correlate very well, so that the replication level of the full-length HCV genome can be easily monitored by quantifying the reporter gene product. it can. Further, it is very useful for functional analysis of HCV including the influence of structural proteins, which is impossible with a subgenomic HCV replicon. That is, by using the cell according to the present invention, it becomes possible to perform HCV function analysis, screening for a substance having an anti-HCV action, and the like simply and rapidly.

  In the present specification, the “full-length genome of HCV” means RNA containing substantially the entire region of the HCV genome shown in FIG. RNA viruses containing HCV undergo spontaneous mutation at a relatively high rate and have a diversity of genomes. It is known that there are 5 to 8% base sequence differences between virus strains even in the same genotype. Therefore, it is not necessary to be identical to the base sequence of the full-length HCV genome registered in the database such as GenBank. That is, in the base sequence of the known full-length HCV genome, some bases may be substituted, deleted or added.

  The RNA introduced into the cell according to the present invention only needs to include a reporter gene sequence, a selectable marker gene sequence, and an HCV full-length genome sequence. The order of these sequences is not particularly limited as long as the reporter gene product, the selectable marker gene product, and the protein encoded by the HCV genome can be expressed. The RNA preferably contains two IRESs, an IRES for translating a reporter gene and a selectable marker gene, and an IRES for translating the HCV ORF. Thereby, the effect that the protein to be translated can be maintained at a high level is obtained. Both of the two IRESs may be HCV-derived IRES, but at least one is preferably an exogenous IRES. By using exogenous IRES, the effect that the replication pattern of the full-length genome is maintained can be obtained. The exogenous IRES is not particularly limited, and examples thereof include IRES derived from encephalomyocarditis virus (EMCV), IRES of bovine viral diarrhea virus (BVDV), IRES of poliovirus, etc. EMCV-derived IRES is preferred because of its high activity and frequent use.

  As an example of the order of each sequence contained in RNA introduced into the cell according to the present invention, from the 5 ′ end, HCV IRES sequence, reporter gene sequence, selectable marker gene sequence, exogenous IRES sequence, HCV ORF sequence HCV 3 ′ untranslated sequence can be mentioned, but is not limited thereto. The HCV IRES is an RNA composed of a 5 'untranslated region and a part of the 5' side of the core. For example, the present inventors use positions 1 to 377 of the nucleotide sequence of the HCV-O strain shown in SEQ ID NO: 1 (positions 1 to 341 are 5 'untranslated regions). However, it is not limited to this.

  The HCV genomic sequence contained in the RNA introduced into the cell according to the present invention may be any derived from HCV. HCV includes not only pathogenic strains that can cause hepatitis C, but also attenuated strains and mutant strains. Moreover, although many genotypes exist in HCV, they may be derived from any genotype of HCV. Preferably it is 1b. This is because about 70% of hepatitis C patients in Japan are infected with type 1b HCV.

  As HCV strains of genotype 1b, HCV-O strain, N strain, Con-1 strain, JT strain and the like are known. Although the present inventors have produced the cells according to the present invention using the genomic RNA of the HCV-O strain, the present invention is not limited to this. The base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the HCV-O strain are registered in GenBank as ACCESSION AB191333.

  The reporter gene contained in the RNA introduced into the cell according to the present invention is not particularly limited, and examples thereof include a luciferase gene, alkaline phosphatase gene, β-lactamase gene, chloramphenicol acetyltransferase gene and the like. Of these, the luciferase gene is preferable in that a reporter assay can be performed easily and with high sensitivity. As the luciferase gene, a firefly-derived luciferase gene or a Renilla luciferase gene is generally used, but any of them may be used. The Renilla-derived luciferase gene is preferred in that the length of the gene is shorter.

  The selectable marker gene contained in the RNA introduced into the cell according to the present invention is not particularly limited, but a drug resistance gene is preferable from the viewpoint of simplicity. The drug resistance gene is not particularly limited, and may be appropriately selected from known drug resistance genes that can be used for selection of transformed cells. Specific examples include a neomycin resistance gene (neomycin phosphotransferase gene), a puromycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene, and the like.

  The origin of the cell according to the present invention, that is, the parent cell into which the RNA is introduced is not particularly limited as long as it can replicate the full-length HCV genome, but is preferably a mammalian hepatocyte, and a human hepatocyte More preferably, it is a human liver cancer cell line. Examples of human hepatoma cell lines include HuH-7, HepG2, Hep3B, C3A, and PLC.

  In the cell according to the present invention, as a parent cell into which the RNA is introduced, an HCV full-length genome replicating cell into which RNA containing a selectable marker gene sequence and an HCV full-length genome sequence has been introduced is added to a medium containing a drug having an antiviral action. It is preferable to use a healing cell obtained by culturing.

  As used herein, the term “healing cell” refers to a subgenomic HCV replicon that is obtained by culturing a cell that replicates a subgenomic HCV replicon or a cell that replicates the full-length HCV genome with an antiviral agent. It means cells that have been eliminated or cells from which the full length HCV genome has been completely eliminated. Here, “completely excluded” means that HCV RNA is not detected by RT-PCR.

  Although it will not specifically limit as a chemical | medical agent which has an antiviral effect, if a healing cell can be obtained by adding to a culture medium, the chemical | medical agent which has an anti- HCV effect | action is preferable. Specific examples include IFN and cyclosporin A, with IFN being particularly preferred. Examples of IFN include IFN-α and IFN-β, and IFN-α is preferable. As an example of a method for treating IFN, a method of treating for 2 weeks using a medium containing IFN-α at a concentration of 500 IU / ml can be mentioned. However, the treatment concentration and the treatment period may be appropriately changed while confirming whether or not the healing cells are obtained. The confirmation of whether or not a cured cell has been obtained may be confirmed by RT-PCR or Northern blotting to determine whether or not the cell contains RNA derived from HCV. Furthermore, it can be confirmed by Western blotting or the like that the HCV-derived protein is not expressed.

  In the cells according to the present invention, it is particularly preferable that Oc cells derived from the human hepatoma cell line HuH-7, which are healing cells prepared by the present inventors, are used as parent cells into which the RNA is introduced. This is because the present inventors have demonstrated that when the Oc cell is used as a parent cell, a cell according to the present invention that replicates the full-length HCV genome and expresses Renilla luciferase can be obtained efficiently.

  Oc cells can be prepared, for example, by the following procedure (see FIG. 2). The specific procedure by which the inventors actually produced Oc cells will be described in detail in the Examples section. Further, in the following explanation, the usual experimental method of molecular biology is, for example, the method described in J. Sambrook et al. “Molecular Cloning, A Laboratory Manual, Third Edition” Cold Spring Harbor Laboratory (2001). Therefore, it can be implemented.

  The ON / 3-3B RNA shown in FIG. 1 is synthesized in vitro and introduced into human hepatoma cell line HuH-7 cells by electroporation or the like. Cells into which ON / 3-5B RNA has been introduced (indicated as “sO” in FIG. 2) can be selected by culturing in a medium containing G418. In addition, the replication of the subgenomic HCV replicon can be confirmed by the RT-PCR method or the Northern blot method. Expression of the HCV protein encoded by the subgenomic HCV replicon can be confirmed by Western blotting. After confirming that the cells are replicating the subgenomic HCV replicon, the cells are cultured in a medium containing IFN-α. For example, the cells are cultured in a medium containing IFN-α at a concentration of 500 IU / ml for 2 to 3 weeks to confirm that cured cells have been obtained. As described above, confirmation may be performed by RT-PCR or Northern blotting to confirm that cells do not contain HCV-derived RNA. Thereby, a healing cell (denoted as “sOc” in FIG. 2) of a subgenomic HCV replicon-replicating cell is obtained.

  Next, ON / C-5B RNA shown in FIG. 1 is synthesized in vitro and introduced into the healing cells (sOc) of the subgenomic HCV replicon-replicating cells by electroporation or the like. Similarly to the above, cells into which ON / C-5B RNA has been introduced (indicated as “O” in FIG. 2) can be selected by culturing in a medium containing G418. In addition, the replication of the full-length HCV genome can be confirmed by RT-PCR or Northern blotting. Expression of the HCV protein encoded by the full-length HCV genome can be confirmed by Western blotting. After confirming that the cell replicates the full-length HCV genome, the cell is cultured in a medium containing IFN-α. For example, the cells are cultured in a medium containing IFN-α at a concentration of 500 IU / ml for 2 to 3 weeks to confirm that cured cells have been obtained. As described above, confirmation may be performed by RT-PCR or Northern blotting to confirm that cells do not contain HCV-derived RNA. Thereby, the healing cell of HCV full length genome replication cell, ie, Oc cell, is obtained.

  The Oc cells produced by the present inventors were deposited on April 26, 2005 at the National Institute of Advanced Industrial Science and Technology, the Patent Organism Depositary, and the deposit number is FERM P-20517. is there.

  It is preferable that the HCV ORF of RNA introduced into the cell according to the present invention contains a mutation with at least one amino acid substitution. By having such a mutation, an improvement in the replication efficiency of the HCV genome in cells is observed. In general, a mutation that provides such an effect is called an “adaptive mutation”. Whether or not it is an adaptive mutation can be confirmed by introducing RNA having a mutation and RNA not having a mutation into a cell and cultivating them in a medium containing G418 for 3 to 4 weeks and then comparing the number of growing colonies. it can.

  The adaptive mutation contained in the RNA introduced into the cell according to the present invention is not particularly limited as long as the above-described effect can be obtained. The adaptive mutation is preferably present in the NS3 (helicase) region. Specifically, for example, a mutation in which lysine at position 1609 in the amino acid sequence of the HCV-O strain (see SEQ ID NO: 2) is substituted with glutamic acid (K1609E) and a mutation in which glutamic acid at position 1202 is substituted with glycine (E1202G) are listed. be able to.

(2) Screening method The present invention provides a method for screening a substance having an anti-HCV action. The screening method according to the present invention includes a step of contacting a test substance with the above-described cell according to the present invention, a step of measuring the level of a reporter gene product, and a level of the reporter gene product when the test substance is not contacted. Including the step of comparing with the level of. By using the screening method according to the present invention, a large number of test substances can be screened easily and rapidly.

  In the screening method according to the present invention, the anti-HCV action is an action that suppresses replication of the full-length genome of HCV. That is, the screening method according to the present invention is a method for screening a substance having an action of suppressing replication of the full-length genome of HCV.

  Further, by using the screening method according to the present invention, it is possible to screen for a virus that suppresses replication of a virus closely related to HCV or a virus having the same replication format as HCV. Examples of viruses closely related to HCV include viruses belonging to the genus Flavivirus and pestivirus in the Flaviviridae family. Viruses belonging to the genus Flavivirus include Japanese encephalitis virus, yellow fever virus, dengue virus, West Nile virus and the like, and pestiviruses include pig cholera virus, bovine viral diarrhea virus and the like.

  Furthermore, if the screening method according to the present invention is used, it is possible to screen for a substance that suppresses the replication of a virus having the same replication format as HCV. A characteristic of HCV replication is that all replication takes place in the cytoplasm and the viral genome is not present in the nucleus. Therefore, by the screening method according to the present invention, a substance that suppresses the replication of a virus having such a replication format can be screened.

  The contact between the test substance and the cells can be performed by dissolving or suspending the test substance in a medium. Therefore, the test substance may be any substance that can be dissolved or suspended in the medium.

  The level measurement of the reporter gene product may be selected from known methods according to the reporter gene used. For example, when a luciferase gene is used as a reporter gene, the amount of luminescence may be measured using a cell lysate obtained by lysing cells with a buffer containing a surfactant or the like, using a device such as a luminometer. At this time, commercially available luciferase measurement reagents and luciferase measurement kits can be suitably used.

  It is possible to determine whether or not the test substance has an anti-HCV action by comparing the level of the reporter gene product measured as described above with the level of the reporter gene product of a cell not contacted with the test substance. it can. It should be noted that if the concentration of the test substance that does not decrease the cell growth ability is lower than the level of the reporter gene product of the cell not contacted with the test substance, it can be determined that it has an anti-HCV action, and preferably at least 50% or less Preferably, it is determined to have an anti-HCV action when it is 10% or less.

  Further preferable criteria include the level when IFN-α, which is the current standard method for treating chronic hepatitis C, is applied to the screening method according to the present invention, or a combination of IFN-α and ribavirin. The level when applied to the screening method according to the invention is used as a criterion. If the level of HCV replication is lower than that used in current standard hepatitis C treatments, very useful treatments for hepatitis C can be found.

  By the screening method according to the present invention, at least a candidate substance for an active ingredient of a therapeutic agent for hepatitis C can be selected. In addition, since only HCV infection model animals are chimpanzees, it is impossible to carry out pharmacological tests using a large number of animals in the current situation, and the screening method according to the present invention can be used to evaluate the efficacy of developing therapeutic agents for hepatitis C. It is expected to become an indispensable method.

(3) Screening kit The present invention provides a screening kit for screening a substance having an anti-HCV action. The screening kit according to the present invention only needs to include the above-described cell according to the present invention. By using the screening kit, the screening method according to the present invention can be carried out simply and efficiently.

  As used herein, the term “kit” intends a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Preferably, an instruction manual for using the material is provided. The instructions for use may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

  The screening kit according to the present invention may be provided with a cell other than the cell that replicates the full-length genome of HCV according to the present invention. A specific configuration other than the cells is not particularly limited, and a necessary reagent, instrument, or the like may be appropriately selected to form a kit configuration. Examples thereof include, but are not limited to, cell lysates, reporter gene product measurement reagents, reporter gene product measurement instruments, and the like.

  Those skilled in the art who have read this specification will easily understand that the method of using the screening kit according to the present invention may be in accordance with the above-described usage of the screening method according to the present invention.

(4) Composition for treating hepatitis C The present invention provides a composition for treating hepatitis C. The composition for the treatment of hepatitis C according to the present invention may be any composition containing as an active ingredient a substance having an anti-HCV action obtained by the screening method according to the present invention described above. Although hepatitis C can be divided into acute hepatitis C and chronic hepatitis C, the composition for treating hepatitis C according to the present invention is suitable for chronic hepatitis C.

  The active ingredient of the composition for treating hepatitis C according to the present invention is selected as a substance having an action of suppressing replication of the full-length genome of HCV by the above-described screening method according to the present invention.

  Using the screening method according to the present invention described above, the present inventors have described anti-statins of HMG Co-A reductase inhibitors that have already been used as therapeutic drugs for hyperlipidemia and hypercholesterolemia. The presence or absence of HCV action was tested. Details will be described in the examples in the latter stage, and among the 6 types of statins used in the test, 5 types of statins showed anti-HCV action.

  The statin subjected to the test is fluvastatin having a structure represented by the following formula (1),

Atorvastatin having the structure represented by the following formula (2),

Simvastatin having a structure represented by the following formula (3),

Lovastatin having a structure represented by the following formula (4),

It is pravastatin having a structure represented by the following formula (5).

And pitavastatin having a structure represented by the following formula (6).

  Fluvastatin, atorvastatin, simvastatin, pravastatin, and pitavastatin are marketed as ethical drugs in Japan under the brand names Locoal (Novartis), Lipitor (Yamanouchi), Lipovas (Ariyu), Mevalotin (Sankyo), and Rivaro (Kowa), respectively. Has been. Lovastatin is not sold in Japan, but has already been approved and sold overseas.

  In tests conducted by the present inventors, fluvastatin, atorvastatin, simvastatin, lovastatin, and pitavastatin were found to have anti-HCV effects, whereas pravastatin was not found to have anti-HCV effects.

  Regarding lovastatin, it has been reported that the expression of HCV protein is suppressed using an HCV full-length genome replication cell line (Patent Document 2, Non-Patent Document 9). However, the replicated RNA is not quantified. In addition, pravastatin having similar structure (see the above formulas (4) and (5)) has an effect (HMG) on the statins used in the test, as is clear from the fact that no anti-HCV action was observed. It is not easy for those skilled in the art to predict whether or not to have an anti-HCV action from the similarity of the Co-A reductase inhibitor) and the structure.

  The structure of ribavirin used clinically as a therapeutic agent for chronic hepatitis C in combination with IFN-α is represented by the following formula (7).

  Ribavirin is a nucleic acid structural analog and has no similarity to the structure of each of the above statins. In addition, ribavirin alone is known to have almost no anti-HCV action. Therefore, it is clear from the structure or action of ribavirin that one skilled in the art cannot easily estimate the anti-HCV action of statins.

  The active ingredient of the composition for treating hepatitis C according to the present invention includes fluvastatin, atorvastatin, simvastatin, lovastatin and pitavastatin, and derivatives thereof.

  As described above, fluvastatin, atorvastatin, simvastatin, lovastatin, and pitavastatin are already used in clinical practice as HMG Co-A reductase inhibitors, so that they are clinically applied as a therapeutic agent for hepatitis C without delay. Has the advantage of being possible.

  The composition for treating hepatitis C according to the present invention can be administered to a mammal by any of oral, parenteral, and topical routes. The content of the active ingredient may be appropriately selected according to the weight and condition of the subject to be treated, the condition of the disease to be treated, and the specific administration route selected.

  The active ingredient can be administered by any of the above routes of administration, alone or in combination with a pharmaceutically acceptable carrier or diluent, and can be administered in single or multiple doses. Can be implemented. More specifically, the composition for treating hepatitis C according to the present invention can be administered in a wide variety of dosage forms. Tablets, capsules, lozenges, troches, hard candy, powders, sprays, creams, paints, suppositories, jelly, gels, pastes, lotions, ointments, aqueous suspensions Can be combined with various pharmaceutically acceptable inert carriers in the form of injection solutions, elixirs, syrups and the like. Carriers include solid diluents or fillers, sterile aqueous media, various non-toxic organic solvents and the like. Furthermore, an appropriate sweetness and / or aroma can be imparted to the oral pharmaceutical composition. Usually, the active ingredient is present in the dosage form at a concentration level ranging from 5% to 70% by weight (preferably 10% to 50% by weight).

  For oral administration, tablets containing various excipients (eg, microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine) can be combined with various disintegrants (eg, starch (preferably corn , Potato, or tapioca starch), alginic acid, and silicate complexes) or with granulating binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and gum arabic). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very useful for tablet formation. The same type of solid composition can also be used as a filler in gelatin capsules. Suitable materials in this regard include lactose and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredients can be combined with various sweetening amounts or flavors, colorings, or dyes, optionally further emulsifying and / or Suspending agents can be combined with the above diluents (eg, water, ethanol, propylene glycol, glycerin, and various combinations thereof).

  For parenteral administration, either sesame or peanut oil or aqueous propylene glycol can be used as a solvent. If desired, the aqueous solution should be appropriately buffered (preferably pH> 8) and the liquid diluent should first be made isotonic. These aqueous solutions are suitable for intravenous injection purposes. Oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmacological techniques well known to those skilled in the art.

  Pharmaceutically acceptable carriers are well known to those skilled in the art and are well described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Merck Pub. Co., NJ 1991).

(5) Hepatitis C therapeutic agent combining statin and other drugs Currently, the standard treatment for chronic hepatitis C is combination therapy of IFN and ribavirin. However, the effectiveness against hepatitis C when this treatment is applied is only up to 50% even when using peg IFN with increased blood stability, and the remaining patients have fatal cirrhosis. , At risk of developing liver cancer. In addition, ribavirin is frequently affected by the side effects of hemolytic anemia in elderly people 65 years of age and older, and there is a situation in which the treatment must be stopped. Therefore, early development of a drug effective for use in combination with a new IFN in place of ribavirin is desired.

  The present inventors examined the anti-HCV effect (HCV replication inhibitory effect) obtained by the combined use of a statin having anti-HCV action and IFN, which was found by the screening method according to the present invention. It was found that a significantly higher anti-HCV effect was expressed as compared with the combined use of and Ribavirin. That is, the therapeutic agent for hepatitis C according to the present invention is at least one selected from the group consisting of fluvastatin or a derivative thereof, atorvastatin or a derivative thereof, simvastatin or a derivative thereof, lovastatin or a derivative thereof, and pitavastatin or a derivative thereof. Any combination of interferons may be used. As IFN, IFN-α or IFN-β is preferable, and IFN-α is particularly preferable. In addition, IFN may be modified such as PEGylation.

  The therapeutic agent for hepatitis C may be in the form of a combination preparation in which a statin and IFN as active ingredients are mixed in one preparation, and as described later, a preparation containing statin as an active ingredient and IFN as an active ingredient It is good also as the form of the kit containing the formulation to do.

  Furthermore, the present inventors also examined the anti-HCV effect of cyclosporin A, which has been reported to have an anti-HCV action, in combination with statins and in combination with statins and IFNs. Here, cyclosporin A is a drug widely used clinically as an immunosuppressant and has a structure represented by the following formula (8).

  As a result, it was found that the combined use of statin and cyclosporin A expresses a higher anti-HCV effect than the combined use of IFN and ribavirin. Furthermore, it has been found that when the three drugs, statin, cyclosporin A and IFN, are used in combination, the anti-HCV effect significantly exceeds the anti-HCV effect obtained by the combined use of statin and IFN. Accordingly, the therapeutic agent for hepatitis C according to the present invention is at least one selected from the group consisting of fluvastatin or a derivative thereof, atorvastatin or a derivative thereof, simvastatin or a derivative thereof, lovastatin or a derivative thereof, and pitavastatin or a derivative thereof. More preferred is a combination of interferon and cyclosporin A.

  The therapeutic agent for hepatitis C may be in the form of a combination drug in which a statin, IFN, and cyclosporin A as active ingredients are mixed in one preparation. As described later, a preparation containing statin as an active ingredient and IFN It is good also as a kit form containing the formulation which makes an active ingredient, and the formulation which uses cyclosporin A an active ingredient.

  Since statins, IFN and cyclosporin A are all already used in clinical practice, they have an advantage that they can be clinically applied as a therapeutic agent for hepatitis C without delay.

(5) Kit for treating hepatitis C The present invention provides a kit for treating hepatitis C comprising the composition for treating hepatitis C according to the present invention described above. As used herein, the term “kit” intends a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Preferably, an instruction manual for using the material is provided. The instructions for use may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

  The kit for treating hepatitis C according to the present invention may contain not only the composition for treating hepatitis C according to the present invention but also a composition containing components different from this. Although it does not specifically limit as a composition containing a component different from the composition for hepatitis C treatment based on this invention, The composition for hepatitis C treatment which uses IFN as an active ingredient is preferable. The present inventors have demonstrated that the active ingredient of the composition for treating hepatitis C according to the present invention suppresses HCV genome replication more strongly when used in combination with IFN than when used alone. (See Examples 11 and 12). As IFN, IFN-α or IFN-β is preferable, and IFN-α is particularly preferable. In addition, IFN may be modified such as PEGylation.

  Moreover, as a composition containing a different component from the composition for hepatitis C treatment based on this invention, the composition for treatment of hepatitis C which uses cyclosporin A as an active ingredient is preferable. The kit for treating hepatitis C according to the present invention further comprises a composition for treating hepatitis C according to the present invention, a composition for treating hepatitis C containing IFN as an active ingredient, and a type C containing cyclosporin A as an active ingredient. It is particularly preferred that three agents for treating hepatitis are included. This is because, by using these three agents in combination, expression of a remarkable anti-HCV effect (inhibition of HCV genome replication) has been demonstrated by the present inventors (see Example 14).

  If the kit contains more than one composition, these may be placed in separate containers (eg, divided bottles, etc.) or in a single undivided container. The kit may also include a container containing a diluent, a solvent, a washing solution or other reagent. Furthermore, the present hepatitis C treatment kit may be provided with an instrument necessary for application to the hepatitis C treatment method.

  The kit form is such that separate components are preferably administered in different dosage forms (eg, oral and parenteral), administered at different dosages, or where the prescribing physician desires a titration of each component of the combination, etc. Is particularly advantageous.

  The hepatitis C treatment composition according to the present invention and the method of using other substances in the kit for treating hepatitis C according to the present invention may be in accordance with the use form of the composition described above. Those of ordinary skill in the art who read will readily understand.

(6) Treatment method for hepatitis C The present invention also provides a treatment method for hepatitis C. The hepatitis C treatment method of the present invention may be any method that includes the step of administering the composition for treating hepatitis C according to the present invention to a subject. A more preferred form is a combination therapy with a composition for treating hepatitis C containing IFN as an active ingredient. Therefore, the subject is preferably a hepatitis C patient to whom IFN is administered. A more preferable form is a triple combination therapy with a composition for treating hepatitis C containing IFN as an active ingredient and a composition for treating hepatitis C containing cyclosporin A as an active ingredient. Therefore, it is preferable that the subject is a hepatitis C patient who is administered IFN and cyclosporin A.

  It has been read from the present specification that the application of the composition for treating hepatitis C according to the present invention and other substances in the method for treating hepatitis C according to the present invention may be in accordance with the use form of the composition described above. Contractors understand easily.

  It should be noted that the specific embodiments and the following examples made in the section of the best mode for carrying out the invention are intended to clarify the technical contents of the present invention, and to such specific examples. It is not to be construed as limiting in any way whatsoever, and those skilled in the art can implement the invention within the spirit of the invention and the scope of the appended claims.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these Examples.

  First, experimental materials and experimental methods are described.

[Reagent used]
Atorvastatin was purchased from Yamanouchi, Simvastatin from Ariyu, pravastatin, fluvastatin and lovastatin from Calbiochem, and pitavastatin from Kowa. Nystatin was purchased from Calbiochem and Fungizone (AMPH-B) was purchased from the Institute for Immunobiology. IFN-α was purchased from Sigma. Cyclosporine A was purchased from Sigma.

[Cultivated cells]
A highly differentiated human hepatoma cell line, HuH-7 cells, was used. HuH-7 cells were cultured in Dulbecco's modified Eagles medium (complete DMEM) (Invitrogen) containing 10% fetal bovine serum, penicillin and streptomycin. The HuH-7 cell line containing the subgenomic replicon, HCV full-length genome, was maintained in a medium containing G418 (Invitrogen) at a concentration of 0.3 mg / ml. Passaging was performed twice a week at a 5: 1 split ratio.

(Construction of plasmid)
Plasmid pON / C-5B contains sequences encoding the neomycin phosphotransferase (Neo) downstream of the IRES (Internal ribosomal entry site) of HCV and the full-length HCV-O protein downstream of the IRES of Encephalomyocarditis virus (EMCV) .

  First, a plasmid pHCV-O containing HCV-O full-length cDNA of genotype 1b from HCV positive serum was constructed using two fragments. The two fragments refer to references (N. Kato, M. Ikeda, K. Sugiyama, T. Mizutani, T. Tanaka, K. Shimotohno, Hepatitis C virus population dynamics in human lymphocytes and hepatocytes infected in vitro, J. Gen. Derived from pBR322 / 16-6 (corresponding to positions 45 to 2528 of the HCV genome) and serum 1B-2 PCR product derived from pBR322 / 16-6 described in .Virol. 79 (1998) 1859-1869.) Mlu I-Spe I fragment (corresponding to positions 2528-3420 of the HCV genome). These were ligated to the EcoR I-Spe I site of pNSS1RZ2RU (see Non-Patent Document 2) having the 1B-2R1 sequence to construct pHCV-O.

  In order to obtain a fragment for constructing pON / C-5B, overlapping PCR was used to fuse the IRES of EMCV and the sequence encoding the core protein. The resulting DNA was digested with Rsr II and Cla I and ligated to the Cla I-Xba I site of pHCV-O together with the Xba I-Rsr II fragment of pNSS1RZ2RU to construct pON / C-5B. .

  The construction of plasmid pON / 3-5B is described in detail in Non-Patent Document 2. That is, as described in Non-Patent Document 2, RNA extracted from sO cells was amplified by RT-PCR in the region corresponding to positions 3474-9185 of the HCV-O gene, and this was amplified with the Spe I- of pNSS1RZ2RU. We ligated to the Bsiw I site and constructed pON / 3-5B.

  In accordance with the method of M. Ikeda et al. (See Non-Patent Document 7), KON6091 mutation was introduced into pON / 3-5B and pON / C-5B respectively using QuickChange mutagenesis (Stratagene), and pON / 3-5B / KE and pON / C-5B / KE were constructed. Specifically, a mutation was introduced to replace adenine (a) at position 5166 with guanine (g) in the base sequence described in SEQ ID NO: 1. The K1609E mutation will be described later in [Example 2].

  Plasmid pORN / 3-5B / KE and plasmid pORN / C-5B / KE are transferred to pON / 3-5B / KE and pON / C-5B / KE, respectively, and the PCR product of Renilla luciferase (Promega) is upstream of the Neo gene. It was constructed by introducing it into the Asc I site. Each of the above plasmids contains a T7 promoter sequence on the 5 'side of the inserted gene.

[RNA synthesis]
Plasmid DNA was linearized by cutting with Xba I, and RNA was synthesized using T7 MEGAscript Kit (Ambion) according to the attached experimental instructions. After precipitation with lithium chloride, RNA was washed with 75% ethanol and dissolved in RNase-free water.

[RNA transfection and selection of G418-resistant cells]
In order to apply electroporation, HuH-7 cells were washed twice with ice-cold PBS and resuspended to 10 ⁷ cells / ml in PBS. 500 μL of the cell suspension and 20 μg of the synthesized RNA were transferred to a 2 mm wide cuvette (Bio-Rad). Electrical stimulation was performed twice with Gene Pulser (Bio-Rad) under the conditions of 1.2 KV, 25 μF, maximum resistance, and RNA was introduced into the cells. The cells were then incubated at room temperature for 10 minutes, and the cells were seeded in a 10 cm culture dish. G418 resistant cells were selected in DMEM medium containing G418 at a concentration of 0.3 mg / ml.

[Northern blot analysis]
Total RNA was extracted from the target cells using RNeasy Mini Kit (Qiagen) according to the attached experimental instructions. The extracted RNA was quantified by measuring the absorbance at a wavelength of 260 nm. HCV RNA and β-actin RNA were detected using 4 μg of RNA. That is, specific detection of RNA was performed using Northern Max Kit (Ambion) according to the attached experimental instructions. The RNA sample was electrophoresed and the gel was blotted onto Hybond-N + nylon membrane (Amersham-Pharmacia Biotech). UV cross-linking (Stratagene) was performed to immobilize RNA on the membrane, and the 28S rRNA portion on the membrane was stained with ethidium bromide. The membrane was cut approximately 1 cm below the 28S rRNA band. The upper part of the cut membrane contains HCV RNA and the lower part contains β-actin mRNA. For specific detection of HCV RNA, a minus-strand riboprobe complementary to the HCV NS5B region labeled with digoxigenine was synthesized and used using a digoxigenine labeing kit (Roche) according to the attached experimental instructions. An alkaline phosphatase-labeled anti-digoxigenine antibody was used to detect a riboprobe specifically bound to HCV RNA. After reacting with CSPD (Roche), X-ray film was exposed to light and specific HCV RNA was detected. β-actin RNA was detected in the same manner. For comparison with the replicon RNA level and full-length HCV RNA levels, the PON / 3-5B / KE and PON / C-5B synthesized RNA has been transferred from each ⁽¹⁰⁸ genome equivalents RNA of normal cells from the synthesis of RNA Used).

[Western blot analysis]
100 μl of sample buffer containing SDS was added to cells cultured in a 6-well cell culture plate, and the cell lysate was collected. After sonication with an ultrasonic crusher for 10 minutes, 10 μl of 2-mercaptoethanol was added to each sample and treated at 100 ° C. for 3 minutes. 10-20 μl of the sample was subjected to 10% SDS-PAGE and transferred to a membrane (PVDF membrane). The membrane to which the protein was transferred was blocked with 0.1% Tris buffer solution (10 mM Tris (ph7.5), 150 mM NaCl, 0.1% Tween20) containing 5% skim milk for 60 minutes. Thereafter, a solution obtained by diluting an antibody against each HCV protein and an antibody against β-actin protein 1000-fold with 0.1% Tris buffer was brought into contact with the membrane and reacted for 60 minutes. The membrane was washed with 0.1% Tris buffer for 5 minutes × 3 times, then contacted with 0.1% Tris buffer containing HRP-labeled mouse secondary antibody diluted 1000 times, and reacted for 60 minutes. The membrane was washed with 0.1% Tris buffer for 20 minutes × 3 times. Chemiluminescence was performed with Renaissance ™ Luminol Western Blot Chemiluminescence Detection Reagent Plus (NEN Life Science) and exposed to X-ray film (KODAK BioMax).

  The antibodies used in this experiment are anti-Core antibody (Institute of Immunology), anti-E1 antibody (provided by Tokyo Clinical Research Institute, Dr. Ohara), anti-E2 antibody (reference: Microbiol. Immunol. 42,875-877, 1998), anti-NS3 antibody (Novocastera Laboratories), anti-NS4A antibody (provided by Osaka University, Dr. Takamizawa), anti-NS5A antibody (provided by Osaka University, Dr. Takamizawa), anti-NS5B antibody (Tokyo Metropolitan Research Institute, This is an anti-β-actin antibody (Sigma).

[Preparation of healing cells by IFN treatment]
A method for producing a healing cell derived from a subgenomic replicon cell is described in Non-Patent Document 2 (in this specification, a subgenomic replicon cell denoted as “1B-2R1” in Non-Patent Document 2 is referred to as “ (The healing cell of the subgenomic replicon cell denoted as “sO” and denoted as “1B-2R1C” in Non-Patent Document 2 is denoted as “sOc” in this specification.) In order to prepare healing cells of HCV full-length genome-replicating cells (indicated herein as “O”), O cells were seeded in 6-well culture plates, and subjected to IFN treatment 24 hours later. Human IFN-α (Sigma) was added to the medium at a final concentration of 500 IU / ml. Every 4 days, the medium was replaced with a fresh medium containing IFN-α (500 IU / ml), and the cells were cultured for 2 weeks without G418. In this specification, the healing cell of O cell is described as “Oc”.

  The healing cells were confirmed by the absence of HCV RNA and / or the absence of expression of the protein encoded by the HCV genome.

[RT-PCR]
RT-PCR was performed on the two separated parts. The first part covers NS3 from 5'UTR of HCV, and an approximately 5.1 kb fragment is amplified. The second part covers the most 3'UTR from NS2, and an approximately 6.1 kb fragment is amplified. These fragments had overlapping NS2 and NS3 regions, and were subcloned into pBR322 and used for base sequence analysis of HCV ORF as described later.

Antisense primers 290ROK and 386R shown below were used for reverse transcription (RT) of the two parts, respectively.
290ROK: 5'-ATTATTCTAGATCGACCTGGTTCCTGTCCCG-3 '(SEQ ID NO: 3)
386R: 5'-AATGGCCTATTGGCCTGGAG-3 '(SEQ ID NO: 4)
The following primer pairs were used for PCR of the first part.
21X: 5'-ATTATTCTAGAGCCAGCCCCCGATTGGGGGCG-3 '(SEQ ID NO: 5)
NS3RXOK: 5'-ATTATTCTAGAGGCCTGTGAGACTAGTGATGATGC-3 '(SEQ ID NO: 6)
The following primer pairs were used for PCR of the second part.
NS2XOK: 5'-ATTATTCTAGACGTGTGGGGACATCATCTTGGGTC-3 '(SEQ ID NO: 7)
9388RX: 5'-ATTATTCTAGAATGGCCTATTGGCCTGGAGTG-3 '(SEQ ID NO: 8)
Forty-five cycles of PCR were performed using KOD-plus DNA polymerase. One cycle was annealed at 64 ° C for 30 seconds, extended at 68 ° C for 7 minutes, and modified at 94 ° C for 15 seconds.

[CDNA cloning and nucleotide sequence analysis]
Two PCR products (5.1 kb and 6.1 kb) were digested with Xba I and subcloned into the Xba I site of pBR322MC. The nucleotide sequence of the region inserted into the plasmid was analyzed in the sense direction and the antisense direction. For analysis, Big Dye Terminator Cycle Sequencing kit (Perkin-Elmer Life Sciences) and ABI PRISM 310 genetic analyzer (Applied Biosystems) were used.

[Quantification of HCV RNA]
Total RNA was extracted from HCV RNA replicating cells using RNeasy Mini Kit (Qiagen) according to the attached experimental instructions. First, using 2 μg of RNA as a template, a reverse transcription reaction (RT) was performed using SuperScript ^™ II reverse transcriptase (Invitrogen) and the following primer 319R according to the attached experimental instructions. HCV RNA was quantified by real-time LightCycler PCR using the obtained cDNA as a template. Real-time LightCycler PCR was performed based on the method previously reported by the present inventors (Reference: Acta Med. Okayama 56, 107-110, 2002) using the following primers 104 and 197R.
319R: 5'-TGCTCATGGTGCACGGTCTA-3 '(SEQ ID NO: 9)
104: 5'-AGAGCCATAGTGGTCTGCGG-3 '(SEQ ID NO: 10)
197R: 5'-CTTTCGCGACCCAACACTAC-3 '(SEQ ID NO: 11)
[Drug treatment]
To observe the effects of IFN-α or various statins on ORN / 3-5B / KE cells and ORN / C-5B / KE cells, seed 2 × 10 ⁴ cells in a 24-well culture plate and incubate for 24 hours did.

  In the case of IFN-α treatment, 24 hours after the start of culture, final concentrations of 0, 1, 10 and 100 IU / ml of IFN-α were added to the medium and treated for 24 hours.

  In the case of various statin treatments, the drug was added to the medium at various concentrations 24 hours after the start of the culture, and cultured for 72 hours.

[Luciferase reporter assay]
Using Renilla Luciferase Assay System (Promega), cells were collected according to the attached experimental instructions, and Renilla luciferase was quantified.

[Example 1: Establishment and characteristics of Oc cells]
As described above, Non-Patent Document 2 describes a method for producing subgenomic replicon cells (sO cells) and healing cells (sOc cells) of subgenomic replicon cells.

  As parent cells for producing full-length HCV full-length genome-replicating cells, sOc cells were used. The reason is that the present inventors have found that the colony formation rate of G418 resistant cells is higher when sOc cells are used as the parent cells of subgenomic replicon cells than HuH-7 cells.

  10μg of ON / C-5B in vitro transcript (ON / C-5B RNA) was introduced into sOc cells by electroporation and selected with G418 for 3 weeks. As a result, only one G418 resistant colony was found. Obtained (O cells). When the same experiment was repeated, the appearance of G418 resistant colonies was 1 or 0. The colony formation rate (ECF) under these conditions was estimated to be 0.5 colonies / μg RNA.

  Subsequently, O cells that were healing cells of O cells were obtained by treating O cells with IFN-α as described above (see FIG. 2).

FIG. 3 shows the results of Northern blot analysis of Oc cells, sO cells and O cells. Lanes 1 and 2 are controls, plus the PON / 3-5B / KE and PON / C-5B synthesized RNA has been transferred from each ⁽¹⁰⁸ genome equivalents RNA from normal cells synthesis of RNA, FIG These are the results using Sub-genome and Genome-length respectively. As is clear from FIG. 3, HCV RNA was not detected in RNA derived from Oc cells.

  FIG. 4 shows the results of Western blot analysis for Oc cells, sO cells, and O cell-derived proteins. As is clear from FIG. 4, none of the seven proteins (core, E1, E2, NS3, NS4A, NS5A and NS5B) encoded by HCV RNA was detected from Oc cells.

  From the above results, it was confirmed that the Oc cells are healing cells without full-length HCV RNA.

  Next, we re-introduced ON / C-5B RNA or ON / 3-5B RNA into sOc cells that are healing cells of subgenomic replicon cells and Oc cells that are healing cells of HCV full-length genome replication cells, and The formation rates were compared. Specifically, RNA-introduced cells were seeded in a φ10 cm culture dish, cultured for 3 weeks in a medium containing G418, and the appearing G418-resistant colonies were stained with Coomassie Brilliant Blue.

  The results are shown in FIG. As is clear from FIG. 5, for ON / C-5B RNA introduction, G418-resistant colonies were obtained in Oc cells even when the RNA amount was 0.02 μg, and the number of colonies increased depending on the RNA dose. The colony formation rate of ON / C-5B in Oc cells was estimated to be about 50 colonies / μg RNA. On the other hand, the ECF of ON / C-5B in sOc cells was less than 0.5 colonies / μg RNA. A number of G418-resistant colonies were obtained from sOc cells and Oc cells into which ON / 3-5B RNA was introduced. From the above results, it was shown that Oc cells are very excellent as parent cells for the full-length HCV RNA replication system.

[Example 2: Detection of adaptive mutation and examination of effect of adaptive mutation]
The ORF base sequence of the HCV genome was analyzed for three independent O cell clones. The results of nucleotide sequence analysis of 3 clones were compared with each other, and mutations possessed by each clone were detected.

  The results are shown in FIG. Mutations that are common to the three clones (Clone1-1, Clone1-2, and Clone1-3 in the figure) are indicated by asterisks, and mutations that are detected individually in each clone are indicated by dots. As apparent from FIG. 6, only one mutation common to the three clones was detected. This mutation was present in the NS3 helicase region and was a mutation (K1609E) in which the lysine at position 1609 in the amino acid sequence encoded by the HCV-O strain (see SEQ ID NO: 2 (GenBank ACCESSION AB191333)) was replaced with glutamic acid. .

  In order to verify the effect of the detected mutation (K1609E) on the colony formation rate, wild-type RNA (C-5B / wt) that does not have the mutation or RNA (C-5B / KE) that has the mutation Introduced into sOc cells and Oc cells, the formation rate of G418-resistant colonies was compared by the same method as described in Example 1 above.

  The results are shown in FIG. As is apparent from FIG. 7, when C-5B / KE RNA was introduced into sOc cells, G418-resistant colonies were obtained (the colony formation rate was about 75 colonies / μg RNA), but ON / C-5B / When wt RNA was introduced, G418 resistant colonies were not obtained. On the other hand, in the case of Oc cells, when C-5B / KE RNA is introduced, a large number of G418-resistant colonies are obtained (the colony formation rate is about 1500 colonies / μg RNA), and when C-5B / wt RNA is introduced But many G418 resistant colonies were obtained.

  From the above results, it was shown that the K1609E mutation has a function as an adaptive mutation, and that the Oc cell is very excellent as a parent cell for the full-length HCV RNA replication system.

[Example 3: Establishment of HCV full-length genome replicating cells expressing luciferase gene]
Electroculate 10 μg of pORN / 3-5B / KE or pORN / C-5B / KE in vitro transcript (ORN / 3-5B / KE RNA or ORN / C-5B / KE RNA, see Figure 1) into Oc cells The cells were introduced by the poration method, the cells were seeded in 5 cell incubators (2 μg RNA / cell incubator), selected with a medium containing G418 (300 μg / ml) for 4 weeks, and G418 resistant cells (ORN / 3- 5B / KE cells and ORN / C-5B / KE cells) were selected. The colony formation rate of ORN / C-5B / KE in Oc cells was about 7 colonies / μg RNA.

  In order to confirm the presence of HCV RNA in ORN / 3-5B / KE cells and ORN / C-5B / KE cells, Northern blot analysis was performed using total RNA derived from these cells.

The results are shown in FIG. Lanes 1 and 2 are controls, pORN / 3-5B / KE and pORN / C-5B synthesized RNA has been transferred from each (10 ⁸ genome equivalents of the synthetic RNA plus the normal cell-derived RNA) use the Is the result. Lane 3 is the result of Oc cell, lane 4 is the result of ORN / 3-5B / KE cell, and lane 5 is the result of ORN / C-5B / KE cell. As is clear from FIG. 8, 9 kb HCV-specific RNA was detected in RNA derived from ORN / 3-5B / KE cells, and 12 kb HCV specific in RNA derived from ORN / C-5B / KE cells. Target RNA was detected.

  Further, FIG. 9 shows the results of Western blot analysis for proteins derived from each of Oc cells, ORN / 3-5B / KE cells and ORN / C-5B / KE cells. As is clear from FIG. 9, NS3 protein and NS5B protein were detected in ORN / 3-5B / KE cells, and core protein, NS3 protein and NS5B protein were detected in ORN / C-5B / KE cells.

  From the above results, it was confirmed that the obtained ORN / C-5B / KE cells were HCV full-length genome-replicating cells containing a Renilla luciferase gene and a Neo gene.

  The ORN / C-5B / KE cell was a polyclonal cell line in which a plurality of G418 resistant colonies gathered. Therefore, in order to establish a more stable and uniform cell line, cloned cells were established from colonies obtained after G418 treatment for about 3 weeks. Among several cloned cell lines, the most stable cell line (which can continue to grow with high viability in the presence of G418) was named “OR6”. Further, for this OR6 cell, a healing cell “OR6c” was prepared by the above-described method (IFN-α treatment).

[Example 4: Verification of correlation between luciferase activity and HCV RNA level]
To verify the correlation between luciferase activity and HCV RNA levels, a luciferase reporter assay and real-time LightCycler PCR were performed. That is, ORN / 3-5B / KE cells and ORN / C-5B / KE cells were treated with IFN-α at final concentrations of 0, 1, 10 and 100 IU / ml for 24 hours, respectively, and then the luciferase reporter was prepared as described above. Assays and real-time LightCycler PCR were performed.

  The results are shown in FIGS. 10 (a) to 10 (d). (A) is a graph showing the results of luciferase activity measurement of ORN / 3-5B / KE cells, (b) is a graph showing the results of HCV RNA quantification by real-time LightCycler PCR of ORN / 3-5B / KE cells. Yes, (c) is a graph showing the luciferase activity measurement results of ORN / C-5B / KE cells, and (d) is the HCV RNA quantification results of real-time LightCycler PCR of ORN / C-5B / KE cells. It is a graph. Both measurement results are shown as relative values with 100% of cells not added with IFN-α.

As is clear from FIGS. 10 (a) to (d), the luciferase activity and the amount of HCV RNA decreased in both cells depending on the concentration of IFN-α, and the luciferase activity correlated very well with the amount of HCV RNA. It was. The IC _{50 of} IFN-α is around 1 IU / ml, and this IC ₅₀ value is based on previous studies (reference: JTGuo, Q.Zhu, C. Seeger, Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subge- nomic replicons, J. Virol. 77 (2003) 10769-10779.).

[Example 5: Examination of inhibitory effect of statins on HCV genome replication]
According to the method described above, ORN / 3-5B / KE cells and OR6 cells were treated with atorvastatin, simvastatin, pravastatin, fluvastatin and lovastatin at a concentration of 0, 5 or 10 μM for 72 hours, respectively, and the cells were collected. The luciferase activity was measured.

  The results are shown in FIGS. 11 (a) and (b). All the results are shown as relative values with 100% being not added with statin. (A) shows the results of ORN / 3-5B / KE cells which are subgenomic replicon cells, and (b) shows the results of OR6 cells which are HCV full-length genome replicating cells. As is clear from (a), when ORN / 3-5B / KE cells were used, the relative luciferase activity was reduced by about 20 to 50% by treatment with each statin except pravastatin. On the other hand, as is clear from (b), when OR6 cells were used, the relative luciferase activity was greatly reduced in a dose-dependent manner by each statin treatment except pravastatin. In particular, the effect of fluvastatin was most remarkable, and even when 5 μM was added, the relative luciferase activity was reduced to about 1/30.

  From the above results, it was revealed that atorvastatin, simvastatin, fluvastatin and lovastatin suppressed the replication of the full-length genome of HCV. It was also shown that in order to screen for drugs having anti-HCV effects, it is necessary to use the HCV full-length genome replication system, not the subgenomic replicon cell line lacking the structural protein coding region of HCV.

[Example 6: Examination of toxicity of statin to OR6 cells]
After 4 × 10 ⁴ OR6 cells were seeded in a 6-well culture plate and cultured for 24 hours, atorvastatin, simvastatin, pravastatin, fluvastatin and lovastatin were added at a concentration of 5 μM and cultured for 72 hours. After completion of the culture, the cells were detached with trypsin and stained with trypan blue, and the number of viable cells was counted. The control was OR6 cells to which no drug was added.

  The results are shown in FIG. The number of viable cells in the control was 100%, and the number of viable cells in each statin treatment group was shown as a relative value. As is clear from FIG. 12, the number of viable cells treated with 5 μM of each statin was similar to the number of viable cells of the control, and each statin of 5 μM did not show cytotoxicity against OR6 cells. From this result, it was confirmed that the HCV genome replication inhibitory effect of each statin shown in Example 5 was not based on cytotoxicity but was due to the anti-HCV effect of statin.

[Example 7: Confirmation of action of statin on EMCV-IRES]
Since the expression of HCV protein in OR6 cells is regulated by the activity of EMCV-IRES, statins may suppress the activity of EMCV-IRES. To negate this possibility, we created an expression plasmid that has the Renilla luciferase gene downstream of EMCV-IRES, and introduced it into OR6c cells (OR6 cell healing cells) to express Renilla luciferase. .

  pEMCV-RL was prepared by ligating two PCR fragments of EMCV and RL to the BamHI-NotI site of pCX / bsr. The EMCV fragment has a BamHI site on the 5 'end side and an XhoI site on the 3' end side so that EMCVBamHIF (5'-cgggatccgcgggactctggggttcg-3 ': SEQ ID NO: 12) and EMCVXhoI (5'-ccgctcgagggtattatcgtgtttttcaaagg-3': sequence It amplified by PCR using the two primers of No. 13). RenilaXhoIF (5'-ccgctcgagatggcttccaaggtgtacgacc-3 ': SEQ ID NO: 14) and RennilaNotIR (5'-atagtttagcggccgcctagacgttgatcctgg-3': SEQ ID NO. So that the RL fragment has an XhoI site at the 5 'end and a NotI site at the 3' end Amplified by PCR using the two primers of 15). pEMCV-RL is a plasmid having a CMV promoter upstream.

  5 μM of various statins were added to the prepared cells, and luciferase activity was measured 72 hours later. Cells without added drug were used as negative controls. As positive controls, fungizone (AMPH-B) and Nystatin, which are known to suppress luciferase activity, were used.

  The results are shown in FIG. The luciferase activity of the negative control was taken as 100%, and the luciferase activity of each drug-treated group was shown as a relative value. As is clear from FIG. 13, the luciferase of each statin treatment group was not inhibited, indicating that the statin used did not inhibit EMCV-IRES activity and Renilla luciferase.

[Example 8: Confirmation of anti-HCV effect of statin by Western blot analysis]
After seeding 4 × 10 ⁴ OR6 cells in a 6-well culture plate and culturing for 24 hours, the cells cultured for 96 hours with the addition of each drug were subjected to Western blot analysis. The drugs used are IFN-α (10 IU / ml), pravastatin (5 μM), lovastatin (5 μM), fluvastatin (5 μM or 10 μM). As controls, OR6c cells that were healing cells and OR6 cells that were not treated with drugs were used.

  The results are shown in FIG. As is clear from FIG. 14, the HCV protein was not expressed in the OR6c cells in lane 1. In addition, strong expression of the core protein, NS3 protein, and NS5B protein was observed in OR6 cells not treated with the drug in Lane 2. In the IFN-α treated cells in lane 3, the expression of each HCV protein was decreased as compared to lane 2. In the pravastatin-treated cells in lane 4, the expression of each HCV protein was not decreased, and the same level of expression as in lane 2 was observed. In lovastatin-treated cells in lane 5, the expression of each HCV protein was decreased. Lanes 6 and 7 are cells treated with 5 μM and 10 μM of fluvastatin, respectively, but the expression of each HCV protein was more strongly reduced than that of cells treated with 5 μM of lovastatin in lane 5.

[Example 9: Confirmation of inhibitory effect of fluvastatin on HCV-O RNA replication]
In addition to the HCV gene, ORN / C-5B / KE RNA replicated in OR6 cells contains three exogenous genes: Renilla luciferase gene, Neo gene and EMCV-IRES for translation of HCV protein. include. In order to deny the possibility that statins suppress these three foreign genes, cells into which only HCV-O RNA containing no foreign genes was introduced were prepared.

  Specifically, HCV-O / KE / EG RNA containing an adaptive mutation of KE / EG that enhances HCV replication efficiency was used as HCV RNA to be introduced into cells. As described above, the KE mutation is a mutation (K1609E) in which lysine at position 1609 of the amino acid sequence encoded by the HCV-O strain is substituted with glutamic acid. EG is also a mutation (E1202G) in which glutamic acid at position 1202 in the NS3 region is replaced with glycine. For the introduction of mutation, QuickChange mutagenesis (Stratagene) was used. For the introduction of K1609E, adenine (a) at position 5166 in the nucleotide sequence shown in SEQ ID NO: 1 was replaced with guanine (g), and E1202G was introduced. Adenine (a) at position 3946 in the base sequence described in SEQ ID NO: 1 was substituted with guanine (g).

  In addition, as a negative control cell, a cell having no RNA polymerase activity, ie, no HCV RNA replication occurred was prepared. Specifically, HCV-O / dGDD RNA lacking 10 amino acids (MLVCGDDLVV) from positions 2732 to 2741 in the NS5B region containing GDD which is a motif for RNA polymerase activity was introduced into cells. For the introduction of the 10 amino acid deletion mutation, QuickChange mutagenesis (Stratagene) was used, and the bases at positions 8535 to 8564 in the base sequence described in SEQ ID NO: 1 were deleted.

  OR6c cells, which are healing cells, were used as cells into which these HCV-O RNAs were introduced. The method for introducing RNA into cells was performed as described in the above section [RNA transfection and selection of G418-resistant cells]. However, since HCV-O / KE / EG RNA and HCV-O / dGDD RNA do not contain the Neo gene, selection with G418 is not necessary.

  HCV-O / dGDD RNA-introduced cell group as negative control, HCV-O / KE / EG RNA-introduced cell group without drug treatment, and fluvastatin-treated HCV-O / KE / EG RNA-introduced cell group are provided. The experiment was conducted. HCV-O / dGDD RNA-introduced cells and non-drug-treated HCV-O / KE / EG RNA-introduced cells are collected at 24, 48, 72, and 96 hours after RNA introduction, and used for Western blot analysis. did. In the fluvastatin treatment group, 5 μM of fluvastatin was added to the medium 24 hours after the introduction of RNA, and the cells were collected after 48, 72 and 96 hours, respectively, and subjected to Western blot analysis.

  The results are shown in FIG. Lanes 1 to 4 are the results of the HCV-O / dGDD RNA-introduced cell group. No expression of core protein or NS3 protein was observed at any time. Lanes 5 to 8 are the results of the HCV-O / KE / EG RNA-introduced cell group without drug treatment. Slight core protein and NS3 protein expression was seen after 24 hours, after which expression levels increased and became strongest after 96 hours. Lanes 9 to 11 are the results of the HCV-O / KE / EG RNA-introduced cell group treated with 5 μM fluvastatin. It was clearly shown that the expression of core protein and NS3 protein was suppressed.

[Example 10: Confirmation of dose-dependent anti-HCV effect of statin]
Atorvastatin, simvastatin, pravastatin, fluvastatin and lovastatin were added to OR6 cells at concentrations of 0, 0.625, 1.25, 2.5 or 5 μM, respectively, and luciferase activity after 72 hours was measured.

  The results are shown in FIG. The luciferase activity of the cells to which no drug is added (0 μM) is shown as 100%. Pravastatin was not shown because it did not show an anti-HCV effect. As is clear from FIG. 16, luciferase activity decreased in four types of statin-treated cells other than pravastati depending on the dose. That is, atorvastatin, simvastatin, fluvastatin and lovastatin were shown to have an effect of suppressing the replication of the HCV genome in a dose-dependent manner.

  Whether there is a significant difference in the anti-HCV effect of various statin drugs was statistically examined using the Student t test (P value less than 0.05 is significant). As a result, fluvastatin (P <0.01 at 0.625 μM-5 μM), atorvastatin (P <0.05 at 1.25 μM, P <0.01 at 2.5 μM and 5 μM) and simvastatin (P <0.05, 2.5 μM at 0.625 μM and 1.25 μM) It was found that P <0.01 at 5 μM had a significantly stronger anti-HCV effect compared to lovastatin.

  In particular, fluvastatin has a significantly stronger anti-HCV effect than other simvastatins (1.25 μM-5 μM P <0.01) and atorvastatin (1.25 μM P <0.05, 2.5 μM and 5 μM P <0.01). I found it.

The IC ₅₀ value of each statin was calculated from the results of this experiment (see Table 1).

[Example 11: Examination of anti-HCV effect by combined use of statin and IFN-α]
After seeding 4 × 10 ⁴ OR6 cells in a 24-well culture plate and culturing for 24 hours, atorvastatin, simvastatin, pravastatin, fluvastatin and lovastatin were added at a concentration of 5 μM respectively, and IFN-α was 0, 2 4 or 8 IU / ml. Thereafter, the cells were cultured for 72 hours, and the cells were collected and luciferase activity was measured. Cells with no statin added and IFN-α added at a concentration of 0, 2, 4 or 8 IU / ml served as controls.

  The results are shown in FIG. The luciferase activity of the cells to which neither statin nor IFN-α was added was taken as 100%, and the luciferase activity of each treatment group was shown as a relative value. When only IFN-α was added at concentrations of 2, 4, and 8 IU / ml, the luciferase activities decreased to 15.2%, 6.2%, and 2.5%, respectively. Luciferase activity when atorvastatin 5 μM is combined with IFN-α at concentrations of 0, 2, 4, and 8 IU / ml is 8.9%, 0.75%, 0.30%, and 0.19%, respectively, which is more than that of IFN-α alone treatment. Declined. The luciferase activity when simvastatin 5 μM was combined with IFN-α at concentrations of 0, 2, 4, and 8 IU / ml was 8.5%, 1.3%, 0.45%, and 0.24%, respectively. The luciferase activity when IFN-α is combined with pravastatin 5 μM at concentrations of 0, 2, 4, and 8 IU / ml is 100%, 18.3%, 6.4%, and 2.2%, respectively. It was almost the same level. The luciferase activity of fluvastatin 5 μM combined with IFN-α at concentrations of 0, 2, 4, 8 IU / ml is 3.5%, 0.42%, 0.29%, 0.15%, respectively. The strongest anti-HCV effect was shown. The luciferase activities when LFNstatin 5 μM was combined with IFN-α at concentrations of 0, 2, 4, and 8 IU / ml were 24.1%, 3.3%, 1.1%, and 0.45%, respectively.

  From the above results, it was revealed that atorvastatin, simvastatin, fluvastatin and lovastatin other than pravastatin combined with IFN-α enhances the anti-HCV effect compared to each treatment alone.

[Example 12: Examination of anti-HCV effect by combined use of fluvastatin and IFN-α]
Regarding fluvastatin, which showed the strongest anti-HCV effect in combination with IFN-α in Example 11 above, the combined effect with IFN-α was examined in more detail.

After seeding 4 × 10 ⁴ OR6 cells in a 24-well culture plate and culturing for 24 hours, add fluvastatin at a concentration of 0, 0.625, 1.25, 2.5 or 5 μM and add IFN-α to 0, 4, 8 16, 32 or 64 IU / ml. Thereafter, the cells were cultured for 72 hours, and the cells were collected and luciferase activity was measured.

  The results are shown in FIG. The luciferase activity of the cells to which no drug was added was defined as 100%, and the luciferase activity of each treatment group was shown as a relative value. The vertical axis of the graph shown in FIG. 18 represents relative luciferase activity (%), and the horizontal axis represents fluvastatin concentration (μM).

  When treated with fluvastatin 2.5 μM alone, it exhibits an anti-HCV effect equivalent to using IFN-α 4 IU / ml alone, and when treated with 5 μM fluvastatin alone, IFN-α 8 IU The anti-HCV effect corresponding to the use of / ml alone was shown. When fluvastatin 1.25 μM, 2.5 μM, or 5 μM was used in combination with IFN-α, an anti-HCV effect of about 2-fold, about 4-fold, and about 8-fold was observed regardless of the IFN-α concentration, respectively. Specifically, for example, the anti-HCV effect when IFN-α 8 IU / ml is combined with fluvastatin 1.25 μM, 2.5 μM or 5 μM is 16 IU / ml, 32 IU / ml of IFN-α alone treatment, respectively. , Corresponding to an anti-HCV effect of 64 IU / ml.

  The maximum dose of clinical IFN-α administration is about 10 million IU / ml from the viewpoint of side effects, etc., but the above results show that when fluvastatin 1.25 μM, 2.5 μM and 5 μM are used in combination, It implies that the effects of IFN-α equivalent to 20 million IU / ml, 40 million IU / ml and 80 million IU / ml can be expected.

  In general, it is known that the cure rate of HCV improves when the dose and dose of IFN-α are increased. Although the administration period can be extended, the burden of medical expenses and the pressure on the patient's social life are problematic. An increase in dose is not realistic because IFN-α 10 million IU / ml causes considerable side effects. If FFN is combined with IFN-α 10 million IU / ml to achieve a higher anti-HCV effect within the range of tolerable side effects, it will not only improve the cure rate of HCV but also shorten the HCV treatment period. It can be expected and the patient's burden may be reduced.

[Example 13: Dose-dependent anti-HCV effect of pitavastatin]
Pitavastatin was added to OR6 cells at concentrations of 0, 0.156, 0.31, 0.625, 1.25, 2.5 or 5 μM, respectively, and luciferase activity was measured after 72 hours.

The results are shown in FIG. The luciferase activity of cells not added with pitavastatin (0 μM) is shown as 100%. As is clear from FIG. 19, luciferase activity decreased depending on the dose of pitavastatin, indicating that it has an effect of suppressing replication of the HCV genome in a dose-dependent manner. The IC ₅₀ value was 0.45 μM.

[Comparative example: anti-HCV effect by combined use of ribavirin and IFN-α]
The anti-HCV effect of combined use of ribavirin and IFN-α currently in clinical use was examined using OR6 cells.

After seeding 4 × 10 ⁴ OR6 cells in a 24-well culture plate and culturing for 24 hours, IFN-α is added at a concentration of 2 IU / ml and ribavirin is added at 0, 5, 10, 15, 20, or 25 μM. Was added at a concentration of Thereafter, the cells were cultured for 72 hours, and the cells were collected and luciferase activity was measured.

  The results are shown in FIG. The luciferase activity of the cells to which ribavirin was not added was taken as 100%, and the luciferase activity of each treatment group was shown as a relative value. As is clear from FIG. 20, the luciferase activity decreased in a dose-dependent manner, and showed an inhibitory effect of about 30% at 25 μM. However, the inhibitory effect was much weaker than the combined effect of statin and IFN-α.

[Example 14: Examination of anti-HCV effect by combined use of fluvastatin, cyclosporin A and IFN-α]
After 4 × 10 ⁴ OR6 cells were seeded on a 24-well culture plate and cultured for 24 hours, each drug of fluvastatin, cyclosporin A, and IFN-α was added. Fluvastatin was added at a concentration of 0, 0.625, 1.25 or 2.5 μM. The concentration of cyclosporin A was 0.25 μg / ml as a concentration at which no cytotoxicity was observed. IFN-α was added at a concentration of 2 IU / ml. The combination of the drugs was a combination of two drugs of fluvastatin and cyclosporin A, a combination of two drugs of fluvastatin and IFN-α, and a combination of three drugs of fluvastatin, cyclosporin A and IFN-α. As a control, no drug was added. After adding the drug, the cells were cultured for 72 hours, and the cells were collected to measure luciferase activity.

  The results are shown in FIG. 21 and Table 2. The control (no drug added) luciferase activity was taken as 100%, and the luciferase activity of each drug-treated group was shown as a relative value. In the graph of FIG. 21, the Y axis indicates relative luciferase activity (%), and the X axis indicates fluvastatin concentration. In addition, (◇) is for fluvastatin alone, (□) is for the combination of fluvastatin and cyclosporin A, (△) is for the combination of fluvastatin and IFN-α, (×) is fluvastatin, The case where cyclosporin A and IFN-α are used in combination is shown.

  The predicted value as an additive effect in Table 2 was calculated as a value obtained by adding the respective anti-HCV effects.

  When IFN-α2 IU / ml was used in combination with fluvastatin, a synergistic anti-HCV effect was observed because the values were lower than expected from the anti-HCV effect when used alone. . In addition, even when cyclosporin A 0.25 μg / ml and fluvastatin were used in combination, the synergistic anti-HCV effect was observed because the values were lower than expected from the anti-HCV effect when used alone. It was done.

  Furthermore, when IFN-α2 IU / ml, cyclosporin A 0.25 μg / ml, and flavastatin were used in combination, the values were significantly lower than expected when used alone. A synergistic anti-HCV effect was observed. For example, in the case of fluvastatin 1.25 μM, the relative luciferase activity expected with the combination of the three agents is 2.96%, but the actual measurement value is 0.58%, which is about one-fifth of the expected value. From the above results, it has been clarified that the anti-HCV effect by the combined use of fluvastatin, cyclosporin A and IFN-α greatly exceeds the effect of the combined use of fluvastatin and IFN-α.

  The maximum dose of IFN-α in the clinic is about 10 million IU / ml from the viewpoint of side effects, etc., but when combined with fluvastatin and cyclosporin A, a large anti-HCV effect can be expected. Further, the combined use of fluvastatin and cyclosporin A makes it possible to reduce the amount of IFN-α and reduce the side effects caused by IFN-α.

  By using the present invention, it is possible to provide a screening method for a substance having an anti-HCV action and a therapeutic agent for hepatitis C, which can contribute to the development of the reagent industry, the pharmaceutical industry, and the like.

HCV genome composition, RNA introduced into subgenomic HCV replicon cells (ON / 3-5B), RNA introduced into HCV full-length genome replicating cells (ON / C-5B), luciferase gene product expressed Of RNA (ORN / 3-5B / KE) introduced into subgenomic HCV replicon cells, and RNA (ORN / C-5B / KE) introduced into HCV full-length genomic replicating cells expressing the luciferase gene product It is a schematic diagram which shows a structure. It is a figure which shows the procedure which produces Oc cell. It is an image which shows the result of the Northern blot analysis of Oc cell, sO cell, and O cell. It is an image showing the results of Western blot analysis of Oc cells, sO cells and O cells. It is a photograph which shows the experimental result which introduce | transduced ON / C-5B RNA into sOc cell and Oc cell, and compared the G418 resistant colony formation rate. It is the figure which showed the mutation location which compared the HCV genome ORF base sequence of three O cell clones. It is a photograph which shows the experimental result which compared the difference in the colony formation rate by the presence or absence of K1609E mutation using sOc cell and Oc cell. It is an image which shows the result of the Northern blot analysis of ORN / 3-5B / KE cell and ORN / C-5B / KE cell. It is an image which shows the result of the Western blot analysis of ORN / 3-5B / KE cell and ORN / C-5B / KE cell. It is a graph which shows the result of having performed IFN- (alpha) treatment of each density | concentration with respect to ORN / 3-5B / KE cell and ORN / C-5B / KE cell, and performing luciferase reporter assay and real-time LightCycler PCR. It is a graph which shows the experimental result which investigated the HCV genome replication inhibitory effect of five types of statins using ORN / 3-5B / KE cell and OR6 cell. It is a graph which shows the result of having examined the cytotoxicity with respect to OR6 cell of five types of statins. It is a graph which shows the result of having examined the EMCV-IRES activity inhibitory effect of five types of statins. It is an image which shows the result which confirmed the anti- HCV effect of IFN- (alpha), pravastatin, lovastatin, and fluvastatin in the OR6 cell line by Western blot analysis. It is a western blot analysis image which shows the experimental result for confirming that fluvastatin does not suppress a foreign gene in OR6 cell line. It is a graph which shows the result which confirmed the dose-dependent anti-HCV effect of atorvastatin, simvastatin, fluvastatin, and lovastatin in OR6 cell line. It is a graph which shows the result of having examined the anti-HCV effect by combined use of 5 types of statins and IFN- (alpha) in OR6 cell line. It is a graph which shows the result of having examined in detail the anti-HCV effect by combined use of fluvastatin and IFN- (alpha) in OR6 cell line. It is a graph which shows the result of having confirmed the dose-dependent anti-HCV effect of pitavastatin in OR6 cell line. It is a graph which shows the result of having examined the anti-HCV effect by combined use of ribavirin and IFN- (alpha) in OR6 cell line. It is a graph which shows the result of having examined the anti-HCV effect by combined use of fluvastatin, cyclosporin A, and IFN- (alpha) in OR6 cell line.

Claims (8)

  A composition for treating hepatitis C, comprising pitavastatin as an active ingredient.   The composition according to claim 1, wherein the subject is a hepatitis C patient to which interferon is administered.   The composition according to claim 1, wherein the subject is a hepatitis C patient to which interferon and cyclosporin A are administered.   A therapeutic agent for hepatitis C comprising a combination of pitavastatin and interferon.   Furthermore, the hepatitis C therapeutic agent of Claim 4 formed by combining cyclosporin A.   A kit for treating hepatitis C, comprising the composition according to claim 1.   Furthermore, the kit of Claim 6 provided with the composition for hepatitis C treatment which uses an interferon as an active ingredient.   Furthermore, the kit of Claim 7 provided with the composition for hepatitis C treatment which uses cyclosporin A as an active ingredient.