Patent Application: US-59190905-A

Abstract:
modulation of the activity of glucocorticoid inducible kinase sgk1 in pancreatic islet cells restores insulin release . also disclosed are methods and compounds useful for the treatment of glucocorticoid induced diabetes mellitus type - 2 .

Description:
the underlying mechanism of glucocorticoid induced diabetes has up to now remained elusive . in this invention it is shown that glucocorticoids such as dexamethasone up - regulate transcription and expression of the serum and glucocorticoid inducible kinase sgk1 in insulin secreting cells , an effect that can be reversed by mifepristone ( ru486 ), an antagonist of the nuclear glucocorticoid receptor . when coexpressed in xenopus oocytes sgk1 increases the activity of voltage - gated k + channel kv1 . 5 . in ins - 1 cells dexamethasone stimulates the transcription of kv1 . 5 , increases the repolarizing outward current and decreases glucose induced insulin release . the latter two effects are reversed by k + channel blockers 4 - ap and tea . dexamethasone virtually abolishes the glucose induced insulin release of islets isolated from wild type mice , an effect significantly attenuated in islets isolated from sgk1 knockout mice . in conclusion , glucocorticoids stimulate the transcription of sgk1 which in turn upregulates the activity of voltage gated k + channels . the subsequent hyperpolarisation counteracts the depolarising effect of glucose and prevents the activation of voltage gated ca 2 + channels , ca 2 + entry and insulin release . the present invention relates to the role of sgk1 and sgk1 dependent channel activity in the regulation of insulin secretion . according to real time pcr the sgk1 transcript level is low in untreated ins - 1 cells ( fig1 a ), a finding paralleling the low transcript levels reported previously for human pancreatic islets ( klingel et al ., 2000 ). however , incubation of ins - 1 cells with 100 nm dexamethasone for 2 to 23 hours increased mrna transcript levels , an effect which was completely abrogated by the glucocorticoid receptor antagonist ru486 ( fig1 a ). within 23 hours dexamethasone increased the cellular sgk1 transcript levels increased in mouse islets following treatment with dexamethasone ( fig1 a ). similarly strong stimulation of sgk1 transcription by glucocorticoids was observed in other cell types ( itani et al ., 2002 ; rozansky et al ., 2002 ). as apparent from western blotting , the sgk1 protein was not detectable in untreated cells but appeared already within 2 hours and increased further within the next 23 hours exposure to dexamethasone ( 100 nm ) ( fig1 b ). the increase in sgk1 protein abundance was fully reversed by ru486 . thus , dexamethasone stimulates the expression of sgk1 in insulin secreting cells . as shown in fig1 d , coexpression of sgk1 and kv channels in xenopus oocytes , upregulates approximately 2 - fold the activity of heterologously expressed kv1 . 5 channels ( fig1 d ). those channels have previously been shown to be expressed in ins - 1 cells ( su et al ., 2001 ) as well as rodent and human β cells ( philipson et al ., 1994 ; roe et al ., 1996 ). in ins - 1 cells the channels are inhibited by the k + channel blocker 4 - ap ( su et al ., 2001 ). as illustrated in fig2 a and 2b , treatment with dexamethasone was indeed followed by an increase in 4 - ap sensitive voltage gated outward current . in untreated cells , the k + channel blocker 4 - ap inhibited only 10 % ( 0 . 1 mm ) and 28 % ( 1 mm ) of the outward current . following a 4 hours treatment with 100 nm dexamethasone , the 4 - ap sensitive current increased to 28 % ( 0 . 1 mm 4 - ap ) and 40 % ( 1 mm 4 - ap ). these data show that kv1 . 5 channel activity is augmented by dexamethasone in insulin secreting cells . glucocorticoids have been found to increase the expression of kv 1 . 5 channels in heart ( takimoto and levitan 1994 ), in skeletal muscle and pituitary but not in hypothalamus and lung ( levitan et al ., 1996 ). furthermore , dexamethasone was necessary for t3 to increase kv1 . 5 mrna levels in the rat left ventricle from adrenalectomized animals rendered hypothyroid ( nishiyama et al ., 1997 ). real time pcr reveals that dexamethasone ( 100 nm ) treatment within 4 hours increases the abundance of kv1 . 5 mrna in ins cells by a factor of approx . 10 . thus , dexamethasone stimulates the expression of sgk1 which in turn increases kv channel activity . additional experiments were performed to elucidate the impact of kv channels and sgk1 on the blunting of insulin release by dexamethasone . as illustrated in fig3 , pretreatment of ins - 1 cells with dexamethasone ( 100 nm ) inhibited glucose - induced insulin secretion by 62 %. this inhibition was reversed by kv channel blockers , tea and 4 - ap , showing that dexamethasone mediated inhibition of insulin secretion depends on kv channel activity . to estimate the contribution of sgk1 to the inhibitory effect of dexamethasone on insulin secretion the effects of dexamethasone on insulin secretion in sgk1 knockout mice ( sgk −/− ) as compared to that in wild type littermates ( sgk1 +/+ ) was studied . without dexamethasone pretreatment insulin secretion following exposure to glucose ( 16 . 7 mm ), activation of adenylate cyclase ( 5 μm forskolin ), or stimulation of protein kinase c ( 100 nm pma ) was not significantly different in islets isolated from sgk1 −/− and from sgk1 +/+ mice ( fig4 a and b , black bars ). dexamethasone treatment , however , decreased the stimulatory effect of glucose , forskolin or pma on insulin secretion significantly more in sgk1 +/+ islets than in sgk1 −/− islets . these data indicate that sgk1 participates in the downregulation of insulin secretion by dexamethasone . in conclusion , the present experiments disclose a novel mechanism in the regulation of insulin secretion . the glucococorticoid dexamethasone enhances the transcription and expression of sgk1 in insulin secreting cells . the kinase upregulates voltage gated k + channels including kv1 . 5 . overexpression of kv channels hyperpolarizes the β - cell plasma membrane thus impeding the activation of voltage gated ca 2 + channels . accordingly , the kinase contributes to the inhibition of insulin release during glucocorticoid excess . fig1 : dexamethasone induces the expression of sgk1 in insulin secreting ins - 1 cells . ins - 1 cells were treated with 100 nm dexamethasone or vehicle ( dmso ) in culture for the indicated periods of time . dexamethasone significantly induced expression of sgk1 within 2 hours . ru486 at 1 μmol / l completely inhibited the dexamethasone effect . ( a ) cellular rna was transcribed into cdna using reverse transcriptase m - mulv ( roche diagnostics gmbh , roche applied science , mannheim , germany ). sgk1 mrna was quantified by real time pcr in a light cycler system ( roche diagnostics gmbh , roche applied science , mannheim , germany ). the primers used were : sgk1 up : 5 ′- ttt ttt ttc cca acc ctt gc - 3 ′; down : 5 ′- aat gaa caa agg ttg ggg gg - 3 . shown are mean ± sem of the indicated number of experiments . ( b ) whole cell lysates were subjected to 1 % sds - page and plotted onto a nitrocellulose membrane ( schleicher and schuell , dassel , germany ). plots were incubated with antibodies against sgk1 ( new england biolabs , beverly , mass ., usa ). bound antibody was visualized using a second antibody coupled to horse radish peroxidase . ( c ) real time pcr for kv1 . 5 was performed using a light cycler system ( roche diagnostics gmbh , roche applied science , mannheim , germany ). the same rna preparations as for the experiments described in fig1 a were analyzed . shown are mean ± sem of 3 independent experiments . ( d ) sgk1 and kv channel coexpression in xenopus oocytes increases k + currents . mrna for human sgk1 ( μg / ml ) and kv 1 . 5 ( μg / ml ) was injected into oocytes and whole cell currents measured using the 2 - voltage clamp method 2 days after injection . shown are representative traces and mean ± sem . fig2 : dexamethasone augments kv channel activity in ins - 1 cells . cells were treated prior to the experiment with 100 nm dexamethasone for 4 h . ( 2 a ) whole cell current was induced by 200 ms voltage pulses increasing by 10 mv steps from − 70 mv to + 50 mv . ( 2 b ) sensitivity to 4 - ap ( 0 . 1 and 1 mm ) and tea ( 1 and 10 mm ) was tested in cells before ( black columns ) and after dexamethasone treatment ( white columns ). voltage pulses of 200 ms duration from − 70 to 50 mv were applied . shown are means ± sem for the indicated number of experiments . * denotes significance ( p & lt ; 0 . 05 ) to current in control , non treated cells at the same inhibitor concentration . ins - 1 cells were cultured as described before ( abel et al ., 1996 ; asfari et al ., 1992 ). the external patch clamp solution contained ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 0 . 5 glucose and 10 hepes , ph 7 . 4 . the internal solution contained ( in mmol / l ): 30 kcl , 95 k + - gluconate , 1 mgcl 2 , 1 . 2 nah 2 po 4 , 4 . 8 na 2 hpo4 , 5 na 2 atp , 1 na 3 gtp , 5 mmol / l egta , ph 7 . 2 . an epc9 patch clamp amplifier ( heka electronic , lambrecht , germany ) was used for current measurements . fig3 : kv channel inhibition reverses dexamethasone - induced inhibition of insulin secretion of ins - 1 cells . prior to the experiment ins - 1 cells were treated in culture with dexamethasone , 100 nm , for 4 h . cells were washed twice and preincubated in hepes buffered salt solution containing ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 0 . 5 glucose , 10 hepes and 5 g / l bovine serum albumin , ph 7 . 4 at 37 ° c . thereafter cells were incubated for 30 min at 37 ° c . in fresh solution containing the test substances at the appropriate concentrations . insulin was measured by radioimmunoassay using rat insulin antiserum ( linco , biotrend chemikalien gmbh , cologne , germany ), i 125 - insulin ( cis diagnostik gmbh , dreieich , germany ) and rat insulin ( novo nordisk , mainz , germany ) as standard or by an insulin elisa kit ( mercodia , uppsala , sweden ). fig4 a and b : dexamethasone did not affect secretion from islets of sgk1 knockout mice . isolated islets were cultured over night in rpmi 1640 containing 11 mmol / l glucose . dexamethasone ( 100 ng / ml ) or dmso ( control ) were added 5 h before the experiment . after culture islets were preincubated for 1 h at 37 ° c . in incubation buffer containing ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 2 . 8 glucose , 10 hepes , ph 7 . 4 and 5 g / l bovine serum albumin ( fraction v , sigma , deisenhofen ). thereafter , batches of 5 islets / 0 . 5 ml were incubated for 30 min at 37 ° c . in the presence of test substances as indicated for each experiment . insulin was measured using an elisa kit ( mercodia , uppsala , sweden ). a conditional targeting vector was generated from a 7 - kb fragment encompassing the entire transcribed region on 12 exons ( wulff et al ., 2002 ). 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 . 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 c57bl / 6 and 129 / svj females . heterozygous sgk1 - deficient mice were backcrossed to 129 / svj wild - type mice for two generations and then intercrossed to generate homozygous sgk1 −/− and sgk1 +/+ littermates . ins - 1 cells ( kindly provided by cb wollheim , university of geneva , switzerland ) derived from a rat insulinoma were cultured in hepes - buffered rpmi 1640 supplemented with 10 % fetal calf serum ( biochrom , berlin , germany ), 1 mmol / l hepes , 1 mmol / l na pyruvate , 10 μmol / l β - mercaptoethanol ( sigma , munich , germany ) and antibiotics as described elsewhere ( abel et al ., 1996 ; asfari et al ., 1992 ). cells were seeded at a cell density of 2 . 0 - 2 . 5 10 5 cells / ml in 24 - well culture plates and cultured for 2 days prior to the experiment . cells were washed twice with hepes buffered salt solution containing ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 0 . 5 glucose , 10 hepes and 5 g / l bovine serum albumin , ph 7 . 4 . and preincubated for 30 min at 37 ° c . thereafter medium was discarded and fresh medium containing the test substances at the appropriate concentrations added . cells were incubated for 30 min at 37 ° c . incubations were stopped on ice , medium removed and frozen at − 20 ° c . until insulin released into the supernatant was measured by radioimmunoassay using rat insulin antiserum ( linco , biotrend chemikalien gmbh , cologne , germany ), i 125 - insulin ( cis diagnostik gmbh , dreieich , germany ) and rat insulin ( novo nordisk , mainz , germany ) as standard or an insulin elisa kit ( mercodia , uppsala , sweden ). insulin content was measured after extraction with acid ethanol ( 1 . 5 ( v / v ) % hcl / 75 % ethanol ) over night at 4 ° c . for isolation of islets from sgk1 ko and wild type littermates mice 3 ml of collagenase solution containing 1 mg / ml collagenase ( serva , heidelberg , germany ) was injected into the pancreas in situ through the ductus coledochus . the entire gland was removed and digested for 10 min at 37 ° c . thereafter the islets were isolated from the exocrine tissue by collecting them into fresh medium under a dissection microscope . islets were cultured over night in rpmi 1640 containing 11 mmol / l glucose and dexamethasone ( 100 ng / ml ) or dmso ( control ). after culture islets were preincubated for 1 h at 37 ° c . in incubation buffer containing ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 2 . 8 glucose , 10 hepes , ph 7 . 4 and 5 g / l bovine serum albumin ( fraction v , sigma , deisenhofen ). thereafter , batches of 5 islets / 0 . 5 ml were incubated for 30 min at 37 ° c . in the presence of test substances as indicated for each experiment . insulin was measured using an elisa kit ( mercodia , uppsala , sweden ). ins - 1 cells were cultured for 2 - 4 days on glass cover slips coated with poly - l - ornithine ( 10 mg / l sigma , munich , germany ) at appropriate cell densities ( 1 . 2 × 10 6 cells / ml ). the cover slips were mounted in a bath chamber on the stage of an inverted microscope ( im , zeiss , jena , germany ). the cells were kept at room temperature or at 34 ° c . as indicated for each experiment and superfused with a solution containing ( in mmol / l ): 140 nacl , 5 . 6 kcl , 1 . 2 mgcl 2 , 2 . 6 cacl 2 , 0 . 5 glucose and 10 hepes , ph 7 . 4 . the patch clamp pipettes ( clark - medical , reading , great britain ) with a resistance of 4 - 6 mω were pulled using a dmz - universal puller ( zeitz , augsburg , germany ). they were filled with an internal solution containing ( in mmol / l ): 30 kcl , 95 k + - gluconate , 1 mgcl 2 , 1 . 2 nah 2 po 4 , 4 . 8 na 2 hpo 4 , 5 na 2 atp , 1 na 3 gtp , 5 mmol / l egta , ph 7 . 2 . an epc9 patch clamp amplifier ( heka electronic , lambrecht , germany ) was used for current measurements . only stable current measurements , i . e . when currents reached at least 90 % of control current after removal of the respective inhibitory drug , were used for analysis . ins - 1 cells were cultured in 70 cm 2 flasks , medium was removed and 600 μl of lysis buffer ( mini kit , qiagen , hilden , germany ) added . lysed cells were scraped and the lysate collected into an eppendorf tube . cellular rna was isolated using the qiagen mini kit and 2 μg of rna transcribed into cdna using reverse transcriptase m - mulv ( roche diagnostics gmbh , roche applied science , mannheim , germany ). an aliquot of cdna , corresponding to the amount of rna as indicated in each experiment was used for quantification of mrna by real time pcr using a light cycler system ( roche diagnostics gmbh , roche applied science , mannheim , germany ) with specific primers for rat kv1 . 5 channel , sense : 5 ′- atc ttc aag ctc tcc cgc cac tcc aag gg - 3 ′; antisense : 5 ′- ggg tta tgg aaa gag gag tta - 3 ′. the primers of rat sgk1 used were : sense : 5 ′- ttt ttt ttc cca acc ctt gc - 3 ′; antisense : 5 ′- mt gm cm agg ttg ggg gg - 3 . isolated mouse islets were cultured and treated with dexamethasone as indicated . thereafter the islets were collected and lysed in lysis buffer ( mini kit , qiagen , hilden , germany ) and by repeatedly sucking of the islets into an insulin syringe . ins - 1 cells were cultured in 70 cm 2 flasks without ( control ) or with 100 ng / ml dexamethasone for the indicated period of time . thereafter , culture medium was removed and cells were lysed in a solution containing 300 mm nacl , 20 mm tris hcl , ph 7 . 4 , 1 % ( v / v ) triton x - 100 , 1 % sodiumdeoxycholate , 0 . 1 % sds , 2 . 5 mm edta , 10 μg / ml pepstatin a , 10 μg / ml aprotinin and 0 . 1 mm pmsf . total cellular protein , 50 μg , quantified by coomassie blue g staining ( bradford dye assay , biorad laboratories gmbh , munich , germany ) was subjected to sds - page ( 1 %), and plotted onto a nitrocellulose membrane ( schleicher and schuell , dassel , germany ). plots were incubated with antibodies against sgk1 ( new england biolabs , beverly , mass ., usa ). bound antibody was visualized using a second antibody coupled to horse radish peroxidase . 6 . 1 . compounds of the general formula i and pharmaceutical useful derivates , salts , solutions and stereoisomeres thereof including mixtures . 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 , 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 , ( 3 - methylsulfonyloxy - phenyl )- 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 )- propionsäure -( 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 , ( 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 , ( 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 . 2 . compounds of the general formula ii and pharmaceutical useful derivates , salts , solutions and stereoisomeres thereof including mixtures . r 4 , r 5 is either h , a , oh , oa , alkenyl , alkinyl , no 2 , nh 2 , nha , na 2 , hal , cn , cooh , cooa , two groups selected from r 1 , r 2 , r 3 , r 4 , r 5 or as well — o — ch 2 — ch 2 —, — o — ch 2 — o — or — o — ch 2 — ch 2 — o —, r 6 , r 7 is either h , a , hal , oh , oa or cn , 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 (═ o ) a alkyl with 1 to 10 c - atoms , wherein 1 - 7h - atoms may be replaced by f and / or chlorine , compound according to formula ii selected from the following group of compounds : 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 - fluor - 5 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 4 - chlor - 5 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 , 4 - difluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 , 6 - difluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 3 - fluor - 5 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 4 - fluor - 5 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 4 - methyl - 5 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 , 3 , 4 , 5 , 6 - pentafluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 , 4 - dibrom - 6 - fluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 - fluor - 6 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 - fluor - 5 - methyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 , 3 , 4 - trifluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 4 - brom - 2 , 6 - difluor - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -( 2 - fluor - 3 - trifluormethyl - phenyl )- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 2 -( 1 - tert .- butyloxycarbonyl - piperidin - 4 - yl )- phenyl ]- harnstoff , n -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 2 , 4 - dichlor - benzamid , n -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 4 - chlor - 5 - trifluormethyl - benzamid , n -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 2 - fluor - 5 - trifluormethyl - benzamid , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 3 - chlor - 5 - trifluormethyl - 2 -( piperidin - 4 - yloxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[( 2 - fluor - 5 -( 2 - dimethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 5 - fluor - 2 -( piperidin - 4 - yloxy )- phenyl ]- harnstoff , 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 ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 2 -( piperidin - 4 - yloxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 2 - fluor - 5 -( 2 - diethylamino - ethoxy )- phenyl ]- harnstoff , 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 ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 4 - fluor - 2 -( 2 - dimethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 4 - fluor - 2 -( 2 - diethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 3 - chlor - 4 -[ 2 -( morpholin - 4 - yl )- ethoxy ]- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 4 - fluor - 2 -[ 2 -( morpholin - 4 - yl )- ethoxy ]- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 3 - chlor - 4 -( 2 - dimethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 3 - chlor - 4 -( 2 - diethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 4 - chlor - 2 -( 2 - dimethylamino - ethoxy )- phenyl ]- harnstoff , 1 -[ 4 -( 4 - amino - 5 - oxo - 5h - pyrido [ 2 , 3 - d ] pyrimidin - 8 - yl )- phenyl ]- 3 -[ 2 - chlor - 5 -( 2 - diethylamino - ethoxy )- phenyl ]- harnstoff , sowie ihre pharmazeutisch verwendbaren derivate , solvate , salze , tautomere und stereoisomere , einschlieβlich deren mischungen in alien verhältnissen . the nucleotide sequence defining intron 6 of facultative hypertensive patients is . . . aattacattg c gcaacccag . . . , whereas the nucleotide sequence representing a healthy population is . . . aattacatt t gcaacccag . . . . both sequences are available through accession number gi 2463200 position 2071 . the exon 8 sequences of facultative hypertensive patients are either homozygotic . . . tactga c ttcggact . . . or . . . tactgatttcggact . . . or heterozygotic . tactga c ttcggact . . . and . . . tactgatttcggact . the sequences are available through accession number nm — 005627 . 2 , position 777 . a homozygotic individual with a tt nucleotide combination is protected even if simultaneously a cc single nucleotide polymorphism is presented in intron 6 . data are presented as mean ± sem . anova for multiple groups and student &# 39 ; s t - tests were used for statistical analysis . p values & lt ; 0 . 05 were accepted to indicate statistical significance . böhmer , c ., wagner , c . a ., beck , s ., moschen , i ., meizig , j ., werner , a ., lin , j .- t ., lang , f ., wehner , f . the shrinkage - activated na + conductance of rat hepatocytes and its possible correlation to renac . cell phys biochem . 2000 ; 10 : 187 - 194 . brenan f e , fuller p j . rapid upregulation of serum and glucocorticoid - regulated kinase ( sgk ) gene expression by corticosteroids in vivo . mol cell endocrinol . 2000 ; 30 ; 166 : 129 - 36 . busjahn a , aydin a , uhlmann r , feng y , luft f c , lang f . serum - and glucocorticoid - regulated kinase ( sgk1 ) gene and blood pressure . hypertension 40 ( 3 ): 256 - 260 , 2002 chen s y , bhargava a , mastroberardino l , meijer o c , wang j , buse p , firestone g l , verrey f , pearce d : epithelial sodium channel regulated by aldosterone - induced protein sgk . proc natl acad sci usa 1999 ; 96 : 2514 - 2519 . cowling r t , birnboim h c . expression of serum - and glucocorticoid - regulated kinase ( sgk ) mrna is up - regulated by gm - csf and other proinflammatory mediators in human granulocytes . j leukoc biol . 2000 ; 67 : 240 - 248 . de la rosa d a , zhang p , naray - fejes - toth a , fejes - toth g , canessa c m : the serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of xenopus oocytes . j biol chem 1999 ; 274 : 37834 - 37839 . hoogwerf b , danese r d : drug selection and the management of corticosteroid - related diabetes mellitus . rheum dis clin north am 1999 ; 25 : 489 - 505 . klingel k , wärntges s , bock j , wagner c a , sauter m , waldegger s ., kandolf r , lang f . expression of the cell volume regulated kinase h - sgk in pancreatic tissue . am j physiol ( gastroint . liver - physiol .) 2000 ; 279 : g998 - g1002 . kobayashi t , cohen p : activation of serum - and glucocorticoid - regulated protein kinase by agonists that activate phosphatidylinositide 3 - 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f619 . staessen j a , wang j , bianchi g , birkenhager w h . essential hypertension . lancet . 2003 ; 361 : 1629 - 1641 . wagner c a , ott m , klingel k , beck s , melzig j , friedrich b , wild n k , bröer s , moschen i , albers a , waldegger s , tumler b , egan e , geibel j p , kandolf r , lang f . effects of serine / threonine kinase sgk1 on the epithelial na + channel ( enac ) and cftr . cell physiol biol 2001 ; 11 : 209 - 218 . warnock d g . liddle syndrome : genetics and mechanisms of na + channel defects . am j med sci . 2001 ; 322 : 302 - 307 . webster m k , goya l , firestone g l : immediate - early transcriptional regulation and rapid mrna turnover of a putative serine / threonine protein kinase . j biol chem 1993a ; 268 : 11482 - 11485 . webster m k , goya l , ge y , maiyar a c , firestone g l : characterization of sgk , a novel member of the serine / threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum . mol cell biol 1993b ; 13 : 2031 - 2040 . 6 . 2 . compounds of the general formula ii and pharmaceutical useful derivates , salts , solutions and stereoisomeres thereof including mixtures . r 4 , r 5 is either h , a , oh , oa , alkenyl , alkinyl , no 2 , nh 2 , nha , na 2 , hal , cn , cooh , cooa , two groups selected from r 1 , r 2 , r 3 , r 4 , r 5 or as well — o — ch 2 — ch 2 —, — o — ch 2 — o — or — o — ch 2 — ch 2 — o —, r 6 , r 7 is either h , a , hal , oh , oa or cn , 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 (═ o ) a alkyl with 1 to 10 c - atoms , wherein 1 - 7h - atoms may be replaced by f and / or chlorine , compound according to formula ii selected from the following group of compounds :