DE10149393A1 - Detecting the expression of serum and glucocorticoid-dependent kinase-1 (sgk1), for diagnosing coagulative diseases, diabetes, tumors, diabetes and autoimmune diseases, comprises using an antibody against sgk1 - Google Patents

Abstract

Use of at least one compound (A) for detecting the expression and/or function of sgk1 (serum and glucocorticoid-dependent kinase-1) in eukaryotic cells, for diagnosing diseases associated with abnormal activity of sgk1, is new. Independent claims are also included for: (1) use of an active agent (B) for modifying, especially inhibiting or activating, the expression and/or function of sgk-1 in eukaryotic cells, for the treatment of diseases associated with abnormal activity of the tissue factor; (2) a diagnostic kit containing (A); and (3) a pharmaceutical composition containing (B) and optionally a carrier.

Description

The present invention relates to the use of a substance for diagnostic evidence of sgk1 (serum and glucocorticoid dependent kinase 1) and the use of an active ingredient for Influencing sgk1 for the therapeutic treatment of diseases, who have an impaired activity of TF (tissue factor) in the Are related, as well as a related diagnostic kit.

A variety of external signals to which a cell is exposed in its environment lead to intracellular phosphorylation / dephosphorylation cascades to enable rapid and reversible transmission of these signals from the plasma membrane and its receptors into the cytoplasm and the cell nucleus. The regulation of individual proteins that are involved in these cascades enables the high specificity and flexibility of the cells, which allow them to react very quickly to extracellular signals. Kinases, ie proteins which transfer a phosphate group to individual substrates, are particularly involved in these regulatory processes. Serum and glucocorticoid dependent kinase (sgk) was originally cloned from rat breast cancer cells (Webster MK, Goya L, Firestone GL. J. Biol. Chem. 268 (16): 11482-11485, 1993; Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL. Mol. Cell. Biol. 13 (4): 2031-2040, 1993). The human kinase hsgk was cloned from liver cells as a cell volume-regulated gene (Waldegger S. Barth P, Raber G, Lang F. Proc. Natl. Acad. Sci. USA 94: 4440-4445, 1997). It was found that the rat kinase (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D. Proc. Natl. Acad. Sci. USA 96: 2514-2519, 1999; Naray-Fejes-Toth A, Canessa C, Cleaveland Es, Aldrich G, Fejes-Toth GJ Biol. Ghem. 274: 16973-16978, 1999) stimulated the epithelial Na ⁺ channel (ENaC). It has also been shown that increased ENaC activity is associated with hypertension (Warnock DG. Kidney Ind. 53 (1): 1824, 1998).

In DE 197 08 173 it could be shown that the hsgk1 in many Diseases caused by pathophysiological changes in cell volume are influenced, such as hypernatremia, hyponatremia, Diabetes mellitus, renal failure, hypercatabolism, hepatic Encephalopathy and microbial or viral infections has considerable diagnostic potential.

DE 199 17 990 contains kinase inhibitors such as, for example Staurosporine, chelerythrine or transdominant inhibitory kinase have been described which are dependent on cell volume in therapy Diseases can be used.

The hsgk is also expressed in the brain (Waldegger S. Barth P, Raber G, Lang F. Proc. Natl. Acad. Sci. USA 94: 4440-4445, 1997), where it regulates the voltage-dependent K ⁺ channels Kv1.3 , It has been shown that these Kv1.3-type K ⁺ channels are involved in the regulation of neuronal excitability (Pongs O. Physiol. Rev. 72: 69-88, 1992), the regulation of cell proliferation (Cahalan MD and Chandy KG. Cur. Opin. Biotech. 8 (6): 749-756, 1997) and the regulation of apoptiotic cell death (Szabo I, Gulbins E, Apfel H, Zhan X, Barth P, Busch AE, Schlottmann K, Pongs O, Lang FJ Biol. Chem. 271: 20465-20469, 1999; Lang F, Szabo I, Lepple-Wienhues A, Siemen D, Gulbins E. News Physiol. Sci. 14: 194-200, 1999). Kv1.3 is also important in the regulation of lymphocyte proliferation and function (Cahalan MD and Chandy KG, Cur. Opin. Biotech. 8 (6): 749-756, 1997). Two other members of the sgk family were cloned, the sgk2 and sgk3 (Kobayashi T, Deak M, Morrice N, Cohen P. Biochem. J. 344: 189-197, 1999). Furthermore, it was shown that the sgk form a family of serine-threonine protein kinases, which can be regulated transcriptionally and post-transcriptionally. Like the sgk1, the sgk2 and sgk3 are replaced by z. B. Insulin and IGF1 activated via the P13 kinase pathway. So far, however, the sgk protein family has not been fully characterized.

Accordingly, the invention has the task that sgk1 for new diagnostic and therapeutic applications can be used do.

Surprisingly, it was shown that overexpression of intact sgk1 compared to the expression of inactive sgk1 leads to increased coagulatory activity. The coagulation was in this experimental system through the so-called tissue Factor (TF) triggered. The TF is a 47 kDa transmembrane glycoprotein, that as the primary link between vascular cells or serves mononuclear cells and the hemostatic system. Thereby TF initiates the blood coagulation cascade (Davie EW, Fujikawa K, Kisiel W. Biochemistry 30: 10363-10370, 1991). TF initiates blood coagulation Binding to factors VII / VIIa with high affinity. The one out of it resulting complex initiates the activation of factors IX and X with the subsequent thrombin generation. Thrombin in turn catalyzes the conversion of fibrinogen to fibrin, resulting in Fibrin storage and blood clotting leads (Nemerson Y. Blood 71: 1-8, 1998).

An increased expression of TF is not necessarily associated with one increased biological activity associated with TF. Functionally active TF depends on the expression of a biologically active form on the Cell surface. In smooth muscle cells (SMC) and Monocytes are only 10-20% of the total cellular TF at the Cell surface available, which is also the biologically active form, while the remaining TF in intracellular pools (approximately 30%) and as latent Surface TF (50-60%) is available (Preissner KT, Nawroth PP, Kanse SM. J. Pathol. 190: 360-372, 2000; Schecter AD, Giesen PL, Taby O, Rosenfield CL, Rossikhina M, Fyfe BS, Kohtz DS, Fallon JT, Nemerson Y, Taubmann MB. J. Clin. Invest: 100: 2276-2285, 1997). It could are shown to be TF in addition to its coagulant effect (Ruef J, Hu ZY, Yin LY, Wu Y, Hanson SR, Kelly AB, Harker LA, Rao GN, Runge MS, Patterson C. Circ: Res .: 24-33, 1997) an important one Role in tumor metastasis and angiogenesis (Lwaleed BA and Cooper AJ. Medical Hypotheses. 55: 470-473, 2000; Verheul HMW, Jorna AS, Hoekman K, Broxterman HJ, Gebbink MFBG, Pinedo HM. Blood: 4216-4221, 2000). This will show the found functional data that the effects of sgk1 are appropriate, the To influence expression and / or function of TF on the cell membrane and thus indirectly the coagulability of the blood, the attachment of Tumor cells with the following metastasis, angiogenesis, as well Diseases in which angiogenesis plays a role influence. By stimulating the sgk1 there is an increased Expression of the tissue factor, by inhibition of sgk1 to a decreased expression of active tissue factor, and thus can indirectly stimulating or inhibiting those described above Indications are taken into account.

Accordingly, the object of the invention is through the subject of independent claims 1, 2, 3, 17 and 20 solved. preferred Designs are in the dependent claims 4 to 16, 18, 19, and Called 21 to 26. The content of all these claims is hereby acknowledged Reference made to the content of the description.

According to the invention, at least one substance for the detection of Expression and / or the function of sgk1 in eukaryotic cells be used. This is also a diagnosis of Diseases related to impaired TF activity stand, possible. This substance could e.g. B. one Act antibodies directed against sgk1 and in one Detection methods known to those skilled in the art, such as, for. B. ELISA (enzyme-linked immunosorbent assay) can be used. In such Immunoassays are directed against the antigen to be determined (sgk1) specific antibodies (or homologous for antibody determinations Test antigens) to a carrier substance (e.g. cellulose, polystyrene) bound to the immune complexes after incubation with the sample form. In a subsequent step, these immune complexes become one labeled antibody supplied. By adding a chromogenic Substrates for the reaction batch can be bound to the immunocomplex Enzyme-substrate complexes made visible Antigen concentration in the sample via a photometric determination of the Immune complex-bound marker enzymes by comparison with standards known enzyme activity can be determined. Other substances for the diagnostic evidence can be used so-called oligonucleotides, which with the help of the so-called polymerase Chain reaction (PCR) are suitable via a molecular genetic Method in which certain sections of DNA are selectively amplified to provide quantitative evidence of sgk1. Further Methods of quantitatively detecting a known target protein can are familiar to the person skilled in the art.

According to the invention, an active ingredient for influencing, in particular the inhibition or activation of the expression and / or function of sgk1 in eukaryotic cells, for the treatment of diseases that associated with impaired TF activity, claimed. Since sgk1, like sgk2 and sgk3, is a kinase, come in particular kinase inhibitors known to the person skilled in the art, such as, for. B. Staurosporine, chelerythrine etc., but also other substances such as B. transdominant negative kinase mutants, in question. The specialist are such substances are known and you can get them from commercial (Sigma, Calbiochem, etc.) as well as non-commercial sources. As activators z. B. genetically modified mutants of sgk1, but also z. B. inhibitors of phosphatases can be used. Phosphatase inhibitors are also known to the person skilled in the art, and they are also partly commercial (Sigma, Calbiochem, etc.) as well not commercially available. When using phosphatase inhibitors would inhibit dephosphorylation, and thereby that of sgk1 activated target (TF) remain in the activated state. To be favoured these active ingredients for the manufacture of a medicament or a pharmaceutical composition used.

In a further preferred embodiment of the invention, the Active ingredient directed against sgk1 itself. It can be the active ingredients z. B. antisense sequences, so-called kinase deficient mutants, but also about kinase inhibitors, such as those mentioned above Staurosporine and / or chelerythrine or their analogs act. Furthermore, the active ingredient can also be a so-called "small molecular compound "or a polynucleotide which codes for a peptide which the expression of sgk1 is influenced, preferably inhibited or activated.

In another preferred embodiment of the invention, the Active ingredient against activators, inhibitors, regulators and / or biological precursors directed by sgk1. These activators, inhibitors, Regulators and / or biological precursors could be up and / or downstream members of the sgk1 signal transduction cascade, Transcription factors relevant for the expression level of sgk1 are responsible for proteases that are responsible for the proteolytic degradation of Activators, inhibitors, regulators and / or biological precursors of sgk1, but also so far unknown molecules, caused by the active ingredient be influenced and on the expression and / or function of sgk1 involved.

According to the invention, it is possible to use known and still unknown To use active ingredients. In a particularly preferred The embodiment is the active ingredient which is active against activators, inhibitors, Regulators and / or biological precursors of sgk1 is directed So-called "small molecular compound", especially one with a Molecular weight (MW) <1,000. Small molecular compounds can e.g. B. Kinase inhibitors, such as B. the imidazole derivatives SB 203580 (MG 377.4, or also SB 202190 (MG 331.3), both of which are known Kinase expression inhibitors are commercially available from Calbiochem to be expelled.

The invention can be used to treat all forms of diseases, associated with impaired TF activity to treat. In particular, here is coagulopathy and / or To think of congenital or acquired angiopathies. Under Coagulopathies are generally understood to be coagulation disorders. Congenital coagulopathies (so-called defect coagulopathies) are e.g. B. Dysfibrinogenemia, hypoproconvertinemia, hemophilia B, Stuart Prower defect etc. Acquired coagulation disorders are e.g. B. Prothrombin complex deficiency, consumption coagulopathy, hyperfibrinolysis, Immune coagulopathy and complex coagulopathies. Both forms of coagulopathies are caused by lack of or Dysfunction of various plasma coagulation factors. According to the different symptoms, coagulopathies become with bleeding tendency (minus coagulopathies) and coagulopathies with Tendency to thrombosis (plus agulopathies) and according to the location of the Cause hepatogenic, cardiogenic and immunocoagulopathies distinguished. Thus, by activating or inhibiting sgk1 reduce or increase the willingness of the blood to clot, and thus adapt to the medical indication. Similar considerations also apply to angiopathies, d. H. Diseases that are among the General term for vascular diseases can be summarized, such as. B. diabetic angiopathy, diabetic microangiopathy etc. the active ingredient in particular can be used congenital and / or treat acquired angiopathies.

In a further preferred embodiment, the active ingredient becomes Stimulation or inhibition of angiogenesis used. Under Angiogenesis is the development of vessel walls, e.g. B. during the Embryonic development understood, and a number of Angiogenesis-dependent diseases are known to the person skilled in the art. B. Diabetes Tumor formation and autoimmune diseases. In another preferred embodiment is the active ingredient for stimulation or Inhibition of wound healing used.

The invention also relates to a diagnostic kit. This includes at least a substance used to demonstrate expression and / or function of sgk1 is suitable for the diagnosis of diseases with a related activity of TF related. Can continue with such a kit, diseases can be diagnosed with a Over or under expression or function of sgk1 are connected. Such diagnostics can be used in a targeted manner in a diagnostic kit to treat diseases such as the ones above described coagulopathies, angiopathies, angiogenesis-dependent Diseases, diseases of wound healing, etc. to be proven. Also Here, the detection of the diseases can be demonstrated by the detection of a impaired expression and / or function of sgk1. In particular this substance can be one that is based on Nucleotide and / or peptide level or polynucleotide and / polypeptide this Provides proof. The other characteristics of such a substance will refer to the corresponding previous text of the description taken.

The invention further comprises a pharmaceutical composition, which contains at least one active ingredient which expression and / or Function of sgk1 influenced, in particular inhibited or activated, and preferably a pharmaceutical carrier if necessary. It can be the active ingredient is a kinase inhibitor, like the one already mentioned above mentioned inhibitors staurosporine, chelerythrine, SB 203580 and SB 202190 or their analogues, but also other substances. Furthermore, the active substance can be a polynucleotide which encodes a peptide, preferably a polypeptide, this Peptide influences the expression of sgk1, preferably inhibits or activated. An example of a polypeptide according to the invention is a so-called kinase deficient mutant. More examples like that Expression and / or function via genetically modified variants of the Target protein can be influenced are known to the person skilled in the art, and can be found in a number of textbooks / specialist books as well as instructions for the Laboratory work (e.g. Maniatis T, Fritsch EF, Sambrook J. Cold Spring Harbor, NY: Cold Spring Harbor Laborator, 1996; Leonard G, Davis PhD, Michael W, Kuehl Md, James F, Battey MD. McGraw-Hill Professional Publishing, 1995). In the invention Active ingredient can also be a so-called "small molecular compound ", preferably a" small molecular compound " with a molecular weight (MW) <1,000. Furthermore, it can Active ingredient is also a so-called antisense sequence, d. H. a sequence that is able to bind to the mRNA Form double strand duplex, and thereby the translation of a target polypeptide prevent. The sequence of sgk1 itself can also be used to to achieve overexpression, e.g. B. by incorporation in vectors or Plasmids, the target sequence also previously being carried out by "carrier" Molecules, e.g. B. promoters can be modified. To further Characteristics of such a composition is based on the corresponding previous text of the description referred.

Finally, the invention includes a pharmaceutical Composition which has an effective amount of at least one active ingredient, the expression and / or function of activators, inhibitors, Regulators and / or biological precursors influenced by sgk1, in particular inhibited or activated. Preferably, this can pharmaceutical composition, possibly also a pharmaceutical composition Carrier included. These activators, inhibitors, regulators and / or biological precursors of sgk1 can e.g. B. be further kinases that are involved in regulating the activity of sgk1, Transcription factors that play a role in the expression level by sgk1, as well as other known or as yet unknown members the sgk1 in a transduction cascade, as well as the one above described molecules. Polynucleotides that encode a peptide, which is the expression of activators, inhibitors, regulators and / or biological precursors influenced by sgk1, preferably inhibited or activated in such a composition be included. Also so-called "small molecular compounds", the preferably have a molecular weight (MW) <1,000, and the against Activators, inhibitors, regulators and / or biological precursors are directed by sgk1, and thereby the expression or the function inhibit or activate this kinase can be used. To further Characteristics of such an active ingredient is based on the corresponding previous text of the description reference taken.

The existing features and other features of the invention emerge from the following description of preferred Embodiments in connection with the subclaims and the figures. The individual features can be used individually or separately several can be realized in combination with each other.

The pictures show:

Fig. 1 stimulation of the procoagulatory activity of smooth vascular muscle cells.

experiment

The procoagulatory activity is plotted in% of the maximum value against the time after recalcification. In the experiment of FIG. 1, the procoagulatory activity of human smooth vascular muscle cells (HAOSMC) was measured by measuring the thrombin formation during the coagulation process in recalcified low-platelet plasma (PPP) (Beguin S. Lidhout T, Hemker HC: Throm. Haemost. 61 : 25-29, 1998). For this purpose, confluent smooth vascular muscle cells were kept in serum-free medium for 24 hours, then washed three times in HEPES-Tyrode solution, and then incubated with human PPP. The formation of thrombin was triggered by adding 16.7 mM CaCl ₂ to the incubation medium. In each case 20 μl of the supernatant were removed every one to two minutes and the thrombin formation was determined therein with the aid of the dye S-2238 (Haemochrom Diagnostica). The optical density was determined in a spectrophotometer (Uvikon, Controninstruments) at 405 nm. The dependence of the surface procoagulatory activity of smooth muscle cells on the availability of the membrane-bound tissue factor was demonstrated by neutralizing antibodies against the human tissue factor (Mab # 4508; American Diagnostica; 10 µg / ml 20 minutes before the recalcification of the PPP).

As shown in FIG. 1, the procoagulatory activity of human smooth vascular muscle cells increases a few minutes after the addition of CaCl ₂ . This increase is slower when the inactive kinase (sgk-MT) is expressed than when the normal kinase (sgk-WT) is expressed. The additional administration of thrombin (Thr) is expected to accelerate the increase in procoagulant activity. Here, too, the effect is stronger and faster in cells which express the intact kinase than in cells which express an inactive mutant. Cells which express intact (wild-type) sgk kinase (sgk-WT) have a higher procoagulatory activity at all times than cells which express an inactive sgk mutant (sgk-MT), regardless of whether thrombin is added (sgk-WT / Thr or Sgk-MTIThr) was or not.

This result clearly shows that overexpression of intact sgk1 in smooth vascular muscle cells to an increased leads to coagulatory activity. Due to the well-known important role of the TF in various cell processes, this result also shows that a Overactivity of sgk1 with an increased expression of the tissue factor promote the coagulability of the blood on the cell membrane, the attachment of tumor cells with the following metastasis as well as the Can increase angiogenesis. This mechanism would be reversed by suppressing sgk1 expression or by pharmacological inhibition of skgl can be suppressed.

Claims (26)

1. Use at least one substance for the detection of Expression and / or function of sgk1 in eukaryotic cells Diagnosis of diseases with an impaired activity of TF (Tissue Factor) related. 2. Use of at least one active ingredient for influencing, in particular for inhibiting or activating expression and / or function of sgk1 in eukaryotic cells, for Treatment of disorders associated with impaired TF activity Related. 3. Use of at least one active ingredient for influencing, in particular for inhibiting or activating expression and / or function of sgk1 in eukaryotic cells, for Manufacture of a drug or pharmaceutical Composition for the treatment of diseases with a related activity of TF related. 4. Use according to claim 2 or 3, characterized in that the active ingredient is directed against SGK1. 5. Use according to one of claims 2 to 4, characterized characterized in that the active ingredient against activators, inhibitors, Regulators and / or biological precursors of sgk1 is directed. 6. Use according to any one of claims 2 to 5, characterized characterized in that the active ingredient is a kinase inhibitor, preferably Staurosporine and / or chelerythrine or one of their analogues, is. 7. Use according to one of claims 2 to 6, characterized characterized in that the active ingredient is a polynucleotide, which is a Peptide, preferably a polypeptide, which peptide encodes the Expression and / or function of sgk1 influenced, preferably inhibited or activated. 8. Use according to one of claims 2 to 7, characterized characterized in that the active ingredient is a "small molecular compound", preferably a "small molecular compound" with a Molecular weight (MW) <1,000. 9. Use according to one of the preceding claims, characterized in that the diseases that especially associated with impaired TF activity stand for coagulopathies. 10. Use according to claim 9, characterized in that it coagulopathies are congenital coagulopathies is. 11. Use according to claim 9, characterized in that it coagulopathies are acquired coagulopathies is. 12. Use according to one of claims 1 to 8, characterized characterized in that the diseases, in particular with related activity of TF related to Angiopathy. 13. Use according to claim 12, characterized in that it angiopathies are congenital angiopathies. 14. Use according to claim 12, characterized in that it the angiopathies are acquired angiopathies. 15. Use according to one of claims 1 to 8, characterized characterized in that the active ingredient for stimulating or inhibiting the Angiogenesis is used. 16. Use according to one of claims 1 to 8, characterized characterized in that the active ingredient for stimulating or inhibiting the Wound healing is used. 17. Diagnostic kit comprising at least one substance for detection of expression and / or function of sgk1, for the diagnosis of Diseases associated with impaired activity of TF in Related. 18. Diagnostic kit according to claim 17 for the diagnosis of diseases, linked to an over- and / or under-function of sgk1 are. 19. Diagnostic kit according to claim 17 or 18 for the diagnosis of Coagulopathies, angiopathies and / or angiogenesis-dependent Diseases. 20. A pharmaceutical composition comprising an effective one Amount of at least one active ingredient, the expression and / or Function of sgk1 influenced, in particular inhibited or activated, and possibly a pharmaceutical carrier. 21. Pharmaceutical composition according to claim 20, characterized characterized in that the active ingredient is a kinase Inhibitor, preferably around staurosporine and / or chelerythrine or one of their analogues. 22. Pharmaceutical composition according to claim 20, characterized characterized in that the active ingredient is a polynucleotide which encodes a peptide, preferably a polypeptide, this Peptide influences the expression and / or function of sgk1, preferably inhibited or activated. 23. Pharmaceutical composition according to claim 20, characterized characterized in that the active ingredient is a "small molecular compound ", preferably a" small molecular compound "with a Molecular weight (MW) <1,000. 24. The pharmaceutical composition according to claim 20, comprising an effective amount of at least one active ingredient which Expression and / or function of activators, inhibitors, Regulators and / or biological precursors influenced by sgk1, in particular inhibited or activated, and possibly one pharmaceutical carrier. 25. Pharmaceutical composition according to claim 24, characterized characterized in that the active ingredient is a polynucleotide which encodes a peptide, preferably a polypeptide, this Peptide the expression and / or function of activators, Inhibitors, regulators and / or biological precursors of sgk1 influenced, preferably inhibited or activated. 26. Pharmaceutical composition according to claim 24, characterized characterized in that the active ingredient is a "small molecular compound ", preferably a" small molecular compound "with a Molecular weight (MW) <1,000.