DE102004028899B4 - Use of a combination for the preventive and / or therapeutic treatment of bacterial infectious diseases or sepsis - Google Patents

Abstract

Using a combination of
a. at least one inhibitor of Rho kinase or of RhoA and
b. at least one inhibitor of glycogen synthase kinase-3β (GSK-3β) and
c. Interferon-γ (IFN-γ)
for the preventive and / or therapeutic treatment of bacterial infectious diseases or sepsis.

Description

The invention relates to the use of inhibitors or activators of certain signaling pathways for increasing the synthesis of the secretory phospholipase A ₂ -IIA (sPLA ₂ -IIA) and / or for increasing the secretion of sPLA ₂ -IA in the bloodstream. Field of the invention is medicine, especially the therapy of bacterial infectious diseases.

State of the art

The human secretory phospholipase A ₂ -IAA (sPLA ₂ -IIA) can be detected in a variety of different cells and tissues, such as platelets ¹ , leukocytes, in the prostate ³ and placenta ⁴ , and also in body fluids such as semen ^5,6 tear fluids ⁷ and synovial fluid ^8,9 , this enzyme occurs. In other cells such as hepatocytes, endothelial cells and smooth muscle cells, expression of the enzyme could ^only be found in vitro after stimulation with interleukin-1β, interleukin-6, tumor necrosis factor-α, and interferon-γ ^10,11,12 . The enzyme activity in serum healthy is rather low. However, it may dramatically increase during acute inflammatory conditions such as sepsis and septic shock ^13,14,15 , acute pancreatitis ^16,17 , peritonitis ¹⁷ or appendicitis ¹⁸ . Thus, 100-1000-fold increased values were described in these diseases compared to the norm. Chronic rheumatoid arthritis ^19,20 and neoplastic diseases ^{21 also} show elevated serum levels of sPLA ₂ -IA. Due to the rapid increase during a local or systemic inflammatory response, sPLA ₂ -IAA is expected to be the acute phase reactant ²² .

Therefore, inhibitors of sPLA ₂ -IIA have been used with great intensity in recent years in order to block the action of sPLA ₂ -IA in severe inflammatory reactions. However, the biological function of sPLA ₂ -IIA is still insufficiently elucidated. In addition to an involvement of the enzyme in processes of cellular signal transduction, apoptosis and / or "remodeling" of cell membranes also an involvement of the enzyme in the body's defense against bacteria and human-pathogenic fungi is discussed.

Singer animals can find a variety of different peptides and polypeptides capable of killing staphylococci and other Gram-positive bacteria. These include Type IIA secretory PLA ₂ , which is an extremely effective antibacterial agent. At the end of the seventies could isolate Elsbach and staff ²³ from rabbit leukocytes a PLA _2, the effective in vitro together with a second protein, the bactericidal / permeability-increasing protein (BPIP), gram-negative strains of Escherichia coli and Salmonella typhimurium kill. Later, an antibacterial effect on Staphylococcus aureus was found in inflammatory exudates, also due to PLA ₂ ^24,25 . Antibacterial properties against gram-positive bacteria were also demonstrated in the following years for the human sPLA ₂ -IIA and the high concentrations of the enzyme in inflammatory exudates ²⁶ and in the tear fluid ^{27 also} speak for the antibacterial function of the enzyme. In addition, the enzyme is released by intestinal ^{28 paneth} cells and macrophages ^29,30 ; both cell types involved in the body's defense against bacterial invaders. It is believed that the ability of sPLA ₂ -IA to attack Staphylococcus aureus and other Gram-positive bacteria is mainly due to the ability of the enzyme to bind to and penetrate the bacterial cell wall and thereby hydrolytically attack the cell wall phospholipids ^31:32 . The initial binding of sPLA ₂ -IIA to the cell surface of S. aureus is based on electrostatic interactions between the sPLA ₂ -IIA and the cell surface. Among the more than 100 structurally similar low molecular weight sPLA ₂ (14 kDa), sPLA ₂ -IA is unique in its extremely high net positive charge, which is between +12 and +17. This high net basicity is considered to be the basis for the potent antibacterial activity of sPLA ₂ -IA against gram-positive bacteria ³³ , ³⁴ , ³⁵ .

Finally, the protective effect of sPLA ₂ -IA against Gram-positive and Gram-negative bacteria could also be demonstrated in vivo by studies on transgenic mice characterized by overexpression of human sPLA ₂ -IIA. Thus, the transgenic mice had compared to the control animals a significantly higher resistance to Staphylococcus areaus on ^36th While the majority of transgenic animals showed only minor symptoms of sepsis after intraperitoneal injection of S. aureus and remained fully alive, 85% of non-transgenic control animals died within 24 hours. The deceased animals showed severe pulmonary, hepatic and renal congestion and showed increased hemorrhagic effusions in the pleural and peritoneal cavities. The same group was able to demonstrate a higher resistance of the sPLA ₂ -IA transgenic animals to the gram-negative E. coli bacteria compared to the control animals ³⁷ .

Every year, 750000 people in the United States contract sepsis, of whom 210000 die from it. In recent years, the understanding of sepsis has changed. So far, sepsis has been viewed exclusively as an out-of-control inflammatory response due to infection, and based thereon an anti-inflammatory prevention and therapy has been established, e.g. B. by application of antibodies to different cytokines or their receptors, so new research shows that not only a damaging, but also a protective effect can be seen in the elevated cytokine levels. Studies in experimental animals with peritonitis showed that the blocking of TNF-α reduced the survival rate of the animals ^38,39 . Combination therapy against TNF-α and IL1 receptor was also fatal for a neuropanic model of sepsis ⁴⁰ . Increased mortality was also detectable in clinical trials in which TNF antagonists were given ⁴¹ . The importance of cytokines, especially those of TNF-α, has also been shown in patients with rheumatoid arthritis. Patients treated with TNF antagonists have increased signs of sepsis or infectious complications ⁴² . What patients ultimately die of with sepsis is still unclear. However, a lack of acute-phase reactants in patients with sepsis is associated with high mortality. While blockade of proinflammatory cytokines did not lead to any major success in the treatment of sepsis patients, but rather worsened, substitution of recombinant activated protein C (APC), an anticoagulant, resulted in more efficient treatment ^43,44 , For example, the relative risk of death for patients with sepsis fell by 19.4%, the absolute risk by 6.1% ⁴³ . APC inactivates the coagulation factors Va and VIIIa and thus prevents the formation of thrombin ⁴⁴ . The fact that the efficiency of the APC is not solely due to the inhibition of the coagulation system was demonstrated by studies with two other anticoagulants, antithrombin III (ATIII) and inhibitors of tissue factor (TF). In contrast to APC, the administration of both anticoagulants did not lead to an improved cure rate of septic patients ⁴⁵ . One explanation for this could be that these inhibitors act at different sites of the coagulation system compared to the APC.

In addition, anti-apoptotic properties could be demonstrated for the APC ⁴⁶ , which was not the case for the other two inhibitors. Apoptosis is considered to be a major cause of sepsis-inducing immune paralysis, which is often associated with a decreased number of lymphocytes and gastrointestinal epithelial cells. Similar to APC, sPLA ₂ -IIA also possesses anti-apoptotic properties as demonstrated in hamster kidney fibroblasts and mast cells ^47,48 . In addition, sPLA ₂ -IIA is also believed to have anti-coagulative properties in that the enzyme competes with coagulation factor Va for binding to factor Xa, thereby inhibiting the formation of thrombin similar to APC ^49-56 . In addition to these properties and the antibacterial effects already mentioned, sPLA ₂ -IIA therefore appears to be a suitable pharmacological target for improving the prevention and therapy of systemic infectious diseases, including sepsis, as well as local and systemic, arterial and venous thrombosis.

Also acute bacterial meningitis is a life-threatening infection, at the annual in Germany About 2,000 people fall ill. Their course is opposite one viral meningitis usually more severe. The main pathogens are Neisseria meningitidis (meningococci), Streptococcus pneumoniae (Pneumococci), Listeria monocytogenes, Borrelia burgdorferi, staphylococci, Streptococci (including hemolytic streptococci group B) and Escherichia coli.

task

The object of the invention is to provide agents for increasing the synthesis of the secretory phospholipase A ₂ -IIA (sPLA ₂ -IIA) and their increased secretion into the bloodstream. By activating the body's own defense, the treatment success of bacterial infectious diseases should be improved. In particular, the treatment success of sepsis should be significantly increased with the invention.

• a. mindestens einem Inhibitor der Rho-Kinase oder von RhoA und
• b. mindestens einem Inhibitor der GlykogensynthaseKinase-3β (GSK-3β) und
• c. Interferon-γ (IFN-γ)

zur präventiven und/oder therapeutischen Behandlung von bakteriell bedingten Infektionserkrankungen oder der Sepsis.According to the invention the object is achieved according to claim 1 by the use of a combination of

• a. at least one inhibitor of Rho kinase or of RhoA and
• b. at least one inhibitor of glycogen synthase kinase-3β (GSK-3β) and
• c. Interferon-γ (IFN-γ)

for the preventive and / or therapeutic treatment of bacterial infectious diseases or sepsis.

advantageous Embodiments of the invention are in the claims 2 to 9 indicated.

Surprisingly, it has been found that the expression of sPLA ₂ -IAA is negatively regulated by at least three signaling pathways, namely by the Rho / Rho-kinase signaling pathway, the Ras / Raf / MEK / ERK signaling pathway and the Ras / PI3 kinase / Akt signaling pathway. If one or more of these negatively regulating signaling pathways (Rho / Rho kinase pathway, Ras / Raf / MEK / ERK pathway, Ras / PI3 kinase / Akt signaling pathway) is inhibited, sPLA ₂ -IIA expression increases and secretion.

The expression and secretion of sPLA ₂ -IAA is also enhanced by inhibiting signaling pathways that activate the listed negative regulatory pathways, or by inhibiting signaling pathways that are activated by these negative regulatory signaling pathways.

Activation of signaling pathways that inhibit the negatively regulatory signaling pathways also increases sPLA ₂ -IAA expression and secretion.

A combination of members of different inhibitor classes or activators leads to a synergistic increase in sPLA ₂ -IAA expression and secretion.

Under bacterial infectious diseases are diseases too understand by gram-positive or gram-negative bacteria caused, for example, acute, subacute and chronic, by pathogens such as Chlamydia, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus-induced bronchitis, acute bacterial meningitis, Acute bacterial abscessing pyelonephritis, acute by Staphylococcus aureus caused skeletal muscle infections (pyomyositides), chronic bacterial infections of the stomach and intestines, endocarditis, Leprosy, tuberculosis and brucellosis. Another chronic-infectious, through Chlamydia-related disease may also present with atherosclerosis so that, where appropriate, the developed drug is applicable is.

In a preferred embodiment The invention will be additional Acetylcholine and / or lysophosphatidic acid (LPA) used.

Interferon-γ is increasingly produced by the body's own T-lymphocytes and released into the bloodstream as soon as the immune system is activated by the invasion of bacterial pathogens. All of the inhibitors or inhibitor combinations tested synergistically induced the synthesis of sPLA ₂ -IIA with IFN-γ, therefore the IFN-γ blood level is preferably controlled in patients with severe infectious diseases and, if necessary, in the absence of detection of the cytokine, for example in patients with immune paralysis in addition to the above-listed drug combinations recombinant IFN-γ given to trigger an optimal, synergistic effect on the body's sPLA ₂ -IIA synthesis and secretion into the bloodstream.

Lysophosphatidic acid (LPA), together with IFN-γ, also leads to a significant up-regulation of sPLA ₂ -IIA expression. The same applies to acetylcholine. The effect of both agents can be further increased in combination with cAMP-increasing agents. LPA acts by activating protein kinase C, which in turn inhibits GSK-3β ⁶⁰ . Similarly, acetylcholine acts by increasing intracellular calcium and activating protein kinase C.

Prefers will therefore be additional to the cited Active ingredient combinations acetylcholine and / or LPA used.

The Rho / Rho-kinase signaling pathway is understood to mean the path of signal transmission from RhoA to Rho-kinase ( 1 ). Rho / Rho kinase signaling pathway inhibitors are all substances that attenuate or inhibit signal transduction through this pathway.

In an embodiment signaling is interrupted by the inhibition of RhoA, in a particularly preferred embodiment, toxin B is knocked out Clostridium difficile used as RhoA inhibitor. Toxin B inhibits specific Rho proteins by glycosylation of threonine residues.

In a further embodiment Rho kinase is inhibited, in a particularly preferred embodiment the Rho kinase inhibitors Y-27632 and H-1152 are used.

Under the Ras / Raf / MEK / ERK signaling pathway, the route of signal transduction from the Ras protein via the Raf protein and via the mitogen activated protein kinase MEK to the extracellular signal-regulated kinase ERK understood ( 1 ). Inhibitors of the Ras / Raf / MEK / ERK signaling pathway are all substances that attenuate or prevent signal transduction via this signaling pathway.

In a preferred embodiment will be added the MEK inhibits, in a particularly preferred form the Inhibitors PD-98059 and U0126 used.

In a further preferred embodiment Raf kinase is inhibited, in a most preferred embodiment the Raf inhibitor ZM336372 is used.

The Ras / PI3 kinase / Akt signaling pathway is understood to mean the pathway of signal transmission starting from the Ras protein via the protein kinase and via protein kinase 13 (Akt) ( 1 ). Inhibitors of the Ras / PI3 kinase / Akt signaling pathway are all substances that attenuate or prevent signal transduction via this signaling pathway.

In a preferred embodiment will be added which inhibits PI3 kinase, in a particularly preferred form the inhibitor Wortmannin used.

Signaling pathways activating the Rho / Rho kinase and / or the Ras / Raf / MEK / ERK signaling pathway and / or the Ras / PI3 kinase / Akt signaling pathway are understood to be pathways upstream of the signal pathways cited, and the signaling pathways Activate the Rho / Rho-kinase RhoA protein and / or the Ras protein of the Ras / Raf / MEK / ERK signaling pathway and the Ras / PI3 kinase / Akt signaling pathway ( 1 ).

Also these include Metabolic pathways whose products are posttranslational modification Ras and / or Rho find use, for example, the post-translational Isoprenylation, through which cell migration, proliferation and the differentiation are controlled. By the farnesyltransferase this is a farnesyl isoprenoid (C15) to a thiol group of a cysteine at the C-terminus of the Ras protein. The corresponding posttranslational modification of the Rho protein with a geranylgeranyl (C20) isoprenoid carried by geranylgeranyl transferase. The used isoprenoids come from the cholesterol metabolism, which is essentially regulated by the HMG-CoA reductase.

inhibitors These signaling and metabolic pathways are all substances that the Signal forwarding over this Attenuate signal paths or block or block these metabolic pathways, for example Inhibitors of HMG-CoA reductase, inhibitors of farnesyltransferase and inhibitors of geranylgeranyltransferase.

In a preferred embodiment be additional Inhibitors of HMG-CoA reductase In a particularly preferred embodiment, the HMG-CoA inhibitors mevastatin, Lovastatin, simvastatin, atorvastatin, fluvastatin or pravastatin used.

Becomes Simvastatin is used to manufacture a drug the preferred dose at 5 to 80 mg a day, is simvastatin in Combination with other inhibitors or activators for the production of a Medicinal product, the preferred dose is a particularly preferred dose is below 5 mg a day.

Becomes Lovastatin is used for the manufacture of a drug the preferred dose at 10 to 80 mg a day, is lovastatin in Combination with other inhibitors or activators for the preparation of a medicinal product, the preferred dose is a particularly preferred dose is below 10 mg a day.

Becomes Atorvastatin used for the manufacture of a drug, so lies The preferred dose at 10 to 80 mg a day is atorvastatin in combination with other inhibitors or activators for the production of a Medicinal product, the preferred dose is a particularly preferred dose is below 10 mg a day.

Becomes Fluvastatin is used for the manufacture of a drug the preferred dose at 20 to 80 mg a day, is fluvastatin in Combination with other inhibitors or

activators used for the preparation of a drug, so is the preferred Dose below, a particularly preferred dose is below 20 mg a day.

Becomes Pravastatin used for the manufacture of a drug, so lies the preferred dose at 10 to 40 mg a day, is pravastatin in Combination with other inhibitors or activators for the production of a Medicinal product, the preferred dose is a particularly preferred dose is below 10 mg a day.

In a further preferred embodiment be additional Inhibitors of farnesyltransferase used in one particular preferred embodiment are the farnesyltransferase inhibitors manumycin A, Cys-Val-2-Nal-Met, FTI-249, FTI-277, FTI-2153, FTI RPR130401, FTI SCH66336, FTI R115777, FTI L-778123 or FTI BMS-214662 used.

Becomes FTI SCH66336 used for the manufacture of a drug, so lies the preferred dose at 400 to 800 mg a day, FTI is SCH66336 in combination with other inhibitors or activators for the production of a medicinal product, the preferred dose is a particularly preferred dose is below 400 mg a day.

Becomes FTI R115777 used for the manufacture of a drug, so lies the preferred dose at 400 to 800 mg a day, is FTI R115777 in combination with other inhibitors or activators for the production of a Medicinal product, the preferred dose is a particularly preferred dose is below 400 mg a day.

In another preferred embodiment be additional Inhibitors of geranylgeranyltransferase used in one particular preferred embodiment are the geranylgeranyltransferase inhibitors GGTI-279, GGTI-286, GGTI-287, GGTI-297, GGTI-298, GGTI-2133 or GGTI-2147.

Under Signaling pathways involving the Rho / Rho-kinase pathway and / or the Ras / Raf / MEK / ERK signaling pathway and / or inhibit the Ras / PI3 kinase / Akt signaling pathway, signaling pathways are understood the signal passing over the Rho / Rho kinase pathway and / or the Ras / Raf / MEK / ERK signaling pathway and / or attenuate or prevent the Ras / PI3 kinase / Akt signaling pathway.

For example, protein kinase A inhibits RhoA to Rho / Rho kinase signal transduction signaling by phosphorylating serine 188 of RhoA. This results in the translocation of the membrane-associated RhoA into the cytosol independent of the isoprenylation state of RhoA ⁵⁷ . Dong et al. ⁵⁸ showed that the reduced biological activity of phosporylated RhoA is caused by the weakened binding of RhoA to its downstream Rho-associated serine / threonine kinase.

Protein kinase A also decreases the availability of Rafl for the Ras protein and thus also inhibits the Ras / Raf / MEK / ERK signaling pathway ^59. In a preferred embodiment, the expression of sPLA ₂ -IAA is enhanced by the activation of protein kinase A. for which the cAMP-enhancing agents forskolin, theophylline, ATP, 8-bromo-cAMP, prostacyclin or β-adrenoceptor agonists are used.

The Protein kinase A is in its inactive form as a tetramer, consisting of two regulatory and two catalytic subunits, where the catalytic center is through the regulatory subunits is covered. By binding cAMP to specific binding sites on the regulatory subunits dissociate the regulatory Subunits and the protein kinase A is in its active form in front. The concentration of cAMP in the cell is dependent on Extent of Synthesis by adenylate cyclase and degradation by phosphodiesterases.

forskolin increases cAMP concentration by activating adenylate cyclase, Theophylline by the inhibition of phosphodiesterase.

The Activation of G-protein coupled receptors leads via the Activation of G proteins as well to activate the adenylate cyclase and thereby increase the cAMP Concentration: ATP activates G protein-coupled purinoceptors P2X and P2Y, prostacyclin and carbacyclin activate the G protein-coupled Prostacyclin receptors, β-adrenoreceptor Agonists, such as isoproterenol, activate the G-protein coupled β-adrenoreceptors.

When forskolin is used in the manufacture of a medicament, the preferred dose is 50 to 100 mg per day, forskolin is used in combination with other inhibitors or activators to produce a medicinal product Used, the preferred dose is below it, a particularly preferred dose is below 50 mg a day.

Becomes Theophylline used for the manufacture of a drug is so The preferred oral dose at 200 to 1200 mg a day becomes theophylline in combination with other inhibitors or activators for the production of a medicinal product, the preferred dose is a particularly preferred dose is less than 200 mg a day.

Signal paths which are activated by the Rho / Rho kinase and / or the Ras / Raf / MEK / ERK signaling pathway and / or the Ras / PI3 kinase / Akt signaling pathway are understood to be pathways downstream of these signaling pathways, for example the GSK-3 signaling pathway that is activated by the Rho / Rho kinase Rho kinase and the Ras / Raf / MEK / ERK pathway ERK ( 1 ).

inhibitors These signaling pathways are all substances that transmit signals via this Attenuate signal paths or inhibit, for example, inhibitors of GSK-3β. In a preferred embodiment Indirubin is used as an inhibitor of GSK-3β. Will be indirubin used for the preparation of a drug, so is the preferred Dose at 20 to 100 mg a day, Indirubin is combined with other inhibitors or activators for the production of a drug used, so the preferred dose is below, a special preferred dose is less than 50 mg a day.

By the combination of different substances, the different signaling pathways blocking in different places, synergistic results Effects that apply much lower concentrations of the Allow substances compared to the individual substances. By the lower concentration of the substances used is also to count with less side effect.

Based the following figures and embodiments the invention will be closer explains:

Figure (fig.) 1 shows an overview of the intracellular signaling pathways, which are influenced by the described inhibitors or activators and lead to an upregulation of sPLA ₂ -IIA expression. The cross-lined lines represent the inhibition by pharmacologically active substances or show a negative regulation. The solid lines indicate known metabolic pathways, the broken line represents a mechanism discussed in the literature. The lines marked with a question mark characterize mechanisms postulated by us, which represent further possibilities for the regulation of sPLA ₂ -IIA.

table 1 shows an overview the various inhibitor or activator substances, their chemical Designation (CAS number), the signaling pathways influenced by it, their function and their sources of supply.

EMBODIMENT ₁ Upregulation of sPLA ₂ -IAA Expression in HASMC and HepG2 Hepatom Cells by Atorvastatin and Mevastatin

cell cultivations

Primary human aortic smooth muscle cells (HASMC) are purchased from Promocell (Heidelberg, Germany) and given the prescribed conditions in Smooth Muscle Cell Growth Medium and 5% fetal calf serum, 0.5 ng / ml human Epidermal Growth Factor, 2 ng / ml basic human fibroblast growth factor, 5 μg / ml bovine insulin, 50 μg / ml gentamycin sulfate and 50 ng / ml amphotericin B cultured at 37 ° C and 5% CO ₂ . The human HepG2 hepatoma cell line was obtained from the American Type Culture Collection (Rockville, Maryland, USA) and RPMI medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin cultured under identical conditions as described for the HASMC. Every second to third day a medium change takes place and before reaching confluence the HASMC and HepG2 cells are passaged with the help of trypsin / EDTA. For RNA isolation and determination of the cell-secreted sPLA ₂ -IIA, the cells are introduced into 6-well plates or 24-well plates at a cell density of 10 ⁴ cells / cm ² . After two days, the medium of HASMC is replaced with medium containing 0.5% fetal calf serum to synchronize the growth of the tent. After a further 48 hours, the medium is again replaced by Smooth Muscle Cell Growth Medium with 5% fetal calf serum. All experiments are performed on exponentially growing subconfluent HASMC and confluent passage 5-8 HepG2 cells. After completion of the incubation, the conditioned cell medium is removed, centrifuged at 800 g for 10 min to remove detached cells and until assayed the sPLA ₂ -IIA is preserved at -80 ° C. The adherent cells are washed twice with ice-cold PBS, lysed in TRI reagent (Sigma-Aldrich, Deisenhofen, Germany) and preserved at -80 ° C until RNA isolation.

Atorvastatin, mevaloxin, mevalonic acid, squaene, cholesterol and forskolin were dissolved in ethanol, manumycin A, GGTI-298, GGTI-286, PD98059, U0126, AG-490 and CAPE in dimethylsulfoxide (DMSO). The final concentration of solvents in each study was less than 0.3%. Ethanol and DMSO were included as controls in all cases. After dissolving mevastatin and D, L-mevalonic acid lactone in ethanol, these compounds were converted to active form by opening the lactone ring by incubating them in 0.1 N NaOH at 37 ° C for 45 min ⁶³ and, after hydrolysis by addition of 0.1 N HCl, the pH of the solutions were readjusted to a value of 7.4.

RNA extraction and reverse transcriptase (RT) PCR analysis

After lysis of the cells in TRI reagent, the RNA is isolated according to the description of the company. Subsequently, using the GeneAmp RNA PCR Kit (Perkin-Elmer), the rewording of the isolated RNA into the cDNA and the amplification in the PCR for the identification of sPLA ₂ -IIA mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) - mRNA. The latter serves as a "house-keeping gene". The following oligonucleotides serve as primer pairs: 5'-GTGATCATGATCTTGGCCTACTGCA-3 '(SEQ_NO.1) and 5'-TCTCCCTCGTGGGGAGCAACGACT-3' (SEQ_NO.2) for sPLA ₂ -IAA (411 bp PCR product) and 5'-CGGAGTCAACGGATTTGGTCGTATTG-3 '(SEQ_3) and 5'-GCAGGAGGCATTGC-TGATGATCTTG-3' (SEQ_4) for the GAPDH (product length of 439 bp). For the sequence of the oligonucleotides used, the published nucleotide sequence of the human placental sPLA ₂ -IIA cDNA ⁶¹ and for the GAPDH mRNA according to the Ercolani et al. ⁶² published nucleotide sequence as a basis. The primer pairs are used in the PCR in a final concentration of 0.8 .mu.mol / l. The amplification conditions are as follows: 40 cycles at 94 ° C for 30 sec, 68 ° C for 30 sec, and 72 ° C for 1 min. The buffer solutions and reagents used are contained in the GeneAmp Kit (Perkin-Elmer). After amplification, the products obtained are analyzed by agarose gel electrophoresis. The determination of GAPDH mRNA served as a control in each approach.

Quantitative determination of sPLA ₂ -IAA and GAPDH mRNA using real-time RT-PCR

The sequences of the primers and molecular beacons for real-time RT-PCR are based on the RNA sequence of the sPLA ₂ -IIA gene ⁶¹ and the GAPDH gene ⁶² . For the analysis of the sPLA ₂ -IIA, the primers used are the oligonucleotides 5'-AATTTGGTGAATTTCCACAG-3 'and 5'-AGTCATGAGTGACACAGCAG-3' and for the GAPDH the oligonucleotides 5'-CAAGCTCATTTCCTGGTATG-3 'and 5'-TGAGGGTCTCTCTCTTCCTC-3' , The beacons are labeled at the 5 'end with 6-carboxyfluorescein (6-FAM) and at the 3' end with the quencher 4-dimethylaminophenylazobenzoic acid (dabcyl). The sequence of the beacons is 5'-CGCTCGCACTCAGTTATGGCTTCTACGGCTGCCACGCGAGCG-3 'for the sPLA ₂ -IAA and 5'-CGCTCGCACATGGCCTCCAAGGAGTAAGACCCCTGGCGAGCG-3' for the GAPDH. The reaction is carried out as a one-step RT-PCR in an iCycler iQ PCR Detection System (Bio-Rad, Germany) using the QuantiTect ^™ kit (QIAGEN) in triplicate. The following reagent batches are used: 15 μl 2 × QuantiTect Probe RT-PCR as master mix, 4 μl each of 4 μmol / l primer pairs, 2.5 μl 4 μmol / l beacon, 0.3 μl QuantiTect Probe RT Mix, 1.2 μl of 25 mM MgCl ₂ solution, 2 μl of template RNA and 1 μl of RNase-free water. Reverse transcription is at 60 ° C for 30 min, followed by a denaturation phase at 95 ° C for 15 min. This is followed by PCR with 10 cycles at 95 ° C for 15 sec, 55 ° C for 15 sec and 72 ° C for 20 sec and up to 70 cycles at 95 ° C for 15 sec, 45 ° C for 30 sec and 72 ° C for 20 sec. The "real-time" measurement takes place during the annealing phase. Batches without an RNA template serve as a negative control. The data obtained in the PCR are then evaluated using the iCycler iQ software. The relative quantification of sPLA ₂ -IAA mRNA is carried out by ΔΔCt method with GAPDH as a reference housekeeping gene according to the "User Bulletin # 2 - Relative Quantitation of Gene Expression - ABI PRISM 7700 Sequence Detection System."

Quantification of sPLA ₂ -IAA by ELISA

Using a specific enzyme-linked immunosorbent assay (ELISA), the cell-secreted sPLA ₂ -IAA according to the instructions of Cayman Chemical, MI, USA) is determined with a few minor modifications. These include the use of additional standard samples at concentrations of 3.9 pg / ml and 7.8 pg / ml and prolonged incubation with the Ellman's Reagent to increase the sensitivity of the assay. The cellular protein content as a reference is determined by bicinchoninic acid assay (Sigma-Aldrich) in which bovine serum albumin acts as an internal standard.

Western blot analysis

Aliquots of conditioned cell culture media are preincubated with 0.2 vol of Laemmli buffer [25% glycerol, 2% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol in 60 mM Tris / HCl, pH 6.8] at 95 ° C for 5 min and then the proteins electrophoretically separated in 7.5% SDS-polyacrylamide gels. This is followed by protein transfer to nitrocellulose membranes by semidry electroblotting [transfer buffer: 48 mM Tris, 39 mM glycine, 0.040% SDS, 20% (v / v) methanol]. Following protein transfer, the nitrocellulose membrane is blocked with NET buffer (150mM NaCl, 50mM Tris / HCl, pH 7.4, 5mM EDTA, 0.05% Triton X-100, 5% low-fat dry mild powder) and specific polyclonal rabbit -Anti-sPLA ₂ -IAA antibodies (diluted 1: 1000 in NET buffer, Cayman Chemical (MI, USA)) overnight at 4 ° C. After washing the membrane with NET buffer, incubation with peroxidase-labeled anti-rabbit IgG (diluted 1: 3000 in NET buffer) for 1 h at room temperature and visualization of sPLA ₂ -IAA by ECL system (Amersham Pharmacia Biotech, Freiburg, Germany).

Statistical evaluations

The Results represent means ± standard deviation of 3 to 6-subject examinations and represent at least 3 independently conducted Experiments. The data obtained are statistically analyzed by unpaired Student's t test evaluated.

2 shows the effects of atorvastatin (Av) on cellular sPLA ₂ -IAA secretion on HASMC after 48 hours incubation with proinflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ, black bars) in one concentration of 25 ng / ml or after incubation with proinflammatory cytokines (25 ng / ml) and simultaneous addition of atorvastatin in a concentration of 6.5 μmol / l (gray bars).

The addition of cytokines or atorvastatin takes place here at time zero, the measurement of cellular sPLA ₂ -IAA secretion via ELISA.

The Results represent the mean ± standard deviation of quadruplicate determinations and are representative for three independent from each other conducted Experiments. p <0.05 compared to the untreated control cells; ** p <0.05 in comparison to the cells after addition of the appropriate cytokines, but without Addition of atorvastatin.

There is a strong synergistic effect of atorvastatin with IFN-γ on sPLA ₂ -IAA expression.

In 3 (A to D) is the concentration and time-dependent up-regulation of sPLA ₂ -IIA expression in HASMC by atorvastin. After incubation of the HASMC (culturing conditions see above) with atorvastatin in different concentrations (0 to 20 μmol / l) for 48 h ( 3A C) and after incubation of the cells with atorvastatin for different time intervals (12 to 72 hours, D) in the presence or absence of IFN-γ (25 ng / ml), both mRNA (by RNA extraction and reverse transcriptase (RT ) -PCR analysis and real-time RT-PCR) as well as at the protein level (by ELISA and Western blot analysis) determines the expression of sPLA ₂ -IAA.

In 3A and B (top) are white bars showing the results of the real-time RT-PCR analysis (see above) as the relative ratio between sPLA ₂ -IIA mRNA and GAPDH mRNA compared to the untreated cells where the ratio of sPLA ₂ -IIA mRNA and GAPDH mRNA was set equal to 1.0.

In 3A and B (below) demonstrates the detection of the ethidium bromide stained amplicons of RT-PCR analysis (see above) in agarose gel electrophoresis (M refers to the 100 bp molecular weight standard).

3C shows the sPLA ₂ -IIA protein release as a function of increasing atorvastatin concentrations together with IFN-γ.

In 3D depicts the time-dependence of sPLA ₂ -IIA mRNA transcription after sole and joint incubation of atorvastatin and IFN-γ.

The results in 3A and B give the mean ± standard deviation of sixfold determinations and in 3C of quadruplicate determinations and represent the results of three independently performed experiments (* p <0.05 compared to the untreated control cells; ** p <0.05 compared to the cells after addition of IFN-γ but without the addition of atorvastatin).

Like atorvastatin, upregulation of sPLA ₂ -IAA expression in HASMC is also found by mevastatin (Table 2).

Table 2 shows the results of determination of sPLA ₂ -IAA expression at mRNA and protein level after incubation of HASMC with mevastatin and IFN-γ.

After 48 hours of incubation with sole and combined addition of mevastatin and IFN-γ, the sPLA ₂ -IAA mRNA and the sPLA ₂ -IA released in the medium were determined (procedure as described in Example 1). The results represent the mean ± standard deviation of quadruplicate determinations and are representative of three independent experiments.

* Ratio of sPLA ₂ -IAA / GAPDH mRNA to untreated control cells equated to 1.0. ^† p <0.005 with respect to the control cells; ^‡ p <0.001 compared to the control cells; ^§ p <0.0005 compared to the control cells; ^∥ p <0.0005 versus the cells incubated with IFN-γ; ^¶ p <0.001 versus control cells; ^# p <0.005 versus the cells incubated with IFN-γ.

If human HepG2 hepatoma cells are incubated with atorvastatin (5 .mu.mol / l) alone and in combination with IFN-y (25 ng / ml), upregulation of sPLA ₂ -IIA expression can also be detected on the HepG2 cells, similar to HASMC , Synergistic effects of atorvastatin and IFN-γ can also be demonstrated with the aid of ELISA technique, Western blotting and real-time RT-PCR ( 4 , A-D).

4 shows the up-regulation of sPLA ₂ -IAA expression after incubation of the human HepG2 cells alone or combined with atorvastatin and IFN-γ for 48 h.

4A shows the results of real-time PCR analysis: sPLA ₂ -IAA mRNA relative to GAPDH mRNA versus atorvastatin alone and in combination with IFN-γ.

In 4B (supra) a western blot of the cells secreted sPLA ₂ -IAA is shown using polyclonal anti-sPLA ₂ -IIA antiserum, with 30 μg protein each being applied.

4B (bottom) shows the quantification of the released sPLA ₂ -IAA by ELISA.

The Results each represent the mean ± standard deviation of triplicate determinations represent and represent the results of three independent performed by each other Experiments (* p <0.05 im Compared to the untreated control cells).

Similar Results are also obtained with mevastatin on HepG2 cells (data are not shown).

embodiment 2:

Effect of mevalonic acid and isoprenoids on statin-mediated upregulation of sPLA ₂ -IAA expression

As 5 shows that the addition of mevalonic acid in a concentration of 100 .mu.mol / l leads to a reduction of the statin-induced secretion of sPLA ₂ -IA in HASMC. If the cells are incubated simultaneously with atorvastatin (5 μmol / l) and IFN-γ (25 ng / ml), the addition of mevalonic acid leads to a reduction of the sPLA ₂ -IAA release to a value that after sole incubation with IFN -γ results.

When HASMC is co-incubated with farnesyl pyrophosphate (FPP) at 10 μmol / L, geranylgeranyl pyrophosphate (GGPP) at 10 μmol / L, squalene at 25 μmol / L and cholesterol at 50 μmol / L , a reduced release of sPLA ₂ -IIA by FPP or GGPP can be detected, but not by squalene or cholesterol.

In 5 these results are summarized. The cellular sPLA ₂ -IIA secretion is set to 1.0 after simultaneous incubation with atorvastatin and IFN-γ without the addition of metabolites. The results each represent the mean ± standard deviation of triplicate determinations and represent the results of three experiments performed independently. The cell culturing and determination of sPLA ₂ -IAA release is carried out as described in Example 1 (* p <0.05 compared to the cells incubated with atorvastatin; ** p <0.05 compared to the cells that were co-administered incubated with atorvastatin and IFN-γ).

embodiment 3:

Up-regulation of sPLA ₂ -IAA expression by inhibitors of farnesyltransferase and geranylgeranyltransferase-I

Cell culture (see Example 1) in the presence of manumycin A, a farnesyl transferase inhibitor, shows a significant and concentration-dependent increase in sPLA ₂ -IAA secretion after 24 hours of incubation (see FIG. 6 ). When IFN-γ (25 ng / ml) is added to the medium simultaneously with manumycin A, sPLA ₂ -IAA expression increases synergistically.

In addition to the induction of sPLA ₂ -IIA by manumycin A, an increased secretion of sPLA ₂ -IA can be detected even after incubation with the GGTase-I inhibitor GTI-298 ( 6 ). GTI-298 also acts synergistically with IFN-γ (25 ng / ml). Similar results are obtained with the FTase inhibitor Cys-Val-2-Nal-Met and the GGTase-I inhibitor GGTI-286 in HASMC as well as in HepG2 cells (data not shown here).

In 6 For example, the effects of the farnesyltransferase inhibitor manumycin A and geranylgeranyl transferase inhibitor GGTI-298 both alone (black bars) and in combination with IFN-γ (25 ng / ml, gray bars) on cellular sPLA ₂ -IAA secretion in HASMC shown after 24 hours incubation.

The Results of the ELISA analysis (procedure as in exemplary embodiment 1) each represent the mean ± standard deviation of triplicate determinations represent and represent the results of three independent performed by each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ were incubated).

embodiment 4:

Up-regulation of sPLA ₂ -IAA expression by toxin B, Y-27632, H-1152, cAMP-increasing agents, PD98059 and U0126

If HASMC are incubated with toxin B (Clostridium difficile toxin B), Y-27632 or H-1152 (cell cultivation as described in Example 1) alone and in combination with IFN-γ, a continuous, and in the case of Y-27632 and H-1152, significant increase in sPLA ₂ -IIA secretion into the cell medium (ELISA analysis as in Example 1) detectable ( 7 ).

At the same time, synergistic effects on sPLA ₂ -IAA secretion occur when these agents are combined with IFN-γ.

Similar Results can also be obtained after incubation of the cells with cAMP-increasing agents like 8-bromo-cAMP and forskolin.

In 7 these effects are summarized and show cellular sPLA ₂ -IAA secretion in HASMC after 24 h incubation with Clostridium difficile toxin B (toxin B), Y-27632, H-1152, 8-bromo-cAMP (Br-cAMP) and forskolin without (black bars) and with (gray bars) simultaneous addition of IFN-γ (25 ng / ml).

The Results of the ELISA analysis each represent the mean ± standard deviation of triplets and represent the results of three independent performed by each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

If the inhibition of MEK as a component of the Ras / Raf / MEK / ERK signaling pathway using the Inhi In the presence of PD98059 and U0126, a significant increase in sPLA ₂ -IIA secretion after single incubation of the cells with PD98059 at a concentration of 25-50 μmol / l ( 8th ). Synergistic effects can also be found in HASMC with PD98059 or U0126 and IFN-γ. 8th Figure 10 shows the effects of MEK inhibitors, PD98059 and U0126, in off-center (black bars) and presence (gray bars) of IFN-γ (25 ng / ml) on sPLA ₂ -IAA release in HASMC after 24 hours of incubation. The results each represent the mean ± standard deviation of triplicate determinations and represent the results of three experiments performed independently.

Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1 (* p <0.05 compared to the control cells, ** p <0.05 compared to the cells with the same concentration of inhibitors, but without IFN -γ were incubated).

embodiment 5

Up-regulation of sPLA ₂ -IIA expression by wortmannin as PI3-kinase inhibitor

In 9 the effect of wortmannin at a concentration of 0.5 μmol / L on sPLA ₂ -IAA expression on HASMC after 24 hours incubation alone and in combination with IFN-γ (25 ng / ml) is shown (white bars). Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

embodiment 6

Effects of lysophosphatidic acid (LPA) alone and in combination with IFN-γ and Br-cAMP on sPLA ₂ -IAA expression in HASMC

10 shows the up-regulation of sPLA ₂ -IAA expression (white bars) after 48 hours incubation of HASMC with lysophosphatidic acid (LPA, 30 μmol / L) alone and in combination with IFN-γ (25 ng / ml) and bromine cAMP (0.5 mmol / l). Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

embodiment 7

Effects of acetylcholine alone and in combination with IFN-γ and Br-cAMP on sPLA ₂ -IAA expression in HASMC

11 shows cellular sPLA ₂ -II secretion (white bars) after 48 hours incubation of HASMC with acetylcholine (10 μmol / L) alone and in combination with IFN-γ (25 ng / ml) and bromine cAMP (0.5 mmol / l). Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

embodiment 8th

Cellular sPLA ₂ -IAA expression in HASMC following co-incubation with MEK inhibitors, statins, and IFN-γ

In 12 is the upregulation of sPLA ₂ -IAA secretion (white bars) in HASMC by combining PD98059 (MEK inhibitor) at a concentration of 50 μmol / l and IFN-γ (25 ng / ml) with mevastatin (30 μmol / l ) after 48 hours of incubation. Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

embodiment 9:

Combinatorial effect of Rho inhibitor, GSK-3β inhibitor and IFN-γ on sPLA ₂ -IAA secretion in HASMC

13 shows the induction of sPLA ₂ -IAA secretion in HASMC (white bars) by incubation with Clostridium difficile toxin B (TcdB) at a concentration of 10 ng / ml and indirubin (50 nmol / l) alone and in combination with IFN-γ (25 ng / mol) for 48 h. Cell cultivation and determination of sPLA ₂ -IAA release are carried out as described in Example 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

embodiment 10:

Block toxin B, PD98059 and statin-induced sPLA ₂ -IIA secretion by inhibiting the Jak / STAT and NFκB signaling pathway in HASMC

14 Figure 2 shows the blocking of the Clostridium difficile toxin B (TcdB) at a concentration of 10 ng / ml, PD98059 (50 μmol / L) and mevastatin (5 μmol / L) mediated induction of sPLA ₂ -IAA release in HASMC by the Jak2 Inhibitor AG-490 (50 μmol / L) and the NFκB inhibitor CAPE (25 μmol / L) alone and in combination with IFN-γ (25 ng / ml) after a 48-hour incubation. Cell culture and determination of sPLA ₂ -IAA release are performed as in Embodiment 1.

The Results represent the mean ± standard deviation of triplicate determinations and are representative for three independently from each other Experiments. (* p <0.05 in comparison to the control cells; ** p <0.05 compared to the cells that with the same concentration of inhibitors, but without IFN-γ incubated were).

AG-490 and CAPE completely block the IFN-γ-dependent up-regulation of sPLA ₂ -IIA. Similar effects can be observed with PDTC and PSI as further NFκB inhibitors (data not shown here). In addition, AG-490 inhibits the effect of toxin B and PD98059 on sPLA ₂ -IAA secretion. The same is true for CAPE. Finally, the synergistic effect of mevastatin and IFN-γ can also be completely inhibited by AG-490 and CAPE.

List of abbreviations

μM:

μMol/l

6-FAM:

6-Carboxyflourescein

AG-490:

Tyrphostin B42

APC:

aktiviertes Protein C

ATP:

Adenosin-triphosphat

cAMP:

zyklisches Adenosin-monophosphat

Bp:

Basenpaare

BPIP:

bactericidal/permeability-increasing protein

Br-cAMP:

6-bromo-cAMP

cAMP:

cyclisches Adenosin-3',5'-monophosphat

CAPE:

Caffeic acid phenethyl ester

CAS:

chemical abstract service

Dabcyl:

4-Dimethylaminophenylazo-Benzoesäure

DNA:

Desoxyribonukleinsäure

E. coli:

Escheichia coli

EDTA:

Ethylendiamintetraessigsäure

ELISA:

enzyme linked immunosorbent assay

ERK:

extracellular signal-regulated kinase

FCS:

fetal calf serum

Fig.:

Figur

FPP:

Farnesylpyrophosphat

FTase:

Farnesyltransferase

FTI:

Farnesyltransferase Inhibitor

GAPDH:

Glycerinaldehyd-3-phosphatdehydrogenase

GGPP:

Geranylgeranylpyrophosphat

GGTase:

Geranylgeranyl Transferase

GGTI:

Geranylgeranyl Transferase-I Inhibitor

GSK-3β:

Glykogensynthasekinase-3β

HASMC:

human aortic smooth muscle cells

HMG-CoA:

3-Hydroxy-3-Methylglutaryl-Coenzym A

IFN-γ:

Interferon-γ

IgG:

Immunglobulin G

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

Jak:

Janus Tyrosinkinase

kDa:

Kilodalton

MEK:

mitogen-activated protein kinase Kinase

mM:

mMol/l

mRNA:

messenger RNA

NFκB:

nuclear factor-κB

p:

probability

PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction, Polymerase-Kettenreaktion

PDTC:

Pyrrolidin Dithiocarbamat

PGI2:

Prostacyclin

PI3-Kinase:

Phosphoinositid-3-Kinase

PKA:

Protein Kinase A

PP:

Pyrophosphat

PSI:

Proteasom Inhibitor

RNA:

Ribonukleinsäure

RT:

Reverse Transkriptase

s.:

siehe

S.

aureus: Staphylococcus aureus

S.

typhimurium: Salmonella typhimurium

SDS:

sodium dodecyl sulfate

sPLA₂-IIA:

secretory phospholipase A₂ of type IIA

STAT:

signal transducer and activator of transcription

TcdB:

Clostridium difficile toxin B

TF:

tissue factor

TNF-α:

Tumor Necrosis Factor-α

Toxin B:

Clostridium difficile Toxin B

z. B.:

zum Beispiel

In the description of the invention the following abbreviations are used:

uM:

.mu.mol / l

6-FAM:

6-carboxyfluorescein

AG-490:

Tyrphostin B42

APC:

activated protein C

ATP:

Adenosine triphosphate

cAMP:

cyclic adenosine monophosphate

bp:

base pairs

BPIP:

bactericidal / permeability-increasing protein

Br-cAMP:

6-bromo-cAMP

cAMP:

cyclic adenosine 3 ', 5'-monophosphate

CAPE:

Caffeic acid phenethyl ester

CAS:

chemical abstract service

Dabcyl:

4-dimethylaminophenylazo-benzoic acid

DNA:

deoxyribonucleic acid

E. coli:

Escheichia coli

EDTA:

ethylenediaminetetraacetic

ELISA:

enzyme linked immunosorbent assay

ERK:

extracellular signal-regulated kinase

FCS:

fetal calf serum

Fig .:

figure

FPP:

farnesyl pyrophosphate

FTase:

farnesyl transferase

FTI:

Farnesyltransferase inhibitor

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GGPP:

geranylgeranyl

GGTase:

Geranylgeranyl transferase

GGTI:

Geranylgeranyl transferase-I inhibitor

GSK-3β:

Glycogen synthase kinase-3β

HASMC:

human aortic smooth muscle cells

HMG-CoA:

3-hydroxy-3-methylglutaryl coenzyme A

IFN-γ:

Interferon-γ

IgG:

Immunoglobulin G

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

Jak:

Janus tyrosine kinase

kDa:

kilodalton

MEK:

mitogen-activated protein kinase kinase

mM:

mmol / l

mRNA:

messenger RNA

NFkB:

nuclear factor-κB

p:

probability

PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction, polymerase chain reaction

PDTC:

Pyrrolidine dithiocarbamate

PGI2:

prostacyclin

PI3 kinase:

Phosphoinositide 3-kinase

PCA:

Protein Kinase A

PP:

pyrophosphate

PSI:

Proteasome inhibitor

RNA:

ribonucleic acid

RT:

Reverse transcriptase

s .:

please refer

S.

aureus: Staphylococcus aureus

S.

typhimurium: Salmonella typhimurium

SDS:

sodium dodecyl sulfate

sPLA ₂ -IAA:

secretory phospholipase A ₂ of type IIA

STAT:

signal transducer and activator of transcription

TcdB:

Clostridium difficile toxin B

TF:

tissue factor

TNF-α:

Tumor Necrosis Factor-α

Toxin B:

Clostridium difficile toxin B

z. B .:

for example

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Claims (9)

Using a combination of a. at least an inhibitor of Rho kinase or RhoA and b. at least an inhibitor of glycogen synthase kinase-3β (GSK-3β) and c. Interferon-γ (IFN-γ) to preventive and / or therapeutic treatment of bacterial infectious diseases or sepsis. Use of a combination according to claim 1, characterized characterized in that the inhibitor of glycogen synthase kinase 3β 5-iodo-indirubin-3'-monoxime (indirubin) is. Use of a combination according to claim 1, characterized characterized in that the inhibitor of Rho kinase is (R) - (+) - trans -N- (4-pyridyl) -4- (1-aminoethyl) cyclohexanecarboxamide (Y-27632) or (S) - (+) - 2-methyl-1 - [(4-methyl-5-isoquinolinyl) sulfonyl] homopiperazine (H1152) or that the inhibitor of RhoA is toxin B (TcdB). Use of a combination according to any one of claims 1 to 3, characterized in that the combination additionally at least an inhibitor of mitogen-activated protein kinase kinase (MEK) and / or at least one inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). Use of a combination according to claim 4, characterized characterized in that the inhibitor of MEK is 2'-amino-3'-methoxyflavone (PD98059) or 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene (U0126) is. Use of a combination according to claim 4, characterized characterized in that the inhibitor of HMG-CoA reductase mevastatin, Lovastatin, simvastatin, atorvastatin, fluvastatin or pravastatin is. Use of a combination according to any one of claims 1 to 6, characterized in that the combination additionally at least an activator of protein kinase A (PKA) selected from forskolin (7β-acetoxy-8,13-epoxy-6β-hydroxy-labd-14-en-11-one), Theophylline, ATP or 8-bromo-cAMP contains. Use of a combination according to any one of claims 1 to 7, characterized in that the combination additionally acetylcholine and / or lysophosphatidic acid Contains (LPA). Use of a combination according to any one of claims 1 to 8, characterized in that the combination additionally at least an inhibitor of farnesyltransferase and / or at least one Inhibitor of geranylgeranyltransferase and / or at least one Inhibitor of PI3 kinase (phosphatidylinositol 3-kinase).