Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/60—Salicylic acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/82—Theaceae (Tea family), e.g. camellia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways

Definitions

• the invention relates to a test system for identifying active ingredients, which are suitable for the prophylaxis and/or treatment of viral diseases, the uses of such active ingredients for preparing a pharmaceutical composition for the prophylaxis and/or treatment of viral diseases, formulations for such pharmaceutical compositions and methods for preparing such pharmaceutical compositions.
• RNA or DNA viruses are a substantial threat for the health of man an animal.
• RNA viruses belong the negative-strand-RNA viruses, such as for instance influenza viruses or the Borna disease virus.
• influenza viruses still belong to the big epidemics of civilization and cause year for year a large number of fatalities. They are an immense cost factor for the economy, for instance by inability to work because of illness.
• BDV Borna disease virus
• attacking horses and sheep which was however already isolated in man, too, and which was connected here with neurological diseases.
• RNA viruses The problem of controlling in particular RNA viruses is the adaptability of the viruses caused by a high fault rate of the viral polymerases, thus the preparation of suitable vaccines as well as the design of antiviral substances being very difficult.
• antiviral substances immediately directed against the functions of the virus, initially at the beginning of the therapy have a fair antiviral effect, but will lead very quickly to the selection of resistant variants, because of mutation.
• An example is the anti-influenza drug amantadine and its derivatives, which is or are directed against a transmembrane protein of the virus.
• resistant variants of the virus are generated.
• These signal transduction pathways have in common that they include at least one kinase, which activates by phosphorylation at least one protein thereafter transducing the signal.
• Newer data shows that the inhibition of the Ras-Raf-MEK-ERK signal transduction pathway or of another signal transduction pathway, the MEKK/SEK/JNK signal transduction pathway, can drastically inhibit by active ingredients, which relatively selectively inhibit one of the kinases involved in this signal transduction pathway, for instance the MEK or the SEK, the intracellular multiplication of intranuclearly replicating negative-strand viruses, for instance of influenza A viruses and the Borna disease virus (BDV) (Pleschka et al., Nature Cell Biol 3, 301-305, 2001; Planz et al., J Virol 10, 4871-4877, 2001; PCT/DE 01/01292; DE 101 38 912).
• BDV Borna disease virus
• influenza viruses preferably use the Raf-MEK-ERK signal transduction pathway or the MEKK/SEK signal transduction pathway for their multiplication, and that therefore an inhibition of these signal transduction pathways would lead to a complete inhibition of the virus multiplication. Since, however, in a cell the signal transduction pathways have hardly any function closed in itself, but with activation of the one signal transduction pathway, further signal transduction pathways are additionally activated by cross linkages, it can in principle not be excluded that an inhibited signal transduction pathway can be bypassed by the cell as well as by the viruses, and the therapeutic effect of an active ingredient inhibiting a virus multiplication by inhibition of a certain signal transduction pathway could be limited thereby.
• antiviral active ingredients which act in addition or as a supplement to those, which inhibit kinases of cellular signal transduction pathways, for instance of the Raf-MEK-ERK signal transduction pathway or of the MEKK/SEK/JNK signal transduction pathway.
• active ingredients have already been described in the documents PCT/DE 01/01292 and DE 101 38 912.
• NF-kB nuclear factor of kappaB
• the central component of this transduction pathway is the heterodimer NF-kB transcription protein complex consisting on the one hand of p50 [formed by proteolysis of NF-kB1 (p105)] or of p52 [formed by proteolysis of NF-kappaB2 (p100)], and on the other hand of p65 (RELA), c-REL or RELB.
• the most common NF-kB complex is composed of p50 and p65.
• NF-kB NFkB
• IkB inhibitor proteins of NFkB
• NIK NF-kB inducing kinase
• kinases such as the “NF-kB inducing kinase” (NIK), the kinase TAK, the kinase AKT and possibly also of the kinase MEKK1.
• IkB inhibitor of kB
• IKK inhibitor of kB
• IKKbeta kinase
• Activated IKK phosphorylates IkB and thus leads to its degradation and permits thereby the release, nuclear translocation and activation of the transcription activity of the p50/p65 heterodimer.
• Another NF-kB complex is composed of p100 and RELB.
• An activation of the cell by lymphotoxines will lead, probably preferably mediated by NIK, to the activation of IKKalpha.
• the activated IKK induces the phosphorylation and thus the proteolysis of p100 to p52, which in turn can translocate in the complex with the RELB into the cell nucleus and act there as a transcription factor.
• NSAIDs nonsteroidal anti-inflammatory drugs
• sulindac Yamamoto et al., J Biol Chem 274, 27307-27314, 1999; Berman et al., Clin Cancer Res 8, 354-360, 2002
• derivatives of sulindac such as sulindac sulphoxide, sulindac sulphone, sulindac sulphide or benzylamide sulindac analogues
• sulindac sulphoxide sulindac sulphone
• sulindac sulphide or benzylamide sulindac analogues Moon and Lerner, Cancer Research 62, 5711-5719, 2002
• acetylsalicylic acid or salicylic acid Yin et al., Nature 396, 77-80, 1998) or curcumin (Oncogene 18, 6013-6020, 1999) by inhibition of the IKKbeta, ii) NEMO binding peptides (
• acetylsalicylic acid known as an inhibitor of the IKK, is also able to inhibit influenza virus infections in the cell culture, however only beginning from concentrations of 5-10 mM (corresponding to 0.9-1.8 mg/ml). Such concentrations are practically not achievable in blood without substantial side effects after oral administration of acetylsalicylic acid (Huang and Dietsch, New Engl J Med 319, 797, 1988). Acetylsalicylic acid is deemed the most toxic of all at present freely available analgesics with the smallest therapeutic width (Jones, Am J Ther 9, 245-257, 2002).
• the invention is based on the surprising findings that i) active ingredients inhibiting the cellular NF-kB signal transduction pathway are able to inhibit the multiplication of viruses in an organism, ii) smaller concentrations of the active ingredient used according to the invention than derivable from the in vitro data are antivirally effective with local administration, iii) the active ingredient acetylsalicylic acid used according to the invention, administered aerogenically in concentrations of 0.1 to 4 mM, causes a distinct inhibition of the influenza virus multiplication in the lung and in the total organism and a systemic healing effect without impairing the general condition, although such concentrations of the acetylsalicylic acid have not shown in vitro any antiviral effectivity.
• acetylsalicylic acid for instance acetylsalicylic acid in concentrations of 10 mM, were comparatively not stronger, even higher doses (up to 50 mM) were clearly less antivirally effective than the lower concentrations according to the invention.
• concentrations over 20 mM turned out to be toxic after areogenic administration.
• Subject matter of the present invention is thus the use of at least one active ingredient for preparing a pharmaceutical composition for the prophylaxis and/or therapy of at least one viral disease, the active ingredient(s) acting on at least one component of the NF-kB signal transduction pathway, so that a virus multiplication in the organism is inhibited.
• Components of the NF-kB signal transduction pathway are for instance: tumor necrosis factor receptor associated factor (TRAF), NF-kB inducing kinase (NIK), mitogen-activated protein kinase kinase kinase 1 (MEKKK1), mitogen-activated protein kinase kinase kinase 3 (MEKKK3), AKR mouse thymoma kinase (AKT), TGF ⁇ activated kinase (TAK1), inhibitor of NF-kB kinase alpha (IKKaplha), inhibitor of NF-kB kinase beta (IKKbeta), NEMO, inhibitor of kB (IkB), RELA (p65), C-REL, RELB, NF-kB1 (p105), NF-kB2 (p100), P50, P52.
• TGF tumor necrosis factor receptor associated factor
• NIK mitogen-
• inhibitors of a kinase of the NF-kB signal transduction pathway include nonsteroidal anti-inflammatory substances inhibiting the NF-kB activation, such as phenylalkyl acid derivatives, for instance sulindac (Yamamoto et al., J Biol Chem 274, 27307-27314, 1999; Berman et al., Clin Cancer Res 8, 354-360, 2002) or derivatives of sulindac such as sulindac sulphoxide, sulindac sulphone, sulindac sulphide or benzylamide sulindac analogues (Moon and Lerner, Cancer Research 62, 5711-5719, 2002), salicylic acid derivatives such as salicylic acid iself or acetylsalicylic acid (Yin et al., Nature 396, 77-80, 1998), salcylamide, salacetamide, ethenzamide,
• An active ingredient in the meaning of the invention is a substance being capable to directly act on at least one component of the NF-kB signal transduction pathway such that a virus multiplication is substantially inhibited.
• active ingredients in the meaning of this invention are derivatives of these active ingredients, which are transformed for instance by enzymatic fission into an active ingredient according to the invention.
• Active ingredients in the meaning of the present invention are moreover pre-stages of active ingredients, which are metabolically transformed into an active ingredient according to the invention.
• RNA or DNA viruses preferably negative-strand RNA viruses, for instance influenza viruses or Borna viruses.
• the active ingredient according to the invention is administered systemically or locally, for instance dermally, nasally, aerogenically, into a body cavity or a tissue.
• acetylsalicylic acid for the prophylaxis or therapy of an influenza viral disease, wherein acetylsalicylic acid is administered nasally or bronchially (aerogenically) in concentrations preferably from 0.1 to 4 mM.
• the total dose per day for man should preferably be in the range of 0.1 to 30 mg (nasal) or 0.1 to 70 mg (bronchial).
• the lower limit may however also be between 0.1 and 20 mg or 50 mg, respectively.
• the upper limit may also be between 1 mg or 2 mg, respectively, and the mentioned maximum values.
• a daily dose is preferably taken in 1 to 8 administrations, which suitably are distributed over a waking time of 16 hours.
• a treatment takes suitably place over a period of time of 1 to 7 days and longer.
• the invention comprises in so far also galenically prepared administration units, and the amount of active ingredient present in an administration unit can easily be calculated according to the above sections of a treatment plan.
• Another embodiment of the present invention relates to a combination preparation for the prophylaxis and/or therapy of a viral disease, comprising at least two antivirally acting active ingredients, and at least one antivirally acting active ingredient inhibits at least one component of the NF-kB signal transduction pathway such that the multiplication of the virus in an organism is inhibited.
• this antivirally acting active ingredient is selected from the active ingredients according to the invention already mentioned above.
• At least one further active ingredient according to the invention and/or at least one antiviral active ingredient, such as: amantadine (1-adamantanamine) and its derivatives, which is or are directed against a transmembrane protein of some influenza A viruses, such as rimantadine, therapeutic agents for influenza infections inhibiting the influenza-viral surface protein neuraminidase.
• at least one antiviral active ingredient such as: amantadine (1-adamantanamine) and its derivatives, which is or are directed against a transmembrane protein of some influenza A viruses, such as rimantadine, therapeutic agents for influenza infections inhibiting the influenza-viral surface protein neuraminidase.
• the combination preparation may be used in the form of a mixture or as individual components for the simultaneous or not simultaneous application at identical or different places, systemically or locally.
• the above explanations with regard to dosage forms apply in an analogous manner.
• the administration of the combination preparation may take place as a mixture of the active ingredients.
• the active ingredients may however also be administered per administration separately from each other at the same place, for instance systemically by intravenous injection or locally for instance by nasal, aerogenic or dermal administration or by injection into a tissue, or also at different places, simultaneously or not simultaneously within a certain period of time, when the substance administered first is still effective, for instance a period of time of three days.
• a special type of administration of the active ingredient according to the invention is the aerogenic, i.e. nasal or bronchial administration of the active ingredient for the prophylaxis or therapy of those virus infections, which aerogenically infect.
• aerogenic auxiliary means and sprayers of the active ingredient are used, as they are sufficiently known to the man skilled in the art.
• Another embodiment of the present invention relates to a test system for identifying active ingredients, which inhibit at lest one component of the NF-kB signal transduction pathway such that a virus multiplication is inhibited, comprising a. at least one cell infectible by at least one virus, said cell containing the NF-kB signal transduction pathway and at least one virus infecting the cells, or b. at least one cell infected by at least one virus, wherein at least one component of the NF-kB signal transduction pathway is missing or is defectively mutated.
• Cells in the meaning of the present invention are cells from different organs and tissues, for instance cells of the blood or lymph vessels, cells coating the body cavities. Further are comprised cell cultures, in particular those, which can be acquired from cell banks, such as the ATCC, in particular permissive eukaryotic cell cultures, for instance A549, 293, 293T and 293T7 ( homo sapiens ), B82, NIH 3T3, L929 from mus musculus , BHK from cricetus cricetus , CHO from cricetulus griseus , MDCK from canis familiaris , Vero, COS-1 and COS-7 from cercopithecus aethiops , and primary embryo fibroblasts from gallus gallus (CEF cells).
• ATCC cell banks
• permissive eukaryotic cell cultures for instance A549, 293, 293T and 293T7 ( homo sapiens ), B82, NIH 3T3, L929 from mus musculus , BHK from
• test system for identifying active ingredients, it is for instance verified, by addition of substances, preferably in concentrations of 0.001 ⁇ M to 100 ⁇ M, and of viruses in a particle number, which is suitable to identify the selected cell, whether a substance is capable to inhibit the virus multiplication, without damaging the cells.
• the virus used in the test system according to the invention is an RNA or DNA virus, preferably an influenza virus.
• the cell a) of the test system according to the invention comprises at least one overexpressed component of the NF-kB signal transduction pathway, also in the form of constitutively active mutants of these components, in particular by introduction of one gene or several genes coding this component.
• an overexpressed component of the NF-kB signal transduction pathway also in the form of constitutively active mutants of these components, in particular by introduction of one gene or several genes coding this component.
• the expression for at least one component of the NF-kB signal transduction pathway is inhibited, for instance by introduction of an antisense DNA or an antisense RNA or by introduction of at least one gene coding for at least one dominant-negative mutant of at least one component of the NF-kB signal transduction pathway.
• Another embodiment of the present invention relates to a method for identifying at least one active ingredient according to the invention for the prophylaxis and/or therapy of viral diseases, said active ingredient(s) inhibiting the multiplication of viruses in the case of viral diseases, comprising the following steps: a. bringing at least one potential active ingredient into contact with at least one test system according to the invention, and b. determination of the effect on the multiplication of the viruses.
• Bringing into contact in the meaning of this invention may for instance take place by addition of the active ingredients into the nutrient medium of a cell culture or by local or systemic administration of the active ingredients in an organism.
• Bringing into contact in the meaning of this invention further comprises the conventional methods of prior art permitting the introduction of substances into intact cells, for instance infection, transduction, transfection and/or transformation and further methods known to the man skilled in the art. These methods are in particular preferred, if the substances are viruses, naked nucleic acids, for instance antisense DNA and/or antisense RNA, viroids, virosomes and/or liposomes, and virosomes and liposomes are also suitable to introduce further active ingredients into the cell, beside a nucleic acid molecule.
• substances are viruses, naked nucleic acids, for instance antisense DNA and/or antisense RNA, viroids, virosomes and/or liposomes, and virosomes and liposomes are also suitable to introduce further active ingredients into the cell, beside a nucleic acid molecule.
• the determination of the effects on the virus multiplication takes for instance place by plaque assays or by determination of the HA units for comparing the virus titer of treated and not treated infected cells.
• Another preferred embodiment of the present invention relates to a method for preparing a drug for the prophylaxis and/or therapy of at least one viral disease, said drug inhibiting the multiplication of viruses in the case of viral diseases, comprising the following steps: a. executing a test system according to the invention, and b. reacting the identified active ingredient(s) dosed in a physiologically effective dosage with at least one auxiliary and/or additional substance and a defined galenic preparation.
• the active ingredient according to the present invention is prepared for the local or systemic administration into an organism by means of the methods and auxiliary and/or additional substances known to the man skilled in the art to a drug.
• auxiliary and additional substances for instance serving for the stabilization or preservation of the drug or diagnostic agent are well known to the man skilled in the art (see e.g. Sucker H. et al., (1991) Pharmazeutician Technologie, 2 nd edition, Georg Thieme Verlag, Stuttgart).
• auxiliary and/or additional substances are physiological common salt solutions, Ringer's dextrose, dextrose, Ringer's lactate, demineralized water, stabilisators, antioxidants, complex-forming agents, antimicrobial compounds, proteinase inhibitors and/or inert gases.
• the local administration may for instance nasally or aerogenically be made on the skin, on the mucous membrane, into a body cavity, into an organ, into a joint or into the connective tissue.
• the systemic administration takes preferably place into the blood cycle, into the peritoneal cavity or into the abdominal cavity.
• the drug preparation comprising the active ingredient according to the invention depends on the type of active ingredient and the way of administration and may for instance be a solution, a suspension, an ointment, a powder, a spray, or another inhalation preparation.
• nucleotide sequences are inserted by methods well known to the man skilled in the art into a viral vector or a plasmid and reacted with auxiliary substances for the cell transfection.
• auxiliary substances belong for instance cationic polymers or cationic lipids.
• Antisense oligonucleotides are derivatized by methods familiar to the man skilled in the art, in order to protect them from enzymatic degradation by DNAses or RNAses.
• the active ingredient according to the invention may be present in the form of a salt, ester, amide or as a pre-stage, and preferably only such modifications of the active ingredient are used, which do not cause any excessive toxicity, irritations or allergic reactions of the patient.
• the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier substance and potential preservation agents, buffers or driving agents, depending on the application.
• a physiologically acceptable carrier substance and potential preservation agents, buffers or driving agents, depending on the application.
• Such carrier substances for the drug preparations are familiar to the man skilled in the art.
• the active ingredient according to the invention is administered in a one-time dose, in particular preferably in several doses, and the individual doses do not exceed the maximum tolerable dose (MTD) of the respective active ingredient for man.
• a dose is selected, which is half the MTD.
• the daily dose may be administered once a day or in several portions over the day, preferably in approximately identical intervals.
• the administration may take place either locally or systemically, only on one day or daily over several days or at every second or third day over several weeks, until a therapeutic effect is visible.
• the respective cDNAs for IKK KD, mIkB and IKK EE were cloned in sense orientation into the retroviral expression vector pCFG5 IEGZ (Kuss et al., Eur J Immunol 29, 3077-3088, 1999).
• the vector DNA Beside the messenger RNA of the ‘gene of interest’, the vector DNA also codes for the messenger RNA of the “green fluorescent protein” (GFP), which during the protein synthesis is expressed by an internal ribosome binding site. This permits the identification of stably transduced cells in the flow cytometry.
• GFP green fluorescent protein
• the vector mediates a resistance against the antibiotic Zeocin.
• the various expression constructs and the empty vector were transfected (Denk et al., J Biol Chem 276, 28451-28458, 2001) by means of the calcium phosphate precipitation method into the virus-producing cell line ⁇ NX (Grignani et al., Cancer Res 58, 14-19, 1998).
• the transfection efficiency was checked after 24 h by means of the GFP expression and was in the order of 70-80%.
• the cells were then selected for approx. 2 weeks with 1 mg/ml Zeocin in the medium.
• the retrovirus-containing medium supernatants of the virus-producing cell lines were filtrated, reacted with 5 ⁇ g/ml Polybrene (Sigma) and given on fresh cells. The infection took place during two centrifugations (1,000 g) of 3 hours each on two successive days. Stably transduced cells were selected 24 h after infection for another two weeks with 400-600 ⁇ g/ml Zeocin in the medium supernatant.
• the latter as well as wild-type influenza A viruses were infected as described below, and the virus titers of cells, which stably carried the vector, IKK KD, mIkB and IKK EE, were determined in comparison to the titer from the supernatant of wild-type cells.
• the virus titer of influenza A virus-infected cells which were transfected either with the empty vector or constructs expressing IKK
• ASA Acetylsalicylic Acid
• Salicylates such as ASA or sulfasalazines are widely clinically used as pain alleviating and inflammatory agents. Newer publications show that these substances are direct and effective inhibitors of the IKK (Yin et al., Nature 396, 77-80, 1998; Weber et al., Gastroenterology 119, 1209-1218, 2000) and can inhibit in vitro the multiplication of influenza viruses (Huang and Dietsch, New Engl J Med 319, 797, 1988). Thus, as a positive control, ASA was used. Lung epithelial cells A549 were treated with increasing concentrations of ASA in the range from 0.01 mM-5 mM. These concentrations remained in the culture medium during the full experiment.
• MDCK cells were treated one hour before the infection or two and four hours after the infection with 5 mM ASA. Infection and detection of the newly formed viruses was made as above.
• Antioxidants such as pyrrolidine dithiocarbamate (PDTC) are sufficiently known as inhibitors of NF-kB activation (a survey can be found in: Piette et al., Biol Chem 378, 1237-1245, 1997). Therefore, it was examined whether this substance class is also inhibiting for the influenza virus multiplication.
• A549 cells were treated one hour before the infection with PDTC in concentrations of 3-24 micromolar. Infection and detection of the newly formed viruses 24 h pi was made as described for ASA in presence or absence of PDTC during the full experiment.
• C57 Bl/6 mice were nasally infected by 5,000 pfu/20 ⁇ l influenza virus (fowl plague virus, FPV).
• PBS PBS was injected or administered.
• Body weight, death rate and survival time were determined. 30 mice per group were treated.
• C57 Bl/6 mice were nasally infected by 5,000 pfu/20 ⁇ l and 10,000 pfu/20 ml, respectively, influenza virus (fowl plague virus, FPV).
• influenza virus fowl plague virus, FPV.
• IP injection 300 ⁇ l Ketamin/Rompun; Serum Werk Bernburg; Bayer AG Leverkusen
• nebulizer Hugo Sachs Elektronik-Harvard App.
• the results of the in vivo experiments according to examples 3.1 and 3.2 show that the single or multiple (up to 3) daily aerogenic administration of ASA in low non-toxic doses, i.e. doses from 0.1 mg/kg to 300 mg/kg body weight, in particular 10 mg/kg to 100 mg/kg, preferably 20 mg/kg to 50 mg/kg, for instance 30 mg/kg, will lead to a distinct therapeutic effect against a fatal disease induced by nasal administration of the influenza virus.
• the formulation should be selected such that the pharmaceutical composition to be aerogenically administered comprises ASA in concentrations below 2 mM, preferably 0.01 mM to 1.99 mM, most preferably 0.1 mM or 0.5 mM to 1.5 mM, for instance 1 mM.
• the liquid phase amount of aerosol has to be calculated and set up according to the above daily doses under consideration of the employed concentration in the liquid phase. The latter may for instance be obtained with conventional spraying devices, which spray defined amounts of a solution as an aerosol.

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