JP2008169116A - Anti-hepatitis C virus agent containing BMP-7 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for use in prevention or treatment of liver disease caused by hepatitis C virus (HCV) infection.
Disclosed is a pharmaceutical composition for preventing or treating liver disease caused by HCV infection, comprising bone morphogenetic protein 7 (BMP-7) as an active ingredient. The pharmaceutical composition of the present invention is particularly useful for reducing the amount of HCV infection in HCV-infected patients. The pharmaceutical composition of the present invention containing BMP-7 as an active ingredient may be administered alone or in combination with a preparation containing interferon (IFN) as an active ingredient or a preparation containing ribavirin as an active ingredient.
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Description

  The present invention relates to a composition for preventing and treating a disease associated with hepatitis C virus (HCV) infection, and more particularly to a pharmaceutical composition used for drug therapy for reducing the amount of HCV infection.

  It is widely known that HCV infection causes onset of diseases such as hepatitis C, cirrhosis and liver cancer (Non-patent document 1: Seeff, Am J Med. 1999, 107: 10S-15S; Non-patent document 2) : Hu and Tong, Hepatology 1999, 29: 1311-1316).

  When HCV disappears after infection, it is recognized that it is effective in preventing or treating diseases caused by HCV infection, and anti-HCV drug treatment for reducing the amount of HCV is performed (Non-patent Document 3: Liang). and Heller, Gastroenterology 2004, 127: S62-71).

  Currently, typical pharmaceuticals used for anti-HCV drug therapy are various interferon (IFN) preparations. It has been known that IFN binds to the membrane of a virus-infected cell and suppresses the growth of various types of viruses by putting the inside of the cell into an antiviral state (Non-patent Document 4: Friedman, Bacteriol Rev 1977). , 41: 453-567). Using the HCV replicon test system, it has been shown that IFN also suppresses proliferation by this mode of action against HCV, and it is clear that NS5A protein of HCV virus is related to the effect of IFN-α (non- Patent Document 5: Blight et al., Science 2000, 290: 1972-1974; Non-Patent Document 6: Frese et al., J Gen Virol 2001, 82: 723-733).

  There are various types of IFN produced by humans, and it is generally recognized that all of IFN-α, IFN-β, and IFN-γ exhibit anti-HCV action (Non-patent Document 7: Okuse). et al., Antiviral Res 2005, 65: 23-34). There are various types of active ingredients in clinically used IFN preparations such as human lymphoblast-derived IFN-α, recombinant IFN-α-2a, recombinant IFN-α-2b, and human fibroblast-derived INF-β. . In addition, there are preparations using modified IFNs instead of these natural IFNs, that is, consensus IFNs with partially modified amino acid sequences, and PEGylated IFNs with polyethylene glycol bound thereto.

  However, IFN causes a high rate of side effects at doses used for anti-HCV therapy. Side effects that may occur include fever, general malaise, loss of appetite, leukopenia, thrombocytopenia, hair loss, and psychiatric symptoms. In addition, antibodies against IFN may be produced. Furthermore, there are many patients who cannot eliminate HCV infection or who do not reduce the amount of HCV by treatment with an IFN preparation alone.

  On the other hand, there is a treatment that increases the effect of reducing the amount of HCV by combining ribavirin, an antiviral agent, with an IFN preparation. The anti-HCV effect of ribavirin alone and in combination with IFN has been confirmed in the HCV replicon test system (Non-patent Document 8: Tanabe et al., J Infect Dis 2004, 189: 1129-1139).

  However, a sufficient clinical effect has not been recognized even when these two agents are used in combination. Herrine SK, for example, listed the combination therapy as a general anti-HCV therapy, and treated patients with chronic infection with subcutaneous injection of PEGylated interferon once a week and oral ribavirin daily. The percentage of patients with persistent virological response was approximately 55% (Non-Patent Document 9: Herrine, Ann Internal Med 2002, 136: 747-757) . That is, this report shows that approximately 45% of patients do not have a sufficient clinical effect.

  In addition, treatment with ribavirin may cause serious anemia as a side effect, in which case the dose of the drug must be reduced or discontinued. In addition, ribavirin needs to be used with caution in women with the possibility of pregnancy, as with IFN.In addition to this, ribavirin adversely affects spermatogenesis, so males should avoid contraception during use. There is a problem of being recommended. In view of the above, there is a need for a drug that replaces ribavirin.

  As described above, the current anti-HCV drug therapy is not fully satisfactory, and a new treatment method is required.

  Bone morphogenetic protein 7 (BMP-7) is a protein that exists in the body called alias osteogenic protein 1 (OP-1) or eptotermin alpha.

  It was found that a protein involved in bone growth exists in the living body, and was named bone morphogenetic factor (BMP). It has been proposed to use this protein as a drug that promotes bone formation and growth (Patent Document 1: US4294753). Later, it was revealed that there were several types of proteins in BMP, one of which was identified as BMP-7. That is, BMP-7 was discovered as a protein that promotes bone growth (Non-patent document 10: Ozkaynak et al., EMBO J 1990, 9 9: 2085-2093; Non-patent document 11: Celeste et al., Proc Natl. Acad Sci USA 1990, 87: 9843-9847; Non-Patent Document 12: Sampath et al., J Biol Chem 1992, 267: 20352-20362).

  BMP-7 is a protein consisting of 139 amino acids that is synthesized as a 431 amino acid precursor protein encoded by the BMP7 gene, forms a homo- or hetero-dimer, is glycated and then cleaved by proteases (Non-patent document 12: Sampath et al., J Biol Chem 1992, 267: 20352-20362; Non-patent document 13: Jones et al., Growth Factors 1994, 11: 215-225), and the nucleotide sequence of the gene And the amino acid sequence of the protein is known.

  Recombinant human BMP-7 already produced by Chinese hamster ovary cells (CHO cells), which is industrially applied with the effect of promoting bone growth of BMP-7, is clinically treated as a drug that promotes fracture repair. It is used for.

  In addition, the involvement of BMP-7 in tooth growth has been investigated (Non-patent Document 14: Rutherford et al., Arch Oral Biol 1993, 38: 571-576), and application of BMP-7 to periodontal disease treatment A method has also been invented (Patent Document 2: US5656593).

  BMP-7 is reported to inhibit cell proliferation and develop osteoblast traits in rat clonal osteoblastic osteosarcoma cell ROS 17 / 2.8, and to inhibit growth of human embryonal carcinoma cells. (Non-patent document 15: Maliakal et al., Growth Factors 1994, 11: 227-234; Non-patent document 16: Andrews et al., Lab Invest 1994, 71: 243-251).

  A study comparing the expression levels of various organs in mice found that the expression level of BMP-7 was higher in the kidney (Non-patent Document 17: Ozkaynak et al., Biochem Biophys Res Commun 1991, 179: 116-123). ). BMP-7 is known to have a protective effect against ischemia-reperfusion injury of the kidney (Non-patent Document 18: Vukicevic et al., J Clin Invest 1998, 102: 202-214).

  In addition, the following actions of BMP-7 are known: Protects against myocardial ischemia / reperfusion injury Protects against cerebral ischemia / reperfusion injury Involved in neogenesis ・ Involved in adhesion and assembly of neurons ・ Stimulated dendritic growth of sympathetic neurons (Non-patent document 19: Lefer et al., J Mol Cell Cardiol 1992, 24: 585- Non-patent document 20: Perides et al., Neurosci Lett 1995, 187: 21-24; Non-patent document 21: Ramoshebi and Ripamonti, Anat Rec 2000, 259: 97-107; Non-patent document 22: Perides et al. Proc Natl Acad Sci USA 1992, 89: 10326-10330; Non-Patent Document 23: Lein et al., Neuron 1995, 15: 597-605).

  In addition, it has been reported that the BMP7 gene is expressed in the prostate by androgen (Non-patent Document 24: Thomas et al., Prostate 1998, 37: 236-245). Furthermore, it is suggested that BMP-7 acts on the ovary and suppresses luteinization (Non-patent Document 25: Shimasaki et al., Proc Natl Acad Sci 1999, 96: 7282-7287).

  In human neutrophils, monocytes, and human fetal dermal fibroblasts, when compared with the action of TGF-β, which has a high structural homology with BMP-7, it was common in that it had a cell migration effect. BMP-7, unlike TGF-β, did not have a fibrogenic effect (Non-patent Document 26: Postlethwaite et al., J Cell Physiol 1994, 161: 562-570). BMP-7 is known to show apoptosis-inducing activity in some cells (Non-patent Document 27: Monroe and Sanders, Mol Cell Biol 2000, 13: 4626-4636).

  It has been shown that BMP-7 is expressed in the embryonic period and is required for normal formation of mesoderm, brain, and eyes (Non-patent Document 28: Hogan, Curr Opin Genet Dev 1996, 6: 432-438 Non-patent document 29: Arkell and Beddington, Development 1997, 124: 1-12; Non-patent document 30: Luo et al., Genes Dev 1995, 9: 2808-2820).

  BMP-7 is also expressed in the liver (Non-patent Document 17: Ozkaynak et al., Biochem Biophys Res Commun 1991, 179: 116-123). It has been reported that activation of a signal transduction pathway via intracellular smad by BMP-7 is suppressed by Glypican 3 in hepatocellular carcinoma cells (Non-patent Document 31: Midorikawa et al., Int J Cancer 2003, 103: 455-465). Therefore, it is suggested that BMP-7 exhibits an action on at least hepatocellular carcinoma cells.

  However, the role of BMP-7 in the liver, in particular, the role of BMP-7 in hepatitis and the role of BMP-7 on HCV proliferation has not been known so far.

US4294753 US5656593 Seeff, Am J Med. 1999, 107: 10S-15S Hu and Tong, Hepatology 1999, 29: 1311-1316 Liang and Heller, Gastroenterology 2004, 127: S62-71 Friedman, Bacteriol Rev 1977, 41: 453-567 Blight et al., Science 2000, 290: 1972-1974 Frese et al., J Gen Virol 2001, 82: 723-733 Okuse et al., Antiviral Res 2005, 65: 23-34 Tanabe et al., J Infect Dis 2004, 189: 1129-1139 Herrine, Ann Internal Med 2002, 136: 747-757 Ozkaynak et al., EMBO J 1990, 9 9: 2085-2093 Celeste et al., Proc Natl Acad Sci USA 1990, 87: 9843-9847 Sampath et al., J Biol Chem 1992, 267: 20352-20362 Jones et al., Growth Factors 1994, 11: 215-225 Rutherford et al., Arch Oral Biol 1993, 38: 571-576 Maliakal et al., Growth Factors 1994, 11: 227-234 Andrews et al., Lab Invest 1994, 71: 243-251 Ozkaynak et al., Biochem Biophys Res Commun 1991, 179: 116-123 Vukicevic et al., J Clin Invest 1998, 102: 202-214 Lefer et al., J Mol Cell Cardiol 1992, 24: 585-593 Perides et al., Neurosci Lett 1995, 187: 21-24 Ramoshebi and Ripamonti, Anat Rec 2000, 259: 97-107 Perides et al., Proc Natl Acad Sci USA 1992, 89: 10326-10330 Lein et al., Neuron 1995, 15: 597-605 Thomas et al., Prostate 1998, 37: 236-245 Shimasaki et al., Proc Natl Acad Sci 1999, 96: 7282-7287 Postlethwaite et al., J Cell Physiol 1994, 161: 562-570 Monroe and Sanders, Mol Cell Biol 2000, 13: 4626-4636 Hogan, Curr Opin Genet Dev 1996, 6: 432-438 Arkell and Beddington, Development 1997, 124: 1-12 Luo et al., Genes Dev 1995, 9: 2808-2820 Midorikawa et al., Int J Cancer 2003, 103: 455-465

  An object of this invention is to provide the pharmaceutical composition used for the anti- HCV therapy which reduces the amount of HCV infection as a prevention or treatment method of the liver disease by hepatitis C virus (HCV) infection.

  The present invention provides a pharmaceutical composition for preventing or treating liver disease caused by HCV infection, comprising bone morphogenetic protein 7 (BMP-7) as an active ingredient. The mRNA sequence of the BMP7 gene encoding the amino acid of the BMP-7 precursor protein is described in GenBank X51801. The structure of the mature form of BMP-7 (BMP-7 precursor protein 293-431; amino acid residue number 139) after being cleaved by protease is shown in Celeste et al. (Proc Natl Acad Sci USA 1990, 87: 9843- 9847). As BMP-7 used in the present invention, a precursor protein produced from the full-length gene of BMP7 may be used, or mature BMP-7 may be used. In addition, a protein with a modified structure may be used as long as it has BMP-7 physiological activity. For example, a shortened mature BMP-7 (38-139) or amino acid obtained by the method disclosed in Patent US 5656593 can be used. Modified proteins may be used.

  Liver diseases due to HCV infection include hepatitis, cirrhosis and viral hepatocellular carcinoma. The pharmaceutical composition of the present invention is particularly useful for reducing the amount of HCV infection in HCV-infected patients.

  In another aspect, the present invention provides anti-HCV therapy by administering a pharmaceutical composition containing BMP-7 as an active ingredient in combination with a preparation containing interferon (IFN) as an active ingredient or a preparation containing ribavirin as an active ingredient. provide.

  The preparation containing IFN as an active ingredient can be selected from IFN preparations that themselves exhibit antiviral effects. Examples of natural IFNs include human lymphoblast-derived IFN-α, recombinant IFN-α-2a, recombinant IFN-α-2b, and human fibroblast-derived INF-β. In addition, preparations using modified IFNs in place of these natural IFNs, such as consensus IFNs with partially modified amino acid sequences, and preparations containing PEGylated IFNs bound with polyethylene glycol can also be used. However, the IFN that can be used in the present invention is not limited to these examples.

  The pharmaceutical composition of the present invention comprises BMP-7 as an active ingredient. BMP-7 contained in the pharmaceutical composition of the present invention is a known substance, and a commercially available BMP-7 protein may be used as long as it is purified to the extent that it can be used as a medicine, and prepared by various methods. A thing may be used. As a method for producing BMP-7, for example, DNA encoding BMP-7 is incorporated into an appropriate expression vector, which is introduced into either a eukaryotic cell or a prokaryotic cell, and a desired protein is obtained. Can be expressed. Examples of host cells that can be used to express BMP-7 include, but are not limited to, prokaryotic hosts such as E. coli and Bacillus subtilis, and eukaryotic hosts such as yeast, fungi, insect cells and mammalian cells. Is mentioned.

  The vector includes a promoter region that drives the expression of DNA encoding BMP-7, and further includes transcriptional and translational control sequences such as a TATA box, capping sequence, CAAT sequence, 3 ′ non-coding region, enhancer, and the like. Also good. Examples of promoters include bla promoter, cat promoter, lacZ promoter when used in prokaryotic hosts, herpesvirus TK promoter, SV40 early promoter, yeast glycolysis system when used in eukaryotic hosts. Examples include enzyme gene sequence promoters. Examples of vectors include, but are not limited to, pBR322, pUC118, pUC119, λgt10, λgt11, pMAM-neo, pKRC, BPV, vaccinia, SV40, 2-micron and the like. Furthermore, the vector can be constructed so that BMP-7 is secreted and expressed using a signal sequence, or BMP-7 is expressed in the form of a fusion protein with another protein. The construction of such expression vectors is well known in the art.

  Vectors constructed to express BMP-7 are introduced into appropriate host cells by transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc. be able to. BMP-7 protein can be obtained by growing cells containing the vector in an appropriate medium to produce BMP-7, and recovering and purifying the desired recombinant protein from the cells or culture supernatant. Purification can be performed using size exclusion chromatography, HPLC, ion exchange chromatography, immunoaffinity chromatography, and the like.

  In addition, proteins expressed in host cells may undergo various post-translational modifications. Therefore, the recombinant BMP-7 protein used in the pharmaceutical composition of the present invention includes a protein having undergone such post-translational modification. Furthermore, BMP-7 used in the pharmaceutical composition of the present invention can also be prepared by a known cell-free protein synthesis system, for example, rabbit reticulocyte lysate or wheat germ extract.

  The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It can be formulated by mixing in a unit dosage form as required for generally accepted pharmaceutical practice, in appropriate combination with a binder and the like.

  For oral administration, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions by mixing BMP-7 with pharmaceutically acceptable carriers well known in the art Etc. can be prescribed as As the carrier, those known in the art can be widely used. For example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like; Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dried starch, sodium alginate, agar powder, laminaran powder, hydrogen carbonate Disintegrating agents such as sodium, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegrating inhibitors such as sucrose, stear cocoa butter, hydrogenated oil; quaternary ammonium salts, lauric Absorption promoters such as sodium sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol Etc. can be used. Furthermore, the tablet can be a tablet coated with a normal coating, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary.

  For parenteral administration, BMP-7 can be formulated according to conventional pharmaceutical practice using pharmaceutically acceptable vehicles well known in the art.

  Examples of water-soluble vehicles for injection include isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing aids. An agent such as an alcohol, specifically ethanol, a polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (TM), HCO-50 may be used in combination.

  Oily vehicles include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  Further, for the purpose of controlling the dispersion of BMP-7, it may be blended with a carrier such as collagen, hydroxyapatite, hyaluronic acid, fibrin, polysaccharide, or liposome. The prepared preparation can be used as an oral preparation or a parenteral administration preparation.

  Suitable routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, rectal, transmucosal, or enteral administration, or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, Intravitreal, intraperitoneal, intranasal, or intraocular injection is included. The administration route can be appropriately selected in consideration of the age and medical condition of the patient, other drugs used in combination, and the like.

  The dosage of the pharmaceutical composition of the present invention can be selected, for example, in the range of 0.01 mg to 1 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.02 to 100 mg per dose per patient, but is not necessarily limited to these values. The dosage and administration method can be appropriately selected in consideration of the patient's weight, age, symptoms, other drugs used in combination, and the like. Moreover, when using the pharmaceutical composition of this invention together with an interferon formulation or a ribavirin formulation, these agents may be administered simultaneously or separately.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Huh 7 / Feo cells described in Tanabe et al., (J Infect Dis 2004, 189: 1129-1139) were used as cells used in the HCV growth suppression replicon test system with BMP-7 in the HCV replicon system . . In this cell, instead of the Neo gene of the replicon plasmid pHCV1bneo-delS created from HCV-N, 1b, an HCV replicon plasmid introduced with chimeric gene coding (Feo) in which firefly luciferase was fused in frame to neomycin phosphotransferase was introduced. This is a cell line established by transfecting the human liver cancer cell line Huh 7 and selecting cells showing constant expression.

Huh7 / Feo cells were seeded at 2.5 × 10 ⁴ cells / well in a 24-well plate 20 hours before BMP-7 treatment, and cultured at 37 ° C. under 5% CO ₂ . The medium is Dulbecco's modified minimal essential medium (Sigma-Aldrich, Co.) with fetal bovine serum 10%, penicillin 100 IU / mL, streptomycin 100 μg / mL, G418 (Wako Pure Chemical Industries, Ltd.) 250 μg / mL What was added was used.

  As BMP-7, a commercial product of recombinant human BMP-7 manufactured by R & D Systems Inc. was used. BMP-7 was dissolved in DMSO, diluted with a medium and added to the medium to a concentration of 50 ng / mL or 100 ng / mL. Cells were collected 72 hours after addition of BMP-7, and luciferase and protein levels were measured. The luciferase assay was quantified using a Luciferase Assay System (Promega Corporation) and a luminometer (Promega Corporation). The amount of protein was measured using Protein assay dye reagent (Bio-Rad Laboratories) and BioPhotometer (Eppendorf AG).

  When the proliferation of HCV is large, the expression of luciferase increases and the luciferase activity increases. On the other hand, if the growth of the host Huh7 is suppressed, the amount of protein decreases. Therefore, in order to evaluate the proliferation ability of HCV replicon per cell, luciferase activity / protein amount was used as the scale.

  FIG. 1 shows the luciferase activity and protein amount after 72 hours of drug treatment. In the figure, the bar indicates the relative light intensity of the fluorescence of Huh7 cells into which the HCV replicon was introduced. The fluorescence intensity reflects the luciferase activity associated with the growth of the HCV replicon inserted with luciferase. A broken line shows the amount of protein. The amount of protein reflects the degree of growth of Huh7 in the HCV replicon host.

  Luciferase activity decreased in a concentration-dependent manner with the addition of BMP-7. The amount of protein also decreased in a concentration-dependent manner with the addition of BMP-7. FIG. 2 shows the luciferase activity / protein amount for evaluating the amount of HCV proliferation per cell. This value reflects the degree of growth of the HCV replicon corrected for the degree of Huh7 growth in the host.

  As is apparent from FIG. 2, the value of luciferase activity / protein amount decreased in a concentration-dependent manner by treatment with BMP-7. Therefore, BMP-7 was shown to suppress the proliferation ability of the HCV replicon.

The test was carried out in the same manner as in Example 1 except for the conditions for treating HCV growth inhibitory drugs with IFN and BMP-7 in the HCV replicon system . As interferon (IFN) α, a commercial product of IFN-α2 manufactured by PBL Biomedical Laboratories was used. IFN-α was dissolved in DMSO and diluted in a medium. IFN-α and BMP-7 alone or both were added to the medium, and after 72 hours, the cells were collected, and luciferase activity and protein amount were measured.

  The luciferase activity / protein amount after 72 hours of drug treatment is shown in FIG. The value of the relative light amount per protein amount reflects the degree of growth of the HCV replicon corrected for the degree of Huh7 growth in the host. Application of BMP-7 100 ng / mL alone reduced the luciferase activity / protein content indicating the degree of HCV proliferation. Addition of 0.5 U IFN-α per well did not show a strong effect. On the other hand, by applying both IFN-α 0.5 U and BMP-7 100 ng / mL, a strong decrease in relative light quantity / protein amount comparable to that when IFN 1 U was added was observed. From the above results, it was shown that BMP-7 enhances the HCV proliferation inhibitory effect of IFN-α.

The test was performed in the same manner as in Example 1 except for the conditions for treating HCV growth inhibitory drugs with ribavirin and BMP-7 in the HCV replicon system . As ribavirin, a commercial product manufactured by Sigma-Aldrich Corporation was used. Ribavirin was dissolved in DMSO and diluted in medium. The drug treatment conditions were as follows: no treatment, ribavirin 100 μM alone treatment, or treatment with both ribavirin 100 μM and BMP-7 100 ng / mL. Cells were collected 72 hours after the drug was added to the medium, and luciferase activity and protein content were measured. When the luciferase activity / protein amount of each drug treatment condition was calculated as a relative value with 1 as the non-treatment condition, it was a ribavirin-only treatment condition of 0.52, and a condition of 0.26 where both ribavirin and BMP-7 were treated. From the above results, it was shown that BMP-7 enhances the inhibitory effect of ribavirin on HCV proliferation.

FIG. 1 shows the effect of BMP-7 on HCV proliferation in the HCV replicon system. FIG. 2 shows the effect of BMP-7 on HCV proliferation in the HCV replicon system. FIG. 3 shows suppression of HCV proliferation by IFN and BMP-7 in the HCV replicon system.

Claims (6)

A pharmaceutical composition for preventing or treating liver disease caused by HCV infection, comprising BMP-7 as an active ingredient. A pharmaceutical composition for reducing the amount of HCV infection, comprising BMP-7 as an active ingredient. A pharmaceutical composition for reducing the amount of HCV infection in combination with an interferon preparation comprising BMP-7 as an active ingredient. A pharmaceutical composition for reducing the amount of HCV infection in combination with a ribavirin preparation comprising BMP-7 as an active ingredient. A pharmaceutical composition for reducing the amount of HCV infection, comprising BMP-7 and interferon as active ingredients. A pharmaceutical composition for reducing the amount of HCV infection, comprising BMP-7 and ribavirin as active ingredients.

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