WO2002084294A2 - Peroxydase de glutathione gastro-intestinale de proteine cellulaire humaine comme cible pour une intervention medicale contre les infections dues au virus de l'hepatite c - Google Patents

Peroxydase de glutathione gastro-intestinale de proteine cellulaire humaine comme cible pour une intervention medicale contre les infections dues au virus de l'hepatite c Download PDF

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WO2002084294A2
WO2002084294A2 PCT/EP2002/004167 EP0204167W WO02084294A2 WO 2002084294 A2 WO2002084294 A2 WO 2002084294A2 EP 0204167 W EP0204167 W EP 0204167W WO 02084294 A2 WO02084294 A2 WO 02084294A2
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gastrointestinal
glutathione peroxidase
cellular protein
agent
cells
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WO2002084294A3 (fr
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Thomas Herget
Matthew Cotten
Sabine Obert
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Axxima Pharmaceuticals Ag
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Priority to EP02730159A priority Critical patent/EP1377833A2/fr
Priority to JP2002581997A priority patent/JP2004533822A/ja
Priority to CA002443525A priority patent/CA2443525A1/fr
Priority to AU2002302535A priority patent/AU2002302535A1/en
Publication of WO2002084294A2 publication Critical patent/WO2002084294A2/fr
Priority to US10/342,054 priority patent/US20030180719A1/en
Publication of WO2002084294A3 publication Critical patent/WO2002084294A3/fr
Priority to US10/723,719 priority patent/US7341717B2/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01009—Glutathione peroxidase (1.11.1.9)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the human cellular protein glutathione peroxidase- gastrointestinal as potential targets for medical intervention against Hepatitis C virus (HCV) infections. Furthermore, the present invention relates to a method for the detection of compounds useful for prophylaxis and/or treatment of Hepatitis C virus infections and a method for detecting Hepatitis C virus infections in an individual or in cells. Also mono- or polyclonal antibodies are disclosed effective for the treatment of HCV infections together with methods for treating Hepatitis C virus infections or for the regulation of Hepatitis C virus production wherein genes or said antibodies may be used.
  • HCV Hepatitis C Virus
  • Liver failure from chronic hepatitis C is the leading indicator for liver transplantation.
  • the CDC estimates that medical and work-loss cost for HCV annually are around $600 million.
  • HCV is transmitted primarily by blood and blood products. Due to routine screening of the blood supplies from mid-1992, new transfusion-related cases are exceedingly rare and have been surpassed by injection drug use as the highest risk factor for acquiring the virus. There is also a sexual, however inefficient, route of transmission, and a 6% rate of transmission from infected mothers to their children, which is higher in case of HIV co-infection. In a certain percentage of infections, the mode of transmission remains unknown.
  • Interferons Intron A, Schering-Plough; Roferon A, Hoffmann-LaRoche; Wellferon, Glaxo Wellcome; Infergen, Amgen
  • ribavirin Rebetol, Schering-Plough
  • Recommended treatment duration is 6 to 12 months, depending on HCV genotype.
  • Experimental forms of slow-release pegylated interferons (Pegasys, Hoffmann-LaRoche; PEG-lntron, Schering-Plough) have shown improvements in response rates (42 to 82% in combination with ribavirin) and application (once- weekly injection) in recent clinical studies (Hepatology 32:4, Pt 2 of 2.
  • Common side effects of interferon therapy include: e.g. fatigue, muscle aches, head aches, nausea, fever, weight loss, irritability, depression, bone marrow suppression, reversible hair loss.
  • the most common side effects of ribavirin are anemia, fatigue and irritability, itching, skin rash, nasal stuffiness, sinusitis, cough. More serious side effects of mono-and combination therapy occur in less than two percent of patients (NIDDK information: Chronic Hepatitis C: Current Disease Management, accessed 09.12.99).
  • Contraindications to interferon are psychosis or severe depression; neutropenia and/or thrombocytopenia; organ transplantation except liver; symptomatic heart disease; decompensated cirrhosis; uncontrolled seizures.
  • Contraindications to ribavirin are end-stage renal failure; anemia; hemoglobinopathies; severe heart disease; pregnancy; no reliable method of contraception (consensus statement EASL).
  • Inhibitors for HCV enzymes such as protease inhibitors, RNA polymerase inhibitors, helicase inhibitors as well as ribozymes and antisense RNAs are under preclinical development (Boehringer Ingelheim, Ribozyme Pharmaceuticals, Vertex Pharmaceuticals, Schering-Plough, Hoffmann-LaRoche, Immusol, Merck etc.). No vaccine is available for prevention or therapeutic use, but several companies are trying to develop conventional or DNA vaccines or immunostimulatory agents (e.g. Chiron, Merck/Vical, Epimmune, NABI, Innogenetics). In addition, antibodies against HCV virion have been developed and entered into clinical trials recently (Trimera Co., Israel).
  • Signal transduction at the cellular level refers to the movement of signals from outside the cell to inside.
  • the movement of signals can be simple, like that associated with receptor molecules of the acetylcholine class: receptors that constitute channels which, upon ligand interaction, allow signals to be passed in the form of small ion movement, either into or out of the cell. These ion movements result in changes in the electrical potential of the cells that, in turn, propagates the signal along the cell.
  • More complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/threonine kinases. Protein phosphorylations change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells.
  • Signal transducting receptors are of three general classes: 1. Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity:
  • Receptors that have intrinsic enzymatic activities include those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors), tyrosine phosphatases (e.g. CD45 [cluster determinant-45 ⁇ protein of T cells and macrophages), guanylate cyclases (e.g. natriuretic peptide receptors) and serine/threonine kinases (e.g. activin and TGF-beta receptors).
  • tyrosine kinases e.g. PDGF, insulin, EGF and FGF receptors
  • tyrosine phosphatases e.g. CD45 [cluster determinant-45 ⁇ protein of T cells and macrophages
  • guanylate cyclases e.g. natriuretic peptide receptors
  • serine/threonine kinases e.g. activin and TGF-beta receptors
  • This class of receptors includes all of the cytokine receptors (e. g. the interleukin-2 receptor) as well as the CD4 and CD8 cell surface glycoproteins of T cells and the T cell antigen receptor.
  • G-proteins Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins (termed G-proteins):
  • Receptors of the class that interact with G-proteins all have a structure that is characterized by seven transmembrane spanning domains. These receptors are termed serpentine receptors. Examples of this class are the adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).
  • Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription:
  • the steroid/thyroid hormone receptor superfamily (e.g. glucocorticoid, vitamin D, retinoic acid and thyroid hormone receptors) is a class of proteins that reside in the cytoplasm and bind the lipophilic steroid/thyroid hormones. These hormones are capable of freely penetrating the hydrophobic plasma membrane. Upon binding ligand the hormone-receptor complex translocates to the nucleus and bind to specific DNA sequences resulting in altered transcription rates of the associated gene. When the message reaches the nucleus via one or several of the pathways described above, it initiates the modulation of specific genes, resulting in the production of RNA and finally proteins that carry out a specific biological function. Disturbed activity of signal transduction molecules may lead to the malfunctioning of cells and disease processes. Specifically, interaction of HCV with host cells is necessary for the virus to replicate.
  • the present invention is based upon the surprising discovery that the human cellular protein glutathione peroxidase-gastrointestinal (P18283) is specifically downregulated as a result of HCV replication in HCV infected host cells.
  • the antiviral therapeutic research approach described herein focuses on discovering the cellular signal transduction pathways involved in viral infections. Identification of the signal transduction molecules, key to viral infection, provides for, among other things, novel diagnostic methods, for example, assays and compositions useful therefore, novel targets for antiviral therapeutics, a novel class of antiviral therapeutics, and new screening methods (e.g. assays) and materials to discover new antiviral agents.
  • processes for finding pharmaceutically effective compounds include target identification.
  • Target identification is basically the identification of a particular biological component, namely a protein and its association with particular disease states or regulatory systems.
  • a protein identified in a search for a pharmaceutically active chemical compound (drug) that can affect a disease or its symptoms is called a target.
  • Said target is involved in the regulation or control of biological systems and its function can be interfered by with a drug.
  • the word disease is used herein to refer to an acquired condition or genetic condition.
  • a disease can alter the normal biological system of the body, causing an over or under abundance of chemical compounds (chemical imbalance).
  • chemical imbalance chemical compounds
  • the regulatory systems for these chemical compounds involve the use by the body of certain proteins to detect imbalances or cause the body to produce neutralizing compounds in an attempt to restore the chemical imbalance.
  • the word body is used herein to refer to any biological system, e.g. human, animal, cells, or cell culture. It is object of the present invention to provide novel targets for medical intervention, prophylaxis and/or treatment of Hepatitis C virus infections in mammals, including humans, and cells or cell cultures together with methods for detecting HCV infections in individuals, cell cultures and cells and methods for detecting compounds useful for prophylaxis and/or treatment of HCV infections.
  • the object of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, and the examples of the present application.
  • the human cellular protein glutathione peroxidase-gastrointestinal (GI-GPx) is also known as glutathione peroxidase-related protein 2 (GPRP) or glutathione hydrogen peroxide oxidoreductase. It has been assigned to the Accession Number P18283 and the EC Number 1.11.1.9.
  • the human cellular protein glutathione peroxidase-gastrointestinal catalyzes the reduction of various organic hydroperoxides, as well as hydrogen peroxide, with glutathione (GSH) as hydrogen donor (2 GSH + H 2 O 2 > GS — GS + 2 H 2 O). It has a molecular weight of 84,000 and 4 subunits per mol of enzyme.
  • the enzyme is useful for enzymatic determination of lipid hydroperoxide.
  • GI-GPx belongs to the family of selenoproteins and plays an important role in the defense mechanisms of mammals, birds and fish against oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. It has been suggested that this enzyme functions in more times as a mechanism of protecting the cellular membrane system against peroxidative damage and that selenium as an essential trace element which may play an important role in this suggested function of the enzyme. It is known that both vitamin E and Se act as antioxidants also in a common mechanism of oxidative stress as an underlying cause of genetic changes.
  • Selenium functions within mammalian systems primarily in the form of selenoproteins.
  • Selenoproteins contain selenium as selenocysteine and perform a variety of physiological roles. Seventeen selenoproteins have been identified: cellular or classical glutathione peroxidase; plasma (or extracellular) glutathione peroxidase; phospholipid hydroperoxide glutathione peroxidase; gastrointestinal glutathione peroxidase; selenoprotein P; types 1 , 2, and 3 iodothyronine deiodinase; selenoprotein W; thioredoxin reductase; and selenophosphate synthetase.
  • antioxidants such as N-acetyl-L-cycteine, N- acetyl-S-famesyl-L-cysteine, Bilirubin, caffeic acid, CAPE, catechin, ceruloplasmin, Coelenterazine, copper diisopropylsalicylate, deferoxamine mesylate, R-(-)-deprenyl, DMNQ, DTPA dianhydride, Ebselen, ellagic acid, (-)- epigallocatechin, L-ergothioneine, EUK-8, Ferritin, glutathione, glutathione monoethylester, ⁇ -lipoic acid, Luteolin, Manoalide, MCI-186, MnTBAP, MnTMPyP, morin hydrate
  • antioxidants may be selected from the group of carboxylic acids such as citric acid and phenolic compounds such as BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), propyl gallate, TBHQ (terf-butyl hydroquinone), tocopherols, lecithin, gums and resin guiac, THBP (trihydroxybutyrophenone), thiodipropionic acid and dilauryl thiodipropionate, and glycines.
  • carboxylic acids such as citric acid and phenolic compounds
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluene
  • propyl gallate propyl gallate
  • TBHQ terf-butyl hydroquinone
  • Oxidative damage is mainly caused by free radicals, preferably reactive oxygen intermediates, derived from normal cellular respiration and oxidative burst produced when phagocytic cells destroy bacteria or virus-infected cells.
  • free radicals preferably reactive oxygen intermediates
  • eukaryotic organisms have evolved many defense mechanisms. These include the above-mentioned antioxidants which act as free radicals scavengers and which may interact with GI-GPx and/or may activate, stimulate, and/or increase the expression and/or production of GI-GPx. This advantageous effect of the radicals on the amount of GI-GPx generated in the cells competes with the HCV- induced down-regulation of GI-GPx and supports the cells in their fight against the Hepatits C viruses.
  • HCV infection studies The only reliable experimental HCV infection studies have been performed with chimpanzees. So far, there is no simple cell culture infection system available for HCV. Although a number of reports have been published describing in vitro propagation attempts of HCV in primary cells and cell lines, questions remain concerning reproducibility, low levels of expression and properly controlled detection methods (reviewed in J. Gen Virol. 8_i, 1631 ; Antiviral Chemistry and Chemotherapy 10, 99). Thus, the replicon system described by Bartenschlager and coworkers (Lohmann et al, Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110. 1999) was used for the studies disclosed herein.
  • This replicon system reproduces a crucial part of the HCV replication cycle which is used as a system for simulating HCV infection.
  • Bartenschlager's group produced bicistronic recombinant RNAs, so-called “replicons”, which carry the neomycin-phosphotransferase (NPT) gene as well as a version of the HCV genome where the sequences for the structural HCV proteins were deleted.
  • replicons bicistronic recombinant RNAs, so-called "replicons” carry the neomycin-phosphotransferase (NPT) gene as well as a version of the HCV genome where the sequences for the structural HCV proteins were deleted.
  • NPT neomycin-phosphotransferase
  • HuH-pcDNA3 cells are HuH7 cells resistant to G-418 by integration of a NPT gene-carrying plasmid (pcDNA3, Invitrogen) and serve as negative control.
  • pcDNA3, Invitrogen a NPT gene-carrying plasmid
  • Three replicon lines were analyzed for changes in cellular RNA expression patterns compared to the control line: ⁇ HuH-9-13: cell line with persistant replicon l377/NS3-3'/wt, described in Science 1999, 285, 110-113,
  • HuH-5-15 cell line with persistant replicon l389/NS3-37wt, described in Science 1999, 285, 110-113,
  • HuH-11-7 cell line with persistant replicon l377/NS2-37wt, described in Science 1999, 285, 110-113.
  • HCV replicon cells serve as a system for simulation of HCV infected cell systems, especially for simulating HCV infected mammals, including humans. Interference of HCV with the cellular signaling events is reflected in differential gene expression when compared to cellular signaling in control cells. Results from this novel signal transduction microarray analysis revealed significant downregulation of GI-GPx. Radioactively labeled complex cDNA-probes from HCV Replicon cells HuH-9-13, HuH-5-15, and HuH-11-7 were hybridized to cDNA- arrays and compared to hybridizations with cDNA-probes from HuH-pcDNA control cells which did not contain HCV Replicons.
  • sample could be used, for instance, blood, biopsies, cells or cell cultures of liver or of any other tissue infected with HCV.
  • one aspect of the present invention is directed to novel compounds useful in the above-identified methods. Therefore, the present invention relates to monoclonal or polyclonal antibodies that bind to GI-GPx.
  • the present invention discloses a method for treating Hepatitis C virus infection in an individual comprising the step of administering a pharmaceutically effective amount of an agent which inhibits at least partially the activity of GI-GPx or which inhibits at least partially the production of GI-GPx in the cells.
  • a similar aspect of the present invention is directed to a method for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in cells or cell cultures comprising the step of administering a pharmaceutically effective amount of an agent which inhibits at least partially the activity of GI-GPx or which inhibits at least partially the production of GI-GPx.
  • Another object of the present invention is to provide a method for regulating the production of Hepatitis C virus in an individual or in cells or cell cultures comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent inhibits at least partially the activity GI-GPx or wherein said agent at least partially inhibits the production GI-GPx in the cells .
  • the above-mentioned monoclonal or polyclonal antibodies directed against GI-GPx may be used as pharmaceutically active agents within said methods.
  • the present invention is also directed to a method for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in an individual comprising the step of administering a pharmaceutically effective amount of an agent which activates at least partially GI-GPx or which activates or stimulates the production of GI-GPx in the cells.
  • Another inventive aspect is related to a method for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in cells or cell cultures comprising the step of administering a pharmaceutically effective amount of an agent which activates at least partially the activity of GI-GPx or which activates or stimulates at least partially the production of GI-GPx.
  • associated diseases refers to, for instance, opportunistic infections, liver cirrhosis, liver cancer, hepatocellular carcinoma, or any other diseases that can come along with HCV infection.
  • GI-GPx The function of GI-GPx is to detoxify peroxides in cells and prevent the cells from oxidative damage.
  • generating artificial oxidative stress conditions allows selective killing of HCV-infected cells.
  • radical forming compounds examples include bipyridyls such as paraquat, 2,2'-bipyridyl and 4,4'-bipyridyl derivatives, bis-6-(2,2'-bipyridyl)- pyrimidines, tris-(2,2'-bipyridyl)-ruthenium, peroxides such as dibenzoylperoxid, diacetylperoxide, hydrogen peroxide, di-tert.-butylperoxide, or diaza compounds such as diazaisobutyronitril.
  • bipyridyls such as paraquat, 2,2'-bipyridyl and 4,4'-bipyridyl derivatives, bis-6-(2,2'-bipyridyl)- pyrimidines, tris-(2,2'-bipyridyl)-ruthenium
  • peroxides such as dibenzoylperoxid, diacetylperoxide
  • Yet another aspect of the present invention is directed to a novel therapeutic composition useful for the prophylaxis and/or treatment of an individual afflicted with Hepatitis C virus and/or associated diseases comprising at least one agent capable of inactivating or inhibiting the activity of GI-GPx or of decreasing or inhibiting the production and/or expression of GI-GPx.
  • Further aspects of the present invention relate to methods either for regulating the expression of the human cellular protein glutathione peroxidase-gastrointestinal in an individual or in cells or cell culture comprising the step of administering either the individual or the cells or cell culture a pharmaceutically effective amount of an agent wherein said agent stimulates or increases at least partially the transcription of DNA and/or the translation of RNA encoding GI-GPx.
  • compositions useful within said methods for prophylaxis and/or treatment of an individual afflicted with Hepatitis C virus and/or associated diseases.
  • Said compositions comprise at least one agent capable of increasing the activity of GI-GPx or of activating or stimulating the production and/or expression of GI-GPx.
  • compositions may further comprise pharmaceutically acceptable carriers, excipients, and/or diluents.
  • GI-GPx inhibitors refers to any compound capable of downregulating, decreasing, inactivating, suppressing or otherwise regulating the amount and/or activity of GI-GPx or its expression.
  • GI-GPx inhibitors may be proteins, oligo- and polypeptides, nucleic acids, genes, small chemical molecules, or other chemical moieties.
  • Small chemical molecules are, for instance, organic compounds with molecular weight typically below 500 g/mol and preferably also with less than 10 heteroatoms.
  • the term "activator” refers to any chemical compound capable of upregulating, activating, stimulating, or increasing the amount and/or activity of Gl- GPx or its expression.
  • said agents may be proteins, oligo- and polypeptides, nucleic acids, genes, small chemical molecules, or other chemical moieties.
  • An example for an activator of glutathione peroxidase is e.g. selenium and retinoic acid (Brigelius-Flohe, R.,1999, Free Radicals in Biology and Medicine, 27, 951-965; Chu et al., 1999, Journal of Nutrition 129, 1846 - 1854).
  • agent refers to any chemical or biological compound capable of down- or upregulating, de- or increasing, suppressing or stimulating, inactivating or activating, or otherwise regulating or effecting the amount and/or activity of GI-GPx and/or the expression of GI-GPx.
  • aptamers which function as regulators of the activity of a wide range of cellular molecules such as GI-GPx.
  • Aptamers are nucleic acid molecules selected in vitro to bind small molecules, peptides, or proteins with high affinity and specificity. Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms.
  • Most famous examples for aptamers are DNA aptamers or RNA aptamers.
  • RNA molecules participate actively in many cell processes. Examples are found in translation (rRNA, tRNA, tmRNA), intracellular protein targeting (SRP), nuclear splicing of pre-mRNA (snRNPs), mRNA editing (gRNA), and X-chromosome inactivation (Xist RNA). Each of these RNA molecules acts as a functional product in its own right, without coding any protein. Because RNA molecules can fold into unique shapes with distinct structural features, some RNAs bind to specific proteins or small molecules (as in the ATP-binding aptamer), while others catalyze particular chemical reactions. Thus, RNA aptamers can be used to interact with GI-GPx and thereby modulate, regulate, activate, or inhibit the activity and biological function of said peroxidase.
  • regulating expression and/or activity generally refers to any process that functions to control or modulate the quantity or activity (functionality) of a cellular component. Static regulation maintains expression and/or activity at some given level. Upregulation refers to a relative increase in expression and/or activity. Accordingly, downregulation refers to a relative decrease in expression and/or activity. Downregulation is synonymous with inhibition of a given cellular component's activity.
  • Further aspects of the present invention relate to methods either for regulating the expression of the human cellular protein glutathione peroxidase-gastrointestinal in an individual or in cells or cell cultures comprising the step of administering either the individual or the cells or cell cultures a pharmaceutically effective amount of an agent wherein said agent inhibits or decreases at least partially the transcription of DNA and/or the translation of RNA encoding said human cellular protein glutathione peroxidase-gastrointestinal.
  • Therapeutics, pharmaceutically active agents or inhibitors, respectively may be administered to cells from an individual in vitro, or may involve in vivo administration to the individual.
  • the term "individual” preferably refers to mammals and most preferably to humans.
  • Routes of administration of pharmaceutical preparations to an individual may include oral and parenteral, including dermal, intradermal, intragastral, intracutan, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical or transdermal application, but are not limited the these ways of administration.
  • the preferred preparations are in administratable form which is suitable for oral application.
  • These administratable forms include pills, tablets, film tablets, coated tablets, capsules, powders and deposits.
  • Administration to an individual may be in a single dose or in repeated administrations, and may be in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier, binder, lubricant, excipient, diluent and/or adjuvant.
  • Pharmaceutically acceptable salt forms and standard pharmaceutical formulation techniques are well known to persons skilled in the art.
  • a "pharmaceutical effective amount" of a GI-GPx activator or GI- GPx inhibitor is an amount effective to achieve the desired physiological result, either in cells or cell cultures treated in vitro or in a subject treated in vivo.
  • a pharmaceutically effective amount is an amount sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the viral infection.
  • the effective amount may vary depending on the specific GI-GPx inhibitor selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the infection.
  • the inhibitor is to be administered in vivo, factors such as the age, weight and health of the patient as well as dose response curves and toxicity data obtained in pre-clinical animal work would be among those considered. If the inhibitor is to be contacted with the cells or cell cultures in vitro, one would also design a variety of pre-clinical in vitro studies to assess such parameters as uptake, half-life, dose, toxicity, etc. The determination of a pharmaceutically effective amount for a given agent is well within the ability of those skilled in the art.
  • detection includes any method known in the art useful to indicate the presence, absence, or amount of a detection target.
  • methods may include, but are not limited to, any molecular or cellular techniques, used singularly or in combination, including, but not limited to: hybridization and/or binding techniques, including blotting techniques and immunoassays; labeling techniques (chemiluminescent, colorimetric, fluorescent, radioisotopic); spectroscopic techniques; separations technology, including precipitations, electrophoresis, chromatography, centrifugation, ultrafiltration, cell sorting; and enzymatic manipulations (e.g. digestion).
  • the present disclosure teaches for the first time the downregulation of GI-GPx specifically involved in the viral infection of Hepatitis C virus.
  • the present invention is also directed to a method useful for detecting novel compounds useful for prophylaxis and/or treatment of HCV infections.
  • Methods of the present invention identify compounds useful for prophylaxis and/or treatment of HCV infections by screening a test compound, or a library of test compounds, for its ability to inhibit or activate GI-GPx, identified herein as characteristically downregulated during HCV growth and RNA replication inside a cell or individual.
  • assay protocols and detection techniques are well known in the art and easily adapted for this purpose by a skilled practitioner. Such methods include, but are not limited to, high throughput assays (e.g., microarray technology, phage display technology), and in vitro and in vivo cellular and tissue assays.
  • the present invention provides, in view of the important role of GI-GPx in the HCV infection and/or replication process, an assay component specially useful for detecting HCV in an individual, in cell cultures, or in cells.
  • the assay component comprises oligonucleotides immobilized on a solid support capable of detecting GI-GPx activity.
  • the solid support would contain oligonucleotides of sufficient quality and quantity to detect all of the above-mentioned human cellular proteins (e.g., a nucleic acid microarray).
  • an assay component specially useful for screening compounds useful for the prophylaxis and/or treatment of HCV infections.
  • One preferred assay component comprises oligonucleotides that encode GI-GPx immobilized on a solid support.
  • the polypeptide product of gene expression may be assayed to determine the amount of expression as well.
  • Methods for assaying for a protein include, but are not limited to, Western Blotting, immuno-precipitation, radioimmuno assay, immuno-histochemistry and peptide immobilization in an ordered array. It is understood, however, that any method for specifically and quantitatively measuring a specific protein or mRNA product can be used.
  • the present invention further incorporates by reference in their entirety techniques well known in the field of microarray construction and analysis. These techniques include, but are not limited to, techniques described in the following patents and patent applications describing array of biopolymeric compounds and methods for their fabrication:
  • Techniques also include, but are not limited to, techniques described in the following patents and patent application describing methods of using arrays in various applications:
  • Replicon cells express less GI-GPx mRNA than control HuH7 cells: HuH7 control cells (pcDNA3) and the HuH replicon cell lines 5-15, 11-7 and 9-13 were plated in 10-cm culture dishes (5x10 5 cells/58 cm 2 ) and harvested after three days when cells were actively progressing through the cell cycle. Total RNA was isolated and 10 ⁇ g separated in a 1.2% agarose gel and used for Northern blot analysis. A: Blots were hybridized with radioactively labeled oligonucleotides (Probe 1 : open bar and Probe 2: filled bar) complementary to the mRNA coding for human gastrointestinal glutathione peroxidase (GI-GPx).
  • GI-GPx radioactively labeled oligonucleotides
  • Membrane was exposed to Kodak x-ray films for one day at -80°C with intensifier screens. The films were scanned and the density of the mRNA coding for GI-GPx calculated. The value for the control cells (pcDNA3) were set as 100% and compared with the values of the three replicon cell lines (+ SEM), as indicated.
  • Cellular activity of glutathione peroxidase is reduced in replicon cell lines: Cultures were plated and harvested as described in the experimental part below. 180 ⁇ g protein of cytosolic extract were used for estimation of glutathione peroxidase activity as described in Materials and Methods. The mean change ( ⁇ SEM) of extinction at 340 nm reflecting glutathione peroxidase activity for each cell line is illustrated.
  • Replicon cells are susceptible towards oxidative stress: Cells were plated in 96-well microtiter plates (5x10 3 cells/0.35cm 2 ) and after three days treated for 24 hours with the concentration of paraquat depicted. Cell viability was measured utilizing an Alamar-Blue assay and is reflected by relative fluorescence units (RFU) at 405 nm. The LD 50 values ( ⁇ SEM) of three independent experiments are shown for each cell line.
  • Fig. 4 Effect of interferon on GI-GPx-, PKR- and genomic HCV-RNA levels:
  • the HuH7 pcDNA3 control cells and the replicon cell lines 5-15, 11-7 and 9-13 were plated as described in legend to Fig. 1 and after three days (Day 0) treated for two (Day 2) and four days (Day 4) with 1000U/ml interferon ⁇ (IFN ⁇ ). Then, cultures were harvested and RNA was prepared. 10 ⁇ g of total RNA were used for Northern blot analysis. For detection of GI-GPx (A) Probe 1 was used (see Fig. 1 ). The membranes were stripped and successively hybridized with probes for PKR (B) and neomycin phosphotransferase (Neo)(C).
  • Exposure time for all blots were two days at -80°C with intensifier screen.
  • the autoradiograms were densitrometrically scanned and the values compared with the maximal value obtained with each probe in the respective experiment.
  • the values depicted ( ⁇ SEM) are obtained from three independent experiments.
  • GI-GPx Overexpression of GI-GPx in replicon cells causes downregulation of HCV:
  • the replicon cell lines 5-15, 11-7 and 9-13 were plated at a density of 10 5 cells per well of a 6-well plate and infected with 10 3 Adenovirus particles/cell containing either the green fluorescent protein (GFP) as negative control, the GI-GPx cDNA without the 3'UTR (- UTR) and with the 3'UTR containing the SECIS (+ UTR), as indicated.
  • GFP green fluorescent protein
  • - UTR 3'UTR
  • SECIS (+ UTR SECIS (+ UTR
  • GI-GPx-specific antiserum a GI-GPx-specific antiserum
  • the x-ray films were densitometrically scanned and the NS5a values compared with untransfected control cells (set as 100 %) (Fig. 5) and the GI-GPx values compared with the maximum expression of the transduced GI-GPx cDNA obtained seven days post infection (set as 100%) (Fig. 6).
  • the data show a drastic down-regulation of the HCV protein NS5a in all replicon cell lines infected with the GI-GPx+3'UTR virus (Fig. 5). Loading efficiency and integrity of proteins was controlled with a tubulin antibody (data no shown). The values depicted ( ⁇ SEM) are obtained from three independent experiments.
  • HuH-pcDNA3, HuH-9-13, HuH-5-15 and HuH-11-7 cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FCS (fetal calf serum), 2 mM Glutamine, Penicillin (100 lU/ml) / Streptomycin (100 ⁇ g/ml) and 1x nonessential amino acids in the presence of 1 mg/ml G-418. Cells were routinely passaged three times a week at a dilution of 1 :3 or 1 :2.
  • HuH-pcDNA3, HuH-9-13, HuH-5-15 and HuH-11-7 cells were seeded at 5 x 10 5 cells per 10 cm plate in medium without G-148.
  • the medium was changed 3 days after plating and cells were harvested 5 days after plating by lyzing the cells directly on the plate with 4 ml of Tri-reagent (Molecular Research Center, Inc., USA).
  • the lysates were stored at room temperature for 5 minutes and then centrifuged at 12000xg for 15 minutes at 4°C. The supernatant was mixed with 0,1 ml of 1-bromo-3-chloropropane per 1 ml of Tri reagent and vigorously shaken.
  • the suspension was stored for 5 minutes at room temperature and then centrifuged at 12000xg for 15 minutes at 4°C.
  • the colorless upper phase was transferred into new tubes, mixed with 5 ⁇ l of polyacryl-carrier (Molecular Research Center Inc., USA) and with 0.5 ml of isopropanol per 1 ml of Tri reagent and vigorously shaken.
  • the samples were stored at room temperature for 5 minutes and then centrifuged at 12000xg for 8 minutes at 4°C.
  • the supernatant was removed and the RNA pellet washed twice with 1 ml of 75% ethanol.
  • the pellet was dried and resuspended in 25 ⁇ l of RNase-free buffer per initial 1 ml lysate.
  • RNA was transcribed into a cDNA-probe in the presence of radioactively labeled dATP. 6 ⁇ g of total RNA was labeled with 100 ⁇ Ci [ 33 P]-dATP (Amersham, UK) according to the protocol provided by Clontech. Subsequently, the reaction was stopped by adding 5 ⁇ l 0.5M EDTA (ethylene diamine tetraacetate) and 25 ⁇ l 0.6M NaOH and incubation for 30 minutes at 68°C.
  • EDTA ethylene diamine tetraacetate
  • the probe was precipitated by centrifugation for 15 minutes at 12000xg after 4 ⁇ l of 5M NaCl, 1 ⁇ l poly-acryl-carrier (Molecular Research Centre, Inc., USA) and 250 ⁇ l ethanol were added. The supernatant was discarded and the pellet dried before starting with the hybridisation.
  • the pellet was resuspended in 10 ⁇ l Cot-1 DNA (1 ⁇ g/ ⁇ l, Roche Diagnostics, Germany), 10 ⁇ l yeast tRNA (1 ⁇ g/ ⁇ l Sigma, USA) and 10 ⁇ l polyA (1 ⁇ g/ ⁇ l, Roche Diagnostics, Germany).
  • Herring sperm DNA was added to a final concentration of 100 ⁇ g/ml and the volume was filled up to 100 ⁇ l with 5 ⁇ l 10% SDS (Sodiumdodecylsulfat), 25 ⁇ l 20x SSC (3 M NaCl, 300 mM Sodium Citrate, pH 7.0) and bidestilled H 2 O.
  • SDS Sodiumdodecylsulfat
  • the mix was put on 95°C for 5 minutes, centrifuged for 30 seconds at 10OOOxg and vigorously shaken for 60 minutes at 65°C. A 1 ⁇ l aliquot of the probe was used to measure the incorporation of radioactive dATP with a scintillation counter. Probes with at least a total of 20x10 6 cpm were used. The arrays were prehybridised for at least 3 hours at 65°C in hybridisation solution in a roller bottle oven. After prehybridisation the radioactively labeled probe was added into the hybridisation solution and hybridisation was continued for 20 hours. The probe was discarded and replaced with wash solution A (2xSSC). The arrays were washed twice in wash solution A at room temperature in the roller oven.
  • wash solution A was replaced by wash solution B (2x SSC, 0.5% SDS) preheated to 65°C and arrays were washed twice for 30 minutes at 65°C.
  • wash solution B was replaced by wash solution C (0.5x SSC, 0.5% SDS) preheated to 65°C and arrays were washed twice for 30 minutes at 65°C.
  • the moist arrays were wrapped in airtight bags and exposed for 8 to 72 hours on erased phospho-imager screens (Fujifilm, Japan). 6. Analysis of cDNA-arrays
  • the exposed phospho-imager screens were scanned with a resolution of 100 ⁇ m and 16bits per pixel using a BAS-1800 (Fujifilm, Japan). Files were imported into the computer program ArrayVision (Imaging Research, Canada). Using the program's features, the hybridisation signals of each target cDNA were converted into numbers. The strength of the hybridisation signals reflected the quantity of RNA molecules present in the probe. Differentially expressed genes were selected according to the ratio of their signal strength after normalization to the overall intensity of the arrays.
  • RNA of each cell line was separated in a 1.2% agarose-formaldehyde gel, transferred on nylon membrane (Amersham) and hybridized with two different oligo-desoxyribonucleotides.
  • Their sequences were derived from the coding (5'- TGG TTG GGA AGG TGC GGC TGT AGC GTC GGA AGG GC-3') and 3'- untranslated (5'-CCT CTC AGA CAC CAC CCA TGA GGG TTT AGG AAG GTG CCA T-3') region of the human gastrointestinal glutathione peroxidase (P18283) gene.
  • Labeling was performed by 3'-end tailing with 32 P-dCTP and terminal transferase (Roche Diagnostics GmbH, Mannheim, Germany). Northern Blotting membranes were hybridized with the labeled oligonucleotides over night at 65°C and unspecifically bound probe washed away.
  • Terf-butyl hydroperoxide was used as substrate and GI-GPx activity was estimated indirectly.
  • Oxidized glutathione produced upon reduction of the peroxide by GI-GPx, is recycled to its reduced state by glutathione reductase by oxidation of NADPH+H + to NADP + .
  • the oxidation of NADPH is accompanied by a decrease in absorbance at 340 nm (A 340 ), which provides a spectra photo metric means of monitoring GI-GPx activity.
  • a 340 absorbance at 340 nm
  • the rate of decrease in the A 3 o (delta E in Fig. 2) is directly proportional to the GI-GPx activity in the sample.
  • Replicon cell lines and control cells were incubated for 24 hours with various concentrations of paraquat (methylviologen) and viability of the cultures were measured using the Alamar Blue assay.
  • the viability assay based on the reduction of tetrazolium salt to formazan by mitochondrial dehydrogenase activity.
  • the assay was performed in 96-well microtiter plates (Greiner, Frickenhausen, Germany) as described previously (T. Herget et al., J. Neurochem. 1998, 70, 47-58) but Alamar Blue (Roche Molecular Biochemicals, Germany) was used instead of MTT.
  • the light absorbance at 405 nm of the medium including all factors but without cells was determined and subtracted from the absorption readings with cells. Eight wells per sample point were analyzed and each experiment was repeated independently at least three times.
  • Replicon cells were incubated with interferon ⁇ (IFN) for two and four days.
  • IFN interferon ⁇
  • Northern blot analyses were performed with 10 ⁇ g total RNA.
  • the IFN treatment 1000 U/ml
  • GI-GPx was up-regulated within four days of interferon treatment.
  • Interferon had no effect on the expression of GI-GPx in mock transfected HuH7 cells (cf. Fig. 4).
  • the cDNA coding for the GI-GPx was cloned by RT-PCR from HuH7 cells. Transient expression of the GI-GPx protein in HEK293 cells caused an increase of glutathione peroxidase activity demonstrating its functionality. The construct was recombined into the genome of Adeno virus. Adeno virus carrying the GI-GPx cDNA was produced and used for transduction of the GI-GPx cDNA into HuH7 and replicon cells. Western blot analyses performed 4 and 7 days after transfection showed a drastic down-regulation of the HCV protein NS5a. Such a down-regulation was not observed when 'empty' or the GFP (green fluorescent protein) gene-containing Adeno virus was used in parallel (cf. Fig. 5 and 6).
  • the adenovirus used here were all E1 , E3 defective derivatives of adenovirus type 5 (W.C. Russell, J. Gen. Virol. 2000, 81 , 2573-2604).
  • the coding region for GI- GPx (0.7 kb) was amplified by PCR using an upstream primer containing an HindlU recognition site (5'-GCG CAA GCT TAT GGC TTT CAT TGC CAA GTC CTT C-3', start codon in italic) and a downstream primer containing an Xbal site (5'-GTT CAT CTA GAT ATG GCA ACT TTA AGG AGG CGC TTG-3') but without stop-codon to allow expression of fusion proteins with HIS- and VSV-tag.
  • RNA isolated from HuH7 cells were reverse transcribed and used as template for PCR.
  • the cDNA coding for human GI-GPx was cloned into the transfer plasmid (pPM7) between the CMV (cytomegalo virus) immediately early promoter/enhancer and the rabbit beta-globin intron/polyadenylation signal.
  • This expression cassette was inserted into a bacterial plasmid bome-adenovirus genome using recombination in bacteria (C. Chartier et al., J. Virol. 1996, 70, 4805-4810).
  • a cloned version of the novel genome was identified, the viral genome was released from the plasmid by restriction enzyme digestion and virus replication was initiated by transfecting the genome into HEK 293 cells using a modified PEI transfection method (A.-l.

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Abstract

L'invention concerne une peroxydase-gastrointestinale de glutathione de protéine cellulaire humaine comme cibles potentielles pour une intervention médicale contre les infections dues au virus de l'hépatite C (HCV). De plus, la présente invention concerne un procédé de détection de composés servant dans la prophylaxie et/ou le traitement des infections dues au virus de l'hépatite C et un procédé de détection d'anticorps des infections dues au virus de l'hépatite C chez un individu ou dans des cellules. L'invention concerne également des anticorps monoclonaux ou polyclonaux efficaces dans le traitement des infections dues au virus de l'hépatite C ainsi que des procédés de traitement des infections dues au virus de l'hépatite C ou afin de réguler la production du virus de l'hépatite dans lesquels on peut utiliser lesdits anticorps.
PCT/EP2002/004167 2001-04-13 2002-04-15 Peroxydase de glutathione gastro-intestinale de proteine cellulaire humaine comme cible pour une intervention medicale contre les infections dues au virus de l'hepatite c WO2002084294A2 (fr)

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EP02730159A EP1377833A2 (fr) 2001-04-13 2002-04-15 Peroxydase de glutathione gastro-intestinale de proteine cellulaire humaine comme cible pour une intervention medicale contre les infections dues au virus de l'hepatite c
JP2002581997A JP2004533822A (ja) 2001-04-13 2002-04-15 Ｃ型肝炎感染に対する医学的介入のためのターゲットとしてのヒト細胞蛋白質胃腸グルタチオンペルオキシダーゼ
CA002443525A CA2443525A1 (fr) 2001-04-13 2002-04-15 Peroxydase de glutathione gastro-intestinale de proteine cellulaire humaine comme cible pour une intervention medicale contre les infections dues au virus de l'hepatite c
AU2002302535A AU2002302535A1 (en) 2001-04-13 2002-04-15 Gastrointestinal glutathione peroxidase in hepatitis c virus infections
US10/342,054 US20030180719A1 (en) 2001-04-13 2003-01-14 Human cellular protein gastrointestinal glutathione peroxidase as target for medical intervention against hepatitis C virus infections
US10/723,719 US7341717B2 (en) 2001-04-13 2003-11-26 Therapeutic targets for treatment of HCV infections, methods of treating HCV infections and compounds useful therefor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005457A2 (fr) * 2002-07-08 2004-01-15 Kylix B.V. Utilisation de genes cibles specifies tcf permettant d'identifier des medicaments pour le traitement du cancer, plus particulierement du cancer colorectal, la signalisation tcf/$g(b)-catenine-wnt jouant un role fondamental
WO2004005457A3 (fr) * 2002-07-08 2005-02-17 Kylix B V Utilisation de genes cibles specifies tcf permettant d'identifier des medicaments pour le traitement du cancer, plus particulierement du cancer colorectal, la signalisation tcf/$g(b)-catenine-wnt jouant un role fondamental
WO2004050101A2 (fr) * 2002-11-29 2004-06-17 Gpc Biotech Ag Formulations utiles pour lutter contre les infections par le virus de l'hepatite c
WO2004050101A3 (fr) * 2002-11-29 2004-09-10 Axxima Pharmaceuticals Ag Formulations utiles pour lutter contre les infections par le virus de l'hepatite c
JP2006514094A (ja) * 2002-11-29 2006-04-27 ゲーペーツェー バイオテック アクチェンゲゼルシャフト Ｃ型肝炎ウイルス感染に対して有用な製剤
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions

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