WO2007121963A1 - Methods for interfering with disease related to impaired mast cell activation - Google Patents

Abstract

A method for interfering with disorders that depend on mast cell activation comprising, contacting mast cells expressing serum and glucocorticoid inducible kinase (SGK) with a substance that modulate glucocorticoid inducible kinase and thereby modulate the PI3 kinase dependent ion channel regulation and function. Furthermore the invention relates to methods for the diagnosis and for the identification of compounds that are useful for the detection or therapy of inflammatory disease.

Description

Methods for interfering with disease related to impaired mast cell activation

Background of the invention

Mast cells express a large number of IGE receptors (FcεRI) and largely account for the IgE dependent allergic reactions [Kawakami and GaIIi 2002; Kawakami and Kitaura 2005], such as allergic rhinitis [Pawankar 2005], asthma [Bradding 2003], anaphylactic and delayed hypersensitivity [Askenase et al., 1983; Conti et al., 1992; GaIIi et al., 2005; Van Loveren et al., 1983]. They are considered to critically participate in the pathophysiology of diverse inflammatory diseases including psoriasis [Cao et al., 2005; Jamieson et al., 2005; Kanda and Watanabe 2003; Kawaguchi et al., 2005; Shepherd et al., 2004; Theoharides et al., 2004], atopic dermatitis [Theoharides et al., 2004], rheumatoid arthritis [Jonsson et al., 2005; Juurikivi et al., 2005], Crohn^'s disease [Gebhardt et al., 2005], irritable bowel syndrome [Gebhardt et al., 2005] and male infertility [Agarwal et al., 1987; Dupont et al., 2000; Frungieri et al., 2002; Nagai et al., 1992; Yamanaka et al., 2000]. Mast cells release a variety of cytokines and thus regulate the function of other inflammatory cells such as neutrophils and T cells [Asada et al., 1997; Biedermann et al., 2000; GaIIi et al., 2005; GaIIi and Nakae 2003; Hill et al., 1996; Nakae et al., 2005; Nakae et al., 2006]. Mast cell function is under tight regulation. Activation of the mast cells involves the stimulation of the PI3 kinase pathway [Andrade et al., 2004; Gilfillan and Tkaczyk 2006; Wymann et al., 2003] and is suppressed by glucocorticoids [Andrade et al., 2004; Collado-Escobar et al., 1990; Fushimi et al., 1998; Krishnaswamy et al., 1997; Matsuda et al., 2005; Mazingue et al., 1978; Robin et al., 1985; Wershil et al., 1995]. Activation of mast cells further involves activation of Ca²⁺ channels [Bradding et al., 2003; Buess et al., 1999; Dernick et al., 2003; Duffy et al., 2001a; Duffy et al., 2001b; Kahr et al., 2004; Mazurek et al., 1980; Stokes et al., 2004], K⁺ channels [Bradding et al., 2003; Bradding 2005; Duffy et al., 2005; Mark et al., 2004] and Cl^" channels [Duffy et al., 2001a; Duffy et al., 2001b].

Signalling molecules downstream of PI3kinase include the serum and glucocorticoid inducible kinase SGK1 [Lang and Cohen 2001], which is expressed in all tissues tested [Waldegger et al., 1997]. SGK1 has been shown to regulate a wide variety of ion channels [Lang et al., 2003]. The kinase contributes to the regulation of salt appetite [Vallon et al., 2005], renal electrolyte excretion [Huang et al., 2004; Wulff et al., 2002] and blood pressure [Huang et al., 2006]. It participates in the regulation of intestinal transport [Grahammer et al., 2006] and peripheral glucose uptake [Palmada et al., 2006]. Several observations point to its pivotal role in fibrosing disease [Feng et al., 2005; Lang et al., 2000; Vallon et al., 2006; Waldegger et al., 1999]. Even though SGK1 is a glucocorticoid sensitive gene [Firestone et al., 2003], nothing is known about its putative role in inflammatory disease. Most recent observations disclosed its role in the regulation of insulin release from pancreatic beta cells [Ullrich et al., 2005].

The present study has thus been performed to elucidate whether PI3 kinase dependent ion channel regulation and function of mast cells similarly involves SGK1. Thus, the function of mast cells has been studied in gene targeted mice lacking SGK1 (sgki^'1') and their wild type littermates (sgk1^+l+).

Summary of the invention

The current invention elucidates the role of SGK1 in mast cell function. It is shown for the first time that SGK1 is involved in the regulation of mast cells by the PI3 kinase pathway and plays a pivotal role in the stimulation of mast cells. Kinases activated through the PI3 kinase pathway include the serum- and glucocorticoid-inducible kinase 1 (SGK1).

Mast cells isolated and cultured from the bone marrow (BMCMC) of SGK1 knockout mice {sgki^^') and their wild type littermates (sgk1^+/+) have been evaluated with respect to their cytosolic Ca²⁺ activity (determined utilizing Fura2 fluorescence), their channel activity by patch clamp technique and furthermore measured by FACS analysis. As a result, forward and side FACS scatter were both significantly smaller in sgk1^'A BMCMC than in sgk1^+/+ BMCMC, and this points to a decrease of cell volume and granulation. Moreover, Ca²⁺ entry following Ca²⁺ depletion was significantly reduced in sgk1^'A BMCMC when compared to sgk1^+/+ BMCMC and this points to a defective activation of the Ca²⁺ release activated Ca²⁺ channel I_CR_AC- Accordingly, Ca²⁺ sensitive K⁺ channels were activated by IgE-DNP in sgk1^+/+ BMCMC but not in sgk1^'A BMCMC. Treatment of the cells with the Ca²⁺ ionophore ionomycin (1 μM) led to similar activation of the K⁺ channels in both genotypes, indicating that the Ca²⁺ sensitive K⁺ channels are similarly expressed and similarly sensitive to activation by Ca²⁺ in sgk1^+/+ BMCMC and sgk1^'A BMCMC. Thus, the defective regulation of K⁺ channels unexpectedly results from blunted increase of cytosolic Ca²⁺ activity. Further studies have been performed to explore whether the impaired stimulation of Ca²⁺ entry in sgkT^A BMCMC affects the in vivo function of mast cells. To this end, Trinitrochlorobenzene- induced contact hypersensitivity reaction has been tested in sgk1^+/+ and sgkT^ mice. As a result, the early ear swelling following stimulation with Trinitrochlorobenzene (TNCB) was completely lacking in sgk1^'A mice, an observation indeed pointing to severe impairment of mast cell function in vivo. The observations unravel for the first time a critical role of SGK1 in the ion channel regulation and function of mast cells, and thus disclose a novel molecular functional switch in the regulation of allergic reaction. The current unexpected findings make serum and glucocorticoid inducible kinase-1 , modulators of SGK-1 activity and especially antagonists of SGK-1 activity important for the therapy of allergic reaction and other diseases driven by mast cell activity. Based on the new and unexpected function of SGK1 in mast cell activation the current invention delivers a method for inhibiting activation of mast cells by contacting said mast cells with substances that inhibit glucocorticoid inducible kinase and thereby modulate the PI3 kinase dependent ion channel regulation and function. An especially useful molecular target for inhibiting activation of mast cells according to this invention are glucocorticoid inducible kinases selected from the group consisting of: SGK1 , SGK2, SGK3.

Moreover a method for treating mast cell activation dependent disorders is readily envisible and this does require to administering substances that inhibit SGK1 to patients in need of such a treatment. The preparation of a medicament on the basis of an SGK1 inhibitor is generally known in the art. Preferred disorders that involve mast cell activation are but are not limited to allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility. The listed disorders would greatly benefit from a treatment with SGK1 inhibiting or modulating compounds. Modulating SGK1 compounds may be prefered in instances where the complete inhibition of SGK1 is not required and controlled residual SGK1 activity is needed. Use of SGK inhibitors selected from the listed compounds having the general formula I or M for the manufacture of a medicament for the treatment of disorders caused by mast cell activation. A list of SGK inhibitors and modulators having the general formula I or Il for the manufacture of a medicament for the treatment of disorders caused by mast cell activation.

The treatment of diseases selected from the group consisting of allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility may greatly benefit from the application of SGK1 modulators.

Prior to the application of SGK directed treatment it may be useful to examine the SGK1 status of a patient suffering from a mast cell activation dependent disorder and this is performed by measuring the up-regulated expression of SGK1 , SGK2 or SGK3 in mast cells derived from tissue samples and specimens of the patient.

It is known in the art that the presence of a specific polymorph SGK1 protein variant predisposes for disease. Therefore a method for determining mast cell activation dependent disorders by measuring the up-regulated expression of a selected single nucleotide polymorph variant of SGK1 in mast cells derived from tissue samples and specimens of risk patients may be used. SGK1 based diagnosis may be especially useful for evaluating human inflammatory disorder or human male infertility. The present invention provides evidence that SGK is involved in the activation of mast cells. Among other aspects of this finding this allows the screening of an inhibitor of serum glucocorticoid inducible kinases (SGK) suitable for the inhibition of mast cell activation wherein the method comprises the following steps: (i) providing a recombinant pre-activated phosphorylated SGK protein (ii) providing an SGK substrate polypeptide together with ATP (iii) providing an inhibitor of glucocorticoid inducible kinases, and (iv) evaluating SGK activity by measuring phosphorylation of the substrate. It is well understood that the SGK protein is selected from the group of SGK1 , SGK2 or SGK3 or that alternatively or in addition the selected single nucleotide polymorph variant of SGK may used as well. Furthermore the invention delivers a method for determining the progression, regression or onset of mast cell activation driven disorders by measuring the up-regulated expression and activation of SGK1 , SGK2 or SGK3 and selected single nucleotide polymorph variants in isolated human tissue samples and specimens.

The expert understands that the claimed methodes are well suited for the diagnosis of disease, wherein the disease is selected from the group consisting of allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility..

Furthermore the expert under stands that the compound selected with the claimed screening system or compounds selected form the lsted compound of Example 7 are well suited for the manufacture of a medicament for the inhibition of SGK1 , SGK2 or SGK3 dependent mast cell activation and that such medicaments are useful for the treatment of disorders selected from the group consisting of allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility.

Detailed description of the invention

The present study reveals a role of the serum and glucocorticoid inducible kinase SGK1 in the regulation of ion channel activity and function of mast cells. Distinct functional differences of mast cells are seen in gene targeted mice lacking SGK1 {sgkV'^') and their wild type littermates (sgk1+/+). Deficiency of SGK1 blunts the capacity of Ca²⁺ entry and reflects that I_CRAC i_s leading to subsequent impairment of Ca²⁺ dependent K⁺ channel activation. Most importantly, the sgki^^' mice completely lack the early, mast cell dependent, response to TNCB, which induces delayed type hypersensitivity reactions

(DTHRs).

The activation of Ca²⁺ channels is critically important for the regulation of mast cell function such as release of inflammatory mediators [Bradding et al., 2003;

Buess et al., 1999; Dernick et al., 2003; Duffy et al., 2001a; Duffy et al., 2001b; Kahr et al., 2004; Mazurek et al., 1980; Stokes et al., 2004]. Activation of mast cells is further paralleled by activation of K⁺ channels [Bradding et al., 2003; Bradding 2005; Duffy et al., 2005; Mark et al., 2004]. K⁺ channels maintain the cell membrane potential, which is required for the function of I_CRAC [Parekh and Penner 1997].

With respect to the molecular identity of the channels that are involved in mast cell activation, PI3 kinase has been suggested to target the TRPV2 Ca²⁺ channel [Tseng et al., 2004]. SGK1 has previously been shown to increase the cell membrane abundance and activity of the Ca²⁺ channel TRPV5 [Embark et al., 2004; Palmada et al., 2005] and SGK1 may have a similar stimulating effect on TRPV2.

The molecular identity of the SGK1 regulated mast cell K⁺ channels is similarly elusive. In other systems, SGK1 has been shown to activate several different K⁺ channels [Baltaev et al., 2005; Embark et al., 2003; Embark et al., 2004; Gamper et al., 2002; Henke et al., 2004; Palmada et al., 2003; Ullrich et al., 2005; Warntges et al., 2002; Yoo et al., 2003; Yun et al., 2002b]. SGK1 function is not limited to the regulation of ion channels but has a known influence on further transport systems, including the NaVH⁺ exchanger NHE3 [Yun et al., 2002a; Yun 2003], several amino acid transporters [Boehmer et al., 2003b; Boehmer et al., 2003a], glucose transporters [Dieter et al., 2004] and the Na⁺/K⁺-ATPase [Henke et al., 2004; Setiawan et al., 2002; Verrey et al., 2003; Zecevic et al., 2004]. Deranged regulation of those transporters maybe the molecular basis for the functional defect of mast cells from SGK1 knockout animals.

SGK1 function is not limited to the basic functions of mast cells but participates in their regulation by hormones and mediators. SGK1 is genomically unregulated by glucocorticoids [Firestone et al., 2003], mineralocorticoids [Chen et al., 1999; Naray-Fejes-Toth et al., 1999; Shigaev et al., 2000], cell shrinkage [Waldegger et al., 1997], gonadotropins [Alliston et al., 1997; Alliston et al., 2000; Gonzalez-Robayna et al., 2000; Richards et al., 1995], and TGFB [Lang et al., 2000; Waldegger et al., 1999]. The kinase is activated by IGF 1 and insulin through the phosphatidyl-inositide 3 (PI3) kinase and phospho-inositide- dependent kinase PDK1 [Alessi et al., 1996; Alessi and Cohen 1998; Divecha et al., 1991 ; Gamper et al., 2002; Kobayashi and Cohen 1999; Kotani et al., 1994; Park et al., 1999]. In conclusion, SGK1 is critically important for the regulation of Ca²⁺ entry and K⁺ channel activation of mast cells and the early phase of TNCB induced delayed type hypersensitivity reaction. The present observations thus disclose a completely novel regulator of mast cell function.

Detailed description of the results

As illustrated in Figure 1 , mast cells cultured from bone marrow (BMCMC) express CD117, CD34 and FcεRI, i.e. the receptors typically expressed by mast cells. No significant difference in receptor abundance is observed between BMCMC from SGK1 knockout mice (sgkT^A) and their wild type littermates (sgk1^+/+).

The forward and side scatter in cultured mast cells from sgk1^+/+ and sgkT^A mice is shown in Figure 2. The forward scatter is slightly but significantly smaller in sgk1^'A BMCMC than in sgk1^+/+ BMCMC, pointing to a slightly smaller cell volume. Similarly, the side scatter is slightly but significantly smaller in sgki^ BMCMC than in sgk1^+/+ BMCMC, pointing to a slight decrease of granulation.

Furo-2 fluorescence reveals that Ca²⁺ entry following Ca²⁺ depletion is significantly smaller in sgkT^ BMCMC than in sgk1^+/+ BMCMC, pointing to defective activation of the Ca²⁺ release activated Ca²⁺ channel I_CRAC in sgkT^A mice. Patch clamp reveals the activation of Ca²⁺ sensitive K⁺ channels by IgE-DNP in sgk1*^/+ BMCMC, an effect lacking completely in sgk1^v' BMCMC. The channels are inhibited by clotrimazole, a known blocker of Ca²⁺ sensitive K⁺ channels. Treatment of the cells with the Ca²⁺ ionophore ionomycin (1 μM) leads to activation of the K⁺ channels to a similar extent in sgk1^+/+ BMCMC and sgkT'^' BMCMC, indicating that the Ca²⁺ sensitive K⁺ channels are similarly expressed and similarly sensitive to activation by Ca²⁺ in sgk1^+/+ BMCMC and sgkT^A BMCMC. Thus, the defective regulation of K⁺ channels presumably results from blunted increase of cytosolic Ca²⁺ activity. Further studies have been performed to explore whether the impaired stimulation of Ca²⁺ in sgk1^'A BMCMC affects the in vivo function of mast cells. To this end, Trinitrochlorobenzene -induced contact hypersensitivity reaction has been tested in both, sgk1^+/+ and sgk1^'A mice. As illustrated in Figs. 5 and 6, the early ear swelling following stimulation with Trinitrochlorobenzene (TNCB) was completely lacking in sgk1^'A mice, an observation indeed pointing to severe impairment of mast cell function in vivo.

Further examples

Examples 1 : Dexamethasone treatment of mice

Mice lacking SGK1 (sgkT^1') have been generated as described previously [Wulff et al., 2002]. A conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons. The neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4-11 , which code for the Sgk1 kinase domain, were "floxed" by inserting a third loxP site into intron 3. Targeted R1 ES cells were transiently transfected with Cre recombinase. A clone with a recombination between the first and third loxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to 129/SvJ females. Heterozygous sg/c^-deficient mice were backcrossed to 129/SvJ wild-type mice for two generations and then intercrossed to generate homozygous sgkr^ and Sgk1^+/+ littermates. The animals were genotyped by PCR using standard methods. The study has been performed in female (n=15) and male (n=5) sgk1^+/+ and sgk1^"7' (6-7 weeks old) mice. For analysis of dexamethasone effects, sgk1^+l+ and sgkT^1' mice were injected with dexamethasonephosphate disodiumsalt (Sigma, Taufkirchen, Germany; dissolved in 0.9% saline) at a dose of 10μg/g BW for four consecutive days at 8 pm. sgk1^~'^~ and sgk1^+l+ mice injected with 0.9% saline alone served as controls. Mice had free access to a standard mouse diet (Altromin diet 1310, Heidenau, Germany) and tap water. On the 4^th day of dexamethasone treatment, the animals were fasted 16 hours on wire grids prior to the experiments with free access to tap water. Where specified the KCNQ1 K⁺ channel inhibitor 293B was applied as a single bolus intravenous injection at a dose of 5μg/g BW 30 minutes before the experiment.

Example 2: Quantitative real-time PCR was used to determine the effect of glucocorticoids on SGK1 transcript levels, gastric tissue was quickly removed and frozen in liquid nitrogen. Automated disruption and homogenization of frozen tissue was performed using the MagNa Lyser Instrument^™ (Roche Diagnostics, Mannheim, Germany). For each sample one-way special tubes were filled with ceramic beads, 20-30 mg of frozen tissue and 600 μl of RLT- buffer (Qiagen, Hilden, Germany). Cleared cell lysate was transferred for further RNA purification process (RNAeasy Mini Kit, Qiagen, Hilden, Germany). Subsequently 1 μg of total RNA was reverse transcribed to cDNA utilizing the reverse transcription system (Bioscience, USA) with oligo(dT) primers according to the manufacturer's protocol. To determine mSGK1 mRNA levels, quantitative real-time PCR with the LightCycler System^™ (Roche Diagnostics, Mannheim, Germany) was established. PCR reactions for mSGK1 were performed in a final volume of 20 μl containing 2 μl cDNA, 2.4 μl MgCI₂ (3 μM), 1 μl primermix (0.5 μM of both primers), 2 μl cDNA Master SybrGreen I mix (Roche Molecular Biochemicals, Mannheim, Germany) and 12.6 μl DEPC treated water. The transcript levels of the housekeeping gene mGAPDH were determined in each sample using a commercial primer kit (Search LC, Heidelberg, Germany). PCR reactions for GAPDH were performed in a final volume of 20 μl containing 2 μl cDNA, 2 μl primer mix (Search LC, Heidelberg, Germany), 2 μl cDNA Master Sybr Green 5 I mix (Roche Molecular Biochemicals, Mannheim, Germany) and 14 μl DEPC treated water.

Amplification of the target DNA was performed during 35 cycles of 95°C for 1 Os₁ 68⁰C for 10s and 72⁰C for 16s, each with a temperature transition rate of 20°C/s and a secondary target temperature of 58°C with a step size of ¹⁰ 0.5⁰C. Melting curve analysis was performed at 95⁰C Os, 58°C 10s, 95°C Os to determine melting temperatures of primer dimers and the specific PCR products. Melting curve analysis confirmed the amplified products, which were then separated on 1.5% agarose gels to confirm the expected size (406 bp). Finally, results were calculated as a ratio of the target vs. house keeping

1¹S gene transcripts.

The following primers for mSGK1 (Gene bank No.: NM_011361) were used: mSGK1 sense: 5' TGT CTT GGG GCT GTC CTG TAT G 3' mSGK1 antisense: 5' GCT TCT GCT GCT TCC TTC ACA C 3'

Example 3: Culture and analysis of bone marrow mast cells

Bone marrow derived cultured mast cells (BMMCs) were isolated from bone marrow of 12 weeks old male sgk1^+/+ and sgk1^'A naive mice. The cells were cultured for 4 weeks in RPMI 1640 (GIBCO, Carlsbad) containing 10% FCS, 1% penicillin/streptomicin, 0.5 μg IL-3 (RD systems, Wiesbaden- Nordenstadt) and 1 μg c-kit ligand (SCF, BIOZOL, Eching, Germany). The maturation of the cells was confirmed by staining the cells against: FceRI (e Bioscience/NatuTec Gmbh, Frankfurt), CD117 kit (BD Pharmingen, Heidelberg), and CD34 (BD Pharmingen, Heidelberg) molecules and analysis on by Flow Cytometry (FACS Calibur, Becton Dickinson). BMMCs were activated by sensitization with monoclonal mouse IgE Ab (1 :100, 12μg/ml/ 1 Ox⁶ cells, clone SPE-7, Sigma Aldrich, Munich) overnight in culture medium and challenged accutely with (100ng/ml) DNP-HSA (dinitrophenyl -human serum albumin, Sigma Aldrich, Munich) [Bradding et al., 2003].

Example 4: Trinitrochlorobenzen (TNCB)-induced contact hypersensitivity reaction (CHSRs)

TNCB (Sigma Aldrich, Munich) was used to induce delayed type hypersensitivity reactions (DTHRs), which are strictly dependent on hapten- specific, type 1 memory T cells and are associated with a strong infiltrate of polymorphic neutrophils PMNs [Biedermann et al., 2000; Pichler et al., 2005]. These memory T cells lead to CHSRs when the hapten is applied to the skin of sensitized mice [Asada et al., 1997]. BALB/c mice were sensitized with 5% TNCB (n=6) (80μl of a 4:1 mixture of acetone/olive oil) at the shaved abdomen, size: 2 per 2 cm. 1 week later, mice were challenged with 1 % TNCB (20 μl of a 1 :9 mixture of acetone/olive oil) on both sides of the ear. In the 5% TNCB solution acetone is an irritant, whereas it is used solely as a solvent in the 1 % TNCB solution. Specific ear swelling was determined by measuring ear thickness with a micrometer (Oditest ®;Kroeplin) before and 4, 8, 12 and 48 h after TNCB challenge. Data are expressed as a change in ear swelling comparing to thickness before treatment, (delta μm). In addition, ear tissue was collected 48 h after TNCB challenge and sections were stained with hematoxylin and eosin.

Example 5: Patch clamp experiments

Patch clamp experiments have been performed at room temperature in voltage-clamp, fast-whole-cell mode according to Hamill et al. [Hamill et al., 1981]. The cells were continuously superfused through a flow system inserted into the dish. The bath was grounded via a bridge filled with NaCI Ringer solution. Borosilicate glass pipettes (8-12 MOhm tip resistance; GC 150 TF-10, Clark Medical Instruments, Pangbourne, UK) manufactured by a microprocessor-driven DMZ puller (Zeitz, Augsburg, Germany) were used in combination with a MS314 electrical micromanipulator (MW, Marzhauser, Wetzlar, Germany). The currents were recorded by an EPC-9 amplifier (Heka, Lambrecht, Germany) using Pulse software (Heka) and an ITC-16 Interface (Instrutech, Port Washington, N. Y., USA). Whole-cell currents were determined at eleven successive 700-ms square pulses from the -20 mV holding potential to potentials between -100 mV and +80 mV. The current values were 3 kHz low-pass filtered.

The pipette solution contained (in mM): 115 Na-gluconate, 10 NaCI, 1 MgATP, 1 EGTA, and 5 HEPES/NaOH (pH 7.4) and was used in combination with NaCI and Cl^'-free Ringer solutions (see above).

The offset potentials between both electrodes were zeroed before sealing. The potentials were corrected for liquid junction potentials as estimated according to Barry & Lynch [Barry and Lynch 1991]. The original whole-cell current traces are depicted without filtering (acquisition frequency of 5 kHz) and currents of the individual voltage square pulses are superimposed. The applied voltages refer to the cytoplasmic face of the membrane with respect to the extracellular space. The inward currents, defined as flow of positive charge from the extracellular to the cytoplasmic membrane face, are negative currents and depicted as downward deflections of the original current traces.

Example 6: Measurement of intracellular Ca²⁺

Fura-2 fluorescence was utilized for cytosolic Ca²⁺ determinations. Intracellular Ca²⁺ measurements were performed as described [Tanneur et al., 2002]. Retinoblastoma cells were loaded with Fura-2 (2.5μM- Molecular Probes, Goettingen, Germany) for 30 minutes at 37°C. Fluorescence measurements were carried out with an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany). Cells were excited alternatively at 340 and 380 nm and the light was deflected by a dichroic mirror into the objective (Fluar 40^χ/1.30 oil, Zeiss, Oberkochen, Germany). Emitted fluorescence intensity was recorded at 505 nm and data acquisition was performed by Axon Imaging Workbench (Axon Instruments, Foster City, USA). Experiments were made prior to, during and following exposure to nominally Ca²⁺ free solution (5 mM EGTA added). In the absence of Ca²⁺ the intracellular Ca²⁺ stores were depleted by inhibition of the vesicular Ca²⁺ pump by thapsigargin (1 μM, Molecular Probes).

Example 7: SGK1 inhibiting and modulating compounds

7.1. Compounds of the general formula I and pharmaceutical useful derivates, salts, solutions and stereoisomeres thereof including mixtures.

wherein

R¹, R⁵ is either H, OH, OA, OAc or Methyl, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰ is either

H, OH, OA, OAc, OCF₃, Hal, NO₂, CF₃, A, CN, OSO₂CH₃,

SO₂CH₃₁ NH₂ Or COOH,

R¹¹ H or CH₃, A Alkyl with 1 , 2, 3 or 4 C-atoms,

X CH₂, CH₂CH₂, OCH₂ or -CH(OH)-, Hal, F, Cl, Br or I

Compound according to formula I selected from the following group of compounds:

(3-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (3-Hydroxy-phenyl)-acidic acid-[1 -(4-hydroxy-2-methoxy-phenyl)-ethyliden]- hydrazid, (3-Methoxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid. Phenylacidic acid-(3-fluor-4-hydroxy-benzyliden)-hydrazid, (4-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3,4-Dichlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, m-Tolyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, o-Tolyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (2-Chlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (3-Chlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (4-Fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (2-Chlor-4-fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(4-hydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(4-hydroxy-2,6-dimethyl-benzyliden)- hydrazid,

(3-Methoxy-phenyl)-acidic acid-(3-fluor-4-hydroxy-benzyliden)-hydrazid, (3-Methoxy-phenyl)-acidic acid-[1 -(4-hydroxy-2-methoxy-phenyl)-ethyliden]- hydrazid,

(S-Methylsulfonyloxy-phenyO-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid, (3,5-Dihydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Fluor-phenyl)-acidic acid-(3-fluor-4-hydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(4-acetoxy-2-methoxy-benzyliden)-hydrazid, (3-Trifluormethyl-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- - hydrazid, 3-(3-Methoxy-phenyl)-propionsaure-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Methoxy-phenyl)-acidic acid-(2,4-dihydroxy-benzyliden)-hydrazid,

(S-Methoxy-phenoxy^acidic acid-^-hydroxy^-methoxy-benzyliden)- hydrazid,

(3-Nitro-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(5-chlor-2-hydroxy-benzyliden)-hydrazid, (3-Methoxy-phenyl)-acidic acid-(2-hydroxy-5-nitro-benzyliden)-hydrazid,

2-Hydroxy-2-phenyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(2-ethoxy-4-hydroxy-benzyliden)-hydrazid,

(3-Brom-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (3-Methoxy-phenyl)-acidic acid-[1-(4-hydroxy-phenyl)-ethyliden]-hydrazid,

(3,5-Difluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methyl-benzyliden)-hydrazid,

(3-Hydroxy-phenyl)-acidic acid-(2-ethoxy-4-hydroxy-benzyliden)-hydrazid,

(S-Hydroxy-phenyO-acidic acid^-methoxy^-hydroxy-e-methyl-benzyliden)- hydrazid,

(2-Fluor-phenyl)-acidic acid-(2-methoxy-4-hydroxy-benzyliden)-hydrazid

7.2. Compounds of the general formula Il and pharmaceutical useful derivates, salts, solutions and stereoisomeres thereof including mixtures.

wherein

R¹, R², R³ R⁴, R⁵ is either H, A, OH, OA, Alkenyl, Alkinyl, NO₂, NH₂, NHA, NA₂,

Hal, CN, COOH, COOA,

-OHet, -O-Alkylen-Het, -O-Alkylen-NR⁸R⁹ or CONR⁸R⁹, two groups selected from R¹, R², R³, R⁴, R⁵ or as well -0-CH₂-CH₂-, -0-CH₂-O- or -0-CH₂-CH₂-O-,

R⁶, R⁷ is either H, A, Hal, OH, OA or CN, R⁸, R⁹ is either H or A,

Het Is a saturated or unsaturated heterocycle with 1 to 4 N-, O- and/or S-atoms, substituted by one or several Hal, A, OA,

COOA, CN or Carbonyloxigen (=0)

A Alkyl with 1 to 10 C-atoms, wherein 1-7 H-atoms may be replaced by F and/or Chlorine,

X₁ X¹ is either NH or is missing

Hal F, Cl, Br or I Compound according to formula Il selected from the following group of compounds:

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-(2-fluor-5- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-(4-chlor-5- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-(2,4-difluor- phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-Qf]pyrimidin-8-yl)-phenyl]-3-(2,6-difluor- phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-(3-fluor-5- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-(4-fluor-5- trifluormethyl-phenyl)-urea,

1-[4-(4-Annino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(4-methyl-5- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(2,3,4,5,6- pentafluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(2,4-dibrom-6- fluor-phenyl)-urea,

1-[4_(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-(2-fluor-6- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-o(]pyrimidin-8-yl)-phenyl]-3-(2-fluor-5- methyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyNmidin-8-yl)-phenyl]-3-(2,3,4-tπfluor- phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(4-brom-2,6- difluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(2-fluor-3- trifluormethyl-phenyl)-urea, 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[2-(1-tert.- butyloxycarbonyl-piperidin-4-yl)-phenyl]-urea,

N-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-f/]pyrimidin-8-yl)-phenyl]-2,4-dichlor- benzamid, N-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-4-chlor-5- trifluormethyl-benzamid,

N-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-2-fluor-5- trifluormethyl-benzamid,

,_ 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cf]pyrimidin-8-yl)-phenyl]-3-[3-chlor-5- b trifluormethyl-2-(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c0pyrimidin-8-yl)-phenyl]-3-[(2-fluor-5-(2- dimethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[5-fluor-2-

(piperidin-4-yloxy)-phenyl]-urea, 10 1 -[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[4-chlor-5- trifluormethyl-2-(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[2-(piperidin-4- yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/|pyπmidin-8-yl)-phenyl]-3-[2-fluor-5-(2- diethylamino-ethoxy)-phenyl]-urea, 15 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[2-fluor-5-[2-

(piperidin-1-yl)-ethoxy]-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-[4-fluor-2-(2- dimethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[4-fluor-2-(2- diethylamino-ethoxy)-phenyl]-urea, on

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[3-chlor-4-[2- (morpholin-4-yl)-ethoxy]-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[4-fluor-2-[2- (morpholin-4-yl)-ethoxy]-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cf]pyrimidin-8-yl)-phenyl]-3-[3-chlor-4-(2- dimethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/|pyrimidin-8-yl)-phenyl]-3-[3-chlor-4-(2- diethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-[4-chlor-2-(2- dimethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[2-chlor-5-(2- 30 diethylamino-ethoxy)-phenyl]-urea, Example 8: SGK1 nucleotide polymorphism

SGK1 nucleotide polymorphism is demonstrated by the sequences ...aattacattgCgcaacccag.., whereas the nucleotide sequence representing a another population is....aattacattgTgcaacccag.... Both sequences are available through accession number Gl 2463200 Position 2071. The exon 8 sequences of facultative patients with mast cell overactivity are either homozygot ..tactgaC_ttcggact..or....tactgaTttcggact....or heterozygot .tactgaC_ttcggact...and...tactgaTttcggact .. The sequences are available through accession number NM 005627.2, Position 777.

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M, Altruda F, Hirsch E, Laffargue M: Phosphoinositide 3-kinase gamma: a key modulator in inflammation and allergy. Biochem Soc Trans 2003;31 :275-

280. Yamanaka K, Fujisawa M, Tanaka H, Okada H, Arakawa S, Kamidono S:

Significance of human testicular mast cells and their subtypes in male infertility. Hum Reprod 2000; 15: 1543-1547.

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2003;278:23066-23075.

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Factor 2 (NHERF2) in Regulation of NHE3. Cell Physiol Biochem

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Figure legends

Figure 1 : Cell surface receptor expression in cultured mast cells from sgk1*^/+ and sgk1^m/' mice

FACS analysis illustrating CD117, CD34 and FcεRI expression in cultured bone marrow mast cells (BMCMC) from SGK1 knockout mice (sgkV') and their wild type littermates (sgk1^+/+).

Figure 2: Forward and side scatter in cultured mast cells from sgk1^+/+ and sgk1~'^~ mice.

Arithmetic means (± SEM; n = 7) of forward scatter (FSC₁ left panel) and side scatter (SSC, right panel) of cultured bone marrow mast cells (BMCMC) in arbitrary units (a.u.) from SGK1 knockout mice (sgkV'^', black bars) and their wild type littermates (sgk1^+/+, white bars).

Figure 3: Ca²⁺ entry into cultured mast cells from sgk1^+/+ and sgk1^m/~ mice. Arithmetic means (± SEM; n = 21) of increase of Fura-2 fluorescence in arbitrary units (a.u.) reflecting cytosolic Ca²⁺ into cultured bone marrow mast cells (BMCMC) from SGK1 knockout mice (sg/c7^"A, black bars,) and their wild type littermates (sgk1^+/+, white bars).

Figure 4: Activation of Ca²⁺ sensitive K⁺ channels in cultured mast cells from sgk1^+/+ and sgkT'^' mice. A. Mean I-V relationships (± SEM, n = 4) of currents in cultured bone marrow mast cells (BMCMC) from SGK1 knockout mice (sgr/cf^Λ, right panel) and their wild type littermates (sgk1^+/+, left panel) prior to (open circles) and following (closed triangles) activation with IgE-DNP in the absence (closed triangles) and presence (closed squares) of clotrimazole.

B. Mean whole-cell conductance (± SEM, n = 4-6) obtained from cultured bone marrow mast cells (BMCMC) from SGK1 knockout mice (sgr/c7^'A, black barsj and their wild type littermates {sgk1^+/+, white bars) prior to (control) and following exposure to IgE-DNP (IgE) and/or clotrimazole (CTZ) * indicates significant difference (p<0.05; ANOVA).

Figure 5: Ear tissue from sgk1^+/* and sgk1^'/m mice prior to and following Trinitrochlorobenzene -induced contact hypersensitivity reaction.

Sections of ear tissue from SGK1 knockout mice (sgk1^'A, left panel) and their wild type littermates (sgk1^+/+, right panel) 8 hours following (lower panels) stimulation with Trinitrochlorobenzene (TNCB).

Figure 6: Ear swelling of sgk1^+/* and sgAT^A mice prior to and following Trinitrochlorobenzene -induced contact hypersensitivity reaction. Time course (arithmetic means ± SEM, n = 6) of the swelling (in μm) of right (left panel) and left (right panel) ears from SGK1 knockout mice (sgk1^'A, closed circles) and their wild type littermates (sgk1^+/+, open circles) following stimulation with Trinitrochlorobenzene (TNCB). ^* indicates significant difference (p<0.05; ANOVA).

Claims

Claims

1. A method for the screening of an inhibitor of serum glucocorticoid inducible kinases (SGK) suitable for the inhibition of mast cell activation wherein the method comprises the following steps: (i) providing a recombinant pre-activated phosphorylated SGK protein

(ii) providing an SGK substrate polypeptide together with ATP (iii) providing an inhibitor of glucocorticoid inducible kinases, and (iv) evaluating SGK activity by measuring phosphorylation of the substrate.

2. A method according to claim 1 , wherein the SGK protein is selected from the group of SGK1 , SGK2 or SGK3.

3. A method according to claim 2, wherein SGK1 , SGK2 or SGK3 represents a selected single nucleotide polymorph variant.

4. A method according to claims 1-3, wherein the SGK inhibitors have the general formula:

R⁹ R10

wherein

_DR5 is either H, OH, OA, OAc or Methyl, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰ Is either

H, OH, OA, OAc, OCF₃, Hal, NO₂, CF₃, A, CN, OSO₂CH₃, SO₂CH₃, NH₂ or

COOH,

R¹¹ H or CH₃,

A Alkyl with 1 , 2, 3 or 4 C-atoms,

X CH₂, CH₂CH₂, OCH₂ or -CH(OH)-,

Hal F, Cl, Br or I

and pharmaceutical useful derivates, salts, solutions and stereoisomeres thereof including mixtures.

10

5. A method according to claim 5 wherein the the SGK inhibitor is selected from the following group of compounds:

(3-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid, 15 (3-Hydroxy-phenyl)-acidic acid-[1 -(4-hydroxy-2-methoxy-phenyl)- ethyliden]-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

Phenylacidic acid-(3-fluor-4-hydroxy-benzyliden)-hydrazid,

(4-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- 20 hydrazid,

(3,4-Dichlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid, m-Tolyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, o-Tolyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(2-Chlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

25

(3-Chlor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(4-Fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, (2-Chlor-4-fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Fluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid, -,_Q (3-Methoxy-phenyl)-acidic acid-(4-hydroxy-benzyliden)-hydrazid, (3-

Methoxy-phenyl)-acidic acid-(4-hydroxy-2,6-dimethyl-benzyliden)- hydrazid, (3-Methoxy-phenyl)-acidic acid-(3-fluor-4-hydroxy-benzyliden)- hydrazid, (3-Methoxy-phenyl)-acidic acid-[1-(4-hydroxy-2-methoxy- phenyl)-ethyliden]-hydrazid,

(3-Methylsulfonyloxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy- benzyliden)-hydrazid,

_ (3,5-Dihydroxy-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-

5 hydrazid,

(3-Fluor-phenyl)-acidic acid-(3-fluor-4-hydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(4-acetoxy-2-methoxy-benzyliden)- hydrazid,

(3-Trifluormethyl-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- 10 hydrazid,

3-(3-Methoxy-phenyl)-propionsaure-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Methoxy-phenyl)-acidic acid-(2,4-dihydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenoxy)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid, 15 (3-Nitro-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(5-chlor-2-hydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-(2-hydroxy-5-nitro-benzyliden)-hydrazid,

2-Hydroxy-2-phenyl-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Methoxy-phenyl)-acidic acid-(2-ethoxy-4-hydroxy-benzyliden)-hydrazid,

20

(3-Brom-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)-hydrazid,

(3-Methoxy-phenyl)-acidic acid-[1-(4-hydroxy-phenyl)-ethyliden]-hydrazid,

(3,5-Difluor-phenyl)-acidic acid-(4-hydroxy-2-methoxy-benzyliden)- hydrazid,

(3-Hydroxy-phenyl)-acidic acid-(4-hydroxy-2-methyl-benzyliden)-hydrazid,

(3-Hydroxy-phenyl)-acidic acid-(2-ethoxy-4-hydroxy-benzyliden)-hydrazid,

(3-Hydroxy-phenyl)-acidic acid-(2-methoxy-4-hydroxy-6-methyl- benzyliden)-hydrazid,

(2-Fluor-phenyl)-acidic acid-(2-methoxy-4-hydroxy-benzyliden)-hydrazid

6. A method according to claim 1-3, wherein the SGK inhibitors have 30 the general formula:

wherein

R¹, R², R³ R⁴, R⁵ is either H₁ A₁ OH, OA₁ Alkenyl, Alkinyl, NO₂, NH₂,

NHA₁ NA₂, HaI₁ CN₁ COOH, COOA₁ -OHet, -O-

Alkylen-Het, -O-Alkylen-NR⁸R⁹ or CONR⁸R⁹, two groups selected from R¹, R², R³, R⁴, R⁵ or as well -0-CH₂-CH₂-, -0-CH₂-O- or -0-CH₂-CH₂-O-,

R⁶, R⁷ is either H, A, Hal, OH, OA or CN, R⁸, R⁹ is either H or A, Het is a saturated or unsaturated heterocycle with 1 to 4

N-, O- and/or S-atoms, substituted by one or several

Hal, A, OA₁ COOA₁ CN or Carbonyloxigen (=0)

A Alkyl with 1 to 10 C-atoms, wherein 1-7 H-atoms may be replaced by F and/or Chlorine,

X₁ X¹ is either NH or is missing Hal, F, Cl, Br or I

and pharmaceutical useful derivates, salts, solutions and stereoisomeres thereof including mixtures.

A method according to claim 6 wherein the SGK inhibitor is selected from the following group of compounds:

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/Ipyrimidin-8-yl)-phenyl]-3-(2- fluor-5-trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-(4- chlor-5-trifluormethyl-phenyl)-urea, 1-[4-(4-Amirio-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3- (2,4-difluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3- (2,6-difluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-(3- o fluor-5-trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/|pyrimidin-8-yl)-phenyl]-3-(4- fluor-5-trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/Ipyrinnidin-8-yl)-phenyl]-3-(4- methyl-5-trifluormethyl-phenyl)-urea, 1 o 1 -[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/Ipyrimidin-8-yl)-phenyl]-3-

(2,3,4,5,6-pentafluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-

(2,4-dibrom-6-fluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-(2- fluor-6-trifluormethyl-phenyl)-urea, 15 1 -[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-(2- fluor-5-methyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/lpyrimidin-8-yl)-phenyl]-3-

(2,3,4-trifluor-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cf]pyrimidin-8-yl)-phenyl]-3-(4- brom-2,6-difluor-phenyl)-urea, on

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-(2- fluor-3-trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[2-

(1-tert.-butyloxycarbonyl-piperidin-4-yl)-phenyl]-urea,

N-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-2,4- _O1- dichlor-benzamid,

N-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-4- chlor-5-trifluormethyl-benzamid,

N-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-2- fluor-5-trifluormethyl-benzamid,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/lpyrimidin-8-yl)-phenyl]-3-[3- 30 chlor-5-trifluormethyl-2-(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/lpyrimidin-8-yl)-phenyl]-3-[(2- fluor-5-(2-dimethylamino-ethoxy)-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[5- fluor-2-(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-[4- chlor-5-trifluormethyl-2-(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-[2-

(piperidin-4-yloxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[2- fluor-5-(2-diethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]-3-[2- fluor-5-[2-(piperidin-1-yl)-ethoxy]-phenyl]-urea,

1 o 1 -[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/|pyriπnidin-8-yl)-phenyl]-3-[4- fluor-2-(2-dimethylamino-ethoxy)-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[4- fluor-2-(2-diethylamino-ethoxy)-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/lpyrimidin-8-yl)-phenyl]-3-[3- chlor-4-[2-(morpholin-4-yl)-ethoxy]-phenyl]-urea,

15 1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c(|pyrimidin-8-yl)-phenyl]-3-[4- fluor-2-[2-(morpholin-4-yl)-ethoxy]-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-djpyrimidin-8-yl)-phenyl]-3-[3- chlor-4-(2-dimethylamino-ethoxy)-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]-3-[3- chlor-4-(2-diethylamino-ethoxy)-phenyl]-urea, on

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-c(]pyrimidin-8-yl)-phenyl]-3-[4- chlor-2-(2-dimethylamino-ethoxy)-phenyl]-urea, 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cdpyrimidin-8-yl)-phenyl]-3-[2- chlor-5-(2-diethylamino-ethoxy)-phenyl]-urea,

8. A method for determining the progression, regression or onset of 25 mast cell activation driven disorders by measuring the up-regulated expression and activation of SGK1 , SGK2 or SGK3 in isolated human tissue samples and specimens.

9. A method according to claim 8, wherein SGK1 , SGK2 or SGK3

JU represents a selected single nucleotide polymorph variant.

10. A method according to claim 9 for the diagnosis of disease, wherein the disease is selected from the group consisting of allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility..

11. A method according to claim 10, wherein the SGK1 , SGK2 or SGK3 represents a selected single nucleotide polymorph variant.

12. Use of a compound as specified in any one of the claims 4-7 for the manufacture of a medicament for the inhibition of SGK1 , SGK2 or SGK3 dependent mast cell activation.

13. Use of a compound according to claim 12 for the manufacture of a medicament for the treatment of disorders selected from the group consisting of allergic reaction, allergic rhinitis, anaphylactic and delayed hypersensitivity, psoriasis, atopic dermatitis, rheumatoid arthritis, Crohn^'s disease, irritable bowel syndrome or male infertility.