Methods for interfering with glucocorticoid induced gastric acid secretion

Background of the invention

Gastric acid is produced by parietal cells in the stomach. Parietal cells contain an extensive secretory network from which the gastric acid is secreted into the lumen of the stomach. These cells are part of epithelial fundic glands in the gastric muocsa. The pH of gastric acid is 2-3 in the stomach lumen, the acidity being maintained by the proton pump a H+/K+ ATPase.

Normally, the lining of the stomach and small intestines have protection against the irritating acids produced in the stomach. For a variety of reasons, the protective mechanisms may become faulty, leading to a breakdown of the lining. This results in inflammation (gastritis) or an ulcer.

Ulcer produces a crater-like lesion on the skin or mucous membrane of the stomach (gastric) or the upper part of the small intestine caused by an inflammatory, infectious, or malignant condition.

Peptic ulcer disease is a mucusal ulcer in the acid- producing zone in the distal stomach or the proximal duodenum. The normal stomach produces enough mucus and alkaline juice to protect the gastric and duodenal mucosa against HCI. In the duodenum the pancreatic bicarbonate creates a pH of 7.5 at the luminal membrane of the mucosa.

Risk factors which may induce peptic ulcer disease are drugs such as ASA, NSAIDs and corticoids, hyperparathyroidism where a high Ca ²⁺ level stimulates gastric acid secretion, gastrin-producing tumours of the pancreas and Helicobacter pylori infection of the stomach. Other contributing factors are increased pepsinogen from the chief cells, increased parietal cell mass, reduced somatostatin secretion from the antral D cells, and damage of the mucosa. Acetylsalicylic acid and steroid or non-steroid anti-inflammatory drugs deplete the gastric mucosa for prostaglandins, which leads to mucosal damage.

Genetic factors must be considered, for instance persons who do not secrete blood group 0 antigen into the saliva and gastric juice, have an increased risk of developing duodenal ulcers. Diagnosis may require gastroscopy of the stomach and duodenum to search for abnormalities. Tissue samples may be obtained to check for H. pylori bacteria and inflammatory factors.

Treatment of ulcer often involves a combination of medications to kill the bacteria, reduce acid levels, and protect the Gl tract. The medications may include one or more of the following: Acid blockers like cimetidine, nizatidine, ranitidine, or famotidine. Proton pump inhibitors such as Omeprazole or medications that protect the tissue lining like sucralfate. Bismuth protects the lining and kills the bacteria and furthermore antibiotics such as Clarithromycin are used to kill H. pylori. Prostaglandin Ei analogues, such as misoprostol, inhibits gastric acid secretion by unspecific inhibition of the second messenger, cAMP, in the parietal cell and elsewhere and hereby promote ulcer healing. All treatment procedures, which work by inhibition of gastric acid secretion, have a common drawback. To the extent that gastric acid secretion is reduced there is no inhibition of the gastrin release from the antral G cells. Accordingly, the blood gastrin increases, and during treatment of the patients this concentration is constantly increased. The high gastrin level counteracts the expected effect on the acid production. Since gastrin is a trophical hormone for the gastric mucosa, long-term treatment with acid suppression might result in mucosal hypertrophy with a further rise in acid production and in cellular modifications. These complications are probably related to the rather high ulcer recurrence rate of most treatment procedures. A new rational strategy for eliminating the cause of the peptic ulcer disease is delivered with the current invention which uses SGK inhibition as a means to interfere with gastric acid imbalance.

Glucocorticoids are well known to support the development of gastric ulcer [Ahamed et al., 1983; Zamora et al., 1975], an effect considered to be due to

both, enhanced H ⁺ secretion [Cooke et al., 1966; Raptis et al., 1976] and impaired defense mechanisms such as prostaglandin release [Bandyopadhyay et al., 1999; Nobuhara et al., 1985]. The mechanisms linking the stimulation of the glucocorticoid receptor to acid secretion are, however not known.

Signalling molecules involved in glucocorticoid actions include the serum and glucocorticoid inducible kinase SGK1, which is highly expressed in gastric tissue [Waldegger et al., 1997]. SGK1 has been shown to regulate a wide variety of transport proteins [Lang et al., 2003]. Coexpression of SGK1 in Xenopus oocytes up-regulates the epithelial Na ⁺ channel ENaC [Alvarez de Ia Rosa et al., 1999; Bόhmer et al., 2000; Chen et al., 1999; Faletti et al., 2002; Lang et al., 2000; Naray-Fejes-Toth et al., 1999; Pearce 2003; Verrey et al., 2003; Wagner et al., 2001 ; Wang et al., 2001], the renal outer medullary K ⁺ channel ROMK1 [Palmada et al., 2003b; Palmada et al., 2003a; Yun et al., 2002b], the voltage gated Na ⁺ channel SCN5A [Boehmer et al., 2003c], the voltage gated K ⁺ channel complex KCNE1/KCNQ1 [Embark et al., 2003], and the voltage gated K ⁺ channels Kv1.2, Kv1.3, Kv1.4 and Kv1.5 [Gamper et al., 2002b; Gamper et al., 2002a; Henke et al., 2004; Warntges et al., 2002]. In addition to channels, SGK1 regulates the Na ⁺ /H ⁺ exchanger NHE3 [Yun et al., 2002a; Yun 2003], the glutamine transporter SN1 [Boehmer et al., 2003b], the glutamate transporter EAAT1 [Boehmer et al., 2003a] the renal and intestinal glucose transporter SGLT1 [Dieter et al., 2004] and the Na ⁺ /K ⁺ -ATPase [Henke et al., 2002; Setiawan et al., 2002; Verrey et al., 2003; Zecevic et al., 2004]. A common (-5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight (Vallon et al., 2005) Jan;14(1 ):59-66. SGK1 may thus contribute to metabolic syndrome. SGK1 further participates in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia. SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior. In conclusion, the SGKs cover a wide variety of physiological

functions and may play an active role in a multitude of pathophysiological conditions.

The present study delivers the unexpected finding that the regulation of gastric acid secretion is dependent on the expression and up- regulation of SGK1 activity.

Summary of the invention

The present invention provides evidence that SGK1 is involved in the regulation of gastric acid secretion. SGK1 stimulates H ⁺ secretion by increasing the K ⁺ /H ⁺ ATPase activity and alternatively, SGK1 enhances the H ⁺ secretion by stimulating KCNQ1 channels which have been shown to be of critical importance for gastric H ⁺ secretion [Vallon et al., 2006] and are known to be upregulated by SGK1. The regulation of KCNE1/KCNQ1 involves the ubiquitin ligase Nedd4-2 which decreases the affinity of the enzyme to its target proteins [Abriel et al., 2000; Debonneville et al., 2001 ;

Snyder et al., 2002; Verrey et al., 2003].

The current results furthermore demonstrate that SGK1 is not required for gastric H ⁺ secretion in general, however represents a highly specific protein target that participates mainly in the stimulation of gastric H ⁺ secretion related to disease or alternatively in the context of glucocorticoid treatment.

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 gastric H ⁺ secretion driven disease such as for instance peptic ulcer disease.

In the current invention a method for screening of an inhibitor of serum glucocorticoid inducible kinases (SGK) suitable for the inhibition of gastric acid secretion comprises the following steps has been described: (i) providing a recombinant pre-activated phosphorylated SGK protein, (ii) providing an SGK substrate polypeptide together with ATP or other phosphate sources, (iii) providing an inhibitor of glucocorticoid inducible

kinases and (iv) evaluating SGK activity by measuring phosphorylation of the substrate. A preferred embodiment of the current invention relates to the use of SGK1 for the screening of inhibitors of serum glucocorticoid inducible kinases. Examples of SGK1 inhibitors available through the claimed screening method are listed in Example 4 of the current invention and the expert recognizes that the compounds and pharmaceutical useful derivates, salts, solutions and stereoisomeres and mixtures thereof are useful drugs for the treatment of gastric acid secretion driven diseases such as peptic ulcer.

The expert further understands that a preferred embodiment of the current invention is SGK1 and inhibitors directed to SGK1 and SGK1 function and diagnosis, however it is obvious to the expert that the claimed methods, use and inhibitory compounds can applied to other disease related isoforms of the protein such as SGK2 and SGK3 and selected single nucleotide polymorph variants that have been described for the three isoforms. The invention delivers and claims compounds which do inhibit the function of SGK. The functional inhibition is characterized by the direct inference with the activity kinase of the SGK enzyme and therefore the mechanism is unique over other known phosphorylation inhibitors.

Besides the claimed therapeutic use of SGK for the treatment of diseases which are characterized by a surplus of gastric acid secretion it is another aspect of the current invention to monitor the function of SGK for the diagnosis of diseases such as peptic ulcer.

The expert recognizes immediately that the determination of progression, regression or onset of gastric acid secretion driven disorders can be measured by monitoring the up-regulated expression and/or functional activity of SGK1 , SGK2 or SGK3 in organs or isolated human tissue samples and specimens taken from a patient.

Furthermore the invention opens the possibility to diagnose a disease by the analysis of a single nucleotide polymorph variant for instance for SGK1 in order to correlate said polymorph phenotype with the presence, severity or

predisposition of gastric acid secretion driven disorders. Diagnosis methods for SGK are not restricted to SGK1 however may as well require the simultaneous analysis of SGK2 and or SGK3.

DETAILED DESCRIPTION OF THE INVENTION

The effect of glucocorticoids on gastic H ⁺ transport has been studied in gene targeted mice lacking SGK1 (sgkϊ ^1' ) and their wild type littermates (sgk1 ^+l+ ). SGK1 transcript levels were determined by real-time PCR and BCECF fluorescence was utilized for determination of H ⁺ secretion (δpH).

It has been found, that gastric SGK1 transcript levels is up-regulated significantly by a 4 day treatment with 10 μg/g BW/day dexamethasone (DEX) in gastric tissue of wild type mice. Following the dexamethasone treatment of those mice the msgk1/mGAPDH*1000 ratio increased in gastric tissue from 20.7 ± 10.1 (n = 3) to 46.2 ± 16.0 (n = 6). No msgki transcripts were detected in SGK1 knockout mice (SGK1 /GAPDH ratio 0.0, n=4).

Detailed measurements of cytosolic pH were utilized to determine proton pump activity. Initial cytosolic pH was similar in untreated sgk1 ^+/+ (7.16 ± 0.01 , n = 6 ) and sgk1 ^v~ (7.18 ± 0.01 , n = 6). Dexamethasone treatment did not significantly alter cytosolic pH in sgk1* ^/+ mice (7.15 ± 0.01, n =8 ) or in sgkϊ ^A mice (7.17 ± 0.01, n =10 ).

Ammonium pulses were utilized to load the cells with H ⁺ [Roos and Boron 1981]. The superfusion with 20 mM NH ₄ CI and simultaneous removal of Na ⁺

(replacement with NMDG) was followed by slight alkalinization (Fig. 3). The subsequent replacement of NH ₄ CI with NMDG Cl ^" in the continued absence of Na ⁺ led to a sharp acidification due to exit of NH3 and retention of H ⁺ within the cells. The acidification allowed the calculation of an apparent buffer capacity (see methods) which was similar in untreated sgk1 ^+/+ mice

(47.51 ± 5.79 mM/pH, n =6) and sgki^ mice (48.2 ± 3.18 mM/pH, n =6).

Dexamethasone did not significantly alter the buffer capacity in neither sgk1 ^+/+

mice (46.38 ± 3.12 mM/pH, n =8) nor sgk1 ^v~ mice (46.44 ± 2.42 mM/pH, n

=10).

Prior to dexamethasone treatment pH recovery in the absence of Na ⁺ was similar in untreated sgk1 ^+/+ mice (0.028 ± 0.008 δpH/min, n = 6), and sgk1 ^'A mice (0.029 ± 0.010 δpH/min, n = 6). Treatment with dexamethasone led to a

~4 fold increase of Na ⁺ independent realkalinization in sgk1 ^+/+ mice (0.133 ± 0.022 δpH/min, n = 8) and to ~2 fold increase in sgkV' ^' mice (0.071 ± 0.012 δpH/min, n = 10). Following dexamethasone treatment, the pH recovery in the absence of Na ⁺ was significantly (p<0.05) smaller in and sgk1 ^'A than in sgk 1 ^+/+ mice.

Addition of 50μM Omeprazole decreased the Na ⁺ independent pH recovery to 0.009 ± 0.003 δpH/min, n = 5 in dexamethasone treated sgk1 ^+/ * mice and to 0.007 ± 0.002 δpH/min, n = 5 in dexamethasone treated sgk1 ^'λ . Following dexamethasone treatment, the omeprazol sensitive realkalinization was again significantly (p<0.05) smaller in and sgk f ^λ than in sgk1 ^+/+ mice.

The results of the present experiments are unexpected since they demonstrate that the lack of SGK1 does not affect H ⁺ secretion in untreated animals but abrogates the stimulating effect of glucocorticoids on gastric H ⁺ secretion selectively. The apparently normal H ⁺ secretion in the untreated sgk1-/- mice call for a mechanism which does replace SGK1 in the absence of glucocorticoid stimulation. SGK1 isoforms SGK2 and SGK3 which have been cloned from homology screening are known in the art [Kobayashi et al., 1999] and share the ability of SGK1 to enhance the protein abundance and/or activity of several channels and transporters [Lang et al., 2003]. Experiments in Xenopus oocytes indeed disclosed the ability of SGK3 to up-regulate KCNE1/KCNQ1 [Embark et al., 2003].

In the absence of glucocorticoids little differences are observed between sgk1 ^~ ' ^' and sgk1 ^+l+ mice in gastricv H ⁺ secretion. Thus, basal H ⁺ secretion does not depend on SGK1. However, the glucocorticoid stimulation of H+ secretion is highly dependent on SGK1.

SGK1 is not only relevant for the effects of glucocorticoids, moreover SGK1 is as well up-regulated by mineralocorticoids [Chen et al., 1999; Naray-Fejes- Toth et al., 1999; Shigaev et al., 2000] and participates in the mineralocorticoid regulation of renal Na ⁺ and K ⁺ excretion. Serum glucocorticoid inducible kinases of the present invention may be used as diagnostic reagents, through detecting mutations (SNP) in the associated gene. Detection of a mutated form of the gene characterized by the polynucleotide polymorphism disclosed in Example 5 of the current application in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art.

Preferentially however, the molecular diagnosis should always be confirmed either by an upper intestinal endoscopy, which allows direct examination of the ulcer. With endoscopy, a biopsy specimen can be removed from the body for examination. The tissue will be examined under a microscope using immuno staining methods and molecular diagnosis serum of glucocorticoid inducible kinases expression to assist in diagnosis. Therefore, only very small samples are needed. Alternatively or in addition the nucleic acids for diagnosis may as well be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.

The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled SGK1 , SGK2, or SGK3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting

temperatures. DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401 ).

An array of oligonucleotides probes comprising SGK1 , SGK2 or SGK3 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Such arrays are preferably high density arrays or grids. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M. Chee et al., Science, 274, 610-613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantification of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

For example the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydridization techniques to clones arrayed on a grid, such as cDNA microarray hybridization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6,

639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.

Antibody based assay technique is another possibility that can be used to determine levels of SGK1 , SGK2 or SGK3 of the present invention. Several SGK antibodies from commercial suppliers such as Sigma or Cell Signalling Technologies are available or have been published by others. Additional antibodies against selected polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc.,

¹⁹⁸⁵⁾ -

The antibodies are useful in assays which include radio-immunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostic kit comprising:

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1 , or a fragment or an RNA transcript thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or

⁵ (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or ^ Q susceptibility to a disease, particularly diseases of the invention, amongst others.

The above-described antibodies may be employed to diagnose isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. Antibodies against polypeptides of the present

-I C invention may also be employed to treat diseases of the invention, amongst others.

EXAMPLES

20

Examples 1 : Dexamethasone treatment of mice

Mice lacking SGK1 (sgk1 ^~ ' ^~ ) 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

25 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

30 C57BL/6 blastocytes. Male chimeras were bred to 129/SvJ females. Heterozygous sg/ct-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.

For analysis of dexamethasone effects, sgk1 ^+l+ and sgk1 ^' ' ^~ 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.

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 MgCI ₂ (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 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 10s, 68°C for 10s and 72 ⁰ C for 16s, each with a temperature transition rate ⁵ of 20°C/s and a secondary target temperature of 58°C with a step size of

0.5 ⁰ C. Melting curve analysis was performed at 95°C 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 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' 15

Example 3: Gastric H ⁺ secretion

For isolation of gastric glands animals were fasted for 16 hours prior to experiments on wire grids with free access to tap water. After sacrificing the stomach was removed and cut longitudinally. After washing with standard Hepes solution the fundic region of the stomach was sliced into 0.3 cm ² sections. The tissues were transferred onto the cooled stage of a dissecting microscope and individual glands were detached from the gastric wall by snapping of the tissue using sharpened microdissection tweezers. Care was taken not to touch the apical part of the glands. The glands were attached to a glass coverslip precoated with Cell-Tak adhesive (BD Biosciences). For digital imaging of cytosolic pHj isolated individual glands were incubated in a HEPES-buffered Ringer solution containing 10 μM BCECF-AM (Molecular Probes, Leiden, The Netherlands) for 15 min at 37°C. After on loading, the chamber was flushed for 5 min with Ringer solution to remove any deesterified dye sticking to the outside of the glands. The perfusion chamber was mounted on the stage of an inverted microscope (Zeiss

Axiovert 135), which was used in the epifluorescence mode with a 40x oil immersion objective (Zeiss Neoplan, Germany). BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm using an intensified charge-coupled device camera (Proxitronic, Germany) and specialized computer software (Metafluor, USA).

Parietal cells were outlined and monitored during the course of the measurement. Intensity ratio data (490/440) were converted into pH values using the high-K ⁺ /nigericin calibration technique [Ganz et al., 1989]. Where indicated 50μM omeprazole (Astra-Zeneca Sweden) was added to the BCECF incubation medium and standard Hepes solution.

The solutions, flow lines and perfusion chamber were maintained at 37°C by a thermostatically controlled heating system. Volume of the perfusion chamber was 600 μl and the flow rate was 4 ml/min for all solutions. For acid loading, cells were transiently exposed to a solution containing 20 mM NH ₄ CI leading to marked initial alkalinization of cytosolic pH (pHj) due to entry of NH ₃ and binding of H ⁺ to form NH ₄ ⁺ [Roos and Boron 1981].The acidification of cytosolic pH upon removal of ammonia allowed to calculate the mean intrinsic buffering power of the cells [Roos and Boron 1981], assuming that NH ₄ ⁺ and NH ₃ are in equilibrium in cytosolic and extracellular fluid and that ammonia leaves the cells as NH ₃ : ^β = A[NH ₄ ⁺ VApHi . where δpHj is the decrease of cytosolic pH (pHj) following ammonia removal and A[NH ₄ ⁺ Ii the decrease of cytosolic NH ₄ ⁺ concentration, which is identical to the concentration of NH ₄ ⁺ immediately before the removal of ammonia. Given the pK for NH ₄ ⁺ /NH ₃ of 8.9 [Boyarsky et al., 1988], an extracellular pH (pHo) of 7.4 and the NH ₄ ⁺ concentration in extracellular fluid ([NH ₄ ⁺ ] _O ) of 19.37 [20/(1 +10 ^pHc>pK )]: [NH ₄ ]ι = 19.37-10 ^pHcH3Hi .

The solutions were composed of (in mM): standard Hepes 115 NaCI, 5 KCI, 1 CaCI ₂ , 1.2 MgSO ₄ , 2 NaH ₂ PO ₄ 10 glucose, 32.2 Hepes; sodium free Hepes 132.8 NMDG, 3 KCI, 1 CaCI ₂ , 1.2 MgSO ₄ , 2 KH ₂ PO ₄ , 32 Hepes, 10 Mannitol, 10 Glucose; sodium free ammonium chloride 10 mM NMDG and mannitol was replaced with 20 mM NH ₄ CI; High K ⁺ for calibration 105 KCI, 1

CaCI ₂ , 1.2 MgSO ₄ , 32.2 Hepes, 10 mannitol 5μM nigericin. The pH of the solutions was titrated to 7.4 or 7.0 with HCI/NaOH, HCI/NMDG and HCI/KOH, respectively, at 37°C.

Example 4: SGK1 modulating compounds

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

wherein

R ¹ , R ⁵ is either H, OH, OA, OAc or Methyl,

R ² , R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ is either

H, OH, OA, OAc, OCF ₃ , Hal, NO ₂ , CF ₃ , A, CN, OSO ₂ CH ₃ , SO ₂ CH ₃ , NH ₂ Or COOH, R ¹¹ H Or CH ₃ , A Alkyl with 1 , 2, 3 or 4 C-atoms,

X CH ₂ , CH ₂ CH ₂ , OCH ₂ or -CH(OH)-,

Hal F ₁ 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,

(S-Methoxy-phenyO-acidic acid^-hydroxy^-methoxy-benzylidenJ-hydrazid. Phenylacidic acid-β-fluoM-hydroxy-benzylidenJ-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-phenyO-acidic acid-ti^-hydroxy^-methoxy-phenylJ-ethyliden]- hydrazid,

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

(S.δ-Dihydroxy-phenyO-acidic acid^-hydroxy^-methoxy-benzyliden)- hydrazid, (S-Fluor-phenyO-acidic acid^S-fluor^-hydroxy-benzylidenJ-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-be nzyliden)- hydrazid, (3-Methoxy-phenyl)-acidic acid-(2,4-dihydroxy-benzyliden)-hydrazid,

(3-Methoxy-phenoxy)-acidic acid-(4-hydroxy-2-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,

(S-Methoxy-phenylJ-acidic acid^-hydroxy-δ-nitro-benzylidenJ-hydrazid,

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

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

(S-Brom-phenyO-acidic add-^-hydroxy^-methoxy-benzylidenJ-hydrazid,

(S-Methoxy-phenylJ-acidic acid-li^-hydroxy-phenyO-ethylidenl-hydrazid,

(S.S-Difluor-phenyO-acidic acid^-hydroxy^-methoxy-benzylidenJ-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

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

wherein

R ¹ , R ² , R ³ ,

R ⁴ , R ⁵ is either H, A, OH, OA, Alkenyl, Alkinyl, NO ₂ , NH ₂ , NHA, NA ₂ , Hal,

CN ₁ COOH, COOA ₁ -OHet, -O-Alkylen-Het, -O-Alkylen-NR ⁸ R ⁹ or

CONR ⁸ R ⁹ , two groups selected from R ¹ , R ² , R ³ , R ⁴ , R ⁵ or as well -0-CH ₂ -CH ₂ -, -0-CH ₂ -O- or

-0-CH ₂ -CH ₂ -O-,

R ⁶ , R ⁷ is either H, A, Hal, OH, OA or CN, R ⁸ , R ⁹ 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 ₁ CN or Carbonyloxigen (=0)

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

X, X ¹ is either NH or is missing

Hal F, Cl, Br or I

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

10

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

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

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/lpyrimidin-8-yl)-ph enyl]-3-(2,4-difluor- 15 phenyl)-urea,

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

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-o(Ipyrimidin-8-yl)-ph enyl]-3-(3-fluor-5- trifluormethyl-phenyl)-u rea ,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/lpyrimidin-8-yl)-ph enyl]-3-(4-fluor-5-

20 trifluormethyl-phenyl)-urea,

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

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

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cf]pyrimidin-8-yl)-phen yl]-3-(2-fluor-6- trifluormethyl-phenyl)-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-dlpyrimidin-8-yl)-pheny l]-3-(2-fluor-5- methyl-phenyl)-urea, 30 1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-d]pyrimidin-8-yl)-phenyl]- 3-(2,3,4-trifluor- phenyl)-urea,

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

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-o(lpyrimidin-8-yl)-phen yl]-3-(2-fIuor-3- trifluormethyl-phenyl)-urea, i-μ^^Amino-δ-oxo-δH-pyridop.S-αflpyrimiclin-δ-yO-phenyl l-S-P-CI-tert.- butyloxycarbonyl-piperidin-4-yl)-phenyl]-urea,

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

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

1.[4.(4-Amino-5-oxo-5H-pyrido[2,3-G(Ipyrimidin-8-yl)-phen yl]-3-[(2-fluor-5-(2- dimethylamino-ethoxy)-phenyl]-urea,

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

(piperidin-4-yloxy)-phenyl]-urea, 15 1 -[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/]pyrimidin-8-yl)-phenyl]- 3-[4-chlor-5- trifluormethyl-2-(piperidin-4-yloxy)-phenyl]-urea, i-^^-Amino-S-oxo-SH-pyridop.S-cdpyrimidin-δ-yO-phenylJ-S-p- Cpiperidin-

4-yloxy)-phenyl]-urea,

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

Of)

1.[4-(4-Amino-5-oxo-5H-pyrido[2,3-GGpyrimidin-8-yl)-pheny l]-3-[2-fluor-5-[2-

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

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-dlpyrimidin-8-yl)-pheny l]-3-[4-fluor-2-(2- dimethylamino-ethoxy)-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5/-/-pyrido[2,3-c/lpyrimidin-8-yl)-ph enyl]-3-[4-fluor-2-(2- „_ diethylamino-ethoxy)-phenyl]-urea,

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

(morpholin-4-yl)-ethoxy]-phenyl]-urea,

1-[4-(4-Amino-5-oxo-5H-pyrido[2,3-cdpyrimidin-8-yl)-pheny l]-3-[4-fluor-2-[2-

(morpholin-4-yl)-ethoxy]-phenyl]-urea,

1.[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/jpyrimidin-8-yl)-phen yl]-3-[3-chlor-4-(2- 30 dimethylamino-ethoxy)-phenyl]-urea,

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

1.[4-(4-Amino-5-oxo-5H-pyrido[2,3-c/lpyrinnidin-8-yl)-phe nyl]-3-[4-chlor-2-(2- dimethylamino-ethoxy)-phenyl]-urea,

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

Example 5: SGK1 nucleotide polymorphism

SGK1 nucleotide polymorphism is demonstrated by the sequences

...aattacattgCgcaacccag.., whereas the nucleotide sequence representing a

10 another population is....aattacattgTgcaacccag....Both sequences are available through accession number Gl 2463200 Position 2071. The exon 8 sequences of facultative gastric acid overproducing patients are either homozygot ..tactgaCttcggact..or....tactgaTttcggact....or heterozygot .tactgaCttcggact...and...tactgaTttcggact .. The sequences are available ₁₅ through accession number NM _005627.2, Position 777.

Further examples

SGK1 assay for screening chemical compounds

The SGK1 assay was performed with activated/phosphorylated SGK1. 20 Activated SGK1 was generated by incubating truncated SGK1 peptide (δ(1- 60)hSGK1 S422D) together with a PDK1 -derivative and ATP.

The preactivated/phosphorylated SGK1 was incubated for 60 min at room temperature together with biotinylated Crosstide peptide (biotin- KGSGSGRPRTSSFAEG) and 5 μM [ ³³ P-ATP] (50-1000 cpm/pmole) in the 25 presence or absence of compounds to be tested.

The assay buffer was 20 mM MOPS pH 7.2, 5 mM EGTA, 0.5 μM substrate peptide, 15 mM MgCb, 25 mM glycerophosphate, 1 mM Na ₃ VO ₄ , 1 mM DTT, 0.01 % Brij-35.

_O0 The reaction was stopped by addition of 200 μl of the assay buffer without ATP and aliquots of the reaction mixture have been transferred into a 96- well streptavidin coated FLASH-plate, and incubated for 20 min. Thereafter,

the solution is removed and the plate washed three times with PBS buffer. For measuring the bound radioactivity the FLASH-plates have been placed in a TOPCOUNT microtiter plate scintillation counter.

The assay formats for the investigation of the SGK-1 , SGK2- and SGK3- isoforms have been identical. Human SGK2 was purchased from Stressgen (No # PPK-455 and hSGK3 has been used a δ(1-60)hSGK3 S422D- derivative.

ELISA assay for screening selected SGK1 inhibitors

For testing SGK inhibitors at physiological ATP-concentrations, the current ELISA protocol has been employed for selected compounds.

96-wells microtiter-plates have been coated by adding 50 μl of a 0.5 μM solution of the GSK-3 fusion protein (Cell Signalling; No. 9278) in 0.2 M carbonate puffer. The MTP was incubated for 60 min at 37 ⁰ C. Thereafter, the MTP-wells have been washed 3 x with 100 μl/well of 140 mM NaCI, 10 mM NaH ₂ PO ₄ (wash buffer).

Blocking of free sides was performed adding 100 μl of the blocking buffer (wash buffer + 3 % (w/v) bovine serum albumin) to each well and incubation for 60 min at 37 °C. After washing, the kinase reaction was carried out in a total volume of 50 μl by incubation of 10 ng of hSGK (δ(1-60)hSGK1 S422D-derivative) in the presence of increasing concentrations of the test compounds at 30 - 300 μM ATP in the ADBI-buffer (ADBI-buffer: 20 mM MOPS pH 7.2, 5 mM EGTA, 15 mM MgCI ₂ , 25 mM glycerophosphate, 1 mM Na ₃ VO ₄ , 1 mM DTT ₁ 0.01 % Brij-35). The incubation is carried out for 120 min at room temperature.

After stopping the reaction the wells have been washed three an subsequently 50 μl of phosphor-GSK3-antibody solution (1 :1000 dilution of

Ser21/9 antibody, Cell Signaling #9331 L) have been added to each well.

The MTP was incubated for 30 min at 37 °C and then three times washed.

Next, 50 μl of a goat-anti-rabbit horse-radish-peroxidase (POD) conjugated antibody solution (1 :15,000 dilution in blocking buffer; Sigma #A-0545) was added to each well. After further incubation and washing the assay was developed with ABTS (2,2'-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid), (Calbiochem # 194434) and H ₂ O ₂ . The read-out was performed with a

SpectraFluor-MTP-reader (TECAN) at 405 nm. Inhibitors SGK1 as claimed in this application have been identified by a decrease in the OD in compared to the control incubations.

Screening of selected SGK1 inhibitors in HeLa-cells

NDRG1 -protein has be described as a specific substrate of the SGK1 expressed in HeLa-cells (see Murray JT, et al. Biochem. J. 2004, 384:477- 88). In the current application HeLa-cells have been used as a cellular test system for the characterization of SGK1 -inhibitors.

HeLa cells are plated in 6-wells MTPs (Costar Corning, # 3506) at a density of 10 - 20 x 10 ³ cells / cm ² in DMEM medium, supplemented with 10 % foetal calf serum (FCS), 2 mM glutamine and 1 mM sodium pyruvate have been incubated for 24 hrs at 37 ⁰ C at 5 % CO ₂ . Then serial dilutions of the compound have been added resulting in the anticipated SGK1 -inhibitor concentration at a 1 % DMSO concentration. The cells have been incubated for another 24 hrs.

Thereafter, the supernatants have been removed and the cells washed washed with BPS. To each well, 250 μl of the ice-cold lysis-buffer was added (50 mM Tris/HCI, 1 mM EDTA, 1mM EGTA, 0.5 mM activated Na ₃ VO ₄ , 10 mM glycerophosphate, 50 mM NaF, 5 mM Na-pyrophosphate, 1 % Triton X100, 1 mM DTT, 0.1 mM PMSF ₁ and 1 μM microcystin and 1 μg/ml each of aprotinin, pepstatin and leupeptin). After crapping off the cells and suspending them with a pipette cells have been homogenised and lysed. The cell lysates have been stored at - 24 ⁰ C.

16 μl aliquots of the cell lysates are transferred to 6 μl of the 4 X NuPage ^® LDS-sample buffer plus 1 μl β-mercaptoethanol and heated. The samples

have been further investigated by SDS-PAGE and Western blot analysis, using NDRG1- and P-NDRG1-antisera. The decrease in the intensity of the band on the Western blot, using the P-NDRG1- antibody was determined and used to assess the intracellular inhibitory potency of the SGK1- inhibitorsclaimed in the current application.

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FIGURE LEGENDS

Fig. 1: SGK1 Transcript levels in gastric tissue

The ratio of the transcript levels of msgki over GAPDH in gastric tissue (control, open columns) and following dexamethasone treatment (dexa, closed columns). Arithmetic means ± SEM (n = 3-7).

Fig. 2: pH recovery in parietal cells following an ammonium pulse

Alterations of cytosolic pH (δpH) in ileum following an ammonium pulse. To load the cells with H ⁺ , 20 mM NH ₄ CI were added and Na ⁺ removed (replaced by NMDG) in a first step (see bars below each original tracing), NH ₄ CI removed in a second step, Na ⁺ added in a third step and nigericin (Nig.) applied in a fourth step to calibrate each individual experiment.

A: Original tracings illustrating alterations of pH in typical experiments in sgk1 ^+/+ (left panels) and sgk1 ^v~ (right panels) in untreated mice. B: Original tracings illustrating alterations of pH in typical experiments in sgk1 ^+/+ (left panels) and sgk1 ^v~ (right panels) in dexamethasone treated mice

5 C: Original tracings illustrating alterations of pH in typical experiments in sgk1 ^+/+ (left panels) and sgkV' ^' (right panels) in dexamethasone treated mice in the presence of 50μM omeprazole

D: Arithmetic means ± SEM of Na ⁺ independent δpH in parietal cells from sgk1 ^+/+ (open columns) and sgki^ (closed columns) mice treated with dexamethasone

-jQ (+Dex) or left untreated (-Dex). Omeprazole inhibition of Na ⁺ independent δpH in parietal cells from dexamethasone treated sgk1 ^+/* and sgk1 ^v~ mice.

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