EP1450778A2

EP1450778A2 - Pharmaceutical composition for the prophylaxis and/or treatment of virus diseases

Info

Publication number: EP1450778A2
Application number: EP02758125A
Authority: EP
Inventors: Stephan Ludwig; Oliver Planz; Hans-Harald Sedlacek; Stephan Pleschka
Original assignee: Medinnova Gesellschaft fuer Medizinische Innovationen aus Akademischer Forschung mbH,
Current assignee: Medinnova Gesellschaft fuer Medizinische Innovationen aus Akademischer Forschung mbH,
Priority date: 2001-08-08
Filing date: 2002-07-26
Publication date: 2004-09-01
Also published as: US20100239563A1; JP2005506975A; EP1707193A3; DE10138912A1; EP1707193A2; US20050129694A1; AU2002325169A1; WO2003015689A2; WO2003015689A3

Abstract

The invention relates to the use of at least one active substance for producing a pharmaceutical composition for the prophylaxis and/or treatment of at least one virus disease. The invention is characterised in that the active substance(s) inhibit(s) either at least two kinases or at least one SEK kinase of a cellular signal transmission path, in such a way that virus multiplication is inhibited.

Description

Pharmaceutical composition for the prophylaxis and / or therapy of viral diseases

Field of the Invention. The invention relates to the use of at least one active substance for the production of a pharmaceutical composition for the prophylaxis and / or therapy of at least one viral disease, and a combination preparation therefor.

Background of the Invention and Prior Art.

Infections with RNA or DNA viruses represent a major health threat for humans and animals. For example, infections with influenza viruses are still one of the greatest diseases of mankind and cause a large number of deaths every year. They are an immense cost factor for the economy as a whole, for example due to sick leave. Infections with the Borna Disease Virus (BDV), which primarily affects horses and sheep, but have also been isolated in humans and are associated here with neurological diseases, are also of important economic importance.

The problem of combating RNA viruses in particular is the versatility of the viruses caused by a high error rate of the viral polymerases, which makes the production of suitable vaccines and the development of antiviral substances very difficult. In addition, it has been shown that the use of antiviral substances which are directed directly against the functions of the virus have a good antiviral effect at the start of therapy, but due to the mutation leads very quickly to the selection of resistant variants. On An example of this is the anti-influenza agent arαantadin and its derivatives, which is or are directed against a transmembrane protein of the virus. Resistant variants of the virus form within a short time after use. Another example are the new therapeutics for influenza infections, which inhibit the influenza viral surface protein neuraminidase. This includes, for example, Relanza. Relanza-resistant variants have already been found in patients (Gubareva et al., J Infect Dis 178, 1257-1262, 1998). The hopes placed in this therapeutic agent could not be fulfilled.

Because of their usually very small genomes and the associated limited coding capacity for functions required for replication, all viruses are heavily dependent on the functions of their host cells. By influencing such cellular functions that are necessary for viral replication, it is possible to adversely affect virus replication in the infected cell. It is not possible for the virus to replace the missing cellular function by adaptation, in particular by mutations, in order to escape the selection pressure. This has already been shown using the example of the influenza A virus with relatively unspecific inhibitors against cellular kinases and methyltransferases (Scholtissek and Müller, Arch Virol 119, 111-118, 1991).

It is known that cells have a multiplicity of signal transmission paths with the aid of which signals acting on the cell are transmitted into the cell nucleus. This enables the cell to react to external stimuli and react with cell proliferation, cell activation, differentiation or controlled cell death. this The signal transmission pathways have in common that they contain at least one kinase, which activate at least one subsequent signal-transmitting protein by phosphorylation. When considering the cellular processes that are induced 5 after virus infections, it is found that a large number of DNA and RNA viruses in the infected host cell preferentially activate a defined signal transmission path, the so-called Raf / MEK / ERK kinase signal transmission path ( Benn et al., J Virol 70, 4978-4985,

10 1996; Bruder and Kovesdi, J Virol 71, 398-404, 1997; Popik and Pitha, Virology 252, 210-217, 1998; Rodems and Spector, J Virol 72, 9173-9180, 1998). This signal transmission path is one of the most important signal transmission paths in a cell. and plays a significant role in proliferation

15 eration and differentiation processes. Growth factor-induced signals are successively phosphorylated from the serine / threonine kinase Raf to the dual-specific kinase MEK (MAP kinase kinase / ERK kinase) and finally to the kinase ERK (extracellular signal regu

20 lated kinase). While only MEK is known as the kinase substrate for Raf and the only substrates for MEK that have been identified are the ERK isoforms, ERK can phosphorylate a whole range of substrates. This includes, for example, transcription factors, whereby the cell

25 lular gene expression is directly influenced (Cohen,

Trends in Gell Biol 7, 353-361, 1997; Robinson and Cobb, Gurr. Opin. Gell Biol 9, 180-186, 1997, Treisman, Curr. Opin Cell Biol 8, 205-215, 1996). Examining the role of this signaling pathway in cellular decision-

30 processes has led to the identification of several pharmacological inhibitors, which the

Inhibit signal transmission path, inter alia, at the level of MEK, i.e. relatively at the beginning of the signal transmission path 03/015689

(Alessi et al., J Biol Chem 270, 27489-27494, 1995; Cohen Trends in Cell Biol 7, 353-361, 1997; Dudley et al., PNAS USA 92, 7686-7689, 1995; Favata et al., J Biol Chem 273, 18623-18632, 1998). Recent data show that the inhibition of the Ras-Raf-MEK-ERK signaling pathway by active substances which relatively selectively inhibit one of the kinases involved in this signaling pathway, in particular the MEK, the intracellular multiplication of intranuclearly replicating negative-strand viruses, for example of influenza A Virus and Borna Disease Virus (BDV) can drastically inhibit (Pleschka et al., Nature Cell Biol 3, 301-305, 2001; Planz et al., J Virol 10, 4871-4877, 2001).

The disadvantage of known antiviral active substances is that they are either directed against a viral component and thus quickly lead to resistance (see Amantadine), or are too broad and unspecific against cellular factors (e.g. methyltransferase inhibitors), with major side effects to be expected , Accordingly, none of the substances acting against cellular factors is known to have been developed to date as a therapeutic agent for viral diseases. On the other hand, the inhibition of other kinases, for example the inhibition of the kinase JNK of the MEKK / SEK / JNK signaling pathway, can increase the virus replication.

It is also known that the increased activation of other kinases, for example protein kinase C (PKC), inhibits the replication of viruses (Driedger and Quick, WO 92/02484).

It was previously thought that negative-strand RNA viruses only use the Raf-MEK-ERK signal transmission path for their multiplication and consequently an inhibition of the Virus multiplication for this signal transmission path, in particular through an inhibition of MEK, leads to a complete inhibition of virus multiplication. However, since the signal transmission paths in a cell hardly have any self-contained functions, but when activating one signal transmission path via cross-links additional signal transmission paths are activated to different extents, it cannot be ruled out that the Raf-MEK-ERK signal transmission path can be used by both the cell as well as the viruses can be bypassed and the therapeutic effect of an active substance which inhibits virus multiplication for the Raf-MEK-ERK signal transmission path could thereby be restricted.

Technical problem of the invention.

There is therefore a great need for finding and using antiviral active substances which act in addition or in addition to those which inhibit virus multiplication in the Raf-MEK-ERK signal transmission path. Finding such active substances is made more difficult by the fact that, for example, inhibitors of kinases outside the Raf-MEK-ERK signal transmission path, for example inhibitors of the kinase JNK, can promote virus multiplication, that is to say they achieve the opposite effect than desired (Ludwig et al., J Biol Chem 276, 1-9, 2001), and that, in turn, the activation of selected kinases, for example PKC, by substances can have an antiviral effect.

Basics of the invention and preferred embodiments.

One object of the present invention is the use of at least one active substance for prophylaxis and / or therapy of at least one viral disease, the active substance (s) either acting on at least two kinases of a cellular signal transmission path in such a way that virus multiplication is substantially inhibited or an SEK kinase is essentially inhibited or inhibited. The present invention furthermore relates to the combination of at least one active substance according to the invention with at least one further antiviral active substance, which is preferably not a kinase inhibitor, for the prophylaxis and / or therapy of a virus infection.

It has now surprisingly been found that inhibition of a SEK kinase in the MEKK / SEK / JNK signal transmission pathway inhibits virus replication. This is so surprising since the inhibition of JNK in the MEKK / SEK / JNK signal transmission path does the opposite, an increased virus replication (Ludwig et al., J Biol Chem 276, 1-9, 2001). Since the JNK-AP-1 signaling pathway is significantly involved in the virus-induced expression of IFNß, a highly antiviral cytokine, the lack of IFNß expression after inhibition of JNK appears to enable an increased virus reproduction. Furthermore, it has surprisingly been found that inhibiting at least two kinases of a cellular signal transmission path by at least one active substance inhibits virus multiplication far more than is the case when only one kinase is inhibited by an active substance. For example, the inhibition of p38 and MAPKAPK3 or the inhibition of MKK6 and p38 or the inhibition of Raf and MEK leads to a significantly stronger antiviral effect than the inhibition of only one, in particular the (the second kinase) activating, kinase. It was also surprisingly found that the inhibition of at least one kinase of a cellular signal transmission path through at least one active substance and the additional administration of an antiviral active ingredient, which is not an inhibitor of a kinase, leads to a strong inhibition of the virus multiplication. This inhibition by the combination was stronger than would have been expected if the individual effects of the components of this combination had been added. For example, cells were infected with influenza A and the cells with the kinase inhibitor U0126, which is known to inhibit MEK kinase in the Raf / MEK / ERK signaling pathway and at the same time to treat it with amantadine. Amantadine is known to act not on all influenza A viruses and not on infuenza B viruses, and furthermore that it leads to the formation of resistant virus variants in a short time. The combination of U0126 and amantadine inhibited the multiplication of influenza A viruses, whereby the combination can have a stronger effect than the two individual components alone. These cellular signal transmission paths include, for example: MEKK2, -3 / MEK5 / ERK5; Raf / MEK / ERK; MEKK / SEK / JN; JAK1, JAK2, JAK3, TYK2 / hetero- and homodimers of JAK1, -2, -3, TYK2; ASK / MKK3, -6 / p38; ASK / MKK4, -7 / JNK; MEKK4 / MKK4, -7; DLK / MKK4, -7; TPL-2 / MKK4, -7; TPL-2 / MEK5 / ERK5; MLK-3 / MKK3, -6; MLK-3 / MKK4, -7; TAK / NIK / IKK; TAK / MKK3, -6; TAK / MKK4, -7; PAK / MKK3, -6; PAK / IKK; Cot, Tpl-2 / IKK; PKC / IKK; PKB / IKK; PKC / Raf; PAK / Raf; Lck / Raf; MEKKs / IKK; PI3K / PDK1 / PKB; JAK / TYK / PLCgamma; JAK / Tyk / Stat complexes; MAP kinases / MAPKAP kinases, for example: ERK / 3pK, ERK / Rskp90, p38 / MAPKAP kinases, p38 / 3pK.

Details of these signal transmission paths have been provided by Sedlacek (Drug 59, 435-476, 2000) and in various other publications (Karin and Ben-Neriah, Ann Rev Immunol 18, 621-663, 2000; Vertegaal et al., Cell Signal 12, 759-768, 2000; Vanhaesebroeck and Alessi, Biochem J 346,, 561- 576, 2000; Ludwig et 5 al., Mol Cell Biol 16, 6687-6697, 1996; New et al., J Biol Chem 274, 1026-1032, 1999; Ni et al., Biochem Biophys Res Commun, 243, 492 -496, 1998; Rebecchi and Pentyala, Physiol Rev 80, 1291-335, 2000; Cohen, Trends in Cell Biol 7, 353-361, 1997; Robinson and Cobb, Curr Opin Cell Biol 9,

10 180-186, 1997; Rane and Reddy, Oncogene, 19, 5662-5679,

2000; Sun et al. , Curr Biol. 10, 281-284, 2000; Garrington and Johnson, Curr Opin Cell Biol 11, 211-218, 1999).

Active substance in the sense of the present invention is a substance which is capable of acting either on

15 to act at least one kinase of a cellular signal transmission path in such a way that a virus multiplication is essentially inhibited or to essentially inhibit a SEK kinase of a cellular signal transmission path. Furthermore, as active substances in the sense of this invention

To understand 20 derivatives of the active substances, which are converted, for example by enzymatic cleavage, into an active substance according to the invention. Active substances in the sense of the present invention are also precursors of active substances which are metabolic

25 can be converted into an active substance according to the invention. For the purposes of the present invention, the active substances include, for example: kinase-inhibiting flaven derivatives or benzpyran derivatives; Kinase-inhibiting derivatives of 4H-1-benzopyran,

30 for example as disclosed in EP 0137193 and EP 366061. The structure-activity relationship for these derivatives has been described, for example, by Sedlacek et al. (Int J Oncol 9, 1143-1168, 1996) described in detail; Flavopiridolderi- vate, for example by Kim et al. , J Med Chem 43 (22), 4126-4134, 2000, in particular thio-flavopiridol and oxy-flavopiridol; 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- (1) benzopyran, for example disclosed by Ebendal (WO 5 00/50030); 7, 12-dihydro-indolo (3,2-d) (l)

Benzazepin-6 (5H) -one (NSC 664704 Sausville et al. Pharmacol. Ther. 82 (2-3), 285-292, 1999); Phosphokinase inhibitors, for example 70H-staurosporins and their phosphokinase inhibiting derivatives, butyrolactones, roscovitines,

10 Purvalanol A, Emodin, anilinoquin azoline (PD-168393 and PD 169414), PD 184352 (Duesbery et al., Nature Medicine 5 (7), 736-737, 1999) and Penylaminopyrididine (STI 571, CGP 78850, CP 358774, CP59326 and CGP 60474)), trioylimidazoles (1-779450) (Meijer et al., Parmacol. Ther .82 (2-3),

15, 297-284, 1999; Sedlacek et al. , Int J Oncol 9, 1143-1168, 1996; Sedlacek Drug 59 (3), 435-476, 2000); Paullone (Zaharevitz et al., Cancer Res. 59, 2566-2569, 1999); SB203580 [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridy 1) lH-imidazole, (Ishikawa et al., J Neurochem 75 (2),

20 494-502, 2000, Harada and Sugimoto, Jpn J Pharmacol 79 (3), 369-378, 1999)]; Kinase-inhibiting buatadiene derivatives, in particular derivatives of diaminodicyano- (R) -thiobutadiene; U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2aminophenylthio) butadiene] (Favata et al., J Biol Chem 273 (29), 18623-

25, 18632, 1998; DeSilva et al. , J Immunol 160, 4175-4181, 1998)]; PD098059 [2-2 'amino-3' methoxyphenyl) oxanaphthalen-4-one)], (Dudley et al., PNAS USA 92, 7686-7686, 1995); PD184352 [2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3, 4-difluorobenzamide], (Sebolt-Leopold et

30 al., Nature Med 5, 810-816, 1999); 3-aminomethylene indoline derivatives (Heckel et al., DE 92 4401, Eberlein et al., DE 84 4003; WO 00/018734); CEP-1347 (KT7515) bis-ethylthiomethyl (Maroney et al., J Neurochem 73 (5), 1901-1912, 1999); Tetrapyrrolic macrocycles, for example 5, 10, 15, 20-tetraarylporphyrin and 5,10,15-triarylcorrol (Aviezer et al., WO 00/27379); Pyrimidone derivatives (Ando et al., WO 00/018758); 3-aminomethylene indoline derivatives (Heckel et al., DE 92 4401, Eberlein et al., DE 84 4003; WO 00/018734); Pyrazolo ^• (3, 4-b) pyridine derivatives (Bursuker et al., WO 99/30710); Pyrazole derivatives (Antanarayan et al., WO 00/031063); 1,4-substituted piperidine derivatives (Caravatti et al., EP 374095); lipoid ammonium salts (Daniel et al., WO 90/04918); 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) IH-imidazole (SB202190); SL 327 (a butadiene derivative); Dominant negative mutants of a kinase in a cellular signaling pathway; Antisense oligonucleotides which attach specifically to the DNA sequence or mRNA sequence and code for a kinase of a cellular signal transmission pathway and inhibit their transcription or translation; ds oligonucleotides which are suitable for the targeted degradation of the mRNAs of kinases of a cellular signal transmission path by the RNAi technology in accordance with the method as described by Tuschl et al (Genes Dev 13: 3191-3197, 1999) and by Za ore et al (Cell 101: 25-33, 2000); Antibodies or antibody fragments specific for a kinase of a cellular signal transmission path, or fusion proteins containing at least one

Antibody fragment, for example an Fv fragment, which inhibit the kinase activity of at least one kinase; Peptides that inhibit the interaction of at least two kinases in a cellular signaling pathway.

At least one active substance according to the invention is preferably used in the case of a viral disease caused by RNA or DNA viruses, preferably negative-strand RNA Viruses, for example influenza viruses, or Borna viruses.

A further embodiment of the present invention relates to a combination preparation for the prophylaxis and / or therapy of at least one viral disease, comprising at least two active substances which either act on at least two kinases of a cellular signal transmission path in such a way that virus multiplication is substantially inhibited or one SEK kinase in general - Significantly inhibit, preferably selected from active substances already listed above, the combination preparation being usable in the form of a mixture or as individual components for simultaneous or temporally different use at the same or different locations.

By administering a combination preparation containing at least two different active substances, the virus multiplication is inhibited by acting on at least two kinases of a cellular signal transmission path. The combination preparation can be administered as a mixture of the active substances. However, the active substances can also be administered separately from one another at the same location, for example intravenously, or else at separate locations, simultaneously or at different times within a period in which the substance administered first is still effective, for example in a period of three Days.

Another embodiment of this invention is the administration of a combination preparation comprising at least one active substance according to the invention and at least one antiviral active substance which is not a kinase inhibitor. To these antivirals Active ingredients include, for example: 1-adamantanamine (amantadine); Ri antadine; Neuraminidase inhibitors such as Relenza; synthetic nucleoside analogs such as 3-deaza adenosine and ribavirin. A further embodiment of the present invention relates to a test system for finding active substances which inhibit either at least two kinases of a cellular signal transmission path or an SEK kinase in such a way that virus multiplication is essentially inhibited, comprising: a. at least one cell infectable with at least one virus, which contains either at least two kinases of a cellular signal transmission path or at least one SEK kinase and at least one virus infecting the cells or b. at least one cell infected with at least one virus, which contains either at least two kinases of a cellular signal transmission path or at least one SEK kinase.

Cells in the sense of the present invention are cells from different organs and tissues, for example cells of the blood and lymph vessels, cells that line body cavities. Also included are cell cultures, especially those that can be purchased from cell banks, for example the ATCC, in particular permissive, eukaryotic cell cultures, for example: 293, 293T and 293T7 {Homo sapiens); B82, NIH 3T3, L929 from Mus muscul us; BHK from Cri cetus cricetus; CHO from Cri cetul us griseus; MDCK from Canis familiaris; Vero, COS-1 and COS-7 from Cercopi thecus aethi ops; as well as primary embryo fibroblasts from gall us gall us (CEF cells).

For example, in the test system according to the invention for finding active substances by adding substances, preferably in concentrations of O.OOlμMol up to 100 μmol, and viruses in a particle number, which can infect the selected cell, checked whether a substance is able to inhibit virus multiplication without damaging the cell. The virus used in the test system according to the invention is preferably an RNA or DNA virus, preferably an influenza virus.

In a preferred embodiment, the cell of the test system according to the invention contains at least one overexpressed kinase, in particular by introducing one or more genes encoding the kinase (s). This overexpression detects substances which both strongly inhibit kinases and can also reach a high concentration intracellularly to inhibit the overexpressed kinase. As a control, expression for at least one kinase is inhibited in a cell of the test system according to the invention, for example: by introducing an antisense DNA or an antisense RNA; by introducing at least one gene coding for at least one dominant-negative mutant of at least one parent kinase; and / or by introducing at least one gene coding for at least one dominant negative mutant of at least one subordinate kinase of a signal transmission path. Another embodiment of the present invention relates to a method for finding at least one active substance for the prophylaxis and / or therapy of viral diseases, which essentially inhibits or inhibits the multiplication of viruses in viral diseases, comprising the following steps: a. Bringing at least one test system according to the invention into contact with at least one potential active substance, and b. Determination of the impact on virus replication. For the purposes of this invention, contacting can take place, for example, by adding the active substances into the nutrient medium of a cell culture or by local or systemic administration of the active substances into an organism. For the purposes of the present invention, contacting also includes the methods customary in the prior art which permit the introduction of substances into intact cells, for example infection, transduction, transfection and / or transformation and other methods known to the person skilled in the art. These methods are particularly preferred if the substance is a virus, naked nucleic acids, for example antisense DNA and / or antisense RNA, viroids, virosomes and / or liposomes, virosomes and liposomes also being suitable, in addition to a nucleic acid molecule, further active substances to bring to the cell.

The effect on virus replication is determined, for example, by means of plaque assays by comparing virus titer of infected and uninfected cells.

A further preferred embodiment of the present invention relates to a method for producing a medicament for the prophylaxis and / or therapy of at least one viral disease which essentially inhibits or inhibits the multiplication of viruses in viral diseases, comprising the following steps: a. Performing a test system according to the invention and b. Adding at least one auxiliary and / or additive to the active substance (s) found. The active substance according to the present invention is preferably used for local or systemic administration in an organism with the aid of those skilled in the art common methods and auxiliaries and / or additives prepared for a drug.

Suitable auxiliaries and additives, which are used, for example, to stabilize or preserve the medicament or diagnostic agent, are generally known to the person skilled in the art (see, for example, Sucker H et al. (1991) Pharmaceutical Technology, 2nd edition, Georg Thieme Verlag, Stuttgart). Examples of such auxiliaries and / or additives are physiological saline solutions, Ringer's dextrose, dextrose, Ringer's lactate, demineralized water, stabilizers, antioxidants, complexing agents, antimicrobial compounds, proteinase inhibitors and / or inert gases.

Local administration can take place, for example, on the skin (for example with a transdermal system), on the mucous membrane, in a body cavity, in an organ (e.g. i.m.), in a joint or in the connective or supporting tissue. Systemic administration is preferably carried out in the bloodstream, for example i. ., in the peritoneal cavity or in the abdominal cavity.

The pharmaceutical preparation containing the active substance according to the invention depends on the type of active substance and the way in which it is administered and can be, for example, a solution, a suspension, an ointment, a powder, a spray form or another inhalation preparation. Nucleotide sequences are preferably inserted into a viral vector or a plasmid using methods known to the person skilled in the art and auxiliary substances for cell transfection are added. These auxiliaries include, for example, cationic polymers or cationic lipids. Antisense oligonucleotides are derivatized using the methods familiar to the person skilled in the art protect them from enzymatic degradation by DNAsen or RNAsen.

The active substance according to the invention can be in the form of a salt, ester, amide or as a precursor, preference being given to using only modifications of the active substance which do not trigger excessive toxicity, irritation or allergic reactions in the patient.

The active substance is mixed under sterile conditions, with a physiologically acceptable carrier and possible preservatives, buffers or propellants, as required. Such excipients for pharmaceutical preparations are familiar to the person skilled in the art.

The active substance according to the invention is preferably administered in a single dose, particularly preferably in several doses, the individual doses not exceeding the maximum tolerable dose (MTD) of the respective active substance for humans. A dose is preferably chosen which is half the MTD. For example, for the infusion of flavopiridol, the MTD in the tumor patient is 50 mg / m ² / dx3 (Senderowicz et al J Clin Oncol 16 (9): 2986-2999, 1998). For human use, in the sense of the present invention flavopiridol is thus in a daily dose of 0.1 50 mg / m ^2, vorzug- sweise of 5-30 mg / m ^2, more preferably from 25mg / m ² to be administered. The daily dose can be administered once a day or divided into several portions over the day, preferably at approximately the same time intervals. According to the present invention, administration can be either local or systemic, only on one day or for several days a day, or every other or third day over several weeks until a therapeutic effect is visible.

Further variants of the invention are specified in claims 17 to 19, the foregoing and following explanations applying analogously to this.

Example 1: Positive Control I (Influenza A Virus)

For the multiplication of influenza A viruses, performance, eukaryotic cell cultures (Madine darby canine kidney (MDCK) cells) are washed with a physiological saline solution in parallel batches with the same number of cells, according to general methods customary for cell cultures, and with the same Amount of the infectious influenza A virus strain WSN-HK (reassortant with seven gene segments of the influenza strain A / WSN / 33 and the NA gene of the

Influenza strain A / HK / 8/68), in a ratio of 0.0025 infectious virus particles per cell for one hour at room temperature. 30 min before the infection, the MDCK cells are placed in a suitable cell culture medium which is used for positive control in different concentrations with the kinase inhibitor U0126 [1, 4-diamino-2, 3-di-cyano-1, 4-bis (2aminophenylthio) butadiene] is added (OuM, 30uM, 40uM, 50uM dissolved in DSMO) at 37 ° C and 5% CO 2 concentration. As a solvent control, MDCK cells are incubated with cell culture medium, which is mixed with corresponding different amounts of DMSO. During the infection, the kinase inhibitor U0126 or as a solvent DMSO is added to the inoculum in the appropriate concentrations. The inoculum is then removed and the infected cells are placed in a suitable cell culture medium containing the kinase inhibitor in different concentrations U0126 is mixed (OμM, 30μM, 40μM, 50uM dissolved in DMSO), incubated for 48h, at 37 ° C and 5% C0 ₂ concentration. As a solvent control, infected MDCK cells are incubated with cell culture medium which has been mixed with corresponding different amounts of DMSO. 24 hours after the infection, 200 .mu.l of the medium supernatant are removed and the same volume of cell culture medium containing inhibitor or DMSO is added again to the medium supernatant. A sample is taken again after 48 hours. The cell culture supernatants of the respective samples for the 24- and 48-hour values are calculated according to the usual virological methods on the amount of haemagglutinating units (HA titer), which represents the total production of virus particles, and on the amount of newly formed infectious virus particles (plaque assay

MDCK cells) examined. As a result, it can be determined in such an experimental approach that with increasing concentration of the kinase inhibitor U0126 in the cell culture medium, there is a significant reduction (approx. 80% at 50μM U0126) in the number of newly formed, infectious virus particles compared to the control approach without Inhibitor U0126 or the solvent controls comes. The macroscopic examination of MDCK cells, which were treated with appropriate concentrations of DMSO or inhibitor U0126 dissolved in DMSO, as well as a cytotoxicity study using propidium iodide staining, shows that neither solvent nor inhibitor have a significant cytotoxic effect on the cells.

Example 2: Positive Control II (Influenza B Virus)

This example shows that at a concentration of 60μM of the MEK inhibitor U0126 in the cell culture medium too the number of newly formed, infectious influenza B virus particles is significantly reduced. For the multiplication of influenza B viruses, permissive, eukaryotic cell cultures (Madine darby canine kidney (MDCK) cells) are washed with a physiological saline solution in parallel batches with the same number of cells, according to generally used methods for cell culture, and with the same Amount of the infectious influenza B virus strain Massachusetts / 6/93, in a ratio of 0.01 infectious virus particles per cell for one hour at room temperature, infected. After the infection, the inoculum is removed and the infected cells in a suitable cell culture medium (containing 2 μg / ml trypsin), which has been mixed with the MEK inhibitor U0126 (60 μm dissolved in DMSO), for 60 h, at 37 ° C. and 5% CO, Concentration, incubated. As a control, infected MDCK cells are incubated with cell culture medium to which the appropriate amount of DMSO has been added. Medium supernatant samples are taken every 12 hours after infection. The respective samples of the cell culture supernatants are examined for the amount of newly formed infectious virus particles (plaque assay for MDCK cells) using customary virological methods. The result can be determined in such an experimental approach that the corresponding concentration of the MEK inhibitor U0126 in the cell culture medium leads to a significant reduction (approx. 90%) in the number of newly formed, infectious virus particles compared to the Control approach (solvent control without MEK inhibitor U0126) comes. The macroscopic examination of MDCK cells, which with appropriate

Concentrations of DMSO or MEK inhibitor U0126 dissolved in DMSO were treated, as well as a cytotoxicity test using propidium iodide staining, that neither solvent nor inhibitor have a significant cytotoxic effect on the cells.

Example 3: Specific inhibition of virus replication in the cellular MEKK-SEK-JNK signaling pathway

The influence of a specific inhibition of SEK / MKK4 was first investigated with the help of transient transfection of MDCK cells with a dominant negative mutant of the kinase SEK KD. In this mutant, a lysine amino acid residue at amino acid position 129 was converted into an arginine amino acid residue by targeted mutagenesis at the DNA level (Ludwig et al. Mol Cell Biol 16,6687-6697 1996), a mutation which determines the ATP binding of the kinase disturbing and the kinase is thus inactive. In the event of overexpression in the cell, this mutant has a predominantly negative effect on the endogenous wild type (Ludwig et al Mol Cell Biol 16,6687-6697 1996). For the experiment, MDCK cells with the empty vector pEBG or the expression construct pEBG SEK KD (Ludwig et al. Mol Cell Biol 16,6687-6697 1996) were used with the help of the transfection reagent

Lipofectamine 2000 (Life Technologies) transfected by standard methods (Ludwig et al. J Biol Chem 276,10990-10998,2001). The transfection efficiencies were over 60%. Infection with the influenza A virus strain fowl plage virus occurred 24 hours after transfection

A / Fpv / Bratislava / 79 with an infection multiplicity of 1 (MOI = 1). A further 24 hours after infection, the titers of the newly formed viruses in the cell culture supernatant were checked in standard plaque assays for MDCK cells. The virus titers of influenza A virus-infected MDCK cells, which had been infected either with the empty vector or with a construct which expressed the dominant negative form of SEK, were compared. Furthermore, with the help of retroviral transduction, MDCK cell lines were produced which stably expressed either SEK KD or a SEK antisense RNA. Overexpression of the dominant negative kinase SEK KD competitively inhibits the endogenous wild type, while the formation of an RNA species complementary to the SEK / MKK4 messenger RNA (antisense RNA) inhibits the synthesis of endogenous SEK / MKK4. To generate these stable MDCK cellins, the cDNA for SEK KD was cloned into the retroviral expression vector pCFG5 IEGZ (Kuss et al. Eur J Immunol 29, 3077-3088, 1999) in both sense and antisense orientation. In addition to the messenger RNA for SEK KD or the antisense RNA, the vector DNA also codes for the messenger RNA of the "green fluorescent protein" (GFP), which is expressed during protein synthesis starting from an internal ribosome binding part. This allows the identification of stably transduced cells in flow cytometry. In addition, the vector imparts resistance to the antibiotic Zeocin. The expression constructs for SEK KD and SEK-antisense RNA as well as the empty vector were transfected into the virus-producing cell line 0NX (Grignani et al. Cancer Res 58, 14-19, 1998) using the calcium phosphate precipitation method. The transfection efficiency was checked after 24 hours on the basis of GFP expression and was in a

The order of 70-80%. The cells were then selected for about two weeks with 1 mg / ml Zeocin in the medium. To infect MDCK cells with the recombinant retroviruses, the retrovirus-containing medium supernatants of the virus-producing cell lines were filtered, mixed with 5 μg / ml polybrene (Sigma-Aldrich, Tauf irchen b. Munich, Germany) and applied to fresh MDCK cells. The infection occurred over two 3-hour periods Centrifugation (lOOOxg) on two consecutive days. Stably transduced MDCK cells were selected 24 hours after infection with 400-600 μg / ml Zeozin in the medium supernatant for two more weeks. After the stable cell lines had been generated in this way, both these and wild-type MDCK cells were infected with influenza A virus and the virus titers of SEK KD and SEK antisense lines as described above compared to the titer from the supernatant of wild-type MDCK Cells determined. The following results were obtained. The comparison of the virus titers of influenza A virus-infected MDCK cells, which had previously been transfected with either the empty vector or a construct which expressed the dominant negative form of SEK, showed that the cells in SEK KD expressing the replication of the viruses 24 hours was inhibited to more than 90%. This result was reproducible in several independent approaches. For further investigation, MDCK cell lines were produced using retroviral transduction, which stably expressed either SEK KD or a SEK antisense RNA (see above). In one case the SEK / MKK4 activity is inhibited by competition, in the other case the kinase is inhibited. In these cell lines, too, the virus titers were greatly reduced 24 hours after infection with two different influenza A virus strains (FPV and WSN-HK) compared to wild-type cells, which demonstrates that the blocking of the virus-activated signaling pathway at the level of SEK has a crucial effect on virus replication. These findings show that specific inhibition of the parent SEK kinase within the cellular MEKK / SEK / JNK signaling pathway inhibits virus replication, at least as effectively as the kinase inhibitor U0126, which is known to be inhibiting signal transmission via Raf-MEK-ERK by inhibiting MEK. In addition, the possibility of generating SEK KD and SEK antisense RNA overproducing cell lines, which cannot be distinguished from vector cells or wild-type MDCK cells with regard to their morphology and growth behavior, shows that the specific inhibition of their function or expression Kinase is not toxic to the host cell.

Example 4: Combination of two active substances for inhibiting virus multiplication in the cellular Raf / MEK / ERK signal transmission path

The specific effect of the inhibition of two kinases within a cellular signal transmission path, which can be activated immediately one after the other, on the influenza virus propagation was investigated using the Raf / MEK / ERK signal transmission path as an example. For this purpose, with the help of transient transfection, a dominant negative mutant of the kinase Raf (RafC4B) and, on the other hand, a dominant negative mutant of the kinase ERK2 (ERK2C3, ERK2B3) were introduced into MDCK cells to inhibit the respective wild-type kinase and overexpressed. RafC4B is a deletion mutant of Raf that lacks the kinase domain (Bruder et al Genes Dev 6: 545-556, 1992.). This interrupts the activating signal on the Raf level.

ERK2B3 is a point mutant of the kinase ERK2, in which a conserved lysine in the ATP binding site at amino acid position 52 of the protein is converted to an arginine amino acid residue by targeted mutagenesis at the DNA level, a mutation which disrupts the ATP binding of the kinase and the kinase is thus inactive (Robbins et al J Biol Chem 268: 5097-5106, 1993). In ERK2C3, a tyrosine amino acid residue at position 185 of the protein in Sequence motif threonine-glutamic acid-tyrosine, which is phosphorylated in the course of the activation of the kinase, is converted into phenylalanine, as a result of which the kinase can no longer be activated (Robbins et al J Biol Chem 268: 5097-5106, 1993). ERK2C3 therefore has a correspondingly competitive inhibitory effect on the endogenous wild type in the event of overexpression. For the experiment, MDCK cells with the empty vector KRSPA or with the expression constructs KRSPA RafC4B, KRSPA ERK2B3 or KRSPA ERK2C3 using the transfection reagent Lipofectamine 2000 (Life

Technologies / Tnvi deceived, Karlsruhe Germany _r) according to standard methods (Ludwig et al. J Biol Chem transfected 276.10990 to 10998.2001). The transfection efficiencies were over 60%. 24 hours after the transfection, the cells for additional inhibition of MEK in the signal transmission path were treated with 30 .mu.M U0126, as already described above, in some of the batches (Pleschka et al. Nat. Cell Biol. 3, 301-305, 2001 ). Thereafter, infection with the influenza A virus strain fowl plage virus occurred in all of the approaches

A / Fpv / Bratislava / 79 (H7N7) with an infection multiplicity of 1 (M.0.I. = 1). The titers of the newly formed viruses in the cell culture supernatant were checked for MDCK cells in standard plaque assays a further 9 hours or 24 hours after the infection. The virus titers of: 1. infected MDCK cells which were transfected with the blank vector, 2. infected MDCK cells which were transfected with either RafC4B, ERK2B3 or ERK2C3, 3. infected MDCK cells which were transfected with the blank vector were compared and additionally treated with U0126 and 4. infected MDCK cells, which were either transfected with RafC4B, ERK2B3 and ERK2C3 and additionally treated with U0126. The following results were receive. The comparison of the virus titers of influenza A virus-infected MDCK cells which had previously been transfected with either the empty vector or constructs which predominantly expressed negative forms of RafC4B, ERK2B3 or ERK2C3 showed that in cells which the kinase mutants expressed that the multiplication of the viruses after 9 hours and 24 hours was significantly inhibited. The same applies to virus multiplication in cells which were transfected with an empty vector and treated with U0126 in comparison to cells not treated with U0126. If, in cells in which Raf or ERK was inhibited by overexpression of RafC4B or ERK2B3 or ERK2C3, the kinase MEK is additionally inhibited as a second kinase within the signal transmission path, there is an increased inhibition of the virus multiplication compared to the approaches which do not involve any additional Have experienced inhibition of kinase MEK by U0126. This shows that the inhibition of two kinases within a signal transmission path inhibits the virus multiplication to a greater extent than is possible by inhibiting only one kinase within the signal transmission path.

Example 5: Specific Inhibition of Virus Propagation in the Cellular Raf / MEK / ERK Signaling Pathway The specific effect of the inhibition of kinases within a cellular signaling pathway on the influenza virus multiplication was investigated using the example of the Raf / MEK / ERK signaling pathway. For this purpose, with the help of transient transfection, a dominant negative mutant of the kinase Raf (RafC4B) and secondly a dominant negative mutant of the kinase ERK2 (ERK2C3, ERK2B3) (see above) were introduced to inhibit the respective wild-type kinase in MDCK cells and overexpressed. For the experiment, MDCK cells with the empty vector KRSPA or with the expression constructs KRSPA RafC4B, KRSPA ERK2B3 or KRSPA ERK2C3 using the transfection reagent Lipofectamine 2000 (Life Technologies / Tnvi trogen, Karlsruhe, Germany) according to standard methods (Ludwig et al. J Biol Chem 276.10990-10998.2001). The transfection efficiencies were over 60%. After 24 hours, all batches were infected with the influenza A virus strain fowl page virus A / Fpv / Bratislava / 79 (H7N7) with a multiplicity of infection of 1 (M.0.I. = 1). The titers of the newly formed viruses in the cell culture supernatant were checked for MDCK cells in standard plaque assays a further 9-24 hours after the infection. The virus titers of: 5. infected MDCK cells which were transfected with the blank vector, 6. infected MDCK cells which were transfected with either RafC4B, ERK2B3 or ERK2C3 were compared. The following results were obtained. The comparison of the virus titers of influenza A virus-infected MDCK cells, which had previously been carried out with either

Blank vectors or constructs which predominantly expressed negative forms of RafC4B, ERK2B3 or ERK2C3 had been transfected, showed that in cells which expressed the kinase mutants, the multiplication of the viruses was significantly inhibited. This shows that the inhibition of different kinases within a signal transmission path can represent the starting points for inhibiting virus multiplication.

Example 6: Combination of two active substances for inhibiting virus multiplication in the cellular MKK6 / p38 / 3pK signal transmission path The specific effect of the inhibition of two kinases within a cellular signal transmission path, which can be activated immediately one after the other, on the influenza virus propagation was also examined using the example of the cellular MKK6 / p38 / 3pK signal transmission path. With the help of transient transfection, a dominant negative mutant of the kinase MKK6 (MKK6 (Ala)) and a dominant negative form of the kinase 3pK (3pK K> M), a kinase substrate of the p38 MAP kinase, were used to inhibit the respective wild-type kinase introduced into MDCK cells and overexpressed. 3pK K> M is a point mutant of the kinase 3pK, in which a conserved lysine in the ATP binding site at amino acid position 73 of the protein was converted against a methionine by targeted mutagenesis at the DNA level (Sithanandam et al. Mol Cell Biol 16: 868- 876, 1996), a mutation which disrupts the ATP binding of the kinase and the kinase is thus inactive. In MKK6 (Ala), the lysine in the ATP binding site at amino acid position 82 of the protein was converted into an alanine by targeted mutagenesis at the DNA level (Raingeaud et al Mol Cell Biol 16: 1247-1255, 1996). Through this inactivation, MKK6 (Ala) has a competitive inhibitory effect on the endogenous wild type in the event of overexpression. For the experiment, MDCK cells with the empty vector KRSPA or with the expression constructs KRSPA MKK6 (Ala) or 3pK K> M using the transfection reagent Lipofecttaine 2000 (Life Technologies) according to standard methods (Ludwig et al. J Biol Chem 276, 10990-10998,2001) transfected. The transfection efficiencies were over 60%. 24 hours after transfection, in some of the batches, the cells for additional inhibition of the kinase in the signal transmission path were treated with 20μM SB202190, a specific inhibitor of p38 MAP kinase (Cohen Trends Cell Biol 7: 353-361, 1997). Thereafter, infection with the influenza A virus strain fowl plage virus A / Fpv / Bratislava / 79 followed with an infection multiplication of 1 (MOI = 1). A further 9 hours or 24 hours after infection, the titers of the newly formed viruses in the cell culture supernatant were checked for MDCK cells in standard plaque assays. The virus titers of: 1. infected MDCK cells which were transfected with the blank vector, 2. infected MDCK cells which were transfected with either MKK6 (Ala) or 3pK K> M, 3. infected MDCK cells which were transfected with the empty vector and additionally treated with SB202190 and 4. infected MDCK cells, which had been transfected with either MKK6 (Ala) or 3pK K> M and additionally treated with SB202190. The following results were obtained. The comparison of the virus titers of influenza A virus-infected MDCK cells, which had previously been transfected either with the empty vector or with constructs which expressed dominant negative forms of MKK6 or 3pK, showed that in cells which expressed these kinase mutants the virus replication was significantly inhibited after 9 hours and 24 hours. The same applies to the virus multiplication in cells which were transfected with an empty vector and treated with SB202190 in comparison to cells not treated with SB202190. If, in cells in which MKK6 or 3pK was inhibited by overexpression of MKK6 (Ala) or 3pK K> M, the kinase p38 as the second kinase within the signal transmission path is additionally inhibited, an increased inhibition of the virus multiplication is found compared to that Approaches that have not experienced additional inhibition of kinase p38 by SB202190. This shows that the inhibition of two kinases within one cellular Signal transmission path inhibits the virus multiplication to a greater extent than is possible by inhibiting only one kinase within the signal transmission path.

Example 7: Effect of the combination of amantadine and U 0126 on the influenza virus infection

Scholtissek and Müller (Arch. Virol. 119: 111-118, 1991) have shown that the treatment of influenza-infected cells with a combination of a methylation inhibitor (3-deaza-adenosine) and a broad-acting toxic kinase inhibitor ( 1- (5-Isoquino-Line-sulphonyl) - 2-methylpiperazine = H7) potentiated the effect of the two individual inhibitors. It was tested whether the combination of an active substance according to the invention with an antiviral substance which inhibits a viral component and is not a kinase inhibitor has a synergistic antiviral activity. Permissive MDCK cells were identified with multiplicity of infection (MOI) of 0.01 with influenza A viruses (A / FPV / Bratislava (H7N7) and A / WSN-HK (H3N1)) and influenza B viruses / Massachusetts / 6/92 ( B / Mass) infected. The infected cells were treated as follows: 1) untreated (for FPV, WSN-HK and B / Mass), 2) U 0126 in optimally antiviral concentrations (Pleschka et al Nature Cell Biol 3: 301-305,2001) (for FPV, WSN-HK and B / Mass), 3) Amantadine in optimally antiviral effect

Concentrations (Hay et al EMBOJ 11: 3021-3024, 1985) (for FPV), 4) combinations of amantadine and U0126 (for FPV). Amantadine is known to i) act in pharmacologically reasonable (micromolar) concentrations only with Influenza A infections, but not with Influenza B infections (Davies et al Science 144: 862-863, 1964), ii) that it does not works with all influenza viruses of subtype A, e.g. not with A / WSN / 33 (Thomas et al J. Virol. 252.54-64.1998) or at A / Puerto Rico / 8/34 (Castrucci et al J.Virol 69: 2725-8.1995), iii) that it initially strongly inhibits sensitive influenza A viruses, subsequently to form resistant virus variants leads (Hay et al EMBO J. 11: 3021-3024, 1985). The titer of infectious virus particles in the cell culture supernatant was determined 48 hours after the infection. The supernatants from untreated and from cells treated with amantadine and / or with U0126 were diluted 1: 1000 (the supernatants from influenza A / WSN-HK and influenza B virus-infected cells which had been treated with U0126 became 1: 100 diluted) and used again to infect fresh cells. These rounds of infection were repeated twice. The following result was achieved: i) after the second round of infection, treatment with optimal concentrations of U0126 resulted in a significant reduction in the influenza virus particles, which in an attempt went so far that there was no longer enough infectious influenza A / WSN-HK or Influenza B virus particles were present to enable a third round of infection, ii) the Influenza A / FPV VirusTiter remained consistently low when treated with an optimal concentration of U0126, although this virus subtype multiplies significantly more in cell culture than, for example, Influenza A / WSN-HK, iii) the titer of the amantadine-sensitive influenza A / FPV virus decreased after the addition of amantadine and subsequently increased again, which indicates the formation of resistant virus variants, iv) the titer of the non-amantadine-resistant influenza A / WSN_HK virus despite the A antadine treatment, which confirms that not all influenza A viruses on Amantad increased react, v) treatment with Amantadine and an optimally antiviral concentration of U0126 in FPV leads to an increased inhibition of virus replication. The results thus show the superior antiviral activity of an active ingredient according to the invention, which acts both against various influenza A viruses (which are partially resistant to amantadine) and against influenza B viruses (which are resistant to amantadine), with no formation of resistance neither with influenza A viruses nor was observed in influenza B viruses and which shows a synergistic effect in the combination of an active ingredient according to the invention with an antiviral active ingredient which is not a kinase inhibitor on influenza A viruses.

Example 8: Resistance formation

This example shows that under the action of the MEK inhibitor U0126, multiple variants of resistant varieties of influenza A and B viruses do not form. For the multiplication of influenza A and B viruses, performative, eukaryotic cell cultures (Madine darby canine kidney (MDCK) cells) are washed with a physiological saline solution in parallel batches with the same number of cells, according to generally used methods for cell culture, and with a quantity of the infectious influenza A virus strain fowl plage virus A / Fpv / Bratislava / 79 (H7N7) or the infectious influenza B virus strain Massachusetts / 6/93, in a ratio of 0.01 infectious virus particles per cell for one hour at room temperature, infected. After the infection, the inoculum is removed and the infected cells are incubated in suitable cell culture medium to which the MEK inhibitor U0126 has been added (50 .mu.M dissolved in DMSO) for 48 h at 37 ° C. and 5% CO ₂ concentration. As a solvent control, infected MDCK cells are incubated with cell culture medium to which the corresponding amounts of DMSO have been added. For the influenza B Virus infection contains the cell culture medium 2μg / ml trypsin. The cell supernatant was harvested after 48 hours for the influenza A virus infection. For the influenza B virus infection, the same amount of inhibitor or solvent was added again 48 hours after the infection. The cell supernatant was harvested after a further 24 hours. 0.1 ml of a 10-2 dilution (Influenza B virus) or a 10-3 dilution (Influenza A virus) of the cell supernatants was used to infect fresh MDCK cells (passage). The protocol was repeated four times and the virus titer determined after each run. The samples of the respective cell culture supernatants are examined for the amount of newly formed infectious virus particles (plaque assay for MDCK cells) using conventional virological methods. In an additional control, FPV-infected MDCK cells were treated with amantadine (5uM, cell supernatants were harvested 48 hours after the infection and 0.1 ml of a 10-3 dilution was used for the passage), which significantly inhibits the multiplication of some influenza A viruses because it inhibits the ion channel activity of the M2 protein. For Amantadine, reference is made to Example 7. As a result, it can be determined in such an experimental approach that, with the appropriate concentration of the MEK inhibitor U0126 in the cell culture medium, there is a significant and constant reduction (approx. 80%) in the number of newly formed, infectious influenza A virus particles compared to the Solvent control without MEK inhibitor U0126 comes without the resistant virus variants forming, which would lead to an increase in the virus titer, as described in

The case of amantadine treatment is clearly recognizable. Furthermore, it can be seen that there is also a significant and constant reduction (approx. 90%) in the number of newly formed, infectious influenza B virus particles, compared to the solvent control without MEK inhibitor U0126 comes without the resistant virus variants, which would lead to an increase in virus titer.

Example 9: Negative control

This example shows that under the effect of the transient expression of constitutively active forms of the kinases Raf (Raf BXB, deletion mutant which only contains the kinase domain are described in: Flory, E., Weber, CK, Chen, P., Hoffmeyer, A ., Jassoy, C. & Rapp, UR (1998) J. Virol. 72, 2788-2794) and MEK (delta Stu MEK, was created by Stu-mediated deletion of an inhibitory alpha-helix, described in: Apoptosis suppression by Raf -1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent Signals. Von Gise A, Lorenz P, Wellbrock C, Hemmings B, Berberich-Siebelt F, Rapp UR, Troppmair J. Mol Cell Biol 2001 Apr; 21 (7) : 2324-36) the multiplication of influenza A virus is significantly increased in MDCK cells. For the multiplication of influenza A viruses, performative, eukaryotic cell cultures MDCK cells, in parallel batches with the same number of cells, are transfected with plasmid DNA of the corresponding expression plasmids or by vector control (see above) according to generally used methods for cell culture. , The transfection efficiencies were about 80%. 24 hours after the transfection, the cells are washed according to generally applicable methods for cell culture, with a physiological saline solution and with an amount of the infectious influenza A virus strain fowl plage virus A / Fpv / Bratislava / 79 (H7N7), in a ratio of 1-10 infectious virus particles per cell for one hour at room temperature, infected. The inoculum is then replaced by a suitable cell culture medium. After a further 24 hours, the number of newly formed infectious virus particles was determined by plaque assay (see 0.). As a result, in such an experimental approach, it can be established that the number of newly formed, infectious virus particles from MDCK cells, which were transfected with the corresponding expression plasmids before the virus infection, in comparison to MDCK cells before the virus infection were only transfected with the vector control for which influenza A viruses increased significantly.

Claims

claims

1) Use of at least one active substance for the production of a pharmaceutical composition for the prophylaxis and / or therapy of at least one viral disease, characterized in that the active substance (s) inhibit either at least two kinases or at least one SEK kinase of a cellular signal transmission path in such a way that virus multiplication is inhibited becomes.

2) Use of at least one active substance according to claim 1, characterized in that the kinases of the cellular signal transmission path can be activated immediately one after the other.

3) Use of at least one active substance according to claim 1 or 2, characterized in that the kinases or the SEK kinase of the cellular signal transmission path are selected from the following signal transmission paths: MEKK2, -3 / MEK5 / ERK5; Raf / MEK / ERK;

MEKK / SEK / JNK; JAK1, JAK2, JAK3, TYK2 and / or hetero- and homodimers of JAK1, -2, -3, TYK2; ASK / MKK3, -6 / p38;

ASK / MKK4, -7 / JNK; MEKK4 / MKK4, -7; DLK / MKK4, -7;

TPL-2 / MKK4, -7; TPL-2 / MEK5 / ERK5; MLK-3 / MKK3, -6; MLK-3 / MKK4, -7; TAK / NIK / IKK; TAK / MKK3, -6; TAK / MKK4, -7;

PAK / MKK3, -6; PAK / IKK; Cot, Tpl-2 / IKK; PKC / IKK; PKB / IKK;

PKC / Raf; PAK / Raf; Lck / Raf; MEKKs / IKK; PI3K / PDK1 / PKB;

JAK / TYK / PLCgamma; and / or MAP kinases / MAPKAP kinases, for example ERK / 3pK, ERK / Rskp90, p38 / MAPKAP kinases and / or p38 / 3pK.

4) Use of at least one active substance according to one of claims 1 to 3, characterized in that the Active substance (s) is selected from the following active substances: Kinase-inhibiting flavone derivative or benzpyran derivative; Kinase inhibiting derivative of 4H-1-benzopyran; Flavopiridolderivat; 2- (2-amino-3-methoxyphenyl) -4-oxo-4H- (1) benzopyran; 7, 12-dihydro-indolo (3,2-d) (l) benzazepin-6 (5H) -one; 70H-staurosporine and / or a phosphokinase inhibiting derivative of 70H-staurosporine; butyrolactone; Roscovitine; Purvalanol A; emodin; quinazolin-Anilinoquin; phenylamino; Trioylimidazol; Paullon;

[4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) 1H-imidazole; [1,4-diamino-2,3-dicyano-1,4-bis (2aminophenylthio) butadiene; Kinase inhibiting derivative of butadiene; [2-2 '-amino-3' -methoxyphenyl) -oxaphthalene-4-one); [2- (2-chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3, 4-difluoro benzamide; CEP-1347 (KT7515) bis-ethylthiomethyl; Tetrapyrrolic macrocycles; pyrimidone; 3-aminomethylene-indoline derivative; Pyrazolo (3,4-b) pyridine derivative; Pyrazol derivative; 1,4-substituted piperidine derivative; lipoid ammonium salt; dominant negative mutant of a kinase of a cellular signaling pathway; Antisense oligonucleotide, which attaches specifically to the DNA sequence or mRNA sequence coding for a kinase of a cellular signal transmission pathway and inhibits its transcription or translation; ds oligonucleotides which are suitable for the targeted degradation of the mRNAs of kinases of a cellular signal transmission path by RNAi technology; Antibodies or antibody fragments, specific for a kinase or a fusion protein, containing at least one antibody fragment, for example an Fv fragment, which inhibits the kinase activity of a kinase module; and / or peptide that the interaction of inhibits at least two kinases of a cellular signal transmission path, which can preferably be activated one after the other.

5) Use of at least one active substance according to one of claims 1 to 4, characterized in that the viral disease is caused by RNA or DNA viruses, preferably influenza viruses.

6) Combination preparation for the prophylaxis and / or therapy of at least one viral disease, comprising at least two active substances which either act on at least two kinases of a cellular signal transmission path in such a way that virus multiplication is essentially inhibited or essentially inhibit a SEK kinase selected from the active substances according to claim 3, wherein the combination preparation is applicable in the form of a mixture or as individual components for simultaneous or temporally different use at the same or different locations.

7) Combination preparation for the prophylaxis and / or therapy of at least one viral disease, comprising at least one active substance according to one of claims 1) -6) and at least one antiviral substance which is not a kinase inhibitor.

8) Combination preparation according to claim 7) in which the antiviral substance which is not a kinase inhibitor is 1-adamantanamine, rimantadine, a neuraminidase inhibitor or a nucleoside analogue such as ribavirin.

9) Active ingredient or combination preparation according to one of claims l) -8) for the prophylaxis and / or therapy of an infection with negative strand RNA viruses, in particular influenza viruses or Bornaviruses

10) Test system for finding active substances which act either on at least two kinases or on a SEK kinase of a cellular signal transmission path in such a way that virus replication is essentially inhibited, comprising: a. at least one cell which can be infected with at least one virus and which contains either at least two kinases of a cellular signal transmission path or at least one SEK kinase and at least one virus which infects the cells or b. at least one with at least one

Virus infected cell that contains either at least two kinases of a cellular signaling pathway or at least one SEK kinase.

11. Test system according to claim 10, characterized in that the virus is an RNA or DNA virus, preferably an influenza virus.

12. Test system according to claim 10 or 11, characterized in that the cell contains at least one overexpressed kinase.

13. Test system according to one of claims 10 to 12, characterized in that it contains a cell in which a. at least one gene coding for at least one dominant negative mutant of at least one parent kinase and / or b. at least one dominant contains negative mutant of at least one subordinate kinase.

14. Test system according to one of claims 10 to 13, characterized in that contains a cell in which the

Expression for at least one kinase is inhibited.

15. Method for finding at least one active substance for the prophylaxis and / or therapy of viral diseases, which contribute to the multiplication of viruses

Essentially inhibits or inhibits viral diseases, comprising the following steps: c. Bringing at least one test system according to one of claims 10 to 13 into contact with at least one potential active substance, and d. Determination of the impact on virus replication.

16. A process for the manufacture of a medicament for the prophylaxis and / or therapy of at least one viral disease, which contributes to the multiplication of viruses

Essentially inhibits or inhibits viral diseases, comprising the following steps: c. Performing a test system according to any one of claims 10 to 13, and d. Transfer of the active substance (s) found with at least one auxiliary and / or additive.

17. Use of one or more of the following active ingredients for the production of a pharmaceutical composition for the prophylaxis or treatment of a viral disease: a) an inhibitor of a kinase, the inhibition of which inhibits virus multiplication, in combination with an antiviral active ingredient which is not a kinase inhibitor, b ) an inhibitor of a first kinase, the sole inhibition inhibits virus multiplication, the inhibitor additionally inhibiting a second kinase different from the first kinase, the sole inhibition of which inhibits virus multiplication, c) a first inhibitor of a first kinase, the sole one

Inhibition inhibits virus multiplication, in combination with a second inhibitor different from the first inhibitor, a second kinase different from the first kinase, the sole inhibition of which inhibits virus multiplication, and / or d) an inhibitor of a SEK kinase.

18. A method for screening for prospective active substances according to claim 17, wherein a prospective active substance, optionally with a previously identified different active substance or with a different further prospective active substance, is contacted with a test system according to one of claims 10 to 14, wherein the inhibition of virus replication is quantified, the value of the inhibition obtained being compared with a reference value of the inhibition which is obtained under the same conditions, but without a prospective active substance or with a reference active substance or a combination of reference substances, and a prospective active substance or a prospective active substance combination be selected if the value of the inhibition is higher than the reference value of the inhibition.

19. A method for the prophylaxis or treatment of viral diseases, wherein a patient is administered a pharmaceutical composition according to claim 17 in a defined and physiologically effective dose.