The present invention relates to the human cellular protein glutathione peroxidase-gastrointestinal as a target 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 compositions, compounds, nucleic acid molecules (such as aptamers), mono- or polyclonal antibodies are disclosed which are effective for the treatment of HCV infections, and methods for prophylaxis and/or treatment of Hepatitis C virus infections or for the regulation of Hepatitis C virus production are disclosed.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based upon the surprising discovery that the human cellular protein gastrointestinal glutathione peroxidase (P18283) is specifically downregulated as a result of HCV replication in HCV infected host cells. The antiviral therapeutic and/or prophylactic research approach described herein focuses on discovering the cellular signal transduction pathways involved in viral infections. Identification of the signal transduction molecules that are key to viral infection provides for, among other things, novel diagnostic methods, for example, assays and compositions useful therefor, 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.

In one aspect, the present invention is directed to a method for detecting compounds useful for the prophylaxis and/or treatment of Hepatitis C virus infections comprising the steps of contacting a test compound with human cellular protein gastronintestinal glutathione peroxidase and detecting gastrointestinal glutathione peroxidase activity.

In another aspect, the present invention is directed to a method for detecting Hepatitis C virus infections in an individual comprising: a) providing a sample from said individual; and b) detecting activity in the sample of gastrointestinal glutathione peroxidase.

In another aspect, the present invention is directed to a method for detecting Hepatitis C virus infections in cells, cell cultures, or cell lysates, comprising: a) providing the cells, cell cultures, or cell lysates; and b) detecting activity in said cells, cell cultures, or cell lysates of human cellular protein gastrointestinal glutathione peroxidase.

In another aspect, the present invention is directed to a method for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection 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 another aspect, the present invention is directed to a method for regulating the production of Hepatitis C virus in an individual, cells, cell culture, or cell lysates comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent inhibits at least partially the activity of human cellular protein gastrointestinal glutathione peroxidase or wherein said agent at least partially inhibits the production of human cellular protein gastrointestinal glutathione peroxidase.

Accordingly, as disclosed in the present application, agents with an inhibitory activity for gastrointestinal glutathione peroxidase include, but are not limited to, monoclonal or polyclonal antibodies that bind to GI-GPx.

In yet another aspect, the present invention is directed to methods 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 the activity of human cellular protein gastrointestinal glutathione peroxidase or which activates or stimulates at least partially the production of human cellular protein gastrointestinal glutathione peroxidase.

In another aspect, the present invention is directed to a method for regulating the production of Hepatitis C virus in an individual, cells, cell culture, or cell lysates, comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent activates at least partially the activity of human cellular protein gastrointestinal glutathione peroxidase or wherein said agent at least partially activates or stimulates the production of the human cellular protein gastrointestinal glutathione peroxidase.

In still another aspect, the present invention is directed to a method for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual, cells, cell culture, or cell lysates, comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent inhibits at least partially the transcription of DNA and/or the translation of RNA encoding the human cellular protein gastrointestinal glutathione peroxidase.

Accordingly, as disclosed in the present application, agents which inhibit the transcription of DNA and/or the translation of RNA include, but are not limited to, oligonucleotides that bind the DNA and/or RNA coding for GI-GPx. Such oligonucleotides may be aptamers or antisense nucleic acid molecules.

The present invention is also directed to a method for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual, cell, cell culture, or cell lysate, comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent activates at least partially the transcription of DNA and/or the translation of RNA encoding human cellular protein gastrointestinal glutathione peroxidase.

In addition, the present invention is directed to a method for regulating the activity of the human cellular protein gastrointestinal glutathione peroxidase in an individual, cell, cell culture, or cell lysate, comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent interacts with the human cellular protein gastrointestinal glutathione peroxidase.

In another aspect, the present invention is directed to a method for the selective killing of HCV infected cells in an individual, cells, cell culture, or cell lysate, comprising the step of administering a pharmaceutically effective amount of a radical initiator which is capable of generating artificial oxidative stress conditions within the cells.

In yet another aspect, the present invention is directed to a method for preventing and/or treating HCV infections in an individual by at least partially compensating for the down-regulation of GI-GPx comprising the step of administering a pharmaceutically effective amount of at least one antioxidant which is capable of supporting the function of GI-GPx present within the cells.

In still another aspect, the present invention is directed to a method for at least partially compensating for the down-regulation of GI-GPx in an individual, cells, cell culture, or cell lysates, comprising the step of administering a pharmaceutically effective amount of at least one antioxidant which is capable of supporting the function of GI-GPx present within the cells.

The present invention is also directed to a composition useful for the prophylaxis and/or treatment of Hepatitis C virus and/or diseases associated with HCV infection in an individual, said composition comprising at least one agent capable of inhibiting activity of human cellular protein gastrointestinal glutathione peroxidase or capable of decreasing the expression of human cellular protein gastrointestinal glutathione peroxidase.

The present invention is also directed to a composition useful for the regulation of GI-GPx activity in an individual, cells, cell culture, or cell lysates, said composition comprising at least one agent capable of inhibiting activity of human cellular protein gastrointestinal glutathione peroxidase or capable of decreasing the expression of human cellular protein gastrointestinal glutathione peroxidase.

In another aspect, the present invention is directed to a composition useful for the prophylaxis and/or treatment of Hepatitis C virus and/or diseases associated with HCV infection in an individual, said composition comprising at least one agent capable of increasing the activity of human cellular protein gastrointestinal glutathione peroxidase or capable of activating or stimulating the expression of human cellular protein gastrointestinal glutathione peroxidase.

The present invention is also directed to a composition useful for the regulation of GI-GPx activity in an individual, cells, cell culture, or cell lysates, said composition comprising at least one agent capable of increasing the activity of human cellular protein gastrointestinal glutathione peroxidase or capable of activating or stimulating the expression of human cellular protein gastrointestinal glutathione peroxidase.

Accordingly, as disclosed in the present application, specific chemical substances and compounds that can be used alone or in combination to upregulate and/or activate the human cellular protein gastrointestinal glutathione peroxidase include, but are not limited to, selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid. Additionally, alpha interferon and/or ribavirin may be included to maximize the antiviral effect.

Because retinoic acid and other retinoids are well known, persons skilled in the art will be aware of various other retinoids which may be useful in practicing the methods of the present invention. For example, additional retinoids suitable for use in the present invention are disclosed in U.S. Pat. Nos. 6,274,747; 6,326,397; and Pat. No. 6,403,554, incorporated herein by reference.

In order to develop new pharmaceutically active compounds, a potential target for medical intervention has to be identified. Thus, 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 with by a drug.

It is an 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, cells, cell cultures, or cell lysates together with methods for detecting HCV infections in individuals, cells, cell cultures and cell lysates, and methods for detecting compounds useful for prophylaxis and/or treatment of HCV infections. It is another object of the present invention to provide compounds, compositions and methods which are effective in the prophylaxis and/or treatment of Hepatitis C virus infections, but which lack the negative side-effects described above. A further object of the invention is to provide alternative, effective therapeutic treatments for HCV-infected patients, particularly patients who fail to respond to current anti-HCV combinatorial therapies, for example, patients who fail to successfully respond to interferon and ribavirin treatment. The object of the present invention is accomplished according to the teachings herein and the methods defined in the following independent claims. Further advantageous features, aspects and details of the invention are evident from the description, the examples, and the dependent claims of the present application.

DETAILED DESCRIPTION OF THE INVENTION

Recent research has revealed how cells communicate with each other to coordinate the growth and maintenance of the multitude of tissues within the human body. A key element of this communication network is the transmission of a signal from the exterior of a cell to its nucleus, which results in the activation or suppression of specific genes. This process is called signal transduction.

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 protein of T cells and macrophages), guanylate cyclases (e.g. natriuretic peptide receptors) and serine/threonine kinases (e.g. activin and TGF-beta receptors). Receptors with intrinsic tyrosine kinase activity are capable of autophosphorylation as well as phosphorylation of other substrates.

Additionally, several families of receptors lack intrinsic enzyme activity, yet are coupled to intracellular tyrosine kinases by direct protein-protein interactions. 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.

2. 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).

3. Receptors that are Found Intracellularly and that 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.

The antiviral therapeutic and/or prophylactic research approach described herein focused on discovering the cellular signal transduction pathways involved in Hepatitis C viral infections. Identification of the signal transduction molecules that are key to HCV infection and persistence provides for, inter alia, novel targets for HCV antiviral therapeutics, novel classes of HCV antiviral therapeutics, and new screening methods (e.g., assays), and materials to discover new antiviral agents, and novel HCV diagnostic methods.

It is now revealed for the first time that the human cellular protein gastrointestinal glutathione peroxidase (GI-GPx) is specifically downregulated in a body as a result of HCV infection. This human cellular protein gastrointestinal glutathione peroxidase has been identified as a novel diagnostic and therapeutic target for HCV infection.

Glutathione Peroxidase:

Four distinct species of glutathione peroxidase have been identified in mammals to date, the classical cellular enzyme, the phospholipid hydroperoxide metabolizing enzyme, the gastroinestinal tract enzyme, and the extracellular plasma enzyme. Their primary structures are poorly related. It has been shown that they are encoded by different genes and have different enzymatic properties. The physiological role of the human plasma enzyme remains still unclear due to the low levels of reduced glutathione in human plasma and the low reactivity of this enzyme.

The human cellular protein glutathione peroxidase-gastrointestinal (GI-GPx) is also known as gastrointestinal glutathione peroxidase, 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.

GI-GPx catalyzes the reduction of various organic hydroperoxides, as well as hydrogen peroxide, with glutathione (GSH) as hydrogen donor (2GSH+H.sub.2O.sub.2.fwdarw.GS-GS+2H.sub.2O). 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 as a mechanism of protecting the cellular membrane system against peroxidative damage and that selenium as an essential trace element, may play an important role in this suggested function of the enzyme. It is known that both vitamin E and selenium (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. Of these, cellular and plasma glutathione peroxidase are the functional parameters used for the assessment of selenium status (Holben and Smith, 1999, J. Am. Diet. Assoc., 99:836-843).

In addition to vitamin E (DL-.alpha.-tocopherol), vitamin C (L-ascorbic acid), co-enzyme Q10, zinc, and selenium, many other antioxidants such as N-acetyl-L-cycteine, N-acetyl-S-farnesyl-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, .alpha.-lipoic acid, Luteolin, Manoalide, MCI-186, MnTBAP, MnTMPyP, morin hydrate, NCO-700, NDGA, p-Nitroblue, propyl gallate, Resveratrol, rutin, silymarin, L-stepholidine, taxifolin, tetrandrine, tocopherol acetate, tocotrienol, Trolox.RTM., U-74389G, U-83836E, and uric acid (all available from Calbiochem, San Diego, Calif., U.S.A.) can be applied within the disclosed methods for preventing and/or treating HCV infections by compensating at least partially for the down-regulation of GI-GPx.

Additional 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 (tert-butyl hydroquinone), tocopherols, lecithin, gums and resin guiac, THBP (trihydroxybutyrophenone), thiodipropionic acid and dilauryl thiodipropionate, and glycines.

Oxidative damage is mainly caused by free radicals, particularly reactive oxygen intermediates, derived from normal cellular respiration and oxidative burst produced when phagocytic cells destroy bacteria or virus-infected cells. In order to cope with the constant generation of potentially damaging oxygen radicals, eukaryotic organisms have evolved many defense mechanisms. These include the above-mentioned antioxidants which act as free radical 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 antioxidants 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 Hepatitis 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., 81: 1631; Antiviral Chemistry and Chemotherapy, 10: 99). For this reason, has been extremely difficult to study how HCV infects cells and to test anti-viral drugs in a model system (the only animals that can be infected are humans and chimpanzees). A major step in devising a culture system for HCV was established by the replicon cell lines (see, Lohmann et al., Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line, Science, 285: 110-113 (1999)). Replication of subgenomic HCV RNAs in cultured hepatocytes were obtained for the first time. These subgenomic replicons are composed of only the part of the HCV genome that encodes the non-structural proteins but are competent to be replicated in cells and to synthesize viral proteins. The replicons described in the scientific article of Lohmann et al., supra, and used in making the discoveries disclosed herein allows studies of HCV replication, pathogenesis and evolution in cell culture. They may also allow for cell-based testing of certain types of anti-viral drugs.

Thus, the replicon system described by Lohmann et al., supra, reproduces a crucial part of the HCV replication cycle which is used as a system for simulating HCV infection. Lohmann et al. 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. After transfection of the subgenomic HCV RNA molecules into the human hepatoma cell line HuH7, cells supporting efficient RNA-dependent RNA replication of the HCV replicons were selected based on co-amplification of the NPT gene and resulting resistance to the antibiotic G-418. Integration of coding information into the cellular genome was an exclusion criteria for functional replicons. Several lines were established from G-418 resistant clones with autonomously replicating HCV RNAs detectable by Northern Blotting. Minus-strand RNA replication intermediates were detected by Northern Blotting or metabolic radio-labeling, and the production of nonstructural HCV proteins was demonstrated by immuno-precipitation after metabolic labeling or Western Blotting.

Possible influences and/or dependencies of HCV's RNA-dependent RNA replication and nonstructural proteins on host cell transcription are accessible to analysis with the Clontech cDNA arrays used in the inventive methods described herein. HuH-pcDNA3 cells are HuH7 cells resistant to G-418 by integration of a NPT gene-carrying plasmid (pcDNA3, Invitrogen) and serve as a negative control. Three replicon lines were analyzed for changes in cellular RNA expression patterns compared to the control line: HuH-9-13: a cell line with persistant replicon I377/NS3-3'/wt, described by Lohmann et al., supra, HuH-5-15: a cell line with persistant replicon I389/NS3-3'/wt, described by Lohmann et al., supra, HuH-11-7: a cell line with persistant replicon I377/NS2-3'/wt, described by Lohmann et al., supra.

These 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.

Based on the surprising results reported herein, one aspect of the present invention is directed to a screening method for detecting compounds useful for the prophylaxis and/or treatment of Hepatitis C virus infections. Specifically, this method involves contacting a test compound with GI-GPx and detecting the GI-GPx activity. Such methods are advantageously carried out using cell-based techniques, where GI-GPx activity (e.g., GI-GPx transcription) can be measured (e.g., by Northern blotting, see Example 14, infra). Test compounds that enhance the activity of GI-GPx are identified as compounds for treating HCV infections. Alternatively, inhibitors of GI-GPx activity may be identified in this manner and may be used to promote the selective killing of HCV infected cells, in which GI-GPx is already downregulated.

Another aspect of the present invention is directed to specific chemical substances and compounds, which, alone or in combination, are useful for the prophylaxis and/or treatment of Hepatitis C virus infections. Specifically, these chemical substances and compounds comprise selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid.

An additional aspect of the present invention is directed to the use of a combination of compounds for the prophylaxis and/or treatment of Hepatitis C virus infection. Such combinations preferably include, but are not limited to, one or more selenium compounds, especially selenium and selenium salts, in combination with one or more retinoid compounds, especially retinoic acid, including all-trans-retinoic acid, 9-cis retinoic acid and/or 13-cis retinoic acid. Also contemplated are combination therapies which include alpha interferon and/or ribavirin in combination with selenium compounds and retinoids. Particularly preferred combination therapeutics include alpha interferon in combination with selenium or selenium salt(s) also in combination with retinoic acid (most preferably all-trans-retinoic acid, 9-cis retinoic acid and/or 13-cis retinoic acid).

Another aspect of the present invention is directed to a diagnostic method, for example, an assay for detecting Hepatitis C virus infections in an individual or in cells. This method involves providing a sample from the individual or providing cells and detecting activity of GI-GPx.

Suitable samples for such methods include, for instance, blood, biopsies, cells, cell cultures, or cell lysates of liver or of any other tissue infected with HCV.

Accordingly, 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.

In addition, 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.

Furthermore, 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 at least one of the specific chemical compounds and substances referred to above, which upregulates at least partially the activity of GI-GPx or which upregulates 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 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.

Another 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 an individual, comprising the step of administering a pharmaceutically effective amount of at least one of the specific chemical compounds and substances referred to above, which upregulates at least partially the activity of GI-GPx or which upregulates 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, cell cultures, or cell lysates comprising the step of administering a pharmaceutically effective amount of an agent wherein said agent inhibits at least partially the activity of 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.

Another aspect of the present invention is to provide a method for regulating the production of Hepatitis C virus in an individual or in cells, cell cultures, or cell lysates comprising the step of administering a pharmaceutically effective amount of at least one of the specific chemical compounds and substances referred to above, which at least partially upregulate the activity GI-GPx or which at least partially upregulate the production of GI-GPx in the cells.

In addition to the above-mentioned 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 of the individual.

In addition to the above-mentioned 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 at least one of the specific chemical compounds and substances referred to above, which activates at least partially GI-GPx or which activates or stimulates the production of GI-GPx in the individual.

Another inventive aspect of the present invention is related 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 the activity of GI-GPx or which activates or stimulates at least partially the production of GI-GPx.

In addition, the present invention is related to a method for regulating the effects of Hepatitis C virus infection and/or diseases associated with HCV infection in cells, cell cultures, or cell lysates comprising the step of administering a pharmaceutically effective amount of at least one of the specific chemical compounds and substances referred to above, which activates at least partially the activity of GI-GPx or which activate or stimulate at least partially the production of GI-GPx.

The function of GI-GPx is to detoxify peroxides in cells and prevent the cells from oxidative damage. As demonstrated in FIG. 3 (see Original Patent), subjecting HCV infected cells to oxidative stress conditions, preferably induced by paraquat or radicals generated from peroxides, leads to a decreased resistance of HCV infected cells in comparison to uninfected cells against toxicity of radicals. Thus, generating artificial oxidative stress conditions allows selective killing of HCV-infected cells.

Examples for useful radical forming compounds (radical initiators) are 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 dibenzoylperoxide, diacetylperoxide, hydrogen peroxide, di-tert-butylperoxide, or diaza compounds such as diazaisobutyronitril.

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.

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 of the specific chemical substances and compounds, alone or in combination, selected from the group consisting of selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid.

A preferred selenium salt is sodium selenite. Moreover, according to a further preferred aspect of the present invention, the composition may contain a certain amount of all-trans-retinoic acid.

Further embodiments of the present invention are represented by methods for regulating the production of Hepatitis C virus in an individual or in cells, cell cultures, or cell lysates comprising the step of administering to an individual or the cells, a pharmaceutically effective amount of an agent wherein said agent activates or increases at least partially the activity of said human cellular protein gastrointestinal glutathione peroxidase or wherein said agent at least partially activates or stimulates the production of said human cellular protein gastrointestinal glutathione peroxidase.

Agents useful for this method include, but are not limited to, specific chemical substances and compounds, alone or in combination, selected from the group consisting of selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid, wherein said substances or compounds activate or increase at least partially the activity of said human cellular protein gastrointestinal glutathione peroxidase or wherein said substances or compounds at least partially activate or stimulate the production of said human cellular protein gastrointestinal glutathione peroxidase.

Further aspects of the present invention relate to methods either for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual or in cells, cell culture, or cell lysates comprising the step of administering to either to the individual or the cells, cell culture or cell lysates, 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.

According to the above-mentioned method another aspect of the present invention is directed to novel therapeutic 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.

Agents useful in said compositions include, but are not limited to at least one of the specific chemical substances and compounds, alone or in combination, selected from the group consisting of selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid, wherein said substances and compounds are capable of increasing the activity of GI-GPx or of activating or stimulating the production and/or expression of GI-GPx.

Said pharmaceutical compositions may further comprise pharmaceutically acceptable carriers, excipients, and/or diluents.

Further aspects of the present invention relate to methods either for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual or in cells, cell cultures, or cell lysates comprising the step of administering to either the individual or the cells, cell cultures, or cell lysates 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 gastrointestinal glutathione peroxidase.

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, percutaneous, rectal, subcutaneous, sublingual, topical or transdermal application, but are not limited to these ways of administration. For instance, preferred preparations according to the invention will be in a form which is suitable for oral administration. These orally administratable forms, for example, 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.

As used herein, 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, cell cultures, or cell lysates treated in vitro or in a mammalian subject such as a human patient treated in vivo. Specifically, 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 or activator 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. For example, if the inhibitor or activator 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 or activator is to be contacted with the cells, cell cultures, or cell lysates 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.

By way of illustration, a contemplated therapy according to the invention would entail administration of 1-100 mg/m.sup.2/day of an oral retinoid such as all-trans-retinoic acid, preferably 20-50 mg/m.sup.2/day, preferably administered in 1-4 doses/day (more preferably 1-3 doses daily, most preferably 2 doses daily). Advantageously, this retinoid administration is combined with interferon therapy, e.g., pegylated alpha interferon administered, e.g., 135-180 .mu.g per week by subcutaneous injection. In a preferred aspect of this invention, a selenium compound such as a selenium salt may be added to this combination therapy (e.g., one 30-50 .mu.g capsule daily).

It is also apparent to a person skilled in the art that detection includes any method known in the art useful to indicate the presence, absence, or amount of a detection target. Such 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, calorimetric, fluorescent, radioisotopic); spectroscopic techniques; separations technology, including precipitations, electrophoresis, chromatography, centrifugation, ultrafiltration, cell sorting; and enzymatic manipulations (e.g. digestion).

The present disclosure also teaches for the first time the downregulation of GI-GPx specifically involved in the viral infection of Hepatitis C virus. Thus, the present invention is also directed to a method useful for detecting novel compounds useful for prophylaxis and/or treatment of HCV infections.

The present disclosure teaches for the first time the upregulation of GI-GPx specifically involved in the viral infection of Hepatitis C virus using specific chemical compounds and substances, or combinations thereof, selected from the group consisting of selenium, selenium salts, Vitamin D.sub.3, and retinoids. Particularly preferred retinoids include all forms of retinoic acid, including, but not limited to, 9-cis retinoic acid, salts of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 9-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl esters of 13-cis retinoic acid, C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, salts of C.sub.1-C.sub.10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C.sub.1-C.sub.10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid.

The present invention provides methods to 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. A variety of 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.

In a related aspect, the present invention provides, in view of the important role of GI-GPx in the HCV infection and/or replication process, an assay component especially useful for detecting HCV in an individual, in cells, cell cultures, or cell lysates. Preferably the assay component comprises oligonucleotides immobilized on a solid support capable of detecting GI-GPx activity. Preferably 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).

Similarly, it is an object of the present invention to provide an assay component especially useful for screening compounds 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: U.S. Pat. Nos. 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752; 5,561,071; 5,559,895; 5,624,711; 5,639,603; 5,658,734; 5,807,522; and 6,087,102; international patent publications WO 93/17126; WO 95/11995; and WO 95/35505; European patent publications EP 742 287 and EP 799 897.

Suitable 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: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,994,076; 6,033,860; 6,040,138; 6,040,140; international patent publications WO 95/21265; WO 96/31622; WO 97/10365; and WO 97/27317; European patent publications EP 373 203 and EP 785 280.
 

Claim 1 of 10 Claims

1. A method for inhibiting or downregulating Hepatitis C viral replication in an individual comprising the step of administering to an individual a pharmaceutically effective amount of an agent wherein said agent activates the activity of said human cellular protein gastrointestinal glutathione peroxidase or wherein said agent activates or stimulates the production of said human cellular protein gastrointestinal glutathione peroxidase, and wherein said agent is a combination of (i) selenium, or a selenium salt, and (ii) a retinoid selected from the group of: 9-cis retinoic acid, salts of 9-cis retinoic acid, C1-C10 alkyl esters of 9-cis retinoic acid, salts of C1-C10 alkyl esters of 9-cis retinoic acid, C1-C10 alkyl amides of 9-cis retinoic acid, salts of C1-C10 alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C1-C10 alkyl esters of 13-cis retinoic acid, salts of C1-C10 alkyl esters of 13-cis retinoic acid, C1-C10 alkyl amides of 13-cis retinoic acid, salts of C1-C10 alkyl amides of 13-cis retinoic acid, retinol, retinoic acid adlehyde, etretinate, N-(4-hydroxyphenyl) retinamide (4-HPR), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), all-trans-retinoic acid, C1-C10 esters and amides of all-trans-retinoic acid, paraquat, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl- ]benzoic acid, 4-hydroxyphenylretinamide, and 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]ben- zoic acid.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.