EP1988972A2 - Proteasom or ups inhibitor for treating infections with influenza viruses - Google Patents

Abstract

The invention relates to compositions for the prophylaxis and/or treatment of viral infections, in particular of infections with influenza viruses which cause influenzal infections. The invention relates to compositions which comprise inhibitors of the ubiquitin-proteasome system, in particular proteasome inhibitors, as active ingredients. The present invention further relates to the systemic and topical, preferably the aerogenic administration of proteasome inhibitors. The active substance employed according to the invention as proteasome inhibitor can be employed with at least one further substance having antiviral activity for the prophylaxis and/or therapy of influenzavirus infections.

Description

 Agent for the treatment of infections with influenza viruses

description

The invention relates to agents for the prophylaxis and / or treatment of virus infections, in particular of infections with influenza virus causing flu-like infections. The invention relates to agents containing as active ingredients inhibitors of the ubiquitin proteasome system, in particular proteasome inhibitors. The present invention further relates to the systemic as well as the topical, preferably the aerogenic administration of proteasome inhibitors. The active substance used according to the invention of a proteasome inhibitor can be used with at least one further antivirally active substance for the prophylaxis and / or therapy of influenza virus infections.

1. Characteristic of the known state

1.1. The influenza virus infection in animals and humans

Infections with influenza viruses, the causative agents of the virus flu, represent a significant health threat to humans and animals and year after year not only require a large number of casualties, but are also macroeconomic, such as sick leave incapacitated by an immense cost factor. However, in addition to the annual epidemics, influenza has a much more threatening dimension, as in the past there have been recurrent global outbreaks known as pandemics, which have claimed many millions of lives. The occurrence of highly pathogenic avian flu viruses of subtype H5N1, which has been observed for several years, immediately illustrates the danger of a new pandemic in the near future against which no effective vaccines are currently available.

Influenza viruses belong to the family of orthomyxoviruses and have a segmented genome negative-stranded orientation encoding at least 11 viral protein. (Lamb and Krug, in Fields, Virology, Philadelphia: Lippincott-Raven Publishers, 1353-1395, 1996). Due to the molecular and serological characteristics of the nucleoproteins (NP) and the matrix proteins (M), influenza viruses are assigned to types A, B and C. Type A viruses have the greatest pathogenic potential for humans and some animal species (Webster et al., Microbiol Rev, 56, 152-79, 1992). An influenza A virus particle consists of 9 structural proteins and a lipid envelope derived from the host cell. The viral RNA segments 1 to 3 encode the components of the RNA-dependent RNA polymerase complex (RDRP), PBl, PB2 and PA, linked to the ribonucleoprotein complex, these components catalyze transcription and amplification of the viral genome. Hemagglutinin (HA) and neuraminidase (NA) are viral surface glycoproteins produced by vRNA segments 4 and 6. At present, 16 different HA and 9 different NA subtypes are known, due to which influenza A viruses are divided into different categories.

Viruses with the HA types Hl, H2 and H3 and NA types Nl and N2 can be epidemic in humans (Lamb and Krug, in Fields, Virology, Philadelphia: Lippincott-Raven Publishers, 1353-1395, 1996). Segment 5 encodes the nucleoprotein (NP), the major component of the ribonucleoprotein complex. The two smallest vRNA segments encode two proteins each. The vRNA segment 7 encodes the matrix protein M1 and the M2 protein. The Ml protein associates with the inside of the lipid double membrane and coats the viral envelope from the inside, M2 is a third transmembrane component that acts as a pH-dependent ion channel. The segment 8 sequence carries the information for the nuclear export protein NS / NEP and the only non-structural protein, NS1. Recently, an eleventh influenza A viral protein has been identified (Chen et al., Bibliography behind the embodiments). It is the PB1-F2 protein formed by an open nucleotide-shifted reading frame of the PB1 gene segment. [0006] PB1-F2 is a mitochondrial protein capable of enhancing the induction of controlled cell death, apoptosis.

The problem of controlling RNA viruses is the mutability of the viruses, caused by a high error rate of the viral polymerases, which makes both the preparation of suitable vaccines and the development of antiviral substances very difficult. It has been found that the application of antiviral substances that are directed directly against functions of the virus, due to mutations leads very quickly to the selection of resistant variants. An example of this is the anti-influenza agent amantadine and its derivatives, which are directed against a transmembrane protein of the virus and already within a few passages lead to the formation of resistant variants.

Also, the new therapeutics for Influenzainfektionen that inhibit the influenza viral surface protein neuraminidase and under the trade name RELANZA or TAMIFLU distributed by Glaxo Wellcome or Roche in Germany have already produced resistant variants in patients (Gubareva et al J Infect Dis 178, 1257-1262, 1998). Resistance to TAMIFLU has also been reported in human H5N1 avian influenza viruses (Qui et al., Nature 437, 1108, 2005). The hopes that were placed in these therapeutics could not be fulfilled for this reason either.

Due to their usually small genomes and thus limited coding capacity for replication-necessary functions all viruses are highly dependent on functions of their host cells. By influencing such cellular functions necessary for viral replication, it is possible to adversely affect virus replication in the infected cell (Ludwig et al., Trends Mol. Med. 9, 46-51, 2003). In this case, it is not possible for the virus to replace the missing cellular function by adaptation. An escape from the selection pressure by mutation is not possible here. This could be demonstrated using the example of the influenza A virus not only with relatively unspecific inhibitors against cellular kinases and methyltransferases (Scholtissek and Muller, Arch Virol 119, 11-11, 118, 1991) but also with kinase inhibitors that selectively attack a pathway required by the virus ( Ludwig et al., FEBS Lett 561, 37-43, 2004).

1.2. Function of the Ubiquitin / Proteasome System (UPS)

Proteasomes are the major proteolytic component in the nucleus and cytosol of all eukaryotic cells. They are multicatalytic enzyme complexes that make up about 1% of total cell proteins. Proteasomes play a vital role in diverse functions of cell metabolism. The main function is the proteolysis of misfolded, non-functional proteins. Another function is the proteasomal degradation of cellular or viral proteins for the T-cell mediated immune response through the generation of peptide ligands for major histocompatibility class I molecules (for review, see Rock and Goldberg, 1999). Proteasome targets are typically labeled for degradation by the attachment of oligomeric forms of ubiquitin (Ub). Ub is a highly conserved, 76 amino acid long protein that is covalently coupled to target proteins. Ubiquitinylation itself is reversible, and Ub molecules can be removed from the target molecule by a variety of Ub hydrolases. The link between ubiquitinylation of target proteins and the proteasomal Proteolysis is commonly referred to as the ubiquitin / proteasome (UPS) system (reviewed by Rock and Goldberg, 1999, Hershko and Ciechanover, 1998).

The 26S proteasome is a 2.5 MDa multienzyme complex consisting of approximately 31 subunits. The proteolytic activity of the proteasome complex is realized by a cylindrical, 700 kDa core structure consisting of four superimposed rings, the 2OS proteasome. The 2OS proteasome forms a complex multienzyme complex consisting of 14 non-identical proteins arranged in two α and two β-rings in an αββα order. The substrate specificity of the 2OS proteasome involves three major activities: trypsin, chymotrypsin, and postglutamyl-peptide hydrolysing (PGPH) or caspase-like activities located in the β, Y, and Z subunits. The 2OS proteasome degrades in vitro denatured proteins independent of their poly-ubiquitinylation. In contrast, in vivo enzymatic activities of the 2OS proteasome are regulated by attachment of the 19S regulatory subunits, which together form the active 26S proteasome particle. The 19S regulatory subunits are involved in the recognition of poly-ubiquitinylated proteins as well as in the unfolding of target proteins. The activity of the 26S proteasome is ATP-dependent and almost exclusively degrades only poly-ubiquitinylated proteins (for review see Hershko and Ciechanover, 1998).

1.3. Proteasome inhibitors

Different classes of compounds are known as proteasome inhibitors. On the one hand there are chemically modified peptide aldehydes, such as the tripeptide aldehyde N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal (zLLL, also referred to as MG 132) and the more effective boric acid derivative MG232. Similar to zLLL, another class of modified peptides, the peptide-vinyl sulfones, have been described as proteasome inhibitors (for review, see Elliott and Ross, 2001). Naturally occurring substances are lactacystin (LC) (Fenteany et al., 1995), which consists of streptomycetes, as well as epoxomycin, which is obtained from actinomycetes (Meng et al., 1999a, b). LC is a highly specific, irreversibly acting proteasome inhibitor which mainly blocks the chymotrypsin and trypsin-like activities of the 26S proteasome particle (Fenteany et al., 1995). LC has no basic peptide structure but consists of a γ-lactam ring, a cysteine and a hydroxy-butyl group. LC itself does not inhibit the proteasome. Rather, in aqueous solution, the N-acetyl Cysteine residue hydrolyzed. The result is the formation of a clastolactacysteine β-lactone capable of penetrating cell membranes. Upon cell uptake, nucleophilic attack of the β-lactone ring occurs and subsequent transesterification of the threonine 1-hydroxyl group of the β-subunit (Fenteany et al., 1995).

In terms of specificity and efficacy, epoxomycin is the most potent of all known natural proteasome inhibitors to date (Meng et al., 1999; a, b). Another and very potent class of synthetic proteasome inhibitors are boric acid-peptide derivatives, in particular the compound pyranozyl-phenyl-leucine-boric acid named "PS-341". PS-341 is very stable under physiological conditions and bioavailable after intravenous administration (Adams and Stein, 1996, Adams et al., 1999, US 1,448,012 TWO1).

1.4. Clinical application of proteasome inhibitors

Inhibition of proteasome activity as the major cellular protease may lead to changes in cell cycle regulation, transcription, overall cellular proteolysis and MHC-I antigen processing (for review see Ciechanover et al., 2000). Consequently, a permanent inhibition of all enzymatic activities of the proteasome is incompatible with the life of a cell and thus of the whole organism. However, certain reversibly acting proteasome inhibitors can selectively inhibit individual proteolytic activities of the 26S proteasome without affecting other cellular proteases. Preliminary clinical trials of proteasome inhibitors (Adams et al., 1999) demonstrate that this class of drugs has tremendous potential as pharmaceuticals with a diverse range of applications (for reviews see Elliot and Ross, 2001). The importance of proteasome inhibitors as a novel therapeutic principle has received increasing attention in recent years, particularly in the treatment of cancer and inflammatory diseases (for review, see Elliot and Ross, 2001). The company "Millennium Inc." (Cambridge, MA, USA) have developed proteasome inhibitors for anti-inflammatory, immunomodulatory and antineoplastic therapies, in particular boric acid derivatives of di-peptides, and in particular the compound PS-341 (Adams et al., 1999).

The use of proteasome inhibitors with the aim to block viral infections has already been described. In particular, Schubert et al. (2000 a, b) showed that Proteasome inhibitors block the assembly, release and proteolytic maturation of HIV-1 and HIV-2. This effect relies on a specific blockade of the proteolytic processing of the Gag polyproteins by the HIV protease without proteasome inhibitors affecting the enzymatic activity of the viral protease itself. Further relationships with the UPS have been reported for Budding by Rous Sarcoma Virus, RSV (Patnaik et al, 2000); Simian immunodeficiency virus, SIV (Strack et al., 2000), and Ebola virus (Harty et al., 2000). In the latter case (Harty et al., 2000), a cellular ubiquitin ligase has been shown to interact with Ebola matrix protein.

1.5. Proteasome inhibitors in the patent literature

Proteasome inhibitors and their medical use are the subject of numerous patents or patent applications. US Pat. No. 5,780,454 A (Adams et al.) Describes boronic acid and ester compounds, their synthesis, and uses as proteasome inhibitors. As a mechanism of proteasome inhibition, inhibition of NF-.kappa.B in a cell is indicated.

The international patent application WO 98/10779 has the use of proteasome inhibitors for the treatment of parasite infections the subject. In the document WO 99/15183 proteasome inhibitors are used for the treatment of autoimmune diseases. It also addresses the role of the UPS in NFKB-mediated activation of the HIV-I LTR promoter and transcriptional processes in the nucleus, which are not essential for replication of HIV. It is not shown that proteasome inhibitors can block the replication of HIV. The NFκB pathway is certainly not suitable for this.

Further medical applications are the treatment of fibrotic diseases (US 2005/222043 A), the prevention of transplant rejection and septic shock (EP 0967976 A), the treatment of blood vessel constrictions (WO02 / 060341 A) or the treatment of an endothelium Malfunction (WO2004 / 012732 A).

Also, the use of proteasome inhibitors in a cardiac indication is mentioned (DE 10040742 A). The use of proteasome inhibitors for the treatment of viral infections is the subject of patent application EP 1430903 Al = US2004 / 0106539A1. The use of proteasome inhibitors as agents for inhibiting the release, maturation and replication of retroviruses is described. Using the example of human immunodeficiency viruses (HIV), it is shown that proteasome inhibitors block both the processing of the Gag proteins and the release of virus particles as well as the infectivity of the released virus particles and thus virus replication. Areas of application are the anti-retroviral therapy and prevention in animals and humans lentivirus infections causing immune deficiency, in particular AIDS or HIV-induced dementia, also in combination with other anti-retroviral drugs.

Patent Application EP 1326632 A1 mentions agents for the treatment, therapy and inhibition of hepatitis viral infections and the hepatopathogenesis and diseases associated therewith. The agents used to inhibit the release, maturation and replication of hepatitis viruses in pharmaceutical preparations contain, as an effective component, classes of substances which have in common that they inhibit the 26S proteasome in cells. These include, in particular, proteasome inhibitors which influence the activities of the ubiquitin / proteasome pathway, in particular the enzymatic activities of the 26S and the 2OS proteasome complex. The application of the invention is in the anti-viral therapy of hepatitis infections, especially in the prevention of the establishment and maintenance of acute and chronic HBV and HCV infection and associated liver carcinomas.

Proteasome inhibitors for the treatment, therapy and inhibition of Flaviviridae virus infections (WO2003 / 084551 Al) are also used. The agents used in inhibiting the release, maturation and replication of flaviviridae in pharmaceutical preparations contain, as active component, classes of substances which have in common that they inhibit the 26S proteasome in cells.

Also for the treatment of virus infections, in particular infections with SARS (severe acute respiratory syndrome) triggering corona viruses, proteasome inhibitors have been proposed (DE 10361945 Al). A meaning of the ubiquitin-proteasome pathway for the replication of influenza viruses or even a use of proteasome inhibitors for the prophylaxis and / or the treatment of infections with influenza viruses has not been shown.

German patent application DE 103 00222 A1 describes the use of active substances for the inhibition of IAV replication, which exclusively inhibit components of the NF-κB signal transmission pathway. In addition to a large number of relatively specific-acting NF-κB inhibitors, proteasome inhibitors are also mentioned as possible active substances in this description of the invention without showing sufficient details or any experimental data. On the contrary, it is purely speculated that proteasome inhibitors also affect the NF-κB signal transduction pathway. The decisive disadvantage of the invention described in DE 103 00222 A1 is the fact that investigations into the effect of proteasome inhibitors on NF-κB activation in connection with antiviral activity have so far been negative with influenza viruses. Thus, it has not been shown that it is possible to prepare pharmaceutically active compositions which contain at least one proteasome inhibitor and / or at least one inhibitor of the UPS and are suitable for the treatment of an IAV infection. An anti-viral effect of proteasome inhibitors with respect to influenza viruses could not be demonstrated in the invention described in DE 103 00222 A1. On the contrary, the results contained in the present invention description will demonstrate that the efficacy of proteasome inhibitors on NF-κB activation requires such a high dose of proteasome inhibitors that are not achievable in vivo by standard applications and, moreover, due to the toxicity of the known side effect of already clinically applied proteasome inhibitors in this concentration range would not be medically acceptable. Accordingly, DE 103 00222 A1 does not teach that proteasome inhibitors are suitable for the production of anti-virally active pharmaceutical compositions against influenza viruses.

International Publication WO 00/33654 Al describes the use of an HIV-I protease inhibitor, ritonavir, as a proteasome inhibitor. This protease inhibitor has a non-specific effect on the proteasome in the concentration of about 10 micrograms, which is 10000 times lower than the effective concentration of a specific proteasome inhibitor. Besides, this is extremely high concentration physiologically unreachable. In addition, the logical conclusion is that such permanent inhibition of UPS by ritonavir, as administered in high-dose anti-retroviral therapy (HAART) in HIV-infected individuals, would have resulted in extremely toxic side effects due to the permanent shutdown of the 26S proteasome have to. This has not been previously reported for all ritonavir-treated patients. Likewise, the published patent application WO 00/33654 A1 lacks any experimental data which could prove such an effect for this purely theoretical and also proven false assumption. Of course, ritonavir, as an HIV protease inhibitor, inhibits HIV activity, but it does not do so via the non-specific effect on the proteasome, but because it selectively and selectively inhibits the HIV protease in the therapeutically administrable concentrations. Ritonavir blocks the 26S proteasome only in high concentrations that are not therapeutically achievable, and this may only be ritonavir, none of the other HIV protease inhibitors known to date. Finally, this bizarre effect of ritonavir on the UPS has been demonstrated only in vitro. In theory, influenza viruses are also mentioned in WO 00/33654 A1, but this mention is made exclusively in connection with the improvement of the immune status, in particular concerning the activity of CD4 ⁺ T cells. A direct anti-viral effect on Infiuenzaviren is not mentioned. Furthermore, this document does not teach that proteasome inhibitors can be used as an anti-virally active agent for the preparation of pharmaceutically active compositions for the treatment of influenza viruses.

The subject of the patent application WO 03/064453 A2 are so-called Trojan inhibitors, which consist of proteasome inhibitors and trojan peptides. These should also be used for the treatment of influenza viruses. However, experimental evidence is also lacking in this publication that anti-viral action against influenza viruses can actually be achieved by means of these Trojan inhibitors. Furthermore, it can not be deduced from this document that a possible effectiveness of these Trojan inhibitors is really due to the specific inhibition of 26S proteasome. Rather, it must be assumed that a specific activity can only be achieved by bringing the proteasome inhibitor to the target cell by means of the Trojan component, in which case concrete evidence is lacking.

The document WO 03/064453 A2 does not teach to use proteasome inhibitors for the treatment of influenza viruses. Ubiquitin ligase inhibitors are the subject of document WO 2005/007141 A2. Anti-viral components, anticancer agents and compounds that can be used to treat neurological disorders are described therein. Influenza viruses are not mentioned. Moreover, there is no evidence in the present specification or any other publications that ubiquitin ligases interfere with the replication of infiuenzaviruses.

In summary it can be stated that in all previous uses of proteasome inhibitors their effect on Infiuenzaviren and certainly not their therapeutic use for the treatment of infiuenzavirus infection has been described. Also, the effect of proteasome inhibitors for the treatment of infiuenzavirus infection has not been discussed so far. Furthermore, it has not yet been tested whether proteasome inhibitors block the assembly and the release of infiuenzaviruses. Similarly, no association has been reported between infiuenzavirus infections and the UPS. Thus, both the use of inhibitors of cellular ubiquitin ligases and of ubiquitin hydrolases is completely novel.

2. The essence of the invention

The invention has for its object to provide means that are suitable for the treatment of infections with influenza viruses, in particular those substances that exert an antiviral effect on Infiuenzavirus infections in animals and humans. The problem has been solved - according to the features of the claims - by the use of inhibitors of the UPS. In particular, both proteasome inhibitors and inhibitors of ubiquitin ligases or ubiquitin hydrolases are used.

There have been developed according to the invention agents having anti-viral activity for the treatment of infiuenzavirus infections containing as active components both proteasome inhibitors and inhibitors of ubiquitin ligases or ubiquitin hydrolases in pharmaceutical preparations. The novel agents according to the invention are suitable for the prophylaxis and / or therapy of infections with influenza viruses, in particular the influenza A virus. The essence of the invention is also apparent from the claims.

Furthermore, the agents used in the invention for the prophylaxis and / or treatment, therapy and inhibition of infection with Orthomyxo viruses can be used. It is shown that the applications of these agents lead to the inhibition of the spread of infection and thus the disease development in vivo, in the animal model. These agents can therefore prevent the establishment of an infection with influenza viruses in animals and humans or cure an already established infection.

The object has been achieved by means of pharmaceutical preparations which are suitable for inhibiting the release, maturation and replication of influenza viruses, in particular of IAV.

These preparations are characterized in that they contain as active component at least one proteasome inhibitor. Furthermore, these medicines may contain other components of the UPS. This concerns ubiquitin ligases and / or ubiquitin hydrolases, ie enzymes that regulate ubiquitinylation of proteins. This task was therefore solved by a combination of proteasome inhibitors on the one hand and by ubiquitin ligases and / or ubiquitin hydrolases on the other hand. In a preferred embodiment of the invention, pure proteasome inhibitors are also used, which are distinguished by high membrane permeability and high specificity for the host cell 26S proteasome.

According to an advantageous embodiment of the invention anti-viral effects can be triggered especially in IAV-infected cells. These relate on the one hand the induction of apoptosis in the influenza virus-infected cells and thus the preferred death of infected cells in the organism. At the same time, the inhibition of the assembly and maturation of influenza viruses disturbs the release and production of infectious virus particles. In the sum total of this effect, a therapeutic effect can be effected by blocking virus replication and removing virus-producing cells in the organism.

In a further embodiment of the invention, classical proteasome inhibitors are to be used for the control of infections with influenza viruses. For this purpose, especially inhibitors are to be used, which interact exclusively only with the catalytically active hydroxyl-threonine group of the beta-subunit of the 26S proteasome and therefore specifically block only the proteasome. Another key component and surprising effect of this development is the observation that the blockade of the UPS preferentially induces death (the apoptosis) of influenza virus-infected cells.

The objects of the invention have been achieved by the use of at least one proteasome inhibitor and / or at least one inhibitor of ubiquitin ligases or ubiquitin hydrolases. There have been developed according to the invention agents for the treatment of viral infections, which contain as an effective component inhibitors of the UPS in pharmaceutical preparations, such as for the inhibition of influenza viruses. According to a preferred embodiment of the invention, substances which inhibit, regulate or otherwise influence the activities of the UPS are used as proteasome inhibitors.

It is also possible that as proteasome inhibitors substances are used which specifically affect the enzymatic activities of the complete 26S proteasome complex and the free, not assembled with regulatory subunits 2OS catalytically active proteasome structure. These inhibitors may inhibit either one or more or all three major proteolytic activities of the proteasome (the trypsin, the chymotrypsin, and the postglutamyl-peptide hydrolysing activities) within the 26S or 20S proteasome complex.

A variant of the invention is to use as proteasome inhibitors substances that are taken up by cells of higher eukaryotes and interact after cell uptake with the catalytic beta subunit of the 26S proteasome and thereby all or some of the proteolytic activities of the proteasome -Complex irreversibly or reversibly block.

As a further variant of the invention, substances are used as specific proteasome inhibitors which selectively block individual enzymatic activities of the 26S proteasome after cell uptake and moreover selectively inhibit certain assembly forms of the proteasome, such as, for example, the immunoproteasome. The immunoproteasome is formed by re-assembly as a particular form of the 26S proteasome, especially after stimulation by interferon treatment. The immunoproteasome may also be formed in response to an IAV infection. In this respect, the specific inhibition of immunoproteasome is a particular embodiment of the antiviral effect of proteasome inhibitors in IAV infections. Therefore, according to the invention, those substances are also used which selectively inhibit the immunoproteasome.

As another form of the invention, agents are used which inhibit the activities of the ubiquitin-conjugating and / or the ubiquitin-hydrolyzing enzymes. These include cellular factors that interact with ubiquitin as a mono- or poly-ubiquitin. Poly-ubiquitinylation is generally considered to be a recognition signal for proteolysis by the 26S proteasome, and manipulation of the ubiquitinylation pathway can also regulate the activity of the proteasome.

According to the invention are also used as proteasome inhibitors substances that are administered in various forms in vivo orally in encapsulated form with or without cell specificity-bearing changes, intravenously, intramuscularly, subcutaneously, by inhalation in aerosol form, or otherwise the application of a particular application and dose regimen low cytotoxicity and / or high selectivity for certain cells and organs, have no or trivial side effects, have a relatively high metabolic half-life and a relatively low clearance rate in the organism.

The decisive difference of the present invention over the solution described in DE 103 00222 Al is that according to the invention it has been shown that the specific effect of proteasome inhibitors on IAV replication does not include the NF-κB signal transmission path, but a completely different one cellular pathway, namely the ubiquitin-proteasome pathway (UPS) on the release of infectious progeny viruses in IAV infection. It was also possible to demonstrate for the first time experimentally and in vivo the antiviral effect of proteasome inhibitors on an IAV infection. This could be refuted in the present invention, as proteasome inhibitors also affect the NF-κB signal transduction pathway. The present results in (see embodiments) clearly show that with an in vivo effective anti-viral concentration of proteasome inhibitors clearly and clearly the NF-κB pathway is not affected.

As proteasome inhibitors further substances are used, which are isolated in natural form from microorganisms or other natural sources, emerge by chemical modifications of natural substances or produced completely synthetically or synthesized by gene therapy methods in vivo or by genetic engineering methods in vitro or in microorganisms. These include a) naturally occurring proteasome inhibitors:

- epoxomicin (epoxomycin) and eponemycin,

Aclacinomycin A (also referred to as Aclarubicin),

Lactacystin and its chemically modified variants, in particular the cell membrane penetrating variant "clastolactacysteine beta-lactone", b) synthetically produced:

modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), the boric acid derivative of which is MG232; N-carbobenzoxy-Leu-Leu-Nva-H (designated MGl 15); N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (referred to as LLnL); N-carbobenzoxy-Ile-Glu (OBut) -Ala-Leu-H (also referred to as PSI);

Peptides, the C-terminal epoxyketones (also referred to as epoxomicin / epoxomycin or eponemycin), vinyl sulphones (for example carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine vinyl sulfone or 4-hydroxy-5-iodo 3-nitrophenylactetyl-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone, also referred to as NLVS), glyoxal or boric acid residues (for example, pyrazyl-CONH (CHPhe) CONH (cisobutyl) B ( OH) ₂ ), also referred to as "PS-431" or benzoyl (Bz) -Phe-boroLeu, phenacetyl-Leu-Leu-boroLeu, Cbz-Phe-boroLeu); Pinacol esters - for example, benzyloxycarbonyl (Cbz) -Leu-Leu-boroLeu-Pinacol esters - carry;

- and

Peptides and peptide derivatives bearing C-terminal epoxy ketone structures are used as particularly suitable compounds; These include for example epoxomicin (molecular formula: C28H86N4O7) and eponemycin (molecular formula: C ₂₀ H ₃₆ N ₂ O ₅ V

certain dipeptidyl boric acid derivatives, in particular the compound PS-296 (8-quinolylsulfonyl-CONH- (CH-naphthyl) -CONH (-CH-isobutyl) -B (OH) ₂ ); the compound PS-303 (NH ₂ (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) ₂ ); the compound PS-321 (morpholine CONH- (CH-naphthyl) -CONH- (CH-phenylalanine) -B (OH) ₂ ); the compound PS-334 (CH 3 -NH- (CH-naphthyl-CONH- (CH-isobutyl) -B (OH) ₂ ); the compound PS-325 (2-quinol-CONH- (CH-homo-phenylalanine) - CONH- (CH-isobutyl) -B (OH) ₂ ); compound PS-352 (phenylalanine-CH ₂ -CH ₂ -CONH- (CH-phenylalanine) -CONH- (CH-isobutyl) l B (OH) ₂ ) Compound PS-383 (pyridyl-CONH- (CHpF-phenylalanine) -CONH- (CH-isobutyl) -B (OH) ₂ ) These include all compounds already described in Adams et al., (1999) In addition to epoxomicin and eponemycin, particularly suitable compounds have been found to be peptidyl boric acid derivatives, which are very potent, very specific for the proteasome, do not block other cellular proteases, and therefore have virtually no side effects.

With the proteasome inhibitors agents are provided according to the invention, which surprisingly

- block the production of infectious progeny viruses by blocking the replication of influenza viruses and thereby prevent the spread of an influenza virus infection in the organism;

- block the release of infectious influenza viruses from infected cells;

- limit the spread of acute influenza virus infection;

suppress the viremia in both a new infection and in recurrent influenza virus RE infections and increase the success of virus elimination by one's own immune system and / or by known agents having similar or other effects.

The features of the invention will become apparent from the elements of the claims and from the description, wherein both individual features as well as several represent advantageous embodiments in the form of combinations for which protection is sought with this document. The invention is also in a combined use of known and novel elements - proteasome inhibitors and Ub ligases on the one hand and Ub hydrolases on the other hand.

According to the invention, the proteasome inhibitors are used

in the treatment of influenza and related human and animal diseases caused by influenza viruses and related negative strand RNA viruses, as agents for influencing, inhibiting or regulating the ubiquitin / proteasome pathway as a means to influence the enzymatic activities of the complete 26S proteasome complex and the free, non-regulatory subunits assembled 2OS catalytically active proteasome structure, and to selectively inhibit the immunopro teasome.

The inventively used inhibitors of the UPS are also used - to prevent the onset of disease and to reduce the spread of infection in the organism of already infected persons;

for the prevention of the establishment of systemic influenza infection immediately after contact with infectious biological samples, infected persons or their immediate vicinity.

The inhibitors of the UPS used according to the invention can be administered both systemically and topically, preferably aerogenically. The active substance used according to the invention of a proteasome inhibitor can be used with at least one further antivirally active substance for the prophylaxis and / or therapy of influenza virus infections.

The invention will be explained in more detail by means of embodiments, without being limited to these examples.

embodiments

Example 1: Proteasomenin inhibitor shows no toxic side effects after aerosol treatment of mice

To investigate whether a proteasome inhibitor is toxic after administration as an aerosol for mice, 6 mice were treated 3 times a day for 5 days with 500 nM proteasome inhibitor. For this purpose 2 ml proteasome inhibitor (50OnM) were aerosolized using a nebulizer (PARI ^®). The treatment duration was 10 minutes each. The treatments were at 9:00, 12:00 and 15:00. As control served 6 mice, which were treated with the solvent of the Proteasomeninhibitors (DMSO / H2O). To measure body temperature and physical activity, mini-transmitters were implanted into the mice. The cages in which the mice were standing stood up Receiver plates. These receivers sent the signals to a computer, which evaluated the data using special software.

After implantation of the transmitter, the health of the mice was observed for 5 days, followed by treatment with proteasome inhibitor for another 5 days. During treatment, no difference in body temperature (Figure IA) and physical activity (Figure IB) was observed between mice treated with the proteasome inhibitor and the solvent. The graphics show an example of the test values on the 5th day of treatment. The studies show that mice treated with the proteasome inhibitor at a concentration of 50 nM and a treatment period of 5 days had no significant toxic effect. Thus, the proteasome inhibitor at said concentration is suitable for studies on antiviral activity against influenza viruses in the mouse model.

Example 2: Proteasomenin inhibitors efficiently inhibit influenza virus proliferation in a concentration-dependent manner and are not significantly toxic to the host cell at antiviral concentrations during the observation period.

To investigate whether inhibitors of the proteasome have a negative effect on the proliferation of influenza viruses, human A549 lung epithelial carcinoma cells (Fig. 2 A, B) and Madine Darby Canine Kidney (MDCKII) dog kidney epithelial cells (Fig Proteasome inhibitors were pre-incubated for one hour at the concentrations indicated and then infected with the avian influenza virus strain A / FPV / Bratislava / 79 (H7N7) (multiplicity of infection (MOI) = 0.01). As a comparison served untreated, infected cells or with the solvent dimethyl sulfoxide (DMSO) treated cells. The substances PS341 (10 nM and 100 nM), PS273 (10 nM and 100 nM), lactacysteine (1 μM and 10 μM) and epoxomycin (10 nM, 100 nM and 1 μM) were used. The virus-containing medium supernatants were collected for 8 and 24 h (FIG. 2A) or 8, 24 and 36 h (FIG. 2 B, C) after the beginning of the infection and the virus titers were determined in plaque assays on MDCKII cells.

The result shows that all inhibitors of the proteasome inhibit the replication of the aggressive influenza virus A / FPV / Bratislava / 79 efficiently and in a concentration-dependent manner. To answer the question whether the antiviral effect is indirectly triggered by a cytotoxic effect of the proteasome inhibitors, MDCKII cells (cell number: 2x10 ⁶ ) were treated with antiviral concentrations of the most effective antiviral proteasome inhibitors PS341, lactacysteine and epoxomycin. The control was the highly cytotoxic apoptosis-inducing substance staurosporine (0.3 μM). After 16 and 24 h, both the adherent and the already detached dying cells were collected, washed with PBS and treated with 50 μg / ml of propidium iodide (PI). PI intercalates into the DNA strands of dying cells. Analysis was by flow cytometry (Becton Dickinson FACScan). In Fig. 3 (A, B and C) is shown in each case the percentage of dead cells compared to the untreated control.

It was found that the proteasome inhibitors have no significant toxic effect in antiviral concentrations in the observation period of 24 h. This makes it possible to exclude that the antiviral effect of the proteasome inhibitors shown in FIG. 1 is due to toxic effects on the cells.

Example 3: Proteasome Inhibitors Act Antivirally Against Influenza Viruses in a NF-κB Independent Mechanism

To investigate whether the antiviral effect of the proteasome inhibitors is due to inhibition of the NF-κB signaling pathway, TNFα-induced NF-κB activation was elicited by degradation of the inhibitory protein IKBCC (inhibitor of KB) in the Western blot in The presence and absence of proteasome inhibitors analyzed. For this purpose, A549- (2x10 ⁶ ) or HEK293 cells (4x10 ⁶ ) were preincubated with the proteasome inhibitors in various concentrations for 1 h. Thereafter, the cells were treated for 15 min with 20 ng / ml TNFα to induce the degradation of IKBCC ZU. The cells were then washed with IxPBS and lysed. The protein concentrations were determined by Bradford protein assay (Biorad) and aligned. The proteins were separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane. IκBα degradation was visualized using an IKBCC-specific antiserum (Santa Cruz Biotechnologies) and a horseradish peroxidase-coupled secondary reagent (Amersham) using an electrochemiluminescent reaction (ECL, Amersham). Surprisingly, antiviral concentrations of the respective proteasome inhibitors were unable to effectively inhibit TNFα-induced degradation of IKBCC and thus to inhibit the NF-KB activation. For example, PS341 (100 nM) was unable to prevent IκBα degradation in A549 or HEK293 cells (Figures 4B and D). Nevertheless, the same concentration of PS341 in infected A549 cells resulted in a reduction in virus titer (over 2 logarithmic levels after 8 h, see Figure 2 A). Similarly, lactacysteine (1 μM) reduced viral titers (Figure 2 A), but was not effective enough to inhibit IκBα degradation (Figures 4B and D).

This clearly demonstrates that the proteasome inhibitors are antiviral via a mechanism other than inhibition of NF-κB.

Example 4: A pharmacological inhibitor of the proteasome, MG 132, interferes with the influenza virus-induced expression of pro-apoptotic genes and efficiently inhibits influenza virus proliferation in vitro and in vivo.

To investigate whether the proteasome inhibitor MG 132 has a negative effect on the proliferation of influenza viruses, the human lung epithelial cell line A549 was infected with the highly pathogenic avian influenza A virus / FPV / Bratislava / 79 (multiplicity of infection = l ) in the presence or absence of various concentrations of the proteasome inhibitor MG132 (1, 5, 10 μM). In the case of the presence of the inhibitor, cells were preincubated 30 minutes prior to infection with the substance. At 24 h after infection, cell supernatants were assayed for the level of infectious progeny virus in plaque assays. As a result, in the presence of MG 132, a concentration-dependent up to 10-fold reduction in virus titer is evident (FIG. 5). To investigate whether the reduction in virus titer is associated with a reduced expression of the pro-apoptotic ligands TRAIL and FasL / CD95L, the A549 lung epithelial cell line was as described with the avian influenza A virus A / FPV / Bratislava / 79 (multiplicity of infection = l) in the presence or absence of the proteasome inhibitor MG 132 (10 μM). About 24 hours after infection, cells were fixed with 4% paraformaldehyde and incubated with specific antibodies to TRAIL and FasL / CD95L. After coupling the antibodies with a fluorescent dye, the cells were subjected to flow cytometric analysis (FACS) to measure the expression of pro-apoptotic factors. As a result, the FACS profiles show markedly reduced virus-induced expression of FasL and TRAIL in the presence of MG132 (Figure 6). To test the antiviral activity of MG132 in an in vivo infection model in the mouse, C57B1 / 6 mice (10 weeks old, self-bred BFAV Tübingen) with 10 ⁴ infectious units of mouse-adapted high-pathogenic avian influenza A virus / FPV / Bratislava / 79 intranasally infected and left untreated (n = 14) or treated in an insulating cage for 5 days daily with 2 ml of an aerosol of ImM MG 132 (n = 8). The treatment was once a day, starting before the infection on day 1. The survival rate of the mice was determined.

As a result, an overall significantly increased survival rate of the infected and MG 132 treated mice was found in comparison to the untreated control animals (FIG. 7).

Example 5: Material and Methods

Treatment of mice with proteasome inhibitors: The mice were treated in an inhalation system. For this purpose, 6 mice were treated in inhalation tubes. These 6 tubes were connected to a central cyclinder with a total volume of 8.1 xlθ ^{~ 4} m ³ . A PARI ^® nebulizer (Aerosol Nebulizer; Art. Nr. 73-1963) was connected Andes central cylinder. Proteasome inhibitors or solvents were atomized into the chamber at 1.5 bar for 10 min (about 2 ml). BALB / c mice were treated 3 times a day at 9:00, 12:00 and 3:00 for 5 days. The general health of the mice was checked twice a day and the animals were weighed once a day.

Mouse Monitoring: Monitor body temperature and physical activity in mice using the Vital View ^® software and hardware system (Mini Mitter USA). This system allows the generation of physiological parameters of the mouse. The hardware consists of a transmitter (E-Mitter) / receiver system. The E-Mitter collects data of the body temperature and the physical activity of the animals. This data is collected every 5 minutes and forwarded to a PC. There, the data is analyzed with the help of the Vital View software.

For implantation of the E-Mitters, the mice were anesthetized by intraperitoneal injection of 150 μl of Ketamine Rompun. The belly of the mice is shaved and there is a 1.5 cm section along the line abla to the opening of the abdomen. Then the E-Mitter is positioned in the abdomen and the opening is closed with wound clips (except to clip 9mm; Becton & Dickinson, Germany). The animals are returned to their cages and the successful implantation is controlled with the help of the Vital View software.

Viral Infection of Cells: Cells are washed and the virus solution diluted in infectious PBS (PBS with 1% penicillin / streptomycin, 1% Ca ²⁺ ZMg ²⁺ , 0.6% Bovine Albumin 35%) is added. The cells were incubated with the virus in the indicated amounts for 30 min at 37 ° C. in the incubator and then washed again to remove the virus particles which had not bound to cells.

After this adsorption phase, the cells were covered with infection medium (MEM with 1% penicillin / streptomycin and 1% Ca ²⁺ ZMg ²⁺ ). The cells were kept in the incubator at 37 ° C until cell harvest or determination of the progeny virus in the supernatant.

Plaque Assay for Detecting Infectious Progeny Viruses: To determine the number of infectious particles in a virus solution, plaque assays were performed on MDCK II cells. The infection of the cells was carried out with 500 .mu.l of a logarithmically diluted in infectious PBS series of virus solution. It was incubated with the virus solution in the incubator for 30 min at 37 ° C, then the virus solution was removed and the cells with a mixture of medium and agar (27 ml ddH ₂ O, 5 ml 10 x MEM, 0.5 ml penicillin / Streptomycin, 1.4 mL sodium bicarbonate, 0.5 mL 1% DEAE-dextran, 0.3 mL bovine albumin, 15 mL 3% Oxoid Agar, 500 μL Ca ²⁺ ZMg ²⁺ ). The cells were kept at room temperature until the medium-agar mixture solidified and then incubated for 2-3 days at 37 ° C. in an incubator until visible plaques of lysed cells were formed. The plaques were stained in the incubator with 1 ml of a neutral red PBS solution for an additional 1-2 hours until the plaques became visible.

Cell staining with propidium iodide: The substance Propidium iodide can penetrate through the cell membrane of dying cells and intercalates into the DNA in the cell nucleus. The number of dying and dead cells can then be determined on the basis of their fluorescence in the flow cytometer. MDCK cells (2x10 ⁶ ) were treated with the indicated concentrations of proteasome inhibitors. As a toxicity control, MDCK cells were treated with the apoptosis-inducing substance staurosporine (0.3 μM). After 16 or 24 h, the adherent cells and cells were collected from the supernatant and stained with 50 ug / ml propidium iodide. Analysis was by flow cytometry (BD FACScan). SDS Gel Selector Phosphor and Western Blot: The cells to be lysed were washed first with PBS and then per well of a 6-well plate with 200 μl of Lysis buffer PJPA (25 mM Tris pH 8, 137 mM NaCl, 10% glycerol, 0.1 % SDS, 0.5% sodium deoxychloro DOC, 1% NP40, 2 mM EDTA pH 8, freshly added: Pefablock 1: 1000, aprotinin 1: 1000, leupeptin 1: 1000, sodium vanadate 1: 100, benzamidine 1: 200) Mistake. The cells were lysed with panning at 4 ° C for 30 min and then centrifuged at 14,000 rpm for 10 min at 4 ° C in the bench top centrifuge to separate the proteins from the cell debris. The protein concentration in the lysates was determined using the Biorad protein staining solution (Biorad) and adjusted the same amounts of protein. The samples were then spiked with a 5X sample buffer (10% SDS, 50% glycerol, 25% β-mercaptoethanol, 0.01% bromophenol blue, 312 mM Tris). The ß-mercaptoethanol in the sample buffer additionally caused the denaturation of the proteins by reducing the disulfide bridges. The negatively charged proteins migrate in the electric field to the positive electrode, whereby larger proteins in the gel are held back stronger. The electrophoresis gel consists of a 5% stacking gel (0.49 ml Rotiphoresis Gel 30, 3.25 ml Stacking Buffer (0.14 M Tris pH 6.8, 0.11% Temed, 0.11% SDS), 45 μl 10% ammonium persulfate), in which the proteins are concentrated and purified from a 10% separating gel (3.375 ml Rotiphorese Gel 30, 2.5 ml Running Buffer (1.5 M Tris pH 9, 0.4% Temed, 0.4% SDS), 4.025 ml of bidistilled water, 200 μl of 10% ammonium peroxodisulfate), in which the proteins are separated according to their molecular weight.

The gel is poured into two glass plates clamped in a casting stand and spaced by spacers, the separating gel being poured first. This was covered with isopropanol for polymerization and then washed with double-distilled water. The collection gel is poured onto the separating gel and a sample comb is used bubble-free. After polymerizing out, the sample comb is pulled and the gel placed in an electrophoresis chamber filled with 1 × SDS-PAGE buffer (5 mM Tris, 50 mM Glycine, 0.02% SDS). The denatured proteins and a marker are filled in the pockets. The gel runs at a constant current flow of 25-40 mA.

Thereafter, the proteins are transferred from the gel to a nitrocellulose membrane by means of an electric field. Proteins immobilized on the nitrocellulose membrane can be detected via a specific antibody. This is recognized by an enzyme-linked second antibody, whereupon the proteins can be visualized by a chemiluminescent reaction. In this case, the substrate luminol is oxidized by a horseradish peroxidase bound to the secondary antibody, whereby it goes into an excited state and emits light, which is on a Naked excited state passes and emits light, which can be made visible on an X-ray film.

The SDS-GeI with the separated proteins is removed from the casting apparatus and placed on two Whatman papers soaked in blotting buffer (3.9 mM Glycine, 4.8 mM Tris, 0.0037% SDS, 10% methanol). The nitrocellulose membrane, which was also impregnated with blotting buffer, was placed on the gel without bubbles before it was clamped in a blotting buffer-filled wet blot chamber (BioRad). The proteins are transferred to the nitrocellulose membrane at a constant current of 400 nA in 50 min. In this case, the proteins migrate from the cathode to the anode.

Following the blotting procedure, unspecific binding sites are blocked with 5% milk powder in 1 × TBST (50 μM Tris, 0.9% NaCl, 0.05% Tween 20, pH 7.6) for at least 45 min to prevent non-specific binding of the antibody to the membrane. Subsequently, the membrane is incubated with the primary antibody (Here -anti IKBCC, dilution 1: 1000, Santa Cruz Biotechnologies) while panning at 4 ° C overnight. The membrane is washed three times for 10 minutes each with 1 × TBST to remove unbound residues of the antibody. Thereafter, the membrane is swirled for 1-2 hours in the secondary antibody at room temperature. After washing three more times with 1 × TBST, the membrane is in the state of increased chemiluminescence by adding a chemo luminescence substrate (250 mM luminol, 90 mM p-cumenaric acid, 1 M Tris / HCl pH 8.5, 35% H ₂ O ₂ ) (Enhanced chemoluminescence ECL) brought by light emitting the substrate contained therein Luminol from the horseradish peroxidase bound to the secondary antibody. The membrane is incubated with the substrate for 1 min, then dried and then placed in an X-ray film cassette in which the increased chemiluminescence is visualized on an X-ray film.

Flow cytometric analysis: TRAIL and FasL expression were detected by intracellular fluorescent staining with coupled antibodies. A549 cells were infected with virus strain A / FPV / Bratislava / 79 (H7N7) (MOI = 5) for 8 hours in the presence of 2μM monensin to prevent protein secretion. The cells were then fixed with 4% paraformaldehyde at 4 ° C for 20 min and then washed with permeabilization buffer (0.1% saponin / 1% FBS / PBS). This was followed by incubation with the primary antibody against TRAIL, FasL or an isotype control (antibody from Becton Dickinson). Subsequently, the cells were biotin-Sp-conjugated goat anti-mouse IgG (Dianova) and Streptavidine-Cy-chrome (Becton Dickinson) stained. Fluorescence was measured in the FL3 channel of a FACScalibur flow cytometer (Becton Dickinson).

Infection and treatment of mice: 10-week-old C57B1 / 6 mice (self-breeding, FLI, Tübingen) were used for infection and treatment. Treatment with about 2 ml of an ImM MGI 32 (Sigma) dilution was carried out once daily by aerosol delivery in a cage inhalation, starting one stage before intranasal infection with 5x10 ³ to 10 ⁴ plaque-forming infectious units (pfu) of the virus strain A / FPV / Bratislava / 79 (H7N7). Nebulisation of the MGI 32 solution was carried out by means of a mouse Minivent system (Hugo Sachs Elektronik-Harvard apparatus) connected to a nebulizer (Hugo Sachs Elektronik-Harvard Apparate).

Legend to the figures

FIG. 1: Aerosol treatment of Balb / c mice with the proteasome inhibitor

Course of temperature (A) and activity (B) of mice treated 3 times a day with either the proteasome inhibitor (red) or the solvent (black). The graphs show the mean of the reading of 6 animals. In total, 288 readings were taken every day (every 5 minutes).

FIG. 2: Proteasome inhibitors efficiently inhibit the replication of the influenza virus

A549 cells (2x10 ⁶ ) (A, B) or MDCK cells (4x10 ⁶ ) (C) were preincubated for 1 h with the indicated concentrations of the respective proteasome inhibitors. After preincubation, the cells were infected with the avian influenza virus A / FPV / Bratislava / 79 (H7N7) (multiplicity of infection, MOI = 0.01). After 8, 24 or 36 h, the medium supernatants were collected and the virus titers were determined by means of plaque assay on MDCK cells.

FIG. 3: Antiviral concentrations of the proteasome inhibitors are not toxic for MDCK cells in the observed time range up to 24 h.

(A, B, C) MDCK cells (2x10 ⁶ ) were treated with the indicated concentrations of proteasome inhibitors. As a toxicity control, MDCK cells were treated with the apoptosis-inducing substance staurosporine (0.3 μM) (black bars in the diagram). After 16 or 24 h, the adherent cells and cells were collected from the supernatant and stained with 50 ug / ml propidium iodide. The analysis was carried out with the help of flow cytometry (BD FACScan). The graph shows the percentage of living cells compared to the untreated control.

FIG. 4: Proteasome inhibitors are not able to prevent TNF.alpha.-induced IκB.alpha. Degradation in antivirally active concentrations.

A549 cells (2x10 ⁶ ) (A, B) or HEK293 cells (4x10 ⁶ ) (C, D) were preincubated for 1 h with the indicated concentrations of proteasome inhibitors. After preincubation, the cells were stimulated for 15 min with 20 ng / ml recombinant TNFα and lysed. The lysates were separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane. IκBα degradation was detected using an IκBα-specific rabbit serum (Santa Cruz Biotechnologies).

FIG. 5: MG132 inhibits influenza virus multiplication. A549 cells were infected with FPV (MOI = I) in the presence of MG132 (10 μM). 24h p.i. the supernatants were collected and the titre of progeny viruses determined by plaque assay.

FIG. 6: MG132 inhibits virus-induced induction of TRAIL and FasL A549 with FPV (MOI = I) and incubated with MG132 (10 μM) in the medium. 24h p.i. the cells were fixed with 4% paraformaldehyde and the anti-TRAIL and anti-CD95L stained.

Figure 7: Antiviral activity of MG132 in infected mice. C57Bl / 6 mice were infected with FPV (10 ⁴ pfu, intranasal), treated with MG 132 (n = 8) (solid line) by inhalation of cages, or received no treatment (n = 14) (dotted line). For the cage treatment, the animals were aerosolized with 2 ml of ImM MG 132 (Sigma) for 5 days. The treatment was performed on a daily basis and started one hour prior to infection for five hours. For control, the weight of the animals was determined daily. Shown are the survival curves of FPV infected MGl 32-treated and untreated mice.

List of abbreviations

DNA desoxyribonucleic acid (deoxyribonucleic acid) kDa kilodaltons (measure of molecular weight)

Ki inhibitory constant

LC lactacystin

MDa Mega Dalton MHC Major Histocompatibility Complex

NLVS Proteasome Inhibitor z-Leucinyl-Leucinyl-Leucinyl-Vinylsulfone (NLVS)

PGPH postglutamyl-peptide hydrolyzing

PI proteasome inhibitor

PCR polymerase chain reaction

RNA ribonucleic acid

RSV Rous Sarcoma Virus

RT reverse transcriptase

Ub ubiquitin

UPS ubiquitin / proteasome system

Vero cells human permanent transformed cells of the line VERO

Vpr HIV-I Protein Vpr, ZLLL Tripeptide aldehyde N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-leucinal

literature

Adams, J., Ma, Y., Stein, R., Baevsky, M., Greiner, L., and Plamondon, L. (1996) Boronic ester and acid compounds, synthesis and uses. US 1,448,012 TWO.

Adams, J., Palombella, V.J., Sausville, E.A., Johnson, J., Destree, A., Lazarus, D.D., Maas, J. Pien, CS., Prakash, S., Elliott, PJ. (1999) Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 59: 2615-2622.

Adams, J., Palombella, V.J., Elliot, PJ. (2000) Proteasome inhibition: a new strategy in cancer treatment. Investigational New drugs 18: 109-121.

Chen et al, Nat. Med. 7, 1306-1312, 2001

Ciechanover, A., Orian, A., Schwartz, AL (2000) The ubiquitin-mediated proteolytic pathway: Mode of action and clinical implications. J. Cell. Biochem. 77: 40-51. Drosten C, Günther S, Preiser W, Van Der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Gryvan K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a

Elliott, PJ, Ross JS; (2001). The proteasome: a new target for novel drug therapies. At the J Clin Pathol. 116: 637-46.

Fenteany, G; Standaert, RF; Lane, WS; Choi, S; Corey, EJ; Schreiber, SL (1995). Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lacta- cysteine. Science 268: 726-731. Gubareva et al J Infect Dis 178, 1257-1262, 1998.

Hershko, A., Ciechanover, A. (1998) The ubiquitin system. Annu Rev Biochem. 67: 425-479. Harty, RN; Brown, ME; Wang, G; Huibregtse, J; Hayes, FP; (2000). A PPxY motif within the VP40 protein of Ebola virus interacting with and functioning with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Be. USA 97: 13871-13876.

Lamb and pitcher, in Fields, Virology, Philadelphia: Lippincott-Raven Publishers, 1353- 1395, 1996.

Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, Ahuja A, Yung MY, Leung CB, To KF, Lui SF, Szeto CC, Chung S, Sung JJ. A Major Outbreak of Severe Acute Respiratory Syndrome in Hong Kong. N Engl J Med 2003 Apr 14; [epub ahead of print].

Ludwig et al., Trends Mol. Med. 9, 46-51, 2003.

Ludwig et al., FEBS Lett 561, 37-43, 2004.

Meng, L; Kwok, BH; Sense; Crews, CM (1999a). Eponemycin exerts its antitumor effect through the inhibition of proteasome function. Cancer Res. 59: 2798-2801.

Qui et al. Nature 437,1108, 2005. Patnaik, A, Chau, V, Wills, JW; (2000). Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Be. USA 97: 13069-13074.

Poutanen SM, Low DE, Henry B, Finkelstein S, Rose D, Green K, Tellier R, Draker R, Adachi D, Ayers M, Chan AK, Skowronski DM, Salit I, Simor AE, Slutsky AS, Doyle PW, Krajden M , Petric M, Brunham RC, McGeer AJ. Identification of Severe Acute Respiratory Syndrome in Canada. N Engl J Med 2003 Apr 10.

Rock, K.L., Goldberg, A.L. (1999) Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 11: 139-119.

Scholtissek and Muller, Arch Virol 119, 111-118, 1991.

Schubert, U., Ott, D.E., Chertova, E.N., Welker, R., Tessmer, U., Princiotta, M.F., Bennink, J.R., Kräusslich, H. -G., Yewdell, J.W. (2000a) Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Be. USA 97: 13057-13062.

Strack, B; Calistri, A; Accola, MA; PaIu, G; Göttlinger, HG (2000). A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Be. USA 97: 13063-13068.

Webster et al, Microbiol Rev., 56, 152-79, 1992

Claims

claims

1. Agent for the treatment of infections with Orthomyxoviridae, characterized in that they contain as active component at least one proteasome inhibitor and / or at least one inhibitor of the ubiquitin-proteasome pathway (UPS) in pharmaceutical preparations.

2. Composition according to claim 1, characterized in that they contain as UPS inhibitors inhibitors of the proteasome and / or of ubiquitin ligases and / or ubiquitin hydrolases.

3. Composition according to claim 1 or 2, characterized in that they have a high specificity for the 26S proteasome of a host cell.

4. Composition according to claim 1 or 2, characterized in that they interact exclusively only with the catalytically active hydroxyl-threonine group of the beta-subunit of the 26S proteasome and specifically block only the proteasome.

5. Composition according to claim 1 or 2, characterized in that they selectively block individual enzymatic activities of the 26S proteasome after cell uptake and additionally selectively inhibit certain forms of assembly of the proteasome, preferably the immunoproteasome.

6. Composition according to claim 1 or 2, characterized in that they inhibit the activities of the ubiquitin-conjugating and / or the ubiquitin-hydrolyzing enzymes.

7. Use of the agents according to any one of claims 1 to 6 for the preparation of antiviral agents and / or pharmaceutical preparations with antiviral activity, characterized in that they lead to a) induction of apoptosis in Infiuenzavirus-infected cells and thus the preferred death of b) disrupting the release and production of infectious virus particles by inhibiting the assembly and maturation of infiuenzaviruses.

8. Use of the agent according to any one of claims 1 to 6 for the treatment of Infiuenzaviren infections.

9. Use according to claim 7 or 8 for 9.1.Treating virus infections

9.2. Influencing, Inhibiting, Regulating or Blocking the Ubiquitin / Proteasome

Pathway in the target cell

9.3. Preventing the spread of viral infections in the organism. 9.4. Inhibiting the release, maturation and replication of influenza viruses 9.5. Induction of apoptosis of influenza virus-infected cells.

10. Use according to one of claims 7 to 9, characterized in that the release, maturation and replication of Infiuenzaviren be inhibited.

11. Use according to one of claims 7 to 9 for the control / treatment of influenza and related diseases, especially pandemic and endemic Influenza- outbreaks in humans and animals.

12. Use according to claim 11 in combination with other, already known antiviral drugs, such as ripavarin, interleukins, nucleoside analogs, protease inhibitors, inhibitors of viral kinases, inhibitors of membrane fusion and viral entry, especially of inhibitors of components of Infiuenzaviren , such as nueraminidase or M2 ion channel portals from IAV.

13. Use according to claim 11 and 12 for preventing the onset of disease and reducing the spread of infection in the organism of acutely infected in Infiuenzaviren humans and animals.

14. Use according to any one of claims 7 to 13 for blocking the ubiquitin / proteasome pathway in certain target cells, wherein the target cells of Inlfuenzaviren infested host cells.

15. Use according to one of claims 7 to 14 for influencing target cells in their cellular mechanisms: cell division, cell cycle, cell differentiation, cell death (apoptosis), cell activation, signal transduction or antigen processing, especially NF-κB activation.

16. Use according to claim 7 for the preparation of agents for inhibiting the release, maturation and replication of influenza viruses, characterized in that it comprises as active component at least one proteasome inhibitor and / or at least one inhibitor of ubiquitin ligases and / or ubiquitin hydrolases in a pharmaceutical preparation.

17. Use according to claim 16 for the treatment of influenza infections, characterized in that as proteasome inhibitors substances are used which are taken up as proteasome inhibitors of cells of higher eukaryotes and after cell uptake interact with the catalytic subunits of the proteasome and all or irreversibly or reversibly block the proteasome proteolytic activities - the trypsin, chymotrypsin and postglutamyl-peptide hydrolysing activities - within the 26S or even the 2OS proteasome complex.

18. Use according to claim 16 or 17, characterized in that the pharmaceutical preparations in addition to proteasome inhibitors also contain other agents that influence, regulate or inhibit the cellular ubiquitin system, such as the activities

18.1. the ubiquitin-conjugating enzymes and / or

18.2. the ubiquitin-hydrolyzing enzymes

18.3. of cellular factors that interact with ubiquitin

18.4. of cellular factors associated with ubiquitin

18.4.1. Mono-ubiquitin or as

18.4.2. Poly-ubiquitin interact.

19. Use according to claim 16 to 18, characterized in that as proteasome inhibitors substances are used in different forms in vivo orally in encapsulated form with or without cell specificity-bearing alterations, intravenously, intramuscularly, subcutaneously, by inhalation in aerosol form, or otherwise administered, have low cytotoxicity due to the application of a particular application and / or dose regimen, no or insignificant side effects have a relatively high metabolic half-life and a relatively low clearance rate in the organism.

20. Use according to claim 16 to 19, characterized in that as proteasome inhibitors substances are used which are a) isolated in natural form from microorganisms or other natural sources or b) emerge by chemical modifications of natural substances c) totally synthetic d) be synthesized in vivo by gene therapy methods e) be prepared by genetic engineering methods in vitro or f) in microorganisms.

21. Use according to claim 16 to 20, characterized in that as proteasome inhibitors substances are used which belong to the following substance classes: a) naturally occurring proteasome inhibitors:

- Peptide derivatives containing C-terminal epoxy ketone structures

β-lactone derivatives

Aclacinomycin A (also referred to as Aclarubicin),

- Lactacystin and its chemically modified variants, such as the cell membrane-penetrating variant "clastolactacysteine β-lactone" b) synthetically produced proteasome inhibitors:

modified peptide aldehydes such as N-carbobenzoxy-L-leucinyl-L-leucinyl-L-leucinal (also referred to as MG 132 or zLLL), the boric acid derivative of which is MG232; N-carbobenzoxy-Leu-Leu-Nva-H (termed MGl 15; N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (referred to as LLnL), N-carbobenzoxy-Ile-Glu (OBut) -la) Carbobenzoxy-Ile-Glu (OBut) -Ala-Leu-H (also referred to as PSI), c) peptides which carry an α, β-epoxyketone C-terminal structure, and also vinylsulfones such as c 1) carbobenzoxy-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone or c2) 4-hydroxy-5-iodo-3-nitrophenyl-actetyl-L-leucinyl-L-leucinyl-L-leucine-vinyl-sulfone (NLVS) d) glyoxal or boric acid radicals such as dl) pyrazyl-CONH (CHPhe) CONH (CHisobutyl) B (OH) 2) as well as d2) dipeptidyl boric acid derivatives or e) pinacol esters such as benzyloxycarbonyl (Cbz) Leu-Leu-boroLeu-pinacol ester.

22. Use according to claim 16 to 21, characterized in that as particularly suitable proteasome inhibitors, the epoxyketones

22.1. Epoxomicin (epoxomycin, molecular formula: C28H86N4O7) and / or

22.2. Eponemycin (eponemicin, molecular formula: C ₂ OH ₃₆ N ₂ OS) can be used.

23. Use according to claim 11 to 17, characterized in that as particularly suitable proteasome inhibitors from the PS series, the compounds

23.1. PS-519 as β-lactone and as lactacystin derivative the compound IR- [IS, 4R, 5S]] - 1- (1-hydroxy-2-methylpropyl) -4-propyl-6-oxa-2-azabicyclo [ 3.2.0] heptane-3,7-diones - molecular formula C ₁₂ H ₁₉ NO ₄ -

23.2. PS-303 (NH ₂ (CH-naphthyl) -CONH- (CH-isobutyl) -B (OH) ₂ ) and / or

23.3. PS-321 as (morpholine-CONH- (CH-Napthyl) -CONH- (CH-phenylalanine) -B (OH) ₂ ); - and or

23.4. PS-334 (CH ₃ -NH- (CH-naphthyl-CONH- (CH-isobutyl) -B (OH) ₂ ) and / or

23.5. Compound PS-325 (2-quinol-CONH- (CH-aomo-phenylalanine) -CONH- (CH-isobutyl) -B (OH) ₂ ) and / or

23.6. PS-352 (phenyalanine-CH ₂ -CH ₂ -CONH- (CH-phenylalanine) -CONH- (CH-isobutyl) l B (OH) ₂ ) and / or

23.7. PS-383 (pyridyl-CONH- (CH /? F-phenylalanine) -CONH- (CH-isobutyl) -B (OH) ₂ )

23.8. be used.

24. Use according to claim 16 as an agent for influencing the enzymatic activities of the complete 26S proteasome complex and the free, not with regulatory subunits assembled 2OS catalytically active proteasome structure.

Use according to any one of claims 7 to 24 for systemic as well as topical, preferably aerogenic, administration of UPS inhibitors.