WO2010044474A1 - C型肝炎治療剤 - Google Patents
C型肝炎治療剤 Download PDFInfo
- Publication number
- WO2010044474A1 WO2010044474A1 PCT/JP2009/067942 JP2009067942W WO2010044474A1 WO 2010044474 A1 WO2010044474 A1 WO 2010044474A1 JP 2009067942 W JP2009067942 W JP 2009067942W WO 2010044474 A1 WO2010044474 A1 WO 2010044474A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hepatitis
- therapeutic agent
- interferon
- hcv
- cells
- Prior art date
Links
- 239000003814 drug Substances 0.000 title claims abstract description 31
- 229940124597 therapeutic agent Drugs 0.000 title claims abstract description 29
- 208000006454 hepatitis Diseases 0.000 title description 2
- 231100000283 hepatitis Toxicity 0.000 title description 2
- 208000005176 Hepatitis C Diseases 0.000 claims abstract description 58
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960001330 hydroxycarbamide Drugs 0.000 claims abstract description 17
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 16
- 239000004480 active ingredient Substances 0.000 claims abstract description 8
- 102000006992 Interferon-alpha Human genes 0.000 claims description 23
- 108010047761 Interferon-alpha Proteins 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 241000711549 Hepacivirus C Species 0.000 abstract description 29
- 230000002195 synergetic effect Effects 0.000 abstract description 7
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 23
- 108060001084 Luciferase Proteins 0.000 description 14
- 230000010076 replication Effects 0.000 description 14
- 239000005089 Luciferase Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 238000012216 screening Methods 0.000 description 8
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 7
- 208000010710 hepatitis C virus infection Diseases 0.000 description 7
- 208000006154 Chronic hepatitis C Diseases 0.000 description 6
- 241000710188 Encephalomyocarditis virus Species 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000002775 capsule Substances 0.000 description 4
- 229960000329 ribavirin Drugs 0.000 description 4
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 3
- 241000242739 Renilla Species 0.000 description 3
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 2
- 108010025815 Kanamycin Kinase Proteins 0.000 description 2
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229940096120 hydrea Drugs 0.000 description 2
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- ZGGHKIMDNBDHJB-NRFPMOEYSA-M (3R,5S)-fluvastatin sodium Chemical compound [Na+].C12=CC=CC=C2N(C(C)C)C(\C=C\[C@@H](O)C[C@@H](O)CC([O-])=O)=C1C1=CC=C(F)C=C1 ZGGHKIMDNBDHJB-NRFPMOEYSA-M 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 206010065051 Acute hepatitis C Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000037621 acute hepatitis C virus infection Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000002832 anti-viral assay Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- -1 elixirs Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 229960003765 fluvastatin Drugs 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000005182 global health Effects 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/212—IFN-alpha
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to a novel therapeutic agent for hepatitis C.
- HCV Hepatitis C virus
- HCV infection is a global health problem, and the number of people infected is thought to exceed 170 million.
- HCV genotype 1 is a major genotype in other countries, mainly in Japan and the United States.
- HCV genotype 1 cure less than 50% with peginterferon-ribavirin combination therapy, which is the current strongest standard of care.
- the present inventors have developed an assay system in which the full length HCV gene replicates (Patent Document 1).
- a screening system for substances having a therapeutic effect on hepatitis C is known as described in Patent Document 1, and it has been found by this screening that statins have a therapeutic effect on hepatitis C. .
- the therapeutic effect of statins on hepatitis C is not always satisfactory.
- An object of the present invention is to provide a novel therapeutic agent for hepatitis C having a high therapeutic effect on hepatitis C.
- hydroxyurea used as a therapeutic agent for chronic myeloid leukemia has a high therapeutic effect on hepatitis C.
- the present invention has been completed. Furthermore, it has also been found that hydroxyurea exhibits a synergistic effect with interferon ⁇ with respect to the effect of treating hepatitis C.
- the present invention provides a therapeutic agent for hepatitis C containing hydroxyurea as an active ingredient.
- the present invention also provides a therapeutic agent for hepatitis C, which further comprises interferon ⁇ in the therapeutic agent for hepatitis C of the present invention.
- the present invention provides use of hydroxyurea for producing a therapeutic agent for hepatitis C.
- the present invention provides a method for treating hepatitis C, comprising administering a therapeutically effective amount of hydroxyurea to a hepatitis C patient.
- a novel therapeutic agent for hepatitis C having a high therapeutic effect on hepatitis C was provided.
- FIG. 1 A plasmid encoding Renilla fluorescence by an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV) is transfected into OR6c cells, which are OR6 cells cured by IFN, using FuGENE6 reagent (trade name, manufactured by Roche Diagnostics)
- FIG. 1 A plasmid encoding Renilla fluorescence by an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV) is transfected into OR6c cells, which are OR6 cells cured by IFN, using FuGENE6 reagent (trade name, manufactured by Roche Diagnostics) FIG.
- IRS internal ribosome entry site derived from encephalomyocarditis virus
- FIG. 4 is a graph showing hydroxyurea concentration and relative luciferase activity when various concentrations of hydroxyurea were added after 24 hours and cultured for 72 hours. It is a figure which shows the relative cell number at the time of culturing OR6 cell in the culture solution which added various concentrations of hydroxyurea. It is a figure which shows the relationship between these density
- the therapeutic agent for hepatitis C of the present invention contains hydroxyurea (hereinafter sometimes referred to as “HU”) as an active ingredient.
- HU hydroxyurea
- HU itself is a well-known compound having the following chemical structure, its production method is well-known, and has been commercially available for a long time as a therapeutic agent for chronic myelogenous leukemia.
- commercially available HU can be advantageously used.
- the hepatitis C therapeutic agent of the present invention may further contain other active ingredients in addition to HU.
- HU has an effect of enhancing the therapeutic effect of interferon ⁇ , which has been conventionally used as a therapeutic agent for hepatitis C, that is, interferon ⁇ and hepatitis C treatment. It became clear that it has a synergistic effect. Therefore, it is preferable that the therapeutic agent for hepatitis C of the present invention further contains interferon ⁇ .
- interferon ⁇ used as a medicine, peginterferon ⁇ , which is obtained by adding a polyethylene glycol chain to interferon ⁇ to improve in vivo stability, is frequently used.
- interferon ⁇ refers to a stabilized derivative of interferon ⁇ , such as peginterferon ⁇ , which has the activity of interferon ⁇ and has increased in vivo stability. It is used in an inclusive sense (except in the examples).
- the other active ingredient that the therapeutic agent for hepatitis C of the present invention may contain is not limited to interferon ⁇ , and for example, ribavirin used for the treatment of hepatitis C may be further added, It may further contain both interferon ⁇ and ribavirin.
- the administration route may be oral administration or parenteral administration, and in the case of parenteral administration, intravenous administration, intramuscular administration, local administration to the liver or its surroundings, transdermal administration, etc. can be exemplified. It is not limited to. Of these, oral administration is preferred.
- the dosage form may be any known dosage form, such as tablets, capsules, lozenges, troches, hard candy, powder, spray, cream, paint, suppository, jelly, Gels, pastes, lotions, ointments, aqueous suspensions, injection solutions, elixirs, syrups and the like can be mentioned, but are not limited thereto.
- the therapeutic agent for hepatitis C of the present invention may contain excipients, carriers, diluents and the like for the preparation in addition to HU and optionally other active ingredients as described above.
- Contains ingredients for. These components are well known in the pharmaceutical field, and examples thereof include, but are not limited to, lactose, starch, cellulose, calcium carbonate, sodium alginate, water and the like.
- other additives commonly used in the pharmaceutical field such as buffering action, salts for adjusting osmotic pressure, solubilizers, dispersants, stabilizers, antioxidants, preservatives, disintegrants, binders
- Ingredients such as flavoring agents, lubricants, coating agents, colorants and the like may be blended.
- the dose is appropriately set according to the patient's condition, medical history, severity, body weight, other active ingredients to be used in combination, etc., but is usually about 1 mg to 10000 mg, preferably 500 mg to 2000 mg as an adult daily HU amount. Degree.
- the dose of peginterferon ⁇ is also set as appropriate.
- the amount of peginterferon ⁇ per day for an adult is about 1 ⁇ g to 1000 ⁇ g, preferably about 10 ⁇ g to 200 ⁇ g.
- the administration period is appropriately set while observing the patient's condition, but is usually about 1 to 1500 days, particularly about 14 to 730 days.
- the therapeutic agent for hepatitis C of the present invention is effective for both acute hepatitis C and chronic hepatitis C.
- the therapeutic agent for hepatitis C of the present invention has been confirmed to be effective for the treatment of chronic hepatitis C, particularly intractable chronic hepatitis C. It is particularly effective when used for the treatment of chronic hepatitis B, especially refractory chronic hepatitis C.
- “Chronic hepatitis C” refers to hepatitis that has persisted for more than 6 months due to hepatitis C virus
- refractory hepatitis C refers to genotype 1 hepatitis C virus infection. This means that the amount of HCV RNA is 5 LogIU / ml or more by the TaqMan (trade name) PCR method, which is a conventional method.
- the 50% replication inhibitory concentration of hepatitis C virus (HCV) by HU in the screening system using the cells described in Patent Document 1 is 60 ⁇ mol / L, It is lower than the same concentration (76-126 ⁇ mol / L) of ribavirin, which is a commercially available therapeutic agent for hepatitis C, and is therefore considered to have a high therapeutic effect on hepatitis C.
- the human HU tolerance dose is 800 mg / mm 2 orally administered every 4 hours, in which case the HU blood concentration reaches 2480 ⁇ mol / L. Therefore, it is considered that HU can be clinically used alone.
- interferon alpha therapy is infected with a genotype of hepatitis C virus, it is considered that the therapeutic effect is further enhanced by using interferon alpha together.
- Formulation Example 1 The following can be illustrated as an example of the composition of the capsule for oral administration. HU 500mg Hydrea Capsules (Bristol Myers) 500mg
- Example 1 Anti-HCV activity of HU A compound having an effect of treating hepatitis C was screened by a screening system using cells described in Patent Document 1 developed by the present inventors.
- Patent Document 1 used for screening are as follows. That is, a plasmid containing a full length HCV genome, a luciferase gene as a reporter gene, and a neomycin resistance gene (neomycin phosphotransferase gene) as a selectable marker gene in an Oc cell derived from human hepatoma cell line HuH-7 (FERM P-20517) It has been introduced.
- a plasmid containing a full length HCV genome a luciferase gene as a reporter gene
- a neomycin resistance gene neomycin phosphotransferase gene
- HCV internal ribosome entry site IRES
- luciferase gene luciferase gene
- neomycin phosphotransferase gene encephalomyocarditis virus (EMCV) -derived IRES sequence
- EMCV encephalomyocarditis virus
- HCV open reading frame This is a cell (OR6 cell) in which a plasmid containing RNA linked in the order of sequence and HCV3 ′ untranslated sequence was introduced into an Oc cell (FERM P-20517) derived from human hepatoma cell line HuH-7.
- Luciferase activity was measured by collecting cells using a commercially available luciferase activity measurement kit (Renilla Luciferase Assay System (Promega)) and quantifying luciferase according to the attached experimental instructions. Monolight 3010 (BD Biosciences) was used for measurement of fluorescence activity.
- Test compounds HU and IFN ⁇ having a purity of 98% or more were purchased from Sigma-Aldrich.
- OR6c cells which are OR6 cells cured with IFN, a plasmid encoding Renilla fluorescence by an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV). 24 hours later, various concentrations of HU were added and cultured for 72 hours.
- IFN internal ribosome entry site
- EMCV encephalomyocarditis virus
- Example 2 Clinical trial Hydrourea (trade name Hydrea Capsule) 1500 mg / day was orally administered to 12 intractable hepatitis C patients (serogroup 1 high viral load) who received informed consent for 4 weeks.
- the serum HCV RNA level (log IU / ml) was measured every week from the start of administration to the end of treatment by a real-time detection PCR method using a conventional TaqMan (trade name) probe.
- HCV RNA viral load
- a novel therapeutic agent for hepatitis C effective for the treatment of hepatitis C is provided.
- the therapeutic agent of the present invention is also useful for the treatment of refractory chronic hepatitis C, and contributes to the treatment of hepatitis C.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
経口投与用のカプセルの組成の1例として次のものを例示することができる。
HU 500mg
ハイドレアカプセル(ブリストルマイヤーズ) 500mg
本願発明者らが開発した、特許文献1記載の細胞を用いたスクリーニング系により、C型肝炎治療効果を有する化合物をスクリーニングした。
(1) 細胞培養と抗ウイルスアッセイ
OR6細胞は、24ウェル中で培養した(培養液:5%牛胎児血清及びG418付加DMEM培地、温度37℃)。HUとインターフェロンα(以下、「IFNα」)の抗ウイルス効果をモニターするため、まず24ウェルプレートを用いて1ウェルあたり15000細胞を24時間培養した。引き続いて種々の濃度のHUやIFNαあるいは両剤を加えさらに72時間培養した。各ウェル中の細胞を回収し、ルシフェラーゼ活性を測定した。ルシフェラーゼ活性は、市販のルシフェラーゼ活性測定キット(Renilla Luciferase Assay System (Promega社))を用い、添付の実験説明書に従って細胞を回収し、ルシフェラーゼの定量を行うことにより測定した。蛍光活性の測定にはMonolight 3010(BD Biosciences社)を用いた。
純度98%以上のHU及びIFNαはシグマアルドリッチ社より購入した。
OR6細胞に対するHUの細胞毒性を検証するため、95mm培養皿にOR6細胞を400000個まき、0、50、100、150μmol/Lの濃度でHUを加え72時間培養した。トリパンブルー染色の後で生存している細胞数をヘマトサイトメーターにて測定した。
特許文献1に従い脳心筋炎ウイルス(EMCV)由来内部リボソーム侵入部位(IRES)によりレニラ蛍光をコードするプラスミドをIFNにより治癒したOR6細胞であるOR6c細胞にFuGENE6試薬(Roche Diagnostics社製)を用いてトランスフェクションし、24時間後に種々の濃度のHUを加え72時間培養した。
(1) HUは単独でHCV遺伝子複製抑制効果を示した
OR6細胞はHCV遺伝子複製をモニターする信頼できる実験系と考えられているため、HUが単独でHCV全長遺伝子の複製を抑制できるか検討した(図1A)。150μmol/LまでのHUを72時間投与して得られた用量依存曲線により、50%複製抑制濃度(無添加の場合の複製を50%抑制する濃度)は60μmol/Lとなった(図1B)。インターフェロンαの50%抑制濃度は1.2 IU/mlとなった。コントロールプラスミドpEMCV-RLをトランスフェクションした治癒OR6c細胞を用いて、HUはレニラ蛍光自身を発光抑制しないことを確認した(図2)。これらの結果より、HUは独立してHCV遺伝子複製を抑制できることが明らかになった。
HCVレプリコンの複製は宿主細胞の増殖に依存するという報告があるため、HUのHCV遺伝子複製抑制効果が細胞毒性効果のため生じている可能性がある。この可能性を除外するため、OR6細胞に対するHUの細胞毒性効果を検証した。その結果、100μmol/LまでのHUで処理した細胞では未処理の細胞と比較して生存細胞数に明らかな差は認められなかった(図3)。
続いてHCV全長遺伝子複製におけるインターフェロンαとHUの併用による抑制効果を検討した。HUを0、24、48マイクロモルに固定した時のインターフェロンαの用量依存曲線が得られた。曲線はHUを加えることにより左にシフトしており(図4A)、HU併用はインターフェロン単独より効果的であることが示された。アイソボログラム解析により両剤併用による相乗的な抗HCV効果が証明された(図4B。図4B中の破線が相乗効果がない場合(相加効果)のラインであり、実測値がそれよりも左下側にあることは、IFNα単独投与よりもHUとの併用の方が抗HCV効果が高まること、すなわち、相乗効果があることを示す)。これらの結果よりHUはインターフェロンαと併用して使用できる抗HCV剤となり得ることが明らかになった。
インフォームドコンセントを施した12名の難治性C型肝炎患者(serogroup 1 高ウイルス量)に対してハイドロキシウレア(商品名ハイドレアカプセル)1500mg/日の経口投与を4週間行った。投与開始前から治療終了時まで1週間毎に血清中HCV RNA量(log IU/ml)を常法である、TaqMan(商品名)プローブを用いたリアルタイム検出PCR法により測定した。
Claims (5)
- ハイドロキシウレアを有効成分として含有するC型肝炎治療剤。
- インターフェロンαをさらに含む請求項1記載の治療剤。
- ハイドロキシウレアのC型肝炎治療剤製造のための使用。
- 治療に有効な量のハイドロキシウレアをC型肝炎患者に投与することを含む、C型肝炎の治療方法。
- 治療効果をさらに高める量のインターフェロンαを併用する請求項4記載の方法。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010533943A JP5527549B2 (ja) | 2008-10-17 | 2009-10-16 | C型肝炎治療剤 |
US13/124,584 US20110200554A1 (en) | 2008-10-17 | 2009-10-16 | Therapeutic agent for hepatitis c |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008-268679 | 2008-10-17 | ||
JP2008268679 | 2008-10-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2010044474A1 true WO2010044474A1 (ja) | 2010-04-22 |
Family
ID=42106638
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2009/067942 WO2010044474A1 (ja) | 2008-10-17 | 2009-10-16 | C型肝炎治療剤 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20110200554A1 (ja) |
JP (1) | JP5527549B2 (ja) |
WO (1) | WO2010044474A1 (ja) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3968249A (en) * | 1963-01-22 | 1976-07-06 | E. R. Squibb & Sons, Inc. | Method of treating malignant neoplastic disease |
WO2005018330A1 (en) * | 2003-08-18 | 2005-03-03 | Pharmasset, Inc. | Dosing regimen for flaviviridae therapy |
-
2009
- 2009-10-16 WO PCT/JP2009/067942 patent/WO2010044474A1/ja active Application Filing
- 2009-10-16 JP JP2010533943A patent/JP5527549B2/ja not_active Expired - Fee Related
- 2009-10-16 US US13/124,584 patent/US20110200554A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
STUYVER L. J., ET AL.: "Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites", JOURNAL OF VIROLOGY, vol. 77, no. 19, October 2003 (2003-10-01), pages 10689 - 10694 * |
Also Published As
Publication number | Publication date |
---|---|
JPWO2010044474A1 (ja) | 2012-03-15 |
US20110200554A1 (en) | 2011-08-18 |
JP5527549B2 (ja) | 2014-06-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5185826B2 (ja) | ウイルス性肝炎の処置 | |
AU2019271958B2 (en) | Therapy for inhibition of single-stranded rna virus replication | |
US20130109647A1 (en) | Methods and compositions for treating hepatitis c virus | |
WO2008024843A2 (en) | Combination therapy method for treating hepatitis c virus infection and pharmaceutical compositions for use therein | |
JP2013082726A (ja) | 鉄が病因に関与する肝臓疾患の処置 | |
KR20170132327A (ko) | Hbv 감염의 치료를 위한 조성물 및 방법 | |
JP2013529627A (ja) | ヒドロキシクロロキンまたはヒドロキシクロロキンおよび抗ウイルス剤の組合せを使用するc型肝炎ウイルス関連疾患の処置 | |
EP2776024A1 (en) | Methods and compositions for treating hepatitis c virus | |
JP2013508425A (ja) | Bi201335、インターフェロンアルファおよびリバビリンを含むhcvの併用療法のための投薬処置計画 | |
TWI631943B (zh) | 治療脂肪肝疾病之方法 | |
US20100086519A1 (en) | Treatment of Hepatitis C Infection With Metalloporphyrins | |
US20230241037A1 (en) | Cross-linked medication for treatment of coronaviral infection and method of treatment | |
JP5527549B2 (ja) | C型肝炎治療剤 | |
JP5114401B2 (ja) | ウイルス性疾患の予防又は治療剤 | |
US11364256B2 (en) | Viral inhibition | |
AU2010363574B2 (en) | Uses of unsaturated fatty acids for inhibiting virus replication and /or infection | |
JP5061352B2 (ja) | 肝臓疾患治療剤及び肝機能改善剤 | |
CN114762694A (zh) | 寡糖转移酶抑制剂在预防和/或治疗新型冠状病毒感染中的应用 | |
EP2117563B1 (en) | Amidinoanthracycline antibiotics for use in the treatment of viral infections | |
WO2022213870A1 (zh) | 通过口服给药抑制CD4+Treg细胞的药物和方法 | |
RU2597795C2 (ru) | Ингибитор скопления жидкости в полостях организма | |
WO2016139740A1 (ja) | 肝線維化改善剤 | |
WO2006093211A1 (ja) | 抗ウイルス剤 | |
WO2016164625A1 (en) | Compositions and methods for the treatment of hcv infection | |
JP2017514834A (ja) | Hcv感染症を治療するための組合せ療法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09820652 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2405/DELNP/2011 Country of ref document: IN |
|
ENP | Entry into the national phase |
Ref document number: 2010533943 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13124584 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 09820652 Country of ref document: EP Kind code of ref document: A1 |