WO2005120479A1

WO2005120479A1 - Use of selenium or a selenium salt and a retinoid acid or a retinoid in the treatment of viral hepatitis c

Info

Publication number: WO2005120479A1
Application number: PCT/EP2005/006226
Authority: WO
Inventors: Thomas Herget; Bert Klebl
Original assignee: Gpc Biotech Ag
Priority date: 2004-06-09
Filing date: 2005-06-09
Publication date: 2005-12-22

Abstract

The present invention relates to combination therapies comprising at least one retinoid or retinoid agonist together with selenium or a selenium salt particularly useful in conjunction with conventional antiviral therapeutics which are synergistically effective against Hepatitis C virus (HCV) infections. In particular, the present invention relates to the synergism between compounds capable of activating or upregulating the gastrointestinal form of glutathione peroxidase for prophylaxis and/or treatment of HCV infections, administered in combination therapies with interferons. The combinations disclosed have proven surprisingly effective even in patients unresponsive to interferon/ribavirin therapies.

Description

USE OF SELENIUM OR A SELENIUM SALT AND A RETINOID ACID OR A RETINOID IN THE TREATMENT OF VIRAL HEPATITIS C

Field of the Invention [0001] The present invention relates to combination therapies comprising at least one retinoid or retinoid agonist together with selenium or a selenium salt particularly useful in conjunction with conventional antiviral therapeutics which are synergistically effective against Hepatitis C virus (HCV) infections. In particular, the present invention relates to the synergism between compounds capable of activating or upregulating the gastrointestinal form of glutathione peroxidase for prophylaxis and/or treatment of HCV infections, administered in combination therapies with interferons.

Background of the Invention [0002] Five distinct human hepatitis viruses have been identified. The hepatitis A virus and hepatitis E virus are enterically transmitted RNA viruses that do not cause chronic liver disease. In contrast, the hepatitis B virus, hepatitis C virus and hepatitis D virus (HBV, HCV and HDV, respectively) are parenterally transmitted and cause chronic infection. They are dangerous contaminants of the blood supply. [0003] Hepatitis C Virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. The World Health Organization (WHO) estimates that approximately 3% of the world population, or 170 million people, have been infected with the Hepatitis C Virus. In the United States, an estimated 3.9 million Americans have been infected (CDC fact sheet Sept. 2000). Over 80% of HCV-infected individuals develop chronic hepatitis, which is associated with disease states ranging from asymptomatic carrier states to repeated inflammation of the liver and serious chronic liver disease. Over the course of 20 years, more than 20% of chronic HCV-patients are expected to be at risk to develop cirrhosis or progress to hepatocellular carcinoma. Liver failure from chronic hepatitis C is the leading indicator for liver transplantation. [0004] 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. Though comparatively inefficient, there is also a sexual 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.

[0005] Despite the significant decline in incidence in the 1990's, the number of deaths (estimated deaths annually at the moment: 8000 to 10,000 in U.S.) and severe disease due to HCV is anticipated to triple in the next 10 to 20 years (sources: CDC fact sheet Dec 2000; Houghton M. Hepatitis C Viruses. In BN Fields, DM Knipe, PM Howley (eds.) Fields Virology. 1996. Lippencott-Raven Pub., Philadelphia; Rosen HR and Gretch DR, Molecular Medicine Today Vol 5, 393, 1999; Science 285, 26, 1999: News Focus: The scientific challenge of Hepatitis C; Wong JB et al, Am J Public Health, 90, 1562, 2000: Estimating future hepatitis C morbidity, mortality, and costs in the United States).

[0006] Combination therapy of alpha interferon and ribavirin is currently the recommended treatment for naive patients. Monotherapy with interferon has also been approved by the FDA, but the sustained response rate (as defined by HCV RNA remaining undetectable in the serum for more than 6 months after end of therapy) is only 15 to 20%, in contrast to 35 to 45% with combination therapy. Interferons (Intron A^®, Schering-Plough; Roferon A^®, Hoffmann-LaRoche; Wellferon , Glaxo Wellcome; Infergen , Amgen) are injected subcutaneously three times a week, ribavirin (Rebetol^® Schering-Plough) is an oral drug given twice a day. Recommended treatment duration is 6 to 12 months, depending on HCV genotype. Experimental forms of slow- release pegylated interferons (Pegasys^®, Hoffmann-LaRoche; PEG-Intron^®, 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, Oct 2000; NEJM 343, 1673, 2000; NEJM 343, 1666, 2000). Common side effects of interferon therapy include fatigue, muscle aches, head aches, nausea, fever, weight loss, irritability, depression, bone marrow suppression, and 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). Some of the 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. Moreover, treatment of Hepatitis C virus infection with interferon-alpha is effective in only a minority of individuals. This suggests that the virus may use various tricks to be resistant to interferon.

[0007] Recent review articles by Vogel (Peginterferon-α_2a [40 kDa]/ribavirin combination for the treatment of chronic hepatitis C infection", Expert Rev. Anti-infect. Ther. 1 (3), 423-431, 2003) and Durantel et al., (Current and emerging therapeutic approaches to hepatitis C infection", Expert Rev. Anti-infect. Ther. 1 (3), 441-454, 2003) provide an overview of the current status of HCV therapy. According to these articles, current treatment involves association of two molecules, standard (i.e., non-pegylated) or pegylated interferons and ribavirin. Although this therapy induces a sustained response in 50 to 60 % of the cases, there are still a high number of so-called "non-responders" and treatment is often limited by the above-mentioned side effects. Particularly for non-responding patients, and more generally, for improving the current clinical practice, it is important to develop alternative and effective therapeutic approaches for HCV treatment.

[0008] It has been proposed that alternative interferons may be effective for HCV treatment, instead of alpha interferons that are currently approved for this indication. Candidate interferons include Actimmune (interferon gamma), Albuferon (interferon alpha fused to human serum albumin), interferon beta, interferon omega (Boehringer Ingelheim) and interferon-tau (PepGen Corp., WO0206343).

[0009] Experimental treatments that are not new forms of interferon are Maxamine (histamine dihydrochloride, Maxim Pharmaceuticals), which will be combined with Interferon in phase III studies, Merimepodib (VX-497, Vertex Pharmaceuticals), an IMP dehydrogenase inhibitor, in a triple combination with pegylated interferon and ribavirin in phase II, and amantadine (Endo Labs), an approved influenza drug, as the third component in triple therapy (phase IV). Inhibitors for HCV enzymes such as protease inhibitors, RNA dependent RNA polymerase inhibitors, helicase inhibitors as well as ribozymes and antisense RNAs are under preclinical development (e.g., Boehringer Ingelheim, Ribozyme Pharmaceuticals, Vertex Pharmaceuticals, Schering- Plough, Hoffmann-LaRoche, Immusol, Merck). 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).

[0010] WO 02/066022 Al to Aronex describes a method of treating hepatitis comprising administering to a subject in need of such treatment a therapeutically effective amount of retinoid such as all-trans retinoic acid. In particular embodiments, the form of hepatitis is Hepatitis A, B, C, D, E and G and the treatment is with liposomal all-trans retinoic acid.

[0011] In a recent publication (Retinoic acid enhances the antiviral effect of interferon on hepatitis C virus replication through increased expression of type I interferon receptor. Hamamoto et al., J. Lab. Clin. Med. 141, 2003, 58-66) it is asserted that treating human cells with all trans retinoic acid (ATRA) and 9-cis RA induces the expression of Interferon Type I receptor subunits, and thus reduces HCV infection.

[0012] US patent application 20020001615 discloses a method of treating cancer, especially renal cancer, by exposing cancer cells to a therapeutically effective amount of a composition which comprises at least one interferon and a where the retinoid is associated with a lipid carrier, preferably a liposomal formulation. The application does not teach or suggest the application of these methods to hepatitis.

[0013] WO 02/084294 discloses the human cellular protein gastrointestinal glutathione peroxidase as a novel target for medical intervention against Hepatitis C virus infections and provides methods of identifying suitable compounds to modulate the activity of this enzyme.

[0014] In summary, the available treatment for chronic Hepatitis C is expensive, effective only in a certain percentage of patients and adverse side effects are not uncommon. There remains an unmet medical need for suitable therapeutic methods that are safe, reliable, efficacious, and cost effective. Summary of the Invention [0015] The present invention provides novel compositions comprising highly effective combinations of antiviral therapeutics. The present invention is directed in particular to combination therapies having improved therapeutic outcome preferably with fewer adverse reactions and lower overall toxicity and morbidity. The present invention further provides methods of treatment and treatment protocols for administration of these combination therapies to an individual in need thereof. In particular, the compositions and treatment protocols of the present invention have proven unusually efficacious in the treatment of HCV.

[0016] Unexpectedly, it is now disclosed for the first time that retinoids act synergistically with conventional modes of antiviral treatment to suppress viral load and decrease detectable virus in HCV patients. Furthermore, it is now disclosed for the first time that retinoids act synergistically with conventional modes of antiviral treatment to provide sustained relief, even after shortened duration of treatment, compared to conventional therapies. Even more surprisingly, it was found that combined therapies comprising a retinoid and an interferon were shown to suppress detectable virus in HCV patients previously designated as non-responsive to conventional interferon and/or ribavirin therapy.

[0017] Without wishing to be bound by any particular theory or mechanism of action the clinical outcome obtained in patients with the combinations disclosed herein is unexpectedly superior to those achieved by currently available combination therapies. The present invention provides clinically beneficial combination therapies irrespective of the precise mechanism of action of the individual components. These therapeutic combinations have demonstrated synergistic efficacy that was not and could not have been anticipated.

[0018] In the specification and the claims that follow, the terms "non-responder(s) to interferon and/or ribavirin therapy" or "non responding patient(s) to interferon and/or ribavirin therapy" are used to denote the portion of the HCV infected individuals (particularly humans) who do not show a positive reaction or total cure when treated with pegylated or non-pegylated (standard) α- , β-, or γ-interferon alone (so-called interferon monotherapy), ribavirin alone (so-called ribavirin monotherapy), or a combination therapy of pegylated or non-pegylated (standard) α-, β-, or γ- interferon and ribavirin. Non-responders can be patients who are non responding to interferon and/or ribavirin treatment from the very beginning of a therapy, or who become non responding after a certain time of an interferon and/or ribavirin treatment. [0019] The present invention is based in part upon the fact that the human cellular protein gastrointestinal glutathione peroxidase (PI 8283) is specifically downregulated as a result of HCV replication in HCV infected host cells. Accordingly, the compositions of the invention comprise at least one agent that activates the enzyme glutathione peroxidase -gastrointestinal (also denoted herein as GI-GPx). It is now disclosed that these agents act synergistically in vivo with conventional antiviral therapies as exemplified by interferon alpha/ribavirin therapy conducted in human clinical trials.

[0020] According to a first aspect the present invention provides therapies comprising specific combinations of active pharmaceutical ingredients effective against HCV. According to another aspect the present invention provides methods of using these combinations for treating HCV.

[0021] According to one embodiment the compositions of the invention comprise at least one agent that upregulates or activates the enzyme glutathione peroxidase-gastrointestinal (also denoted herein as GI-GPx, assigned Accession No. PI 8283 and EC No. 1.11.1.9), in combination with one supplementary compound selected from selenium or a pharmaceutically acceptable selenium salt. This combination is particularly beneficial possibly due to the fact that GI-GPx is a selenoprotein, i.e., a protein that contains selenium in the form of selenocysteine. Nevertheless, it cannot be ruled out that the selenium is acting independently as an anti-oxidant apart from its role in selenoproteins.

[0022] According to particular embodiments the activator or regulator of GI-GPx is a retinoid. According to some embodiments the retinoid is selected from a retinoic acid, the latter term including all isomeric forms of retinoic acid, particularly all trans retinoic acid, salts of all trans retinoic acid, Ci - do alkyl esters of all trans retinoic acid, salts of Ci - C,o alkyl esters of all trans retinoic acid, d - Cio alkyl amides of all trans retinoic acid, salts of Q - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Q - Cio alkyl esters of 9- cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13- cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13- cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid. According to alternative embodiments the retinoid is retinol or a retinoid agonist. The term "retinoid" is intended to encompass any chemical substance capable of evoking a response mediated by a retinoic acid responsive element of the genomic material of a cell.

[0023] According to another embodiment the compositions of the invention further comprise at least one interferon. According to particular embodiments the interferon is selected from pegylated and non-pegylated (i.e., standard) α-, β-, and γ-interferons. According to preferred embodiments the term interferon refers to any interferon approved for use in humans currently or in the future. According to more preferred embodiments the term interferon refers to any interferon approved for treatment of hepatitis in humans currently or in the future. According to most preferred embodiments the term interferon refers to any interferon approved for treatment of hepatitis C in humans currently or in the future.

[0024] According to yet further embodiments the compositions of the invention further comprise at least one anti- viral agent approved for use in conjunction with interferon therapy. According to currently preferred embodiments the at least one anti-viral agent is ribavirin.

[0025] According to certain embodiments the individual components are administered in separate pharmaceutical compositions. According to other embodiments the individual components may be combined in a single treatment kit. According to alternative embodiments the individual components may be combined into a single pharmaceutical composition. The problems associated with formulating retinoids, which are highly lipophilic and highly susceptible to oxidation and light are well known in the art to which the invention pertains. In some embodiments the pharmaceutical compositions comprise lipid carrier particles comprising a retinoid, a lipid, and a triglyceride, wherein the composition is stable in an aqueous environment. These pharmaceutical compositions may be selected from liposomes, micelles, emulsions or microemulsions as are known in the art of pharmaceutical formulations.

[0026] According to certain embodiments, a composition of the present invention comprises a therapeutic treatment kit for the treatment of HCV comprising an interferon, a retinoid and a selenium compound and instructional materials for the combined use of said interferon, said retinoid and said selenium compound. In some embodiments instructional materials include such information as dosage, indication and contraindication and storage parameters. [0027] According to another aspect the present invention provides a method of inhibiting or preventing the replication of HCV in an individual infected with this virus comprising administering a therapeutically effective amount of a composition which comprises at least one agent that upregulates or activates the enzyme GI-GPx, in combination with one supplementary compound selected from selenium or a pharmaceutically acceptable selenium salt.

[0028] According to another aspect the present invention provides a method of inhibiting or preventing the growth of HCV in an individual infected with this virus comprising administering a synergistically effective amount of a composition which comprises at least one retinoid and at least one interferon, wherein said retinoid is associated with lipid carrier particles. In some embodiments the compositions comprising the lipid carrier particles comprise a retinoid, a lipid, and a triglyceride, wherein the composition is stable in an aqueous environment. These compositions may be selected from liposomes, micelles, emulsions or microemulsions as are known in the art of pharmaceutical formulations.

[0029] According to another aspect of the invention the methods of treatment provide protocols for administration of the individual components of the combined therapies at predetermined intervals, and for predetermined durations of treatment.

[0030] In specific embodiments the method of treatment comprises administering said retinoid composition in doses administered orally. In alternative embodiments the method of treatment comprises administering said retinoid composition parenterally. According to some embodiments, the retinoid composition is administered parenterally over a period of at least one- half hour, and, optionally, administering said retinoid composition is repeated at a frequency of about every other day or less frequently.

[0031] It is therefore the 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 do not show the negative side-effects described above or at least not to the extent reported for known products and methods. 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. Brief Description of the Figures

[0032] Figure 1. Treatment of human HCV replicon cells with ATRA resulted in suppression of HCV RNA and up-regulation of the selenocysteine protein gastrointestinal glutathione peroxidase (GI-GPx) gene.

[0033] Figure 2. Treatment of human HCV replicon cells with combinations of retinoic acid, sodium selenite and interferon showed additive effects of these agents in vitro.

[0034] Figure 3. Treatment of HCV patients with all trans retinoic acid monotherapy.

[0035] Figure 4. Treatment of HCV patients with all trans retinoic acid monotherapy.

[0036] Figure 5. Treatment of HCV patients with all trans retinoic acid monotherapy.

[0037] Figure 6. Treatment of HCV patients with all trans retinoic acid, selenium and pegylated interferon.

[0038] Figure 7. Treatment of HCV patients with all trans retinoic acid, selenium and pegylated interferon.

Detailed description of the invention

[0039] It has been shown previously that the human cellular protein glutathione peroxidase- gastrointestinal is specifically downregulated in replicon cells as a result of HCV infection. This human cellular protein glutathione peroxidase-gastrointestinal has been identified as a diagnostic and therapeutic target for dealing with HCV infection. The teachings of WO 02/084294 are incorporated by reference as if set forth fully herein.

[0040] The antiviral prophylactic and/or therapeutic approach described herein focuses on specific chemical substances and compounds that can be used to upregulate the human cellular protein glutathione peroxidase-gastrointestinal. These specific chemical substances and compounds are selenium, selenium salts, Vitamin D3, pegylated and non-pegylated (standard) α- , β-, and γ-interferon, ribavirin, and retinoids. According to particular embodiments the retinoid is selected from a retinoic acid, the latter term including all isomeric forms of retinoic acid, particularly all trans retinoic acid, salts of all trans retinoic acid, Q - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9- cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9- cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Q - Cio alkyl esters of 13-cis retinoic acid, salts of Q - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Q - Cio alkyl amides of 13-cis retinoic acid, as well as retinol or (E)-4-[2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB), (4- [5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N- (4-hydroxyphenyl) retinamide (4-HPR) and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2 -naphthalene carboxylic acid (CD437; AHPN).

[0041] Preferred is the use of all trans retinoic acid or 13-cis retinoic acid for the treatment of non-responders. Also preferred is the use of compositions comprising all trans retinoic acid or 13-cis retinoic acid with one of the chemical substances mentioned above for the treatment of HCV infections or HCV infection related diseases. Particularly, (i) all trans retinoic acid or 13- cis retinoic acid is used with selenium and/or selenium salts, (ii) all trans retinoic acid or 13-cis retinoic acid is used with pegylated and/or non-pegylated (standard) α-, β-, and γ-interferon, (iii) all trans retinoic acid or 13-cis retinoic acid is used with pegylated and/or non-pegylated (standard) α-, β-, and/or γ-interferon and ribavirin, (iv) all trans retinoic acid or 13-cis retinoic acid is used with pegylated and/or non-pegylated (standard) α-, β-, and/or γ-interferon and with selenium and/or a selenium salt, (v) all trans retinoic acid or 13-cis retinoic acid is used in combination with ribavirin and selenium and/or a selenium salt, (vi) all trans retinoic acid or 13- cis retinoic acid and ribavirin, and (vii) all trans retinoic acid or 13-cis retinoic acid, selenium and/or a selenium salt, an interferon and ribavirin. In case of combination (v), all trans retinoic acid or 13-cis retinoic acid is preferably used at a concentration of 0.1 to 10 μM, more preferably 0.5 to 2.5 μM, and particularly 1 μM, selenium or selenium salts are preferably used at a concentration of 1 to 200 nM, more preferably 10 to 100 nM, and particularly 50 nM, ribavirin is preferably used at a concentration of 1 to 500 μM, more preferably 10 to 100 μM, and particularly 50 μM. The above-mentioned combinations (i), (ii), (iii), (iv), and (v) can be used both for the treatment of responders and non-responders. [0042] In addition, the following compounds shall be encompassed by the term "retinoids" as used within the present application. In particular, the further retinoids as understood according to the present application also refer to retinol, etretinate, amides of the all-trans-retinoic acid or 13- cis-retinoic acid with 2-aminoethanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine containing phosphate groups. [0043] The structure of these further retinoids is covered by the following general formula: R-CONH-X-OPO(OH)₂, wherein R is selected from

and X is selected from -CH₂ -CH₂ - ; -CH(CO₂ H)— CH₂ - ; -CH(CO₂ H)-CH(CH₃)-; and -CH(CO₂ H)— CH₂ -C₆ H₅ -.

[0044] Thus the amino group of 2-aminoethanol or the alpha-amino group of amino acid forms an amide bond with the carboxylic group of all-trans-retinoic acid or 13-cis retinoic acid. At the same time the hydroxyl group of 2-aminoethanol and the amino acid is modified by a phosphate residue.

[0045] Retinoids falling under the further retinoids according to the present invention are e.g. described in US 6,326,397 Bl and US 6,403,554 B2, which are incorporated herein by reference in their entirety. In these two US patents, among other substances amides of all-trans-retinoic acid or 13-cis-retinoic acid with 2-aminoethanol, alpha-L-serine, alpha-L-threonine, alpha-L- tyrosine are disclosed. At the same time hydroxyl groups of amino acids and 2-aminoethanol are modified by phosphate residues. The all-trans-retinoic acid or 13-cis retinoic acid have been derived by various procedures from naturally-occurring products. It is however possible, within the scope of the present invention, to produce these compound synthetically. Examples how to synthesize the further retinoids according to the present invention are described in the above- mentioned patents US 6,326,397 Bl and US 6,403,554 B2.

[0046] The main characteristic among the synthesized compounds is the phosphorylation of the hydroxyl groups of N-acyl derivatives of amino acids and 2-aminoethanol.

[0047] Further retinoids (retinoic acid derivatives) according to the present invention include the following compounds:

1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol 1 a. N-(l 3-cis-retinoyl)-o-phospho-2-aminoethanol

2. N-(all-trans-retinoyl)-o-phospho-L-serine 2a. N-( 13 -cis-retinoyl)-o-phospho-L-serine

3. N-(all-trans-retinoyl)-o-phospho-L-threonine 3 a. N-( 13 -cis-retinoyl)-o-phospho-L-threonine

4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine 4a. N-(l 3-cis-retinoyl)-o-phospho-L-tyrosine

[0048] The structural formulas of the above-mentioned compounds are presented below:

[0049] As used herein the term "alkyl" means straight-chain, branched or cyclic alkyl residues, in particular those containing from 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, t-butyl, decyl, dodecyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. The term "lower alkyl" means alkyl groups containing from 1 to 7, preferably 1-4 carbon atoms. Most preferred lower alkyl groups are methyl and ethyl.

[0050] In the scope of the present invention, the "pharmaceutically acceptable salts" includes any salt chemically permissible in the art for retinoids and particularly retinoid agonists and applicable to human patients in a pharmaceutically acceptable preparation. Any such conventional pharmaceutically acceptable salt of retinoids or retinoid agonists can be utilized. Among the conventional salts which can be utilized, there are the base salts included, for example, alkali metal salts such as the sodium or potassium salt, alkaline earth metal salts such as the calcium or magnesium salt, and ammonium or alkyl ammonium salts.

Glutathione peroxidase:

[0051] Four distinct species of glutathione peroxidase have been identified in mammals to date, the classical cellular enzyme, the phospholipid hydroperoxide metabolizing enzyme, the gastrointestinal 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.

[0052] The human cellular protein gastrointestinal glutathione peroxidase (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 PI 8283 and the EC Number 1.11.1.9.

[0053] The human cellular protein gastrointestinal glutathione peroxidase (GI-GPx) catalyzes the reduction of various organic hydroperoxides, as well as hydrogen peroxide, with glutathione

(GSH) as hydrogen donor (2 GSH + H₂O₂ > GS— GS + 2 H₂O). It has a molecular weight of

84,000 and consists of 4 subunits. The enzyme is useful for enzymatic determination of lipid hydroperoxide. [0054] 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.

[0055] 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 (D. H. Holben, A. M. Smith, J. Am. Diet. Assoc. 1999, 99, 836-843).

[0056] Beside vitamin E (DL-α-tocopherol), vitamin C (L-ascorbic acid), co-enzyme Q10, zinc, and selenium a lot of further antioxidants such as N-acetyl-L-cysteine, 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, α-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^®, U- 74389G, U-83836E, and uric acid (all available from Calbiochem, San Diego, CA, U.S.A.) can be applied for preventing and/or treating HCV infections by compensating at least partially the down-regulation of GI-GPx.

[0057] Further 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 resins, THBP (trihydroxybutyrophenone), thiodipropionic acid and dilauryl thiodipropionate, and glycines. [0058] 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 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:

[0059] The only reliable experimental animal 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 JO, 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. After transfection of the subgenomic HCV RNA molecules into the human hepatoma cell line HuH-7, 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.

[0060] 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 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 negative control. Three replicon lines were analyzed for changes in cellular RNA expression patterns compared to the control line: ^■ HuH-9-13: cell line with persistent replicon IRES377/NS3-3'/wt, described in Science 1999, 285, 110-113, ^■ HuH-5-15: cell line with persistent replicon IRES389/NS3-37wt, described in Science 1999, 285, 110-113, ^■ HuH-11-7: cell line with persistent replicon IRES377/NS2-3'/wt, described in Science 1999, 285, 110-113.

[0061] 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 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.

[0062] Based on the surprising results reported herein, one aspect of the present invention is directed to specific chemical substances and compounds useful for the prophylaxis and/or treatment of Hepatitis C virus infections. Specifically, these specific chemical substances and compounds comprise a plurality of agents selected from selenium, selenium salts, Vitamin D₃, pegylated and non-pegylated (standard) α-, β-, and γ-interferon, ribavirin, and retinoids, particularly all forms of retinoic acid, all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, like 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Q - Cio alkyl esters of 9-cis retinoic acid, d - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid as well as retinol and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl-1-propenyl] benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphtalenyl) carboxamido] benzoic acid (AM-580), N-(4-hydroxyphenyl) retinamide (4-HPR) and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2 -naphthalene carboxylic acid (CD437; AHPN).

[0063] Furthermore, the present invention discloses a method for treating Hepatitis C virus infection in an individual. Preferably the individual is a non-responder to interferon and/or ribavirin therapy. The method comprises the step of administering a pharmaceutically effective amount a plurality of the specific chemical compounds and substances referred to above, which upregulate at least partially the activity of GI-GPx or which upregulate at least partially the production of GI- GPx in the cells.

[0064] A related 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 a plurality of the specific chemical compounds and substances referred to above, which upregulate at least partially the activity of GI-GPx or which upregulate at least partially the production of GI-GPx.

[0065] 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 or cell cultures comprising the step of administering a pharmaceutically effective amount of a plurality 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. Preferably, the individual is a non-responder to interferon and/or ribavirin therapy. [0066] 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 a plurality 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. Again, preferably the individual is a non-responder to interferon and/or ribavirin therapy.

[0067] 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 at least one of the specific chemical compounds and substances referred to above, which activate at least partially the activity of GI-GPx or which activate or stimulate at least partially the production of GI-GPx.

[0068] The term "associated diseases" refers to, for instance, opportunistic infections, liver cirrhosis, liver cancer, hepatocellular carcinoma, HIV or any other disease that is known to be associated with HCV infection.

Combinations with additional antiviral therapies

[0069] 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 a plurality of the specific chemical substances and compounds selected from the group consisting of selenium, selenium salts, Vitamin D₃, pegylated and non- pegylated (standard) α-, β-, and γ-interferons, ribavirin, and retinoids, particularly all isomeric forms of retinoic acid, like all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9- cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, as well as retinol, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4- hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN). A preferred selenium salt is selected from sodium selenate, sodium selenite, sodium selenide, and mixtures thereof. The preferred individual is a non-responder to interferon and/or ribavirin therapy.

[0070] Further embodiments of the present invention are represented by methods for regulating the production of Hepatitis C virus in an individual or in cells or cell cultures comprising the step of administering an individual or the cells a pharmaceutically effective amount of a plurality of the specific chemical substances and compounds selected from the group consisting of selenium, selenium salts, Vitamin D₃ and retinoids, including all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9- cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, retinol, 4-[E-2-(5,6,7,8- tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl)-l-propenyl]benzoic acid, and/or 4-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido benzoic acid, N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), wherein said substances or compounds activate or increase at least partially the activity of said human cellular protein gastrointestinal glutathione peroxidase or wherein said agents at least partially activate or stimulate the production of said human cellular protein gastrointestinal glutathione peroxidase. Preferably, the individual is a non-responder to interferon and/or ribavirin therapy.

[0071] 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 a plurality of the specific chemical substances and compounds selected from the group consisting of selenium, selenium salts, Vitamin D₃ and retinoids, including all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, retinol, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl- 2-naphthalenyl)-l-propenyl] benzoic acid, and/or 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido benzoic acid, N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l- adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), capable of increasing the activity of GI-GPx or of activating or stimulating the production and/or expression of GI-GPx. Preferred individuals are non-responders to interferon and/or ribavirin therapy.

[0072] According to still further aspect of the present invention, the novel therapeutic compositions contain from 0.01 to 50 % by weight, particularly from 0.02 to 10 % by weight of the specific chemical substances and compounds or "agent(s)". The above mentioned chemical substances and compounds may be combined with further compounds like known antiviral compounds including ribavirin or VX-497 (Vertex Pharmaceuticals), a less toxic ribavirin substitute, or HCV RNA polymerase inhibitors.

[0073] Said pharmaceutical compositions may further comprise pharmaceutically acceptable carriers, excipients, and or diluents.

[0074] In case the pharmaceutical composition is for oral application, according to a further embodiment of the present invention, the therapeutic agents are administered in the form of tablets or capsules. Such tablets or capsules may contain from 1 to 300 mg, preferably from 1 to 150 mg, more preferably from 1 to 100 mg, and particularly from 1 to 50 mg of the agent or agents.

[0075] Another possible way of applying a therapeutically effective amount of at least one of the above-mentioned specific substances to an individual (patient) is by means of including the substances into liposomes and administering the liposomes to the individual. Liposomes are spherical particles having typically a diameter of about 25 nm to about 5 μm. Liposomes usually comprise one or more concentric lipid double layers having an aqueous interior compartment (so-called "lipid vesicles"). Liposomes are known as carriers for pharmaceutical substances, which can be selectively enriched in certain organs and cellular tissues by means of the liposomes, see e.g. Adv. Drug Deliv. Rev. 19, 425 to 444 (1996) and Science 267, 1275 et seq. (1995). [0076] As used herein, the term "activator" refers to any chemical compound capable of upregulating, activating, stimulating, or increasing the amount and/or activity of GI-GPx or its expression.

[0077] As used herein, the term "inhibitor" refers to any compound capable of downregulating, decreasing, inactivating, suppressing or otherwise regulating the amount and/or activity of GI-GPx or its expression. Generally, GI-GPx inhibitors may be proteins, oligo- and polypeptides, nucleic acids, such as RNAi, genes, small chemical molecules, or other chemical moieties.

[0078] The term "agent" is used herein as synonym for regulator, inhibitor, and/or activator. Thus, the term "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.

[0079] In addition to the role in transmitting genetic information from DNA to proteins, RNA molecules participate actively in many cell processes. Examples are found in translation (rRNA, tRNA, rnRNA), 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.

[0080] As used herein, the term "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.

[0081] 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 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 gastrointestinal glutathione peroxidase. Again, preferred individuals are non-responders to interferon and/or ribavirin therapy.

[0082] 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. Humans particularly preferred are non-responders to interferon and/or ribavirin therapy. Routes of administration of pharmaceutical preparations to an individual may include oral and parenteral, including dermal, intradermal, intragastral, intracutaneous, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutaneous, rectal, subcutaneous, sublingual, topical or transdermal application, but are not limited the these ways of administration. For instance, the preferred preparations are in a dosage form which is suitable for oral application. These dosage 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.

[0083] As used herein, a "pharmaceutical effective amount" of a GI-GPx activator is an amount effective to achieve the desired physiological result, either in cells or cell cultures treated in vitro or in a subject (e.g. individual, particularly human being) 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 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 activator 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. As mentioned above, a "therapeutically effective amount" of the substances and compounds according to the present invention may be 0.01 to 0.15 % by weight of a pharmaceutical composition. In case a tablet or capsule is used as administrative form, the amount of the effective substance or compound in the tablet or capsule may be 1 to 300 mg, preferably 1 to 150 mg, more preferably 1 to 100 mg, and particularly 1 to 50 mg.

[0084] 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 selected from the group consisting of selenium, selenium salts, Vitamin D₃, pegylated and non-pegylated (standard) α-, β-, and γ-interferon, ribavirin, and retinoids, particularly all isomeric forms of retinoic acid, like all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, as well as retinol, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl)-l-propenyl]benzoic acid, and/or 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido benzoic acid, N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2- naphthalene carboxylic acid (CD437; AHPN). The above mentioned chemical substances and compounds may be combined with known anti-viral compounds.

[0085] 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, radioimmunoassay, immuno-histochemistry, enzymatic assay 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.

[0086] A robust cell culture system for replication of the hepatitis C virus (HCV) has not been established. For this reason, it is 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 (Lohmann, V., Korner, F., Koch, J.-O., Herian, U., Theilmann, L., and Bartenschlager, R. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 285: 110 - 113). 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 synthesize viral proteins. The replicons described in the scientific article of Lohmann et al. cited above and used for the present investigation 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.

[0087] Recently, gastrointestinal-glutathione peroxidase (GI-GPx) could be validated as drug target in HCV-replication (see WO 02/084294). As mentioned above, GI-GPx belongs to the family of selenoproteins and plays an important role in the defense mechanisms of eukaryotic cells 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. Selenium as a necessary trace element suggests the essential function of this enzyme.

[0088] 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 (D. H. Holben, A. M. Smith, J. Am. Diet. Assoc. 1999, 99, 836 - 843).

[0089] GI-GPx is drastically down-regulated in HCV replicon cells compared with mock- transfected HuH7 cells. Forcing replicon cells to re-express GI-GPx (e.g. by infection with GI- GPx containing adenovirus) results in reduction of subgenomic HCV RNA and of the HCV protein NS5a, indicating the degree of HCV RNA replication, to hardly detectable levels (see WO 02/084294). According to the present invention the knowledge of this inverse correlation was used to develop a method to up-regulate the expression of the cellular, endogenous GI-GPx gene. This up-regulation in replicon cells causes a depletion of HCV. [0090] It is readily apparent to those skilled in the art that other suitable modifications and adaptations of the compositions and methods of the invention described herein are evident and may be made without departing from the scope of the invention or the embodiments disclosed herein. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to limit the invention.

EXAMPLES

[0091] Reference is made to the Examples of WO 02/084294, which are incorporated herein by reference.

[0092] Moreover, as model system for HCV replication there were utilized three replicon cell lines provided by Prof. R. Bartenschlager (University of Heidelberg, FRG). Cultures were treated for various periods of time with all trans retinoic acid (RA) for comparative purposes and the other agents selenium, selenium salts, Vitamin D₃ and retinoids, like 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, N-(4- hydroxyphenyl) retinamide (4-HPR) and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2 -naphthalene carboxylic acid (CD437; AHPN) (obtained from Sigma). Levels of expression of GI-GPx was measured on protein level by Western Blotting using antibodies provided by Prof. Brigelius- Flohe (University of Potsdam, FRG) and on RNA level by Northern blotting using GI-GPx- specific oligonucleotides as probes. Levels of HCV RNA were investigated by Northern Blotting using a DNA oligonucleotide complementary to the neomycin phosphotansferase gene as probe. Concentration of the viral protein NS5a was determined by Western Blotting with an NS5a- specific antibody (Biogenesis, UK).

[0093] Treatment of replicon cells for three days with all trans retinoic acid (1 μM) had hardly an effect on GI-GPx and HCV expression. However, after seven days of incubation, a drastic up- regulation of GI-GPx on RNA- and protein level (three- to ten-fold) was observed. Concomitantly expression of subgenomic HCV RNA and of viral protein NS5a was downregulated two- to five-fold, depending on the cell line investigated. Furthermore, surprisingly it was found that a further downregulation of HCV-RNA and -NS5a protein was dependent on the addition of selenium or a selenium salt, e.g. sodium selenite (50 nM). This fact implies, that downregulation of HCV was promoted firstly by activation of the GI-GPx gene on transcriptional level by retinoic acid and secondly by the synthesis of selenoprotein(s) for which sodium selenite was needed. Indeed it could be shown that all trans retinoic acid-induced downregulation of HCV is independent of the innate immune response induced by interferon. Thus, all trans retinoic acid did not induce the transcription of PKR (double strand RNA- dependent protein kinase). Severe cytotoxic effects were neither observed for all trans retinoic acid nor for sodium selenium salts, or both in combination.

[0094] The presented findings show that retinoids (in combination with selenium or selenium salts like sodium selenite and/or cAMP and/or analogues thereof) can be used for the treatment of HCV-positive patients. Especially the usage of retinoids with high specificity for induction of the GI-GPx, like N-(4-hydroxyphenyl) retinamide (4-HPR) and 6-[3-(l-adamantyl)-4- hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), are preferred. 4-HPR and AHPN display significant potential as therapeutic agents in the prophylaxis and treatment of a number of premalignant and malignant conditions in the context of HCV infections. Indeed, the obtained data show that next to all trans retinoic acid other nuclear receptor ligands, like 9-cis retinoic acid as well as salts thereof, 9-cis retinoic acid Ci to Cio alkyl esters as well as salts thereof, 9-cis retinoic acid Ci to Cio alkyl amides as well as salts thereof, and Vitamin D_3j are also capable of reducing HCV load.

[0095] All-tr «5 retinoic acid on replicon cells for six days led to an upregulation of GI-GPx RNA and protein due to the fact that the GI-GPx -promoter contains three retinoic acid receptor recognition elements. In the presence of selenium or a selenium salt like sodium selenite a two- to five-fold reduction of HCV-RNA and HCV-NS5a protein was observed in the absence of toxic effects. Moreover, also the specific retinoids, like N-(4-hydroxyphenyl) retinamide (4- HPR) and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437; AHPN), 9-cis retinoic acid, 9-cis retinoic acid Ci to Cio alkyl esters, 9-cis retinoic acid Ci to Cio alkyl amides, and Vitamin D₃ alone or in combination with each other or with selenium or a selenium salt showed a similar effect.

[0096] Moreover, preliminary results have shown that 9-cis retinoic acid and its above- mentioned alkyl and amide derivates downregulate HCV RNA very efficiently.

[0097] The following examples describing administrative forms for a lotion (solution), gel, cream, soft gelatin capsules, hard gelatine capsules, tablets, and sachets containing 9-cis retinoic acid are taken from US- A-5 ,428,071 and EP-B1-0 552 624.

EXAMPLE 1 Lotion (solution) preferred

9-cis-Retinoic Acid 0.02-0.30 g Propylene Glycol 5.00-20.00 g 10.00 g PEG-Glyceryl Cocoate* 0.00-20.00 g 10.00 g dl-alpha-Tocopherol 0.001-0.50 g 0.02 g Ascorbyl Palmitate 0.01-0.20 g 0.10 g Propyl Gallate 0.001-0.02 g 0.002 g Citric acid, anhydrous** 0.00-0.20 g 0.01 g Isopropanol*** 40.00-90.00 g 50.00 g Water, dem. ad 100.00 g 100.00 g (resp. ml)

*or other tensides **or other complexing agents, e.g. EDTA ***or other alcohols, e.g. Ethanol

EXAMPLE 2 Gel preferred

9-cis-Retinoic Acid 0.02-0.30 g O 2005/120479

Propylene Glycol 5.00-20.00 g 10.00 g

PEG-Glyceryl Cocoate* 0.00-20.00 g 10.00 g dl-alpha-Tocopherol 0.001-0.50 g 0.02 g

Ascorbyl Palmitate 0.01-0.20 g 0.10 g

Propyl Gallate 0.001-0.02 g 0.002 g

Citric acid, anhydrous** 0.00-0.20 g 0.01 g

Isopropanol*** 40.00-90.00 g 50.00 g

HPMC**** 0.50-5.00 g 3.0 g

Preservative* * * * * q.s. q.i s.

Water, dem. ad 100.00 g 100.00 g

*or other tensides **or other complexing agents, e.g. EDTA ***or other alcohols, e.g. Ethanol ****Hydroxypropyl Methylcellulose or other polymers e.g. neutralized Carbomer, Methyl Cellulose, Sodium Carboxymethylcellulose *****Preservatives, e.g. Paraben esters (methyl, ethyl, propyl, butyl), Sorbic Acid, Benzoic, Acid

EXAMPLE 3

Cream preferred

9-cis-Retinoic Acid 0.02-0.30 g

Glycerol 0.00-10.00 g 5.00 g

Na.sub.2 EDTA 0.001-0.50 g 0.03 g

Glycerides* 5.00-20.00 g 10.00 g

Cetyl Alcohol 0.50-5.00 g 1.00 g

Stearyl Alcohol 0.50-5.00 g 1.00 g

Glycerol mono Stearate 1.00-8.00 g 4.00 g

Cetaereth** 0.50-5.00 g 2.00 g dl-alpha-Tocopherol 0.001-0.50 g 0.02 g

Preservative*** q.s. q.s.

Water, dem. ad 100.00 g 100.00 g

*e.g. Caprylic/Capric/Triglyceride, Caprylic/Capric/Linoleic Triglyceride natural glycerides, as well as e.g., Propylene Glycol, Dicaprylate/Dicaprate and waxes such as Stearyl Stearate, Oleyl Oleate, Isopropyl Myristate. **Ceteareth 5-30, or other emulsifiers such as Polysorbate 20-80, Sorbitane esters of fatty acids, fatty acid esters of PEG. ***Preservatives e.g., Paraben esters (methyl, ethyl, propyl, butyl), Sorbic Acid, Benzoic Acid.

EXAMPLE 4

Fill mass for soft gelatin capsules

9-cis-Retinoic Acid 5.00-50.00 mg

Oil* 1-3 parts

Wax mixture** 1-5 parts

Fill volume 1-6 minims

*natural vegetable oils, e.g., soy oil, peanut oil, and artificial glycerides * Composition of natural and artificial waxes or partially hydrated fats

EXAMPLE 5

1. Hard Gelatine capsules containing 20 mg active substance: Composition: One Capsule contains:

9-cis-Retinoic acid 20.0 mg.

Gelatine Bloom 30 70.0 mg.

Maltodextrin MD 05 108.0 mg. dl-alpha-Tocopherol 2.0 mg.

Sodium ascorbate 10.0 mg.

Microcrystalline cellulose 48.0 mg.

Magnesium stearate 2.0 mg.

(weight capsule content) 260.0 mg.

Procedure:

The active substance is wet milled in a solution of gelatine, maltodextrin, dl-alpha-Tocopherol and sodium ascorbate.

The wet milled suspension is spray-dried.

The spray-dried powder is mixed with microcrystalline cellulose and magnesium stearate.

260 mg. each of this mixture are filled into hard gelatine capsules of suitable size and color.

EXAMPLE 6

2. Tablet containing 20 mg active substance:

Composition: Tablet kernel:

9-cis-Retinoic acid 20.0 mg

Anhydrous lactose 130.5 mg

Microcrystalline Cellulose 80.0 mg dl-alpha-Tocopherol 2.0 mg

Sodium ascorbate 10.0 mg

Polyvinylpyrrolidone K30 5.0 mg Magnesium stearate 2.5 mg

(Kernel weight) 250.0 mg

Film coat:

Hydroxypropyl methylcellulose 3.5 mg

Polyethyleneglycol 6000 0.8 mg

Talc 1.3 mg

Iron oxide, yellow 0.8 mg

Titanium dioxide 0.8 mg

(weight of the film) 7.4 mg

Procedure:

9-cis-Retinoic acid is mixed with anhydrous lactose and micro- crystalline cellulose.

The mixture is granulated in water with a solution/dispersion of polyvinylpyrrolidone, dl-. alpha. -Tocopherol and sodium ascorbate. The granular material is mixed with magnesium stearate and afterwards pressed as kernels with 250 mg. weight. The kernels are film coated with a solution/suspension of above-mentioned composition.

EXAMPLE 7

Sachet containing 50 mg active substance

Composition:

9-cis-Retinoic acid 50.0 mg

Lactose, fine powder 990.0 mg

Microcrystalline Cellulose 1400.0 mg Sodium Carboxymethyl -cellulose 14.0 mg dl-alpha-Tocopherol 5.0 mg

Sodium ascorbate 20.0 mg Polyvinylpyrrolidone K30 10.0 mg Magnesium stearate 10.0 mg Flavoring Agents 1.0 mg (Fill weight of a sachet) 2500.0 mg Procedure: 9-cis-Retinoic acid is mixed with lactose, microcrystalline cellulose and sodium carboxymethyl cellulose. The mixture is granulated in water with a solution/dispersion of polyvinylpyrrolidone, dl-alpha-Tocopherol and sodium ascorbate. The granule is mixed with magnesium stearate and flavoring agents. It is filled into sachets of suitable size.

EXAMPLE 8

In vitro studies with combined therapies

[0098] In the following, results of tests are presented relating to retinoic acid and derivatives thereof for treatment of HCV infected cells non-responding to interferon treatment.

[0099] Treating the HCV replicon cell lines with all trans retinoic acid (ATRA), or some derivatives thereof, resulted in suppression of HCV RNA and of NS5a protein expression (Fig. 1, Panel A). Many findings demonstrate that the underlying mechanism is due to the up-regulation of the selenocysteine protein gastrointestinal glutathione peroxidase (GI-GPx) gene (Fig. 1, Panel B). E.g., this effect was most prominent when sodium selenite (50 nM) was given to the cell culture medium (Fig. 1, Panel A), which is needed for the synthesis and thus up-regulation of the GI-GPx protein (Fig. 1, Panel B).

Retinoic acid (RA)- and interferon (IFN)-dependent pathways

[0100] In the course of retinoic acid studies it was investigated whether the RA-effect may also be mediated by activating an interferon response. A typical interferon-inducible gene codes for the double-stranded RNA-dependent protein kinase (PKR). Mock-transfected HuH7 cells (pcDNA3) and replicon cell lines were treated with ATRA (1 μM), but no up-regulation of the PKR mRNA levels monitored by Northern blotting was observed. IFN as a control, however, caused a dramatic up-regulation of PKR mRNA. An example for a replicon cell line is shown in Fig. 1, Panel C. [0101] These results show that RA acts independently of IFN on the replicon system. These results furthermore imply that HCV-patients, who do not react on IFN treatment, so called non- responders, may be cured by RA-treatment.

[0102] Unfortunately, there is no IFN-resistant replicon system described in the literature to test this hypothesis directly.

Effect of RA in Combination with IFN

[0103] In a recent publication (Retinoic acid enhances the antiviral effect of interferon on hepatitis C virus replication through increased expression of type I interferon receptor. Hamamoto et al., J. Lab. Clin. Med. 141, 2003, 58-66) it is asserted that treating HuH7 cells with ATRA and 9-cis RA induces the expression of Interferon Type I receptor subunits (max. 2-fold after 24 hrs on RNA level, TaqMan analysis). Interestingly, this effect was independent of the dose (see for example Fig. 2 of the article by Hamamoto cited above). IFN treatment decreased the concentration of transfected HCV (replicon-) RNA, and this effect was enhanced by treatment with RAs. The authors conclude that RAs increase the anti-HCV replication effect of IFN-alpha through up-regulation of type I IFN-receptors in HuH-7 cells.

[0104] The expression of interferon receptors after treatment of replicon cells with ATRA was now analyzed by western Blotting in HuH7 and replicon cells, and using this method no upregulation of interferon receptors on protein level was observed by the present inventors.

[0105] When using sub-therapeutic concentrations of IFN alpha, we found a dose-dependent reduction of the HCV protein NS5a (Fig. 2, left Panel). In the presence of RA, the HCV down- regulation was slightly enhanced (center Panel). The strongest effect was observed, when IFN was applied with ATRA and sodium selenite together (Fig. 2, right Panel).

[0106] The data show that the IFN and RA-effects are additive in this in vitro system and imply that their mechanisms to down-regulate HCV are independent. This finding further substantiates the hypothesis that RA can act in IFN-resistant patients (non-responders).

[0107] Comparing the results presented by Hamamoto with the results described herein reveals the important implications for the therapy of human patients. While Hamamoto asserts that the RA effect is due to the up-regulation of IFN Type I receptor (a finding which could not be reproduced herein), according to the present invention it is believed that the RA-effect is due to the up-regulation of GI-GPx. According to the present invention these two therapeutic modalities are not necessarily working through a single shared pathway and therefore they may act synergistically. [0108] Furthermore, the ability of several retinoids (i.e. all trans retinoic acid (ATRA); 9-cis retinoic acid (9-cis RA); 13-cis retinoic acid (13-cis RA); (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB); (4-[5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2 -naphtalenyl) carbox-amido] benzoic acid (AM-580); and N-(4-hydroxyphenyl) retinamide (4-HPR)) to act as ligands for nuclear receptors (RAR = Retinoic Acid Receptor; RXR = Retinoid X Receptor) was tested and compared with a non retinoid substance (11 -methoxy- 3,7,1 l-trimethyl-2E,4E-dodecadienoic acid = methoprene acid). The results are shown in the following Table 1. Table 1

* Kd = dissociation constant ** EC₅o = effector concentration (concentration showing 50% effect) +++ = very strong inhibitory effect; ++ = medium inhibitory effect; - = no inhibitory effect

[0109] The data imply that RARs, but not RXRs are involved in up-regulation of the glutathione peroxidase-gastrointestinal (GI-GPx) promoter. The data furthermore show that inhibition of HCV replication was linked to up-regulation of GI-GPx mRNA. [0110] Preliminary studies have shown that retinoic acids (e.g. Vesanoid , Roche Pharmaceuticals, Nutley, NJ, USA) or retinoids may be administered to a patient in amount of about 1 to 100 mg/m²/day, preferably 20 to 80 mg/m²/day, more preferably 30 to 60 mg/m²/day, and particularly 40 to 50 mg/m² /day. Suitable doses are 1 to 4 times per day, preferably 1 to 3 times per day, and particularly 2 times a day.

[0111] If a combination therapy is applied, in addition to the retinoic acid or retinoid in the amounts mentioned above, an interferon (e.g. pegylated interferon α, e.g. Pegasys®, Hoffmann-La Roche) may be administered in an amount of about 135 to 180 μg/week (preferably 1 dose per week).

[0112] Specifically preferred is a therapy with ATRA alone for the treatment of HCV infections of non responders. Moreover, the treatment with ATRA plus interferon (pegylated or non-pegylated α, β, or γ-interferon), ATRA plus interferon (pegylated or non-pegylated α, β, or γ-interferon) plus selenium or selenium salt, or ATRA plus selenium (and/or selenium salt) and ribavirin is particularly preferred.

EXAMPLE 9

Pilot study to test the efficacy of retinoic acid as monotherapy or in combination with

PEG- Interferon alpha 2a for treatment of Interferon-alpha -resistant chronic hepatitis C

[0113] Study Design:

This is a simple blind, randomized, monocenter phase II pilot study. In the short 12-week treatment period it should initially be established whether the rationale observed in vitro could also be extended to the situation in vivo, i.e. whether ATRA possesses an antiviral efficacy against hepatitis C virus in patients. The 3-month follow-up observation phase, in the case of patients showing a response, should additionally provide information as to whether the ATRA- effect is reversible.

Primary Objectives:

[0114] It should be tested whether treatment with all-trans retinoic acid/selenium alone or in combination with interferon-alpha has an antiviral activity against hepatitis C virus in vivo. The target parameter of the primary study objective is assessed by HCV-PCR after 12 weeks of ATRA treatment.

Secondary Objectives:

[0115] The effect all-trans retinoic acid on virus kinetics should be investigated as well as the tolerability of such a therapy in patients with chronic hepatitis C. The target parameter here is monitored by quantitative PCR, at various time points during and after completion of ATRA treatment. Furthermore, the influence of ATRA on hepatic activity should be investigated and here the target parameter is measuring transaminase levels (GPT) at the time points mentioned above. Finally, the side effects described by the patients or determined in laboratory tests should be documented.

Trial Product:

Vesanoid Capsule:

[0116] 1 capsule contains 10 mg Tretinoin (All-trans-retinoic acid) as active ingredient, as well as the adjunct substances yellow wax, hydrated soy bean extract, soy bean oil, gelatin, glycerol, sorbitol solution 70% (non-crystalline), titanium dioxide (E 171), iron oxide hydrate (E 172) as well as iron (III) oxide (E 172).

Pegasys:

[0117] 1 pre-prepared syringe contains 180 μg PEG-IFNα2a produced as recombinant protein in Escherichia coli, linked to bis-monomethoxy-polyethyleneglycol with a molecular mass of 40 kDa in 0.5 ml solution. Adjuvants sodium chloride, polysorbate 80, benzylalcohol, sodium acetate, acetic acid and water are added for injection purposes.

Selen 30 ALLACT:

[0118] 1 capsule contains 30 μg sodium selenate (selenium) as the active ingredient.

Patients:

[0119] The patients were recruited from the study outpatients' clinic at the I. Medizinischen Klinik, Mainz, Germany. The patients had genotype 1 infection, primary non-responders to a previous therapy involving interferon-alpha and ribavirin in adequate doses over at least three months. The patients are then to be randomly allocated to one of the study regimens.

Previous and Concomitant Medication:

[0120] The patients had not undergone any antiviral or immunosuppressive therapy in the 6 months before commencing the treatment, and this applies to the entire study and follow-up period. Furthermore, no therapeutics should be taken in parallel for chronic liver diseases, such as ursodeoxycholic acid or silymarit. Selenium should not be taken together with vitamin C.

Safety Measures:

[0121] In addition to screening, the patients are to present themselves at the study outpatients clinic at the start of treatment, at week 2, week 4, week 8, week 12 as well as 12 weeks after completion of the therapy, where they are physically examined and asked about side effects of the treatment. At these time points control laboratory tests are carried out (in addition to the HCV-PCR, differential blood count, renal and liver values, blood sugar, CK, lipid status, serum protein and serum electrophoresis, a coagulation status, urine status, as well as in week 0, 4, 12 and 24, a TSH- value). In the screening phase a pregnancy test should be carried out. Moreover, all patients are to undergo an ECG and an abdominal sonography before being included in the trial.

Handling Adverse Events - Dosage Modifications

[0122] Handling adverse events generally involves a dosage reduction. Appearance of life- threatening adverse events, diagnosis of a cardiovascular disease or development of a cardiac dysfunction, pregnancy, lack of compliance, including the stipulated contraception practiced both by men and women, require that the interferon as well as the selenium and Vesanoid therapy is discontinued completely. Patients whose medication has been discontinued are to be monitored within the framework of follow-up observation.

[0123] The decisions concerning instigating dosage reduction are based on the following guidelines. Laboratory Tests:

[0124] At admission to the study, in addition to ECG, sonography, the patient's history and physical examination, the following laboratory tests should be performed:

Hematology: blood count

Serum chemistry: albumin, GPT, GOT, γGT, bilirubin, electrolyte, creatine, urea, CRP, BZ, only at admission: ureic acid, protein + serum electrophoresis, iron, ferritin, differential blood count; every 4 weeks: lipid status, coagulation, urine status, TSH

Serology: HCV-PCR, quantitative General (before admission): HlV-serology, HBsAg, anti-HCV-Ab, HCV-geno- or serotype, pregnancy test, TSH, ANA, alpha-fetoprotein, HbAlc, thyroid-autoantibodies

[0125] During treatment and the follow-up observation period the examinations are to be carried out according to the following scheme.

* with women of childbearing age before, at the end (week 12) of treatment and at the end of the therapy ** TSH before the start of therapy then after 3 months of therapy *** quantitative HCV-RNA measurements twice before admission, then at 1, 2, 4, 8 and 12 weeks as well as at the end of the follow-up period Procedures in the Event of Pathological Laboratory Values

[0126] Interferon or Vesanoid treatment is to be discontinued if, despite applying a lower dosage, repeated laboratory values represent an adverse event. The patient is observed for 12 weeks following termination of the therapy.

Recording and Reporting Adverse Events

[0127] Adverse events (AE) are all undesirable changes in conditions observed during a clinical trial, subjective and objective medical symptoms (including changes in laboratory values), intervening diseases and accidents unrelated to a possible cause correlation with the administered study medication. Also to be described as AEs are such events occurring within the framework of the clinical study in the preceding and follow-up periods, under placebo and in a comparative group under medication or non-medication treatment and previously not included in the study information.

[0128] All adverse events including pathological laboratory values are to be recorded directly after the patient's visit on the documentation forms provided. The description of the adverse events must include the nature of the event, the time point of onset, the duration, severity, intensity, the outcome, the relationship to the treatment and possible required therapy.

Evaluation and Statistical Methods

[0129] The small number of cases, i.e. 20 patients, was initially intended to determine if a possible biological effect of ATRA on HCV replication in vivo can be detected. The biometric evaluation is carried out according to the simple study plan at I. Medizinischen Klinik of the University of Mainz. The course of virus load should be presented as multiple graphs before, under and after treatment in order to detect quantitative effects of the study medication on the virusemia. Reduction of the plasma virusemia by at least 2 factors of 10 over several time points under monotherapy and/or IFN-combination therapy, compared to that before or after treatment can be evaluated as a therapeutic success, since all the patients are defined as twice interferon non-responders. Each regimen should be statistically evaluated separately for the plasma virusemia levels before and under treatment (various time points) as well as under and after treatment with Student t-test for linked random samples (StatView Software, Abacus Concepts, CA). In addition, any possible differences between the two therapy regimens, represented as changes in the plasma virusemia in comparison to individual starting values, should be checked using the same statistical test.

Results

[0130] At the interim analysis of the results of the ongoing trial at 8 weeks, it was clearly established that the effects of interferon and retinoid plus selenium are synergistic to the regimen of each of these therapies as a monotherapy. The combination therapy lowered viral loads significantly, in some cases by two orders of magnitude or even greater. It is noteworthy that these were patients previously shown to be interferon non-responsive. Therefore it appears that interferon potentiates the effects of retinoic acid, which by itself showed a trend to decreased viral load.

[0131] The combined treatment provided a high degree of response, over shortened periods of treatment compared to the monotherapy, and with few side effects. Thus the combined therapy proved superior to conventional IFN/ribavirin treatment with no evident toxicity.

The data to date can be summarized in the following tabulated form. wk 2 wk 4 wk 8 Virological response cohort A 0/6 0/6 0/2 cohort B 1/4 3/4 1/2

Biochemical response cohort A 0/6 0/6 0/2 cohort B 0/4 1/4 1/2

Claims

CLAIMS 1. A combination therapy for the treatment of an individual afflicted with a Hepatitis C virus (HCV) infection or at least one disease associated with a HCV infection, said combination comprising at least two agents, each agent selected from one of the groups consisting of: (i) selenium or a selenium salt; and (ii) a retinoic acid or a retinoid.

2. The combination therapy of claim 1 wherein the retinoic acid or retinoid is selected from the group consisting of all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, retinol, (E)-4-[2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB), (4-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N- (4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4-hydroxyphenyl]-2- naphthalene carboxylic acid (AHPN).

3. The combination therapy according to claim 1, wherein the selenium salt is selected from sodium selenate, sodium selenite and sodium selenide.

4. The combination therapy according to claim 1 in unit dosage form for oral administration, wherein an oral dosage unit of said composition contains from 1 to 300 mg, preferably 1 to 150 mg, more preferably from 1 to 100 mg, and particularly from 1 to 50 mg of the agents.

5. A combination therapy for the treatment of an individual afflicted with a Hepatitis C virus (HCV) infection or at least one disease associated with a HCV infection, said combination comprising a plurality of agents, each agent selected from one of the groups consisting of: (i) selenium or a selenium salt; (ii) a retinoic acid or a retinoid; and (iii) an interferon selected form the group consisting of pegylated α-, β-, γ-interferon, and non-pegylated (standard) α-, β-, and/or γ-interferon

6. The combination therapy according to claim 5, wherein the interferon is selected from pegylated or non-pegylated alpha or beta interferon.

7. The combination therapy of claim 5 further comprising ribavirin.

8. The combination therapy of claim 5 wherein the retinoic acid or retinoid is selected from the group consisting of all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, retinol, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl- 1 -propenyl] benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l- adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN).

9. The combination therapy of claim 5 wherein the selenium salt is selected from sodium selenate, sodium selenite and sodium selenide.

10. The combination therapy of claim 5 wherein the individual components are administered in separate pharmaceutical compositions.

11. The combination therapy of claim 5 wherein the individual components are combined in a single treatment kit.

12. The combination therapy of claim 5 wherein the individual components are combined into a single pharmaceutical composition.

13. The combination therapy of claim 12 wherein the pharmaceutical compositions comprise lipid carrier particles.

14. A therapeutic treatment kit for the treatment of HCV comprising an interferon, a retinoid and a selenium compound and instructional materials for the combined use of said interferon, said retinoid and said selenium compound.

15. A kit according to claim 10 wherein the instructional materials include such information as dosage, indication and contraindication and storage parameters.

16. A method for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in an individual, the method comprising the step of administering a pharmaceutically effective amount of a combination therapy, said combination comprising at least two agents, each agent selected from one of the groups consisting of: (i) selenium or a selenium salt; and (ii) a retinoic acid or a retinoid.

17. The method of claim 16 wherein the retinoic acid or retinoid is selected from the group consisting of all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, retinol (E)-4-[2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB), (4-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4-hydroxyphenyl]- 2 -naphthalene carboxylic acid (AHPN).

18. The method according to claim 16, wherein the selenium salt is selected from sodium selenate, sodium selenite and sodium selenide.

19. The method according to claim 16, wherein the individual is a non-responder to interferon and/or ribavirin therapy.

20. The method according to claim 16 in unit dosage form for oral administration, wherein an oral dosage unit of said composition contains from 1 to 300 mg, preferably 1 to 150 mg, more preferably from 1 to 100 mg, and particularly from 1 to 50 mg of the agents.

21. A method for treating an individual afflicted with a Hepatitis C virus (HCV) infection and or at least one disease associated with a HCV infection, said combination comprising a plurality of agents, each agent selected from a one of the groups consisting of: (i) selenium or a selenium salt; (ii) a retinoic acid or a retinoid; and (iii) an interferon selected form the group consisting of pegylated α-, β-, γ- interferon, and non-pegylated (standard) α-, β-, and/or γ-interferon

22. The method according to claim 21, wherein the interferon is selected from a pegylated or non-pegylated alpha or beta interferon.

23. The method according to claim 21 further comprising ribavirin.

24. The method of claim 21 wherein the retinoic acid or retinoid is selected from the group consisting of all trans retinoic acid, salts of all trans retinoic acid, Ci - Cio alkyl esters of all trans retinoic acid, salts of Ci - Cio alkyl esters of all trans retinoic acid, Ci - Cio alkyl amides of all trans retinoic acid, salts of Ci - Cio alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, Ci - Cio alkyl esters of 9-cis retinoic acid, salts of Ci - Cio alkyl esters of 9-cis retinoic acid, Ci - Cio alkyl amides of 9-cis retinoic acid, salts of Ci - Cio alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoic acid, Ci - Cio alkyl esters of 13-cis retinoic acid, salts of Ci - Cio alkyl esters of 13-cis retinoic acid, Ci - Cio alkyl amides of 13-cis retinoic acid, salts of Ci - Cio alkyl amides of 13-cis retinoic acid, retinal, (E)-4-[2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl-l-propenyl] benzoic acid (TTNPB), (4-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl) carboxamido] benzoic acid (AM-580), N-(4 -hydroxyphenyl) retinamide (4-HPR), and 6-[3-(l-adamantyl)-4-hydroxyphenyl]- 2-naphthalene carboxylic acid (AHPN).

25. The method of claim 21 wherein the selenium salt is selected from sodium selenate, sodium selenite and sodium selenide.

26. The method of claim 21 wherein the individual compone ^are administered in _. separate pharmaceutical compositions.

27. The method of claim 21 wherein the individual components are combined in a single treatment kit.

28. The method of claim 21 wherein the individual components are combined into a single pharmaceutical composition.

29. The method of claim 21 wherein the pharmaceutical compositions comprise lipid carrier particles.

30. The method of any one of claims 21-29, wherein the individual afflicted with a HCV infection and/or at least one disease associated with HCV infection is a non-responder to interferon and/or ribavirin therapy.

31. A method for regulating the production of Hepatitis C virus in an individual, the method comprising the step of administering a pharmaceutically effective amount of a combination therapy according to claim 1.

32. The method of claim 31, wherein the individual is a non-responder to interferon and/or ribavirin therapy.

33. A method for regulating the production of Hepatitis C virus in an individual, the method comprising the step of administering a pharmaceutically effective amount of a combination therapy according to claim 5.

34. The method of claim 33, wherein the individual is a non-responder to interferon and/or ribavirin therapy.

35. A method for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual comprising the step of administering to the individual a pharmaceutically effective amount of a combination therapy according to claim 1.

36. The method of claim 35, wherein the individual is a non-responder to interferon and/or ribavirin therapy.

37. A method for regulating the expression of the human cellular protein gastrointestinal glutathione peroxidase in an individual comprising the step of administering to the individual a pharmaceutically effective amount of a combination therapy according to claim 4.

38. The method of claim 37, wherein the individual is a non-responder to interferon and/or ribavirin therapy.