Patent Application: US-32291405-A

Abstract:
the present invention relates to the use of pteridine derivatives as nitric oxide synthase activators . in particular , the derivatives find use in the treatment of diseases associated with endothelial dysfunction such as cardiovascular diseases .

Description:
compounds used in accordance with the present invention were synthesised according to known procedures as set out in al - hassan et . al ., j chem . soc . perkin trans i , 1985 , pp 1645 - 1659 ; cameron et . al ., j . chem . soc . perkin trans . i , 1985 , pp 2133 - 2143 ; and al - hassan et . al . j . chem . soc . perkin trans . i , 1985 , pp 2145 - 2150 all of which are incorporated herein by reference . table 1 lists a number of compounds which were synthesised and tested according to the following procedures . rings ( 5 mm long ) of rat pulmonary artery and aorta , were supported on a pair of intraluminal wires at 1 gm tension for one hour and then contracted with their ec 50 phenylephrine concentration , which for pulmonary artery was 3 . 6 × 10 − 8 m ( derived from preliminary experiments ) and for aorta was 11 . 8 × 10 − 8 m ( from the previously published literature ). the rings were then relaxed with carbachol ( 10 μm ). if the relaxation to carbachol was & gt ; 75 %, then the tissues were used to generate dose responses by addition of tetrahydrobiopterin ( bh 4 ) or the analogues . stock bh 4 was stored at − 20 ° c . and the dilutions were prepared using saline just before adding the drug into the bath . solutions of analogues were stored at 4 ° c . cumulatively increasing concentrations of the analogues were added to the bath and the extent of relaxation was measured when it had stabilised . another ring from the same rat was treated in parallel with the solvent for the analogue . the solvent used was nh 4 oh . the results are shown graphically in fig1 and 2 . effects on bh 4 depleted rat pulmonary artery and aorta relaxation rat pulmonary artery or aorta rings ( as above ) were placed in 15 ml tubes containing 2 - 3 ml of mem ( minimal essential medium ) solution containing 0 . 1 % bovine serum albumin , 100 u / ml penicillin and 100 μm / ml streptomycin . some of the tubes in addition received diamino - hydroxy - pyrimidine ( dahp ) 10 − 2 m , an inhibitor of gtp cyclohydrolase , the rate - limiting step in tetrahydrobiopterin synthesis . the tubes were incubated at 37 ° c . for six hours . this treatment has been demonstrated to deplete endogenous tetrahydrobiopterin to 4 % of the control level in canine basilar artery . after the incubation with dahp or the control incubation with the medium ( mem ) alone , the rings were removed and then their relaxation in response to the analogue ( or solvent nh 4 oh ) was tested as above . dose response curves of dahp - treated tissues were compared to the control tissues treated in mem alone for 6 hrs . the results are shown graphically in fig3 and 4 . further experiments demonstrated that the arterial relaxation was caused by no formation , since this action was inhibited by the no synthase blocker , l - name ( fig5 ). thus wsg1002 is acting to enhance the ability of the endothelium to form no and is not acting merely as a no donor . the above procedures were used to mimic cardiovascular disease , by treating aorta and pulmonary artery rings with an inhibitor of the enzyme gtp cyclohydrolase , which is the rate - limiting step in the formation of tetrahydrobiopterin by cells . treatment with this inhibitor , dahp , for 6 hours reduced endogenous tetrahydrobiopterin levels in arteries to 4 % of the normal content . in dahp - treated artery rings , addition of the compound having reference code wsg1002 caused a significant and physiologically important vasodilatation . this was found in both pulmonary artery and aorta ( fig3 and 4 ). in contrast , no action was found in arteries that had not been depleted of their tetrahydrobiopterin , thus demonstrating that this action is specific for diseased arteries in this model . from this result , wsg1002 appears to be able to cross the cell membrane and enter the cells in the artery , and to substitute for the normal cofactor ( tetrahydrobiopterin ) in the formation of nitric oxide . wsg1002 was superior to tetrahydrobiopterin itself . addition of tetrahydrobiopterin did not produce any relaxation of rat aorta or rat pulmonary artery rings ( fig6 ). the only effect seen was a contraction — the opposite to the action of wsg1002 — at the highest concentration of tetrahydrobiopterin . the superior action of wsg1002 in comparison with tetrahydrobiopterin is probably due to improved cell permeation , stability and affinity for the target site . wsg1002 has an ec 50 of approximately 10 nm . the arterial assay is sensitive but slow to perform , and therefore a wider range of compounds was investigated in whole cell preparations . tests have been carried out using endothelial cells that produce enos and stimulated macrophages that produce inos . this is a cell based assay with lipopolysaccharide stimulated macrophages in which nitric oxide is produced by the action of inos . primary cultures of macrophage cells isolated from mouse femur were produced over a 7 day period . at this stage the cells were seeded into 96 - well culture plates at a density of 2 × 10 5 cells per well . after further incubation for 24 hrs , the well media was changed and the relevant treatment was introduced . the cells were then incubated for a further 72 hrs before 50 μl was removed from each well and combined with 100 μl of greiss reagent for no 2 determination . all treatments were performed in triplicate . in each plate , 3 wells were designated control i . e . media change only - no lps . these samples represent basal no 2 output over 72 hrs . 3 wells were used for lps ( 0 . 5 μg / ml ) stimulation alone and 3 wells were used for lps + 0 . 05 % dmso . this dmso concentration was present in all subsequent analogue treated wells . all analogues were tested at 1 . 0 and 10 μm . the results are shown in fig7 . in another set of experiments , the assay was repeated with the inclusion of a step to deplete endogenous tetrahydrobiopterin as in the artery ring studies . dahp pre - treatment was employed to deplete the macrophage cells of endogenous tetrahydrobiopterin . in these experiments , dahp ( 10 − 2 m ) was added to the cells for 24 hrs prior to seeding into 96 - well plates and for 24 hrs following the seeding procedure . thereafter , dahp was present throughout the analogue treatment period of 72 hrs . the results are shown in fig8 . endothelial cells were isolated from porcine pulmonary artery and grown to confluency in t75 flasks . subsequently , the cells were seeded into 6 - well plates . at confluency , the medium ( 1 ml / well ) was changed and the relevant treatment introduced to all wells in each plate ( one treatment / plate ). the cells were incubated for a further 7 - day period and the experiment was terminated . the media from the wells of respective plates was combined (˜ 6 mls ) and evaporated to approximately 500 μl (˜ 10 - fold ). a 50 μl aliquot was removed and any no 3 was converted to no 2 using the no 3 conversion method ( calbiochem cat no 482702 ). these treated samples were then assayed using the greiss reaction as described above giving the content of the combination of ( no 2 + no 3 ). wsg1002 , as a representative compound , was incubated with a preparation of endothelial cells over seven days in order to accumulate sufficient nitric oxide for assay as described above . controls included vehicle ( 0 . 5 % dmso ) and l - name ( an established nos inhibitor ). wsg1002 caused a doubling of nitric oxide compared with vehicle control . these results , which are shown in fig9 , confirm the ability of wsg1002 to act as a stimulator of nitric oxide production . whilst the use of a gtp cyclohydrolase i inhibitor such as dahp can reduce tetrahydrobiopterin levels sufficiently to establish effects of test compounds , as shown in the tissue assays described above , it cannot completely remove endogenous tetrahydrobiopterin . macrophages were initially depleted of tetrahydrobiopterin by incubation with dahp . under these conditions , it was possible however to measure both promotion and inhibition of nitric oxide production in these cells . the activity shown in cells after treatment with dahp can be ascribed to residual endogenous tetrahydrobiopterin in the cells . fig8 shows the behaviour of wsg1001 and wsg1002 in this assay . the increases were statistically significant . the activity for all compounds tested is summarised in table 1 in which p = promotes nos activity , when tested at 1 microm . rats were maintained in a chamber containing atmospheric air at 550 mbar for 14 days . these animals were confirmed to have pulmonary hypertension , as demonstrated by right ventricular hypertrophy and increased lung perfusion pressure . age - matched control animals were maintained at normal atmospheric pressure , and these did not have pulmonary hypertension . a group of normotensive and a group of pulmonary hypertensive rats received daily subcutaneous injections of wsg1002 in a dose of 14 . 1 mg / kg / day during the time that they were in the hypoxic chamber . other groups of normotensive and pulmonary hypertensive rats had no drugs and are used as control comparison groups . these studies used lungs from the four groups of rats ( 1 ) pulmonary hypertensive and treated with wsg1002 , ( 2 ) normotensive and treated with wsg1002 , ( 3 ) pulmonary hypertensive and not treated with wsg1002 ( 4 ) normotensive and not treated with wsg1002 . effects on calcium ionophore - induced pulmonary vasodilation — administration of wsg1002 to pulmonary hypertensive rats improves pulmonary endothelium function : the rats lungs were perfused at constant flow through the pulmonary artery with measurement of perfusion pressure and maintained at 37 ° c . the perfusate was 30 ml of krebs - henseleit solution supplemented with albumin and flurbiprofen and was recirculated . the lungs were also ventilated with either normoxic gas ( 20 % o 2 , 5 % co 2 and 75 % n 2 ) or hypoxic gas ( 0 % o 2 and 100 % n 2 ) during hypoxic challenge . the perfusate was equilibrated with the same gas mixture as was used for ventilation . the lungs were precontracted with the thromboxane a 2 mimetic , 9 , 11 - dideoxy - 11 α , 9 α - epoxymethanoprostaglandin f 2α ( u46619 , 3 × 10 − 5 m ) given as a bolus dose via an injection port . then the gas supply was changed from 20 % o 2 to 0 % o 2 for 10 minutes to record hypoxic vasoconstriction . following return to 20 % o 2 , the lungs were precontracted again with a bolus dose of u46619 and relaxation responses to the endothelium - dependent vasorelaxant , calcium ionophore were measured by adding it in increasing doses to the perfusate . the data was plotted as concentration - response curves from which ic 50 values were calculated using biograph program by fitting the curves to the hill equation y = rmax /( 1 +( x / ec 50 ) ˆp ). pulmonary vasodilation produced by the endothelium - dependent vasorelaxant calcium ionophore , were greater in rats that had received treatment with wsg1002 , compared to untreated rats ( fig1 and 11 ). wsg1002 improved endothelium - dependant relaxation in both pulmonary hypertensive and normotensive rats . the ic 50 values for calcium ionophore in pulmonary hypertensive rats was reduced from 1 . 7 μm to 1 . 2 μm ( p ≦ 0 . 01 ) and in the normotensive control group the ic 50 was reduced from 1 . 5 μm to 1 . 1 μm ( p ≦ 0 . 05 ). effect of administration of wsg1002 to pulmonary hypertensive rats — enos in pulmonary vascular endothelium is increased : the rat lungs were fixed with 4 % formaldehyde , wax embedded , and 3 μm thick sections were cut . the sections were mounted on silanated slides , rehydrated and treated with 0 . 3 % h 2 o 2 to block endogenous peroxidase activity . antigens were unmasked by treatment in a microwave oven . the sections were then treated with 20 % normal goat serum followed by incubation with anti - enos mouse monoclonal antibody at a dilution of 1 : 2000 for 1 hr at room temperature . then biotinylated secondary antibody was added to the sections followed by streptavidin - horse raddish peroxidase complex and then 3 , 3 - diaminobenzidinetetrahydrochloride . the sections were then counter - stained with haematoxylin to locate the cell nuclei . the slides were then dehydrated to xylene and mounted in dpx . slides were coded and viewed under magnification 400 × to identify pulmonary arteries (≦ 200 μm diameter ). in the endothelium , the intensity of staining was quantified on a scale of 0 - 3 , where 0 = no staining or the same as the negative control , 1 = faint staining or staining in a few target cells , 2 = moderate staining in most target cells , 3 = the maximum staining observed with that antibody in the positive control slides . the endothelium of small pulmonary arteries stained weakly for enos in normotensive ( control ) rats , however this was markedly increased in pulmonary hypertensive rats ( fig1 ). rats that had been treated with wsg1002 had increased staining for enos in normotensive rats , and there was no further increase when the rats were made pulmonary hypertensive ( fig1 ). 1 . presta a , siddhanta u , wu , c , sennequier n , huang l , abu - 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