DE10255861A1 - Preventing or treating hepatitis C virus infection comprises administering an agent that inhibits or stimulates the activity or production of human gastrointestinal glutathione peroxidase - Google Patents

Abstract

Preventing or treating hepatitis C virus infection in individuals, cells, cell cultures or cell lysates comprising administering an agent that inhibits (I) or stimulates (II) the activity or production of human gastrointestinal glutathione peroxidase (GI-GPx), is new. Independent claims are also included for: (1) detecting compounds useful for preventing or treating hepatitis C virus (HCV) infection by contacting a test compound with human gastrointestinal glutathione peroxidase (GI-GPx) and detecting GI-GPx activity; (2) detecting HCV infections by detecting GI-GPx activity in a sample from an individual or in cells, cell cultures or cell lysates; (3) selectively killing HCV-infected cells by administering a radical initiator (III) capable of generating artificial oxidative stress conditions; (4) regulating the production of HCV in an individual, cells, cell cultures or cell lysates comprising administering an agent that inhibits or stimulates the activity or production of GI-GPx or inhibits or stimulates the transcription of DNA and/or the translation of RNA encoding GI-GPx; (5) preventing or treating HCV infection in individuals or cells by administering an antioxidant (IV) capable of supporting GI-GPx function; (6) regulating GI-GPx activity in individuals, cells or cell cultures by administering an agent (V) that interacts with GI-GPx; and (7) composition for preventing or treating HCV infection, comprising (I) or (II).

Description

The present invention relates to chemical compounds and substances that fight hepatitis C virus (HCV) infections are effective. In particular, the present concerns Invention compositions containing these compounds and / or. substances include, methods of preventing HCV infection as well Use of the compounds and / or substances for the production of compositions for prophylaxis against and for treatment of HCV infections are suitable.

background the invention

The hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. The WHO estimates that in about 3% of the world's population, or 170 million people infected with the hepatitis C virus. In the US, it is estimated that 3.9 million Americans have been infected (CDC data sheet, September 2000). about 80% of HCV-infected individuals develop chronic hepatitis, what about disease states is associated, from asymptomatic carrier states to repeated inflammation the liver and severe chronic liver disease. It is expected to over a period of 20 years more than 20% of chronic HCV patients endangered are developing chronic cirrhosis, or that development to the hepatocellular Carcinoma occurs. Liver failure due to chronic hepatitis C is the most common reason for a liver transplant. With the exception of transplants, she estimates CDC that medical and lost time costs due to HCV yearly is about $ 600 million. HCV is mainly about blood and transmit blood products. Because of the routine investigations since the middle of 1992 of blood stocks are new cases related to transfusion extremely rare and were from drug use through injections as the highest Risk factor for infection with the virus exceeded. It there is also a sexual transmission route that however, is inefficient, and a 6% transmission rate from infected mothers on their children, which is higher in the event of co-infection with HIV. To a specific one Percentage, the type of transmission remains unknown. It will despite a significant decline the frequency of occurrence In the 1990s, the number of deaths (estimated annual mortality currently: 8,000 to 10,000 in the United States) and serious illnesses due to of HCV itself within the next Tripled 10 to 20 years [Sources: CDC data sheet (accessible 12/12/00); Houghton M. Hepatitis C Viruses. BN Fields, DM Knipe, PM Howley (Ed.) Fields Virolgy. 1996. Lipcott-Raven Pub., Philadelphia; roses HR and Gretch DR, Molecular Medicine Today, Vol. 5, 393, Sept. 99; Science 285, 26, July 99: News Focus: The scientific challange of Hepatitis C; Wong JB et al, Am. J. Public Health, 90, 1562, Oct. 2000: Estimating future hepatitis morbidity, mortality, and costs in the United States).

According to an announcement by the EASL (European Association for the Study of the Liver) International Consensus Conference on Hepatitis C (February 26-28 1999, Paris, France) becomes a combination therapy with alpha interferon and ribavirin are recommended for the treatment of untreated patients. Monotherapy with interferon has also been approved by the FDA, the permanent success rate (HCV RNA remains for more than 6 months after the end of the Therapy in the serum undetectable) is only 15 to 20% compared with the 35 to 45% in combination therapy. Interferons (intron A, Schering-Plow; Roferon A, Hoffmann-LaRoche; Wellferon, Glaxo Wellcome; Infergen, Amgen) are injected subcutaneously three times a week, Ribavirin (Rebetol, Schering-Plow) is an orally administered Medicines given twice a day. The recommended duration of treatment is 6 to 12 months, depending of the HCV genotype. Experimental forms of slow release Pegylated interferons (Pegasys, Hoffmann-LaRoche; PEG-Intron, Schering-Plow) showed improved Success rates (42 to 82% in combination with ribavirin) and applicability (1x weekly Injection) in recent clinical Studies (Hepatology 32: 4, Part 2 of October 2, 2000; NEJM 343, 1673 December 2000; NEJM 343, 1666, December 2000). Frequently occurring Side effects of interferon therapy include, for example, fatigue, Muscle pain, headache, nausea, fever, weight loss, Irritability, depression, bone marrow suppression and reversible Hair loss. The most common Side effects of ribavirin include anemia, fatigue and irritability, itching, Rash, blocked nasal passages, sinusitis and cough. More serious Side effects of monotherapy and combination therapy occur in fewer than two percent of patients (NIDDK information: Chronic Hepatitis C: Current Disease Management, accessible December 9, 1999). Some of the Contraindications to interferon are psychosis or severe depression, Neutropenia and / or thrombocytopenia, organ transplantation except the liver, symptomatic heart disease, decompensated cirrhosis and uncontrolled seizures. Contraindications to ribavirin are end stage renal failure, Anemia, hemoglobinopathies, severe heart disease, pregnancy, not a reliable method of contraception (Consensus statement of the EASL). In addition, the treatment of Hepatitis C virus infections with interferon alpha only in one Minority of individuals effective. This suggests that the virus uses various tricks to become resistant to interferons his.

Experimental treatments that don't new forms of interferon are maxamines (histamine dihydrochloride, Maxim Pharmaceuticals), which are combined with interferon in phase III studies, VX-497 (Vertex Pharmaceuticals), an IMP dehydrogenase inhibitor, as a less toxic ribavirin replacement in Phase II and Amantadine (Endo Labs), an approved anti-influenza drug, as a third component in triple therapy (Phase II). Inhibitors for HCV enzymes such as protease inhibitors, RNA polymerase inhibitors, helicase inhibitors, as well as rybozyme and antisense RNAs are in preclinical development (Boehringer Ingelheirn, Ribozyme Pharmaceuticals, Vertex Pharmaceuticals, Schering-Plow, Hoffmann-LaRoche, Immusol, Merck etc.). No vaccine is available for prevention or therapeutic use, but several companies are trying to develop conventional or DNA vaccines or immunostimulating agents (e.g. Chiron, Merck / Vical, Epimmune, NABI, Innogenetics). Antibodies against HCV virion have also been developed and have recently entered clinical trials (Trimera Co., Israel).

In summary, the one available Treatment for chronic hepatitis C expensive, only with a certain percentage the patient's effectiveness and adverse side effects are not unusual.

description the invention

Recently conducted Research has shown how cells communicate with each other, to within the human body the growth and maintenance of the variety of tissues to coordinate. A key element of this Communication network is the sending of a signal from the outside of one Cell to its core that is an activation or suppression of special genes. This process becomes signal transduction called.

Signal transduction at the cellular level refers to the movement of signals from outside the cell inside. The transfer the signals can be simple, like those with the receptor molecules of the Acetylcholine class related transmission: Receptors, the channels represent the passage of signals in ligand interaction in the form of movement of small ions, either in the Cell or out of the cell. These ion movements cause changes in the electrical potential of the cell, which in turn is the signal propagated along the cell. Refers to more complex signal transduction the coupling of ligand-receptor interactions with many intracellular events on. These events include phosphorylation by tyrosine kinases and / or serine / threonine kinases. Protein phosphorylations change that enzyme activities and protein conformations. The end result is a change in cellular activity and changes in the program of genes that are expressed in the target cell.

Signal transduction receptors divided into three general classes:

• 1. Receptors that penetrate the plasma membrane and have an intrinsic, enzymatic activity: receptors which have an intrinsic, enzymatic activity include those which are tyrosine kinases (e.g. PDGF, insulin, EGF- and FGF receptors), tyrosine phosphatases (for example CD45 [cluster determinat-45] proteins from T cells and macrophages), guanylate cyclases (e.g. natriuretic Peptide receptors) and serine / threonine kinases (e.g. Activin and TGF beta receptors). Receptors with intrinsic tyrosine kinase activity can both autophosphorylation as well as phosphorylation of other substrates cause. Furthermore, several receptor families do not have any intrinsic enzyme activity on, but are still over direct protein-protein interactions on intracellular tyrosine kinases coupled. This class of receptors includes all of the cytokine receptors (e.g. interleukin-2 receptor) as well as the CD4 and CD8 cell surface glycoproteins of the T cells and the T cell antigen receptor.
• 2. Receptors that bind to GTP in the cell and hydrolyze Proteins (called G proteins) are coupled: Receptors of Classes that interact with G proteins all have a structure which is characterized by seven domains, which are spread over one Extend membrane: These receptors are called "serpentine" receptors. Examples from this Class are the adrenergic receptors, odorant receptors and certain Hormone receptors (e.g. glucagon, angiotensin, vasopressin, and bradykinin).
• 3. Receptors that are found intracellularly and migrate to the cell nucleus when ligand binding occurs, where the ligand-receptor complex directly influences gene transcription: The superordinate steroid / thyroid-hormone-receptor family (e.g. glucocorticoid, vitamin D, retinoic acid, and thyroid- Hormone receptors) is a class of proteins that reside in the cell plasma and bind the lipophilic steroid / thyroid hormones. These hormones are able to penetrate freely into the hydrophobic plasma membrane. After binding of the ligand, the hormone-receptor complex migrates to the nucleus and binds to specific DNA sequences, which leads to a change in the transcription rates of the associated gene. If the message is at the core Achieved via one or more of the routes described above, it initiates the modulation of specific genes, which leads to the production of RNA and ultimately of proteins which fulfill a specific biological function. A disturbed activity of the signal transduction molecules can lead to a malfunction of the cells and to disease processes. In particular, an interaction of the HCV with host cells is necessary so that the virus can multiply.

The present invention is based on the fact that human, cellular, gastrointestinal protein glutathione peroxidase (P18283) is specifically down-regulated as a result of HCV replication in the HCV-infected host cells. The antiviral, prophylactic, and / or therapeutic use described in this application focuses on specific chemical substances and compounds that can be used to up-regulate human, cellular, gastrointestinal protein glutathione peroxidase. These special chemical substances and compounds are selenium, selenium salts, vitamin D ₃ and retinoids such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid , N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN).

To new, pharmaceutically effective Developing connections had to be a potential target for medical Impact can be identified. That is, the process of finding it of pharmaceutically active compounds includes target identification. details for the Finding suitable targets to treat HCV infections are described in WO 02/084294.

Target identification is essentially the identification of a specific biological component, namely one Proteins and its association with certain disease states and regulatory systems. A protein that, when looking for a pharmaceutically effective, chemical compound (drug) is identified, becomes a target called. This target is in the regulation or control of biological systems, and in its effect can by means of medication.

The term "disease" is used here to mean that an acquired condition or genetic condition is described. A disease can change the body's normal biological systems, causing an excess or deficit on one chemical compounds (chemical imbalance). The regulatory systems for these chemical compounds involve the use of certain Proteins through the body, to detect imbalances and induce the body to neutralize compounds in an attempt to restore chemical balance.

The term "body" is used here to mean that any biological system, for example humans, animals, cells or Cell culture is described.

It is therefore the object of the present invention To provide compounds, compositions and methods that for the prophylaxis and / or treatment of hepatitis C virus infections are effective, but not the negative side effects described above have or at least not to the extent as for known products and methods have been reported. The object of the present invention is through the teaching of independent claims solved. Further advantageous features, aspects and details of the invention result from the dependent Claims of Description and examples of the present application.

Precise description the invention

It was previously shown that the human, cellular, gastrointestinal protein glutathione peroxidase in the body due to HCV infection is specifically downregulated. This human, cellular, gastrointestinal Protein glutathione peroxidase has been used as a diagnostic and therapeutic Target identified for the treatment of HCV infections.

Glutathione peroxidase:

So far, have been in mammals identified four different species of glutathione peroxidase, the classic, cellular Enzyme, the phospholipid hydroperoxide metabolizing enzyme, the gastrointestinal tract enzyme and the extracellular plasma enzyme. Their primary structures are little related. It has been shown through different genes are encoded and different enzymatic Have properties. Because of the small amount of reduced glutathione in human plasma and its low reactivity Enzyme, the physiological role of human plasma enzyme continues not clear.

The human, cellular, gastrointestinal Protein glutathione peroxidase (GI-GPx) is also linked as glutathione peroxidase Protein 2 (GPRP) or glutathione hydrogen peroxide oxidoreductase are known. You became the Accession number P18283 and the EC number 1.11.1.9. assigned.

Human, cellular, gastrointestinal protein glutathione peroxidase (GI-GPx) catalyzes the reduction of various organic hydroperoxides and hydrogen peroxide with glutathione (GSH) as a hydrogen donor (2 GSH + H ₂ O ₂ → GS-GS + 2 H ₂ O). It has a molecular weight of 84,000 and 4 subunits per mole of enzyme. The enzyme is useful Lich for the enzymatic determination of lipid hydroperoxide.

GI-GPx belongs to the selenoprotein family and plays in the defense mechanisms of mammals, birds and Fishing against oxidative damage a big Role by taking the reduction of various hydroperoxides under Catalyzed by the use of glutadione as a reducing substrate. It has been suggested that this enzyme is used in most cases a mechanism for protecting the cellular membrane system against peroxidative damage acts and that selenium is an essential trace element that plays a big role could play in this proposed function of the enzyme. It is known to have both vitamin E and Se as antioxidants even in a conventional one Mechanism of oxidative stress as the underlying cause of genetic changes Act.

Selenium acts primarily in mammalian systems in the form of selenoproteins. Selenoproteins contain selenium as Selenocyctein and lead a variety of physiological functions. Seventeen selenoproteins were identified: Cellular or classic glutathione peroxidase, plasma (or extracellular) glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, gastrointestinal Glutathione peroxidase, selenoprotein P, iodothyronine deiodinase from Types 1, 2 and 3, selenoprotein W, thioredoxin reductase and selenophosphate synthetase. Of these, the are cellular and Plasma glutathione peroxidase the functional parameters for determining the selenium status (D.H. Holben, AT THE. Smith, J. Am. Diet. Assoc. 1999, 99, 836-843).

In addition to vitamin E (DL-α-tocopherol), vitamin C (L-ascorbic acid), co-enzyme Q10, zinc and selenium, there are many other antioxidants that help prevent and / or treat HCV infections by at least partially compensating for the down regulation of GI-GPx can be used, for example N-acetyl-L-cyctein, N-acetyl-S-farnesyl-L-cysteine, bilirubin, caffeine acid, CAPE, catechin, ceruloplasmin, coelenterazine, copper diisopropyl salicylate, deferoxamine mesylate, R- ( -) - Deprenyl, DMNQ, DTPA dianhydride, Ebselen, ellagic acid, (-) - epigallocatechin, L-ergothioneine, EUK-8, ferritin, glutahione, glutathione monoethyl ester, α-lipoic acid, luteolin, manoalid, MCI-186, MnTBYP, M , Morin Hydrate, NCO-700, NDGA, p-Nitro Blue, Propyl Gallate, Resveratrol, Rutin, Silymarin, L-Stepholidin, Taxifolin, Tetrandrin, Tocopherol Acetate, Tocotrienol, Trolox ^® , U-74389G, U-83836E and Uric Acid (all available from Calbiochem , San Diego, CA, USA).

Other antioxidants can 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 guaiac resin, THBP (Trihydroxybutyrophenone), thiodipropionic acid and dilaurylthiodipropionate and glycine.

Oxidative damage is essentially caused by free radicals, preferably reactive oxygen intermediates, caused by normal cellular respiration or oxidative bursting arise when phagocytic cells are infected with bacteria or viruses Destroy cells. To deal with the ongoing generation of potentially harmful Eucariotic organisms have to cope with oxygen radicals developed a variety of protective mechanisms. These include the Antioxidants mentioned above, which act as free radical scavengers can and which can interact with GI-GPx and / or which the expression and / or activate, stimulate and / or produce GI-GPx increase can. This beneficial effect of the radicals on the amount of GI-GPx, which is generated in the cells competes with the HCV-induced Down-regulating the GI-GPx and supporting the cells in their struggle against the hepatitis C virus.

HCV infection studies:

The only reliable experimental HCV infection studies were done on chimpanzees. So far, no simple cell culture infection system is available for HCV. Although a number of reports have been published describing attempts to in vitro increase HCV in primary cells and cell lines, questions regarding reproducibility, low expression levels and reliably controlled detection methods remain open (discussed in: J. Gen Virol. 81, 1631 ; Antiviral Chemistry and Chemotherapy 10, 99). Therefore, the replication system described by Bartenschlager and co-workers (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 here. This replication system reproduces a critical part of the HCV replication cycle that is used as a system for simulating HCV infections. Bartenschlager's group produced bicistronic, recombinant RNA, so-called "replicons", which carry the neomycin phosphotransferase (NPT) gene and a version of the HCV genome in which the sequences for the structural HCV proteins were removed. After the transfection of the HCV subgenomic RNA molecules in the human hepatoma cell line HuH-7 became cells that support the efficient, RNA-dependent RNA replication of the HCV replicons, based on the co-amplification of the NPT gene and the resulting resistance to it Antibiotic G-418 selected. The incorporation of coding information into the cellular genome was an end final criterion for functional replicons. Several lines were created from G-418-resistant clones with autonomously replicating HCV-RNAs detectable by Northern blotting. Negative strand RNA replication intermediates were detected by Northern blotting or metabolic radiolabeling, and the production of non-structural HCV proteins was demonstrated by immunoprecipitation after metabolic radiolabeling or by Western blotting.

Possible influences and / or dependencies on RNA-dependent RNA replication of HCVs and non-structural proteins on host cell transcription are for analysis with the Clontech cDNA arrays described in the here Procedures used are accessible. HuH-pcDNA3 cells are HuH7 cells that are integrated by an NPT gene carrying plasmids (pcDNA3, Invitrogen) are resistant to G-418, and serve as a negative control. Three replicon lines were made for changes in the cellular Expression patterns compared to the control lines examined:

• - HuH-9-13: Cell line with persistent replicon I377 / NS3-3 '/ wt in Science 1999, 285, 110-113.
• - HuH-5-15: Cell line with persistent replicon I389 / NS3-3 '/ wt, described in Science 1999, 285, 110-113.
• - HuH-11-7: Cell line with persistent replicon I377 / NS2-3 '/ wt in Science 1999, 285, 110-113.

These HCV replicon cells serve as a system for simulating HCV-infected cell systems, especially for the simulation of HCV-infected mammals, including People. Influencing cellular signaling HCV is compared to the differential gene expression cellular Signaling reproduced in control cells. The results of this Signal transduction microarray analysis showed significant Down regulation of GI-GPx. Radioactive labeled complex cDNA samples of HCV replicon cells HuH-9-13, HuH-5-15 and HuH-11-7 were added cDNA arrays hybridized and with hybridizations with cDNA samples from HuH pcDNA control cells that did not contain HCV replicons compared.

On the basis of the surprising results reported here, one aspect of the present invention relates to special chemical substances and compounds which are suitable for the prophylaxis and / or treatment of hepatitis C virus infections. In particular, these special substances and compounds selenium, selenium salts, vitamin D ₃ and retinoids include, such as 9-cis-retinoic acid, C ₁ -C ₁₀ alkyl esters of 9-cis retinoic acid, C _{1- 10} alkyl amides of 9-cis retinoic acid , N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN).

Furthermore, the present disclosure Invention a method for the treatment of hepatitis C virus infections in an individual, comprising the step of administering a pharmaceutically effective amount of at least one of the special, above mentioned compounds and substances in the cells at least partly the activity up-regulate from GI-GPx or which at least partially regulate the production of GI-GPx.

A similar aspect of the present The invention relates to a method for prophylaxis against and / or Treatment of HCV infection and / or associated with HCV infection Diseases in cells or cell cultures, including the step of adding a pharmaceutically effective amount of at least one of the special, above compounds and substances that at least partially activity of GI-GPx or at least partially regulate production up-regulate from GI-GPx.

Another aspect of the present Invention is to provide a method of regulation the production of hepatitis C virus in an individual or in Cells or cell cultures comprising the step of administration a pharmaceutically effective amount of at least one of the special, above compounds or substances in the cells at least partly the activity of GI-GPx or at least partially regulate production up-regulate from GI-GPx.

In addition to the above The present invention also relates to methods for prevention methods and / or treatment of a hepatitis C virus infection and / or diseases, associated with HCV infection in an individual, comprising the step of administering a pharmaceutically active Amount of at least one of the special compounds mentioned above or Substances that at least partially activate GI-GPx in the individual or which activates or stimulates the production of GI-GPx.

Another inventive aspect relates to a method for preventing and / or treating Hepatitis C virus infections and / or diseases related to HCV infections stand, in cells or cell cultures, comprising the step of administration a pharmaceutically effective amount of at least one of the special, above compounds or substances at least partially the activity activate GI-GPx or which activate the production of GI-GPx or stimulate.

For example, the term "related diseases" refers for opportunistic infections, cirrhosis, liver cancer, hepatocellular carcinoma or any other illness associated with an HCV infection can occur.

The function of the GI-GPx is that it detoxifies peroxides in cells and protects the cell from oxidative damage. If HCV-infected cells are exposed to oxidative stress conditions, preferably triggered by paraquat or peroxides generated from peroxides, this leads to a reduced resistance of the HCV-infected cells to the toxicity of radicals compared to non-infected cells. Thus, the creation of artificial, oxidative stress conditions allows the selective killing of HCV-infected cells.

Examples of suitable radical-forming Compounds (radical initiators) are bipyridyls such as paraquat, 2,2'-bipyridyl and 4,4'-bipyridyl derivatives, bis-6- (2,2'-bipyridyl) pyrimidines, Tris (2,2-bipyridyl) ruthenium, peroxides such as dibenzoyl peroxide, diacetyl peroxide, Hydrogen peroxide, di-tert-butyl peroxide or diaza compounds such as diazaisobutyronitrile.

Yet another aspect of the present invention relates to a novel therapeutic composition which is suitable for the prophylaxis and / or treatment of an individual suffering from a hepatitis C virus (HCV) infection and / or at least an illness associated with an HCV infection, wherein the composition selected at least one of the special chemical substances and compounds from the group consisting of selenium, selenium salts, vitamin D ₃ and retinoids, such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR), and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN ) includes. A preferred selenium salt is sodium selenite. Furthermore, the composition according to a further preferred aspect of the present invention can comprise a certain amount of all-trans-retinoic acid.

Further embodiments of the present invention are illustrated by methods for regulating the production of hepatitis C virus in an individual or in cells or tent cultures, comprising the step of administering to an individual or adding to the cells a pharmaceutically effective amount of at least one of the specific chemical substances and compound selected from the group consisting of selenium, selenium salts, vitamin D ₃ , and retinoids, such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9- cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR), and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN), the substance or compound being at least partially activates or increases the activity of the human, cellular, gastrointestinal protein glutathione peroxidase or wherein the agent at least partially activates the production of the human, cellular, gastrointestinal protein glutathione peroxidase oxidase activated or stimulated.

A further aspect of the present invention relates to novel therapeutic compositions which are suitable for use in the methods for the prophylaxis and / or treatment of an individual suffering from hepatitis C virus and / or an associated disease. These compositions comprise at least one of the special chemical substances and compound selected from the group consisting of selenium, selenium salts, vitamin D ₃ , and retinoids, such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C. ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR), and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN) that are able to increase the activity of GI-GPx or to activate or stimulate the production and / or expression of GI-GPx.

The pharmaceutical compositions can furthermore pharmaceutically suitable carriers, pharmaceutical carriers and / or Include diluents.

The term “activator” as used here denotes any chemical compound that up-regulates, activates, Stimulation or increase the amount and / or the activity of GI-GPx or its expression.

The term "agent" is used here as a synonym for regulator, Inhibitor, and / or activator used. Thus, the Term "agent" on any chemical or biological compound that regulates down or up, Lowering or increasing, oppression or stimulation, deactivation or activation or otherwise Regulating or influencing the amount and / or activity of GI-GPx and / or the expression of GI-GPx can cause.

In addition to the role in the transfer of Genetic information from DNA to proteins actively take RNA molecules participate in many processes in the cell. Find examples of this in translation (rRNA, tRNA, tmRNA), in intracellular protein targeting (SRP), at core splicing of pre-mRNA (snRNPs), mRNA editing (gRNA) and X chromosome inactivation (Xist RNA). Each of these RNA molecules acts as a functional product itself, without coding a protein. Because RNA molecules in unique shapes with distinctive structural features can fold some RNAs bind to special proteins or small molecules (such as in ATP-binding aptamers), while others have certain chemical Can catalyze reactions. Thus RNA aptamers used to interact with GI-GPx and thereby the activity and modulate, regulate biological function of this peroxidase, activate or inhibit.

As used herein, the term "regulating expression and / or activity" generally refers to any process whose function is to control the amount or activity (functionality) of a cellular component troll or modulate. Static regulation maintains expression and / or activity at a certain level. Up-regulation refers to a relative increase in expression or activity. Accordingly, down-regulation refers to a relative decrease in expression and / or activity. Downregulation is synonymous with the inhibition of the activity of a particular cellular component.

Other aspects of the present Invention relate to methods to either express human, cellular, gastrointestinal protein glutathione peroxidase in an individual or to regulate cells or cell cultures, comprising the step the administration or addition of a pharmaceutically effective amount an agent to the individual or the cells or cell cultures, wherein the agent at least partially transcribes DNA and / or the translation of RNA, which is the human, cellular, gastrointestinal Protein glutathione peroxidase encodes, inhibits or lowers.

Therapeutics, pharmaceutically active Agents or inhibitors can cells of an individual are administered in vitro or can they Include in vivo administration to the individual. The term "individual" preferably refers on mammals, and particularly preferably on people. Routes of administration a pharmaceutical preparation to the individual can oral and parenteral, including dermal, intradermal, intragastric, intracutaneous, intravascular, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutaneous, rectal, subcutaneous, sublingual, topical or transdermal administration, but are not limited to these routes of administration. For example are preferred preparations in one for oral administration suitable form. These forms of administration include, for example Pills, tablets, film-coated tablets, coated tablets, capsules, Powders and deposits. Administration to an individual can be by means of a single dose or repeated administrations in any of a variety of physiologically acceptable forms of salts and / or with a suitable pharmaceutical carrier, binder, lubricant, Drug carriers, diluents or auxiliary substances. Pharmaceutically acceptable salt forms and standard pharmaceutical formulation techniques are familiar to the expert.

In the context of this application means a “pharmaceutical effective amount "of a GI-GIPx activator Amount sufficient to either in the in vitro treated cells or cell cultures or in the subject treated in vivo to achieve physiological effect. In particular, one is pharmaceutical effective amount an amount sufficient to make up one or more of the clinically defined associated with the viral infection pathological processes for inhibit a certain period of time. The effective amount can depending on the selected special inhibitor or activator may be different, and also depends of different, with the subject being treated and the strength of the Infection related factors and conditions. If for example, the activator administered in vivo would become factors like age, weight, and patient's health, as well dose-response curves determined in preclinical animal experiments and toxicity data be among those considered. When the activator comes into contact with cells or cell cultures in vitro would be brought to develop a variety of pre-clinical in vitro studies, around such parameters as intake, half-life, dose, toxicity, etc. estimate. The determination of a pharmaceutically effective amount of a particular Agent falls without further ado of the specialist.

For the first time, the present disclosure teaches the up-regulation of GI-GPx, which is specifically involved in viral infections with hepatitis C viruses, by means of special chemical compounds and substances selected from the group consisting of selenium, selenium salts, vitamin D ₃ , and retinoids, such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR), and 6- [3- (1-Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN).

The polypeptide product of gene expression can can also be examined to determine the level of expression. Investigation methods for Proteins include, but are not limited to, Western blotting, Immunoprecipitation, Radioimmunoassay, immunohistochemistry and peptide immobilization in an ordered array. However, it is said that each method for specific and quantitative determination of a specific protein or mRNA can be used.

The present invention further includes, by reference, all known methods in the field of microarray design and analysis. These techniques include, but are not limited to, the techniques described in the following patents and patent applications that describe arrays of biopolymer compounds and methods of making them:
U.S. Patent No. 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752; 5,561,071; 5,559,895; 5,624,711; 5,639,603; 5,658,734; 5,807,522; 6,087,102;
WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287 and EP 799 897 ,

The techniques also include, but are not limited to, those in the following Pa techniques and patent applications which describe methods in which arrays are used in different applications:
U.S. Patents No. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,994,076; 6,033,860; 6,040,138; 6,040,140;
WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203 and EP 785 280 ,

A robust cell culture system for the Hepatitis C virus (HCV) has not been established. For this reason it’s extremely difficult to study how HCV cells get infected, and testing antiviral drugs in model systems (the only mammals, that can be infected are humans and chimpanzees). An essential step towards disposal a cultivation system for HCV was established through 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 could be preserved for the first time. These subgenomic replicons are composed only of the part of the HCV genome that is for the non-structural Proteins encoded but are able to to be replicated in cells and to synthesize viral proteins. The replicons mentioned in the scientific article cited above by Lohmann et al. are described and for the present investigations were used the study of HCV replication, Pathogenesis and Evulotion in Cell Culture. You can also the implementation of cell-based tests for enable certain types of antiviral drugs.

Recently could target gastrointestinal glutathione peroxidase (GI-GPx) in HCV replication (see WO 02/084294) can be validated. As mentioned above, GI-GPx belongs to the selenoprotein family and plays in the defense mechanisms of eukaryotic cells against oxidative damage play an important role in which they use glutathione as the reducing Catalyze the reduction of a variety of hydroperoxides. It has been suggested that this enzyme acts as a mechanism for Protection of cellular membrane systems across from peroxidative damage acts. Selenium as a necessary trace element points to the essential Function of this enzyme.

Selenium acts primarily in mammalian systems in the form of selenoproteins. Selenoproteins contain selenium in the form of selenocysteine and lead a variety of physiological functions. Seventeen selenoproteins were identified: cellular or classic glutathione peroxidase, plasma (or extracellular) glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, gastrointestinal Glutathione peroxidase, selenoprotein P, iodothyronine deiodinase from Types 1, 2 and 3, selenoprotein W, thioredoxin reductase and selenophosphate synthetase. Of these are cellular and plasma glutathione peroxidase the functional parameters for determining the selenium status (D.H. Holben, A.M. Smith, J. Am. Diet. Assoc. 1999, 99, 836-843).

Compared to HuH7 cells transfected to sham GI-GPx is drastically down-regulated in HCV replicon cells. Become Replicon cells forced to re-express GI-GPx (e.g. by Infection with an adenovirus containing GI-GPx) leads to this to reduce the subgenomic HCV RNA and the HCV protein NS5a to levels that are barely detectable (see WO 02/084294). According to the present Invention was the knowledge of this inverse correlation to development a method for upregulating the expression of the cellular, endogenous GI-GPx gene used. This upregulation in the replicon cells causes depletion of HCV.

It is straightforward for a person skilled in the art apparent that other suitable modifications and adjustments of the compositions and methods of those described herein Invention are obvious and can be made without within the scope of the present invention or those disclosed herein embodiments departing. Having described the present invention in detail has become clearer with reference to the following examples are understood, which are only listed for the purpose of illustration, and not as a limitation of the present invention.

Examples

Reference is made to the examples of WO 02/084294, which are incorporated by reference herein. Furthermore, three cell lines were used as a model system for HCV replication, which were made available by Prof. R. Bartenschlager (Heidelberg University, FRG). The cultures were mixed for various periods with all-trans-retinoic acid (RA) for comparison purposes and the other agents selenium, selenium salts, vitamin D ₃ and retinoids such as 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis retinoic acid, C _{1- 10} alkyl amides of 9-cis retinoic acid, N- (4-hydroxyphenyl) retinsäureamid (4-HPR), and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid ( CD437; AHPN) (purchased from Sigma). The levels of GI-GPx expression were determined at the protein level using antibodies provided by Prof. Brigelius-Flohe (University of Potsdam, FRG) by Western blotting and at the RNA level using GI-GPx-specific oligonucleotides measured as probes by Northern blotting. The levels of HCV RNA were probed using a DNA oligonucleotide complementary to the neomycin phosphotransferase gene as a Northern Blotting probe. The concentration of the viral protein NS5a was determined by Western blotting with an NS5a-specific antibody (Biogenesis, UK).

Treatment of replicon cells over three Days with all-trans retinoic acid (1μm) little effect on GI-GPx and HCV expression. After seven Days of incubation, however, became a drastic up-regulation the GI-GPx on the RNA and Protein level (three to ten times) determined. At the same time the expression of subgenomic HCV-RNA and the viral protein NS5a by two to five times downregulated, depending the cell line examined in each case. Furthermore, surprisingly found that further downregulation of the HCV RNA and the NS5a protein from the addition of selenium or a selenium salt, for example Sodium selenite (50 nM). This fact suggests that the downregulation of HCV firstly through activation of the GI-GPx gene at the level of transcription by the retinoic acid and secondly through the synthesis of selenoprotein (s) for the sodium selenite needed got promoted has been. In fact could are shown to downregulate induced by all-trans retinoic acid independent of HCV of the intrinsic immune response induced by interferon. Thus, the all-trans retinoic acid did not induce the transcription of PKR (double-stranded, RNA-dependent protein kinase). severity cytotoxic effects were neither for all-trans retinoic acid nor for sodium selenite or for both observed together in combination.

The present results show that retinoids (in combination with selenium or selenium salts such as sodium selenite) can be used to treat HCV-positive patients. In particular, the use of retinoids with a high specificity for inducing GI-GPx, such as N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2- naphthalene carboxylic acid (CD437; AHPN) preferred. 4-HPR and AHPN show significant potential as therapeutic agents for the prophylaxis and treatment of a number of pre-malignant or malignant conditions associated with HCV infections. In fact, the present data show that in addition to all-trans retinoic acid, other core receptor ligands, such as, for example, 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides 9-cis-retinoic acid and vitamin D _{3 are} also able to reduce the HCV load.

All-trans retinoic acid on replicon cells over a period of six days leads 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 such as sodium selenite, a two to five-fold reduction in HCV-RNA and HCV-NS5a protein was observed with no toxic effects. Furthermore, the special retinoids such as N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (CD437; AHPN), 9-cis-retinoic acid , C ₁ -C ₁₀ alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid and vitamin D ₃ , either alone or in combination with one another or with selenium or a selenium salt, have a similar effect.

In conclusion, have preliminary results shown that 9-cis-retinoic acid and its above-mentioned alkyl and amide derivatives HCV-RNA essential down-regulate better than all-trans retinoic acid alone.

Claims (20)

Composition suitable for the prophylaxis and / or treatment of an individual suffering from a hepatitis C virus (HCV) infection and / or at least from an illness associated with an HCV infection, the composition consisting of at least one agent selected from the group from selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN). The composition of claim 1, wherein the selenium salt Is sodium selenite. A composition according to claim 1 or 2, wherein the composition further comprises all-trans retinoic acid. Composition according to one of claims 1 to 3, further comprising at least one pharmaceutically acceptable one Carrier, excipient and / or diluents. Use of at least one of the agents selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- ( 4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN) for the manufacture of a pharmaceutical composition for the treatment of a hepatitis C virus infection and / or disease related to HCV infection and / or prophylaxis against hepatitis C virus infection and / or disease related HCV infection. Use according to claim 5, wherein the Selenium salt is sodium selenite. Use according to claim 5 or 6, the composition further comprising all-trans retinoic acid. A method of preventing and / or treating hepatitis C virus infection and / or HCV infection related diseases in an individual comprising the step of administering a pharmaceutically effective amount of selenium, selenium salts, vitamin D ₃ , 9-cis retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN) to the individual. A method of preventing and / or treating hepatitis C virus infection and / or HCV infection related diseases in cells or cell cultures comprising the step of adding a pharmaceutically effective amount of selenium, selenium salts, vitamin D, 9-cis-retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN) to the individual. A method of regulating the production of hepatitis C virus in an individual comprising the step of administering a pharmaceutically effective amount of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl ester of 9-cis-retinoic acid , C ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN ) to the individual. A method of regulating the production of hepatitis C virus in a cell or cell culture comprising the step of adding a pharmaceutically effective amount of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl ester of 9-cis -Retinoic acid, C ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN) to the cells or cell culture. A method of preventing and / or treating hepatitis C virus infection and / or HCV infection related diseases in an individual comprising the step of administering a pharmaceutically effective amount of selenium, selenium salts, vitamin D ₃ , 9-cis retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), which at least partially triggers the activity of the human, cellular, gastrointestinal protein glutathione peroxidase or which at least partially triggers or stimulates the production of the human, cellular, gastrointestinal protein glutathione peroxidase. A method of preventing and / or treating a hepatitis C virus infection and / or diseases related to HCV infection in cells or cell cultures comprising the step of adding a pharmaceutically effective amount of selenium, selenium salts, vitamin D ₃ , 9-cis Retinoic acid, C ₁ -C ₁₀ alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- ( 1-adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN) which at least partially triggers the activity of the human, cellular, gastrointestinal protein glutathione peroxidase or which at least partially triggers or stimulates the production of the human, cellular, gastrointestinal protein glutathione peroxidase , A method for regulating the production of hepatitis C virus in an individual comprising the step of administering to an individual a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ - C ₁₀ alkyl esters of 9-cis-retinoic acid, C _{1- 10} alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4 -hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), wherein the agent at least partially triggers the activity of human, cellular, gastrointestinal protein glutathione peroxidase or which at least partially triggers or stimulates the production of human, cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the production of hepatitis C virus in cells or a cell culture comprising the step of adding a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C _10- alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-adamantyl) -4 -hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), wherein the agent in the cells or the cell culture at least partially triggers the activity of the human, cellular, gastrointestinal protein glutathione peroxidase or wherein the agent in the cell or cell culture at least partially triggers the production of the human, Triggers or stimulates cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the expression of human, cellular, gastrointestinal protein glutathione peroxidase in an individual comprising the step of administering to an individual a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), the agent at least partially transcribing DNA and / or translating RNA encoding human, cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the expression of human, cellular, gastrointestinal protein glutathione peroxidase in an individual comprising the step of administering to an individual a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), the agent at least partially transcribing DNA and / or translating RNA encoding human, cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the expression of human, cellular, gastrointestinal protein glutathione peroxidase in cells or a cell culture comprising the step of adding to the cells or the cell culture a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), the agent at least partially transcribing DNA and / or translating RNA encoding human, cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the activity of human, cellular, gastrointestinal protein glutathione peroxidase in an individual comprising the step of administering to the individual a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid , C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1 -Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), the agent interacting with the human, cellular, gastrointestinal protein glutathione peroxidase. A method for regulating the activity of human, cellular, gastrointestinal protein glutathione peroxidase in cells or a cell culture comprising the step of adding to the cells or the cell culture a pharmaceutically effective amount of an agent selected from the group consisting of selenium, selenium salts, vitamin D ₃ , 9-cis-retinoic acid, C ₁ -C ₁₀ -alkyl esters of 9-cis-retinoic acid, C ₁ -C ₁₀ -alkylamides of 9-cis-retinoic acid, N- (4-hydroxyphenyl) retinoic acid amide (4-HPR) and 6- [3- (1-Adamantyl) -4-hydroxyphenyl] -2-naphthalene carboxylic acid (AHPN), the agent interacting with the human, cellular, gastrointestinal protein glutathione peroxidase.