cho - wt cells : cho ( chinese hamster ovary ) cells are fibroblasts transfected with the wild - type cftr gene ( cftr - wt ). these cells will therefore overexpress the cftr protein . culture medium : mem alpha medium ( gibco )+ 7 % foetal calf serum + 0 . 5 % penicilline / streptomycin + 100 μm methotrexate ( amethopterine , sigma ). cf15 cells : cf15 cells are human nasal epithelial cells expressing the δf508 - cftr gene . culture medium : dmem + ham f12 medium + 10 % fcs + 0 . 6 % penicilline / streptomycin + growth factors ( 5 μg / ml insulin , 5 μg / ml transferrin , 5 . 5 μm epinephrin , 0 . 18 mm adenine , 10 ng / ml egf , 2 nm t3 , 1 . 1 μm hydrocortisone ). calu - 3 cells : calu - 3 cells are human lung epithelial cells expressing the wild - type cftr gene . culture medium : dmem / f12 medium with glutamax + 7 % foetal calf serum + 1 % penicillin / streptomycin . immunolabelling allows visualising the cellular localisation of the cftr protein by means of an anti - cftr primary antibody ( ab ), and a secondary cy3 - labelled fluorescent antibody directed against the primary antibody . the cells are first seeded on cover slips in an appropriate culture medium . the cells are then washed 3 times with tbs ( 157 mm nacl , 20 μm tris base , ph 7 . 4 ) for 5 min . each time and then fixed by adding 3 % tbs - paraformaldehyde for 20 min . after 3 washes with tbs ( 5 min . each ), the cells are incubated with 0 . 1 % tbs - triton ( 10 min ) to make holes in the cell membrane , and then washed 3 times with tbs before being exposed to 0 . 5 % tbs - bsa - 0 . 05 % saponin for 1 hr . the cells are then incubated with the primary anti - cftr c terminal antibody ( 2 μg / ml ) for 1 hr . the cells are washed 3 times ( 5 min . each ) with tbs - bsa - saponin before incubating with the gam - cy3 secondary antibody ( 1 / 400 ) for 1 hr . after 2 tbs washes ( 5 min . each ), the nuclei are labelled by incubating with topro3 ( 1 / 1 , 000 ) for 5 min . finally , the cover slips can be mounted on a glass slide after 3 final tbs washes ( 5 min . each ). the slides are analysed with a confocal microscope ( bio - rad ) using laser excitation at appropriate wave - lengths . in order to differentiate cy3 from topro3 labelling , the colour of the topor3 fluorescence has been changed to blue ( colour of the nuclei ). measurements of chloride ion transport in the cells were performed using the radioactive iodide efflux technique ( becq and al ., 1999 ; dormer and al ., 2001 ). the 125 i tracer is incorporated into the intracellular milieu . the quantity of radiotracer coming out of the cell is then measured after adding various pharmacological agents . iodide is used as a tracer of chloride ion transport . 125 i has the advantage of being short - lived compared to other markers such as 35 ci ( respective half - lives : 30 days and 300 , 000 years ). the cells are incubated in an appropriate medium in 24 - well plates . the cells are rinsed twice with efflux medium ( 136 . 6 mm nacl , 5 . 4 mm kcl , 0 . 3 mm kh 2 po 4 , 0 . 3 mm nah 2 po 4 , 4 . 2 mm nahco 3 , 1 . 3 mm cacl 2 , 0 . 5 mm mgcl 2 , 0 . 4 mm mgso 4 , 10 mm hepes , 5 . 6 mm d - glucose ) in order to eliminate dead cells , which release radioactivity in an anarchic fashion . the cells are then incubated with a 500 - μl load ( 1 μci / ml 125 ina ) for 30 min for cho - wt or 1 hr for cf15 and calu - 3 cells . the iodide equilibrates on either side of the cell membrane . the following steps are performed using a multiprobe probe ( packard ): the loading medium is rinsed with efflux medium in order to eliminate extracellular radioactivity . the supernatant is collected every minute into haemolysis tubes and the medium is replaced by an equivalent volume of medium ( 500 μl ). no drug is added to the samples taken in the first three minutes in order to obtain a stable baseline characterising the passive exit of i ions . the 7 samples that follow are obtained in the presence of the molecule to be tested . at the end of the experiment , the cells are lysed by adding 500 μl of 0 . 1 n naoh / 0 . 1 % sds ( 30 min ), which allows determining the level of radioactivity remaining inside the cell . the radioactivity present in the haemolysis tubes is measured as counts per minute ( cpm ) using a cobra ii gamma counter ( packard ). the results ( cpm ) are expressed as velocity of radioactive iodide efflux ( r ) according to the formula : r ( min − 1 )=[ in ( 125 i t 1 )− in ( 125 i t 2 )]/( t 1 − t 2 ) where 125 i t 1 = at time t 1 and 125 i t 2 = at time t 2 . this iodide flux is represented by a graph . in order to quantify the iodide efflux due to administration of the tested molecule , the relative flux is calculated as follows in order to ignore the basal flux : relative velocity ( min − 1 )= rpeak − rbasal . finally , these results are normalised in order to compare the effect of one drug versus another . the results are presented in the form of a mean +/− sem . student &# 39 ; s statistical test is used to compare the effect of the various drugs with the controls ( the corresponding p & lt ; 0 . 01 values are considered to be statistically significant ). the mtt cytotoxicity test is a calorimetric test based on the capacity of mitochondrial dehydrogenases to metabolise mtt ( a yellow tetrazolium salt ) into formazan ( purple ). the absorbance is proportional to the concentration of converted dye and can then be measured by spectrophotometry . the cells are incubated in 96 - well microplates in the presence of the agent to be tested for 2 hours . three controls are performed : 100 % live cells ( cells without any agent ); 0 % live cells ( cells left under normal atmosphere ); blank ( medium without cells ). the cells are rinsed with rpmi medium without phenol red in order for the colour of the medium not to interfere with absorbance measurements . the cells are then incubated for 4 hours with 100 μl rpmi solution supplemented with mtt ( 0 . 5 mg / ml ). the medium is eliminated and 100 μl dmso is added to dissolve the converted dye ( formazan ). the absorbance is measured by spectrophotometry at 570 nm ( purple ); 630 nm ( background noise ). in order to ignore the background noise , the following calculation is performed : do reelle = do 570nm − do 630nm . the results are then normalised with respect to the controls ( 100 % and 0 % live cells ) and presented in the form of a mean +/− sem . the study of delf508 - cftr protein addressing is performed in the laboratory by using a combination of pharmacological , cellular imagery , biochemical and electrophysiological tests on cf15 human lung epithelial cells homozygous for the delf508 deletion . results of immunoprecipitation and western - blot analyses of non mutated cftr from wild - type cho cells and delf508 cells treated for 2 hrs with 100 μm of the tested compound ( fig3 c ) or untreated cells ( control ). cf15 cells treated for 24 hrs at 27 ° c . ( fig3 b ) or for 2 hrs with 10 μm thapsigargin ( tg ) were used as a positive control . for each experiment , the addition of a cocktail ( 10 μm forskoline + 30 μm genisteine ) allows activation of the cftr when the latter is localised in the membrane . this allows an iodide efflux to be observed if delf508 addressing has been restored . the results , presented in the form of a histogram , were normalised with respect to a standard treatment ( treatment of the cells with 250 μm mpb - 91 for 2 hrs ), considered to represent 100 % cftr activity . the results obtained show that treatment of cf15 cells with the two forms ( r and s ) of roscovitine for 2 hrs at 37 ° c . restores addressing of the delf508 protein and allows it to function as an ion transporter ( fig1 ). when the cells are not treated , the delf508 protein is not localised in the membrane and no iodide efflux is observed as a result of stimulation with the 10 μm forskoline + 30 μm genisteine cocktail . the ec50 ( molecular concentration giving 50 % of maximal efficiency ) for roscovitine was found to be 34 ± 1 . 9 μm ( r - roscovitine , fig1 a , c ) and 32 ± 1 . 4 ( s - roscovitine , fig1 b , c ) ( n = 4 , in each case ). cellular imagery showed that the delf508 protein was localised in the plasma membrane compartment after treatment with roscovitine . fig3 a to 3c also illustrate the immunolocalisation of delf508 - cftr after 2 hrs of treatment with roscovitine or in the absence of treatment . this involves confocal visualisation of cftr - delf508 in cf15 cells using a mouse anti - cftr monoclonal antibody . cf15 cells treated for 24 hrs at 27 ° c . were used as a positive control ( fig3 a ). fig4 a illustrates the activation of delf508 - cftr in cf15 cells after treatment with roscovitine . the iodide efflux was observed after 2 hrs of incubation with 100 μm of the tested compound or in the absence of treatment . cf15 cells treated for 24 hrs at 27 ° c . were used as a positive control and untreated cf15 cells as a negative control ( 37 ° c .). fig4 b shows that the response to roscovitine depends on the incubation time . roscovitine ( 100 μm ) was used for treating cf15 cells . the cells were then stimulated with 10 μm fsk + 30 μm gst . fig5 a shows the dose - response results after 2 hrs of treatment with roscovitine . fig5 b shows the pharmacological profiles of cftr channels in cf15 cells after 2 hrs of incubation with 100 μm of roscovitine . these results show that after treatment with roscovitine , the following is observed : maturation of delf508 - cftr ( band c appears in the western - blot ) after treatment with cftr - t1 , correct delf508 - cftr relocation to the plasma membrane , and maximal correction of delf508 - cftr chloride ion channel function after 2 hrs of treatment . the competition between cftr - t1 and the er chaperone machinery is illustrated by fig6 a and 6b . fig6 a illustrates the inhibition of cftr - t1 action by brefeldine a ( bfa ), an inhibitor of the ergic vesicular traffic , which shows that roscovitine induces readdressing of the delf508 - cftr protein . fig6 b shows that no modulation is observed in the presence of mg132 , a proteasome inhibitor , which shows there is competition between roscovitine and mg132 . the table below summarises the roscovitine and er chaperone machinery competition experiments performed by the iodide efflux technique . — inhibition , ** p & lt ; 0 . 01 , * p & lt ; 0 . 1 , ns p & gt ; 0 . 1 ( student &# 39 ; s t - test these results show that roscovitine induces delf508 - cftr readdressing , inhibits the cftr - t1 degradation pathway and is able to modulate the interaction between cftr - t1 and calnexin ( calcium - dependent mechanism ). in order to show that the effect of roscovitine is specific for delf508 addressing and does not alter other chloride channels , roscovitine was tested as a potential activator in calu - 3 cells . these results were obtained using the iodide efflux technique . the controls used were forskoline ( 5 μm , n = 8 ) and mpb - 91 ( 250 μm , n = 8 ). roscovitine ( n = 8 ) was not found to be an activator of wild - type cftr or any other anionic transporter in these cells ( no significant difference ). in order to show that the effect of roscovitine is specific to delf508 addressing , roscovitine was tested as a modulator of wild - type cftr addressing in calu - 3 cells . these results were obtained by measuring iodide efflux in calu - 3 cells treated for 2 hrs with roscovitine ( 100 μm ). the cftr activity under such experimental conditions is not significantly different from the controls . these results demonstrate that roscovitine does not affect the addressing pathway of the wild - type cftr or other chloride channels , nor does it alter cftr activity in non - cf human lung epithelial cells . in order to test the cytotoxicity of roscovitine , cho - wt cells were incubated for 2 hrs with different concentrations of inhibitors before being tested for viability with mtt . the results show that the cells are viable at all concentrations of roscovitine . therefore , this molecule does not present any cell cytotoxicity . efflux tests revealed that roscovitine allows relocation of the delf508 - cftr protein to the membrane and therefore represents a pharmacological mean of re - addressing delf508 in human lung epithelial cells . a 2 - hr treatment with 100 μm of roscovitine results in the appearance of the mature cftr band ( as shown by the immunoprecipitation and western - blot techniques ), indicating that roscovitine allows the liberation of the delf508 - cftr protein from the er and its maturation in the golgi apparatus . immunofluorescence experiments confirmed that a 2 - hr treatment with 100 μm of roscovitine allowed relocation of the delf508 - cftr protein to the membrane . finally , iodide efflux and patch - clamp experiments on whole cells showed that the readdressed protein was functional . a 2 - hr treatment with roscovitine gives an ec 50 of 56 μm and allows maximal readdressing . in addition , competition experiments to determine the mechanism of action of roscovitine have shown that roscovitine competed with inhibitors of calnexin / delf508 - cftr binding as well as inhibitors of the degradation pathway . these various results show that roscovitine can correct the delf508 - cftr protein addressing defect . treatment of cf cells with roscovitine should therefore allow interference with the capacity of the control machinery to interact with and retain the delf508 - cftr protein in the endoplasmic reticulum , via inhibition of calnexin and the chaperon molecules involved in the degradation pathway . an inhalation solution is prepared with an ampoule spray starting with sodium chloride , dehydrated calcium chloride and water for injectable preparations . roscovitine is then added as active ingredient . the solution is formulated in 2 . 5 - ml ampoules . ampoules including 5 , 10 mg or 20 mg of roscovitine can be prepared in this way . becq et al . ( 1999 ) journal of biological chemistry 274 , 27415 - 27425 . dormer et al . ( 2001 ) journal of cell science 114 , 4073 - 4081 .
the invention relates to the use of purine derivatives for the production of medicaments for the treatment of mucoviscidosis and diseases related to protein addressing errors in cells , said derivatives being of formula : where r2 , r6 and r9 , independently = halogen , a r — nh —, r — nh — nh —, nh 2 — r ′— nh or r — nh — r ′— nh — group , where r = straight or branched chain saturated or unsaturated alkyl , aryl , cycloalkyl , or heterocyclyl group , r ′= straight or branched chain , saturated or unsaturated alkylene , arylene or cycloalkylene , r and r ′ each include 1 to 8 carbon atoms optionally substituted with one or more — oh , halogen , amino or alkyl groups , r2 furthermore may be a heterocycle optionally with a straight or branched chain saturated or unsaturated alkyl , aryl or cycloaryl or a heterocycle optionally substituted by one or more — oh , halogen , amino or alkyl groups , r9 furthermore may be a straight or branched chain saturated or unsaturated alkyl , aryl or cycloalkyl and r2 and r9 furthermore may be hydrogen with the exception of 2 -- 6 - benzylamino - 9 - methylpurine .