Patent Application: US-201213980533-A

Abstract:
a compound having the structural formula and pharmaceutically acceptable salt and / or hydrates thereof , wherein y is an arylester or an c 1 - c 8 alkylaryl ester , selected from the group consisting of : benzene , toluene , xylene , benzoic acid , benzoate , nicotinate , isonicotinate and halobenzene , which can be unsubstituted or substituted with at least one nitric oxide releasing group ; and / or at least one of hydroxide , — cl , — br , a c 1 - c 8 alkyl , benzyl , a c 1 - c 8 alkoxy , benzyloxy , — nhcr , — nh 2 , — no 2 — ono 2 , — n ono 2 , — oc n ] cyclic ono 2 , — ocoarono 2 , — ocoar n ono 2 or a c 1 - c 5 haloalkyl ester , wherein r is a c 1 - c 8 alkyl or a c 1 - c 8 alkoxy group , n = 1 - 8 and m = 3 - 10 , to produce a super - aspirin effect .

Description:
the present inventors have demonstrated that the compounds used within the context of the present address aspirin resistance problems in 3 major ways : ( i ) the prodrug compounds used herein demonstrates significantly better gi tolerability when compared to aspirin ; ( ii ) in the case of st0702 ( isana or the “ nicotinate ”), data obtained in a non - human primate study indicate that there are surprisingly good effects of the drug in - vivo ; and ( iii ) in the case of the prodrugs used within the context of the present invention , and exemplified by the data on the nicotinate compound st0702 , there are advantageous and desirable non - aspirin antiplatelet effects produced by a metabolite of the prodrug compounds . it has been found that because platelets are activated by multiple pathways , these non - aspirin antiplatelet effects supplement the aspirin effects to reduce platelet activation , particularly in disease states with high levels of inflammation or in patients with genetic polymorphisms that render them aspirin resistant . each of these beneficial mechanisms of dealing with the clinical problem of aspirin resistance are described in the following studies outlined below . furthermore , the present inventors report on a pharmacological investigation into the platelet inhibitory properties of isosorbide - based aspirin prodrugs ( for example , compounds st0701 - 05 ). the study focuses on prodrug activation by plasma and platelet esterases . the following has been examined : the relative potency of the compounds as inhibitors of aggregation in prp and washed platelets ( where buche is absent ), the involvement of platelet receptors , the effect of buche and other esterase inhibitors on potency and efficacy , the relative roles of aspirin and nitric oxide in the effects of compounds st0703 - 05 , the time course for activation and platelet inhibition , and the role of platelet esterases in aspirin prodrug activation . furthermore still , the present inventors have produced compounds that that are potent inhibitors of adp and collagen - induced platelet aggregation . two in vitro experimental models for the study of tcipa ( under static and flow conditions ) have been established . using both models , the effect of different aspirin pro - drugs in tcipa has been studied . st0702 inhibits tcipa under both static and flow conditions , and therefore it seems to be an effective platelet - modulating drug for cancer metastasis . st0702 therefore is a compound showing enhanced aspirin effects or what is termed herein “ a super aspirin effect ” which means it is a more potent inhibitor of adp and collagen induced platelet aggregation than would be expected from aspirin release alone and that this additional antiplatelet aggregation effect inhibits tcipa under both static and flow conditions . this data demonstrates significantly better gi tolerability of the prodrugs compared to aspirin . poor gi tolerability is a known major driver of patient non - compliance and aspirin resistance . the precise mechanisms by which aspirin causes mucosal damage have not been fully explained . although nsaids can induce gi damage via both topical and systemic effects , several lines of evidence indicate that the direct irritant effect is the major mechanism leading to ulcers and ulcer complications : there is not a direct link between cox activity / pg synthesis and gastric injury and pg synthesis can be markedly suppressed without ulcers forming . cox - 1 selective nsaids , while inhibiting cox - 1 activity , do not cause ulcers . although selective cox - 2 inhibitors and synthetic pg analogues attenuate nsaid induced gi toxicity , then do not abolish it . although parenteral aspirin inhibits cox - 1 activity , ulcers only form when aspirin is given orally . in this study , rats are fed aspirin 30 mg / kg daily or molar equivalent doses of isas , isana (“ nicotinate ”), isano “ meta ” nitrate or isamna in oral gavage daily for 3 days ( n = 3 per group ). at the end of the dosing period , all animals were sacrificed , stomachs removed and were immersed in 2 % formalin for at least 10 min to fix the gastric tissue wall , and will opened along the greater curvature for scoring for irritation or hemorrhagic lesions or ulcers developed in the corpus mucosa under a dissecting microscope on a 5 - point scale . the percentage of stomach surface area showing ulcers was recorded for each animal and expressed as a percentage . the results ( figure a ( i )) demonstrate that aspirin , as expected , has a significant gastric ulcerogenic effect in this rat model . conversely , none of the aspirin pro - drugs demonstrated ulcerogenic effects and were significantly better than conventional aspirin at equimolar doses . this means the prodrug compounds may advantageously used in patients with poor gi tolerability for aspirin or patients with sensitive gi conditions aggravated by aspirin . in the case of isana (“ nicotinate ” st0702 ), there are surprisingly good effects of the drug in - vivo . these data are obtained in a non - human primate study . in - vivo data on the gastro - intestinal benefits of the series of aspirin pro - drugs suggest that gastro - protection is a feature shared by all of the pro - drugs and will contribute to the improvement of gastro - intestinal tolerability , compliance with therapy and therefore will help to resolve the cause of aspirin resistance in large proportion of patients . in earlier in - vitro data , it was demonstrated that isas is the most effective aspirin prodrug studied to date , by releasing 85 % of the expected amount of aspirin in human plasma . conversely , st0702 ( isana —“ nicotinate ”) is only a moderately effective aspirin prodrug in the previous in - vitro studies , releasing approximately 40 % of the available aspirin ( wo 2009 / 080795 ). surprisingly , it has now been found that the relative in - vitro efficacy of isana is significantly better than expected . a series of studies in non - human primates to reflect as closely as possible the in - vivo behaviour of aspirin and its prodrugs in human primates has been carried out . the details of the general methods and animal housing applied to each phase of the study are described later . the objectives of this study are to determine the comparative clinical efficacy as determined by txb2 suppression with single oral gavage dose administration of aspirin , st0702 and st0702 micronised to the cynomolgus monkey . aspirin pharmacokinetics were compared using area under the curve ( auc ) calculations . results demonstrate that in terms of suppression of efficacy , denoted by txb2 levels as a percentage of baseline , st0702 and aspirin were effective at 1 and 24 hours post dosing with statistical differences between crystalline non - micronised and miconised st0702 . there was no difference between aspirin and micronized st0702 . the modeled auc of aspirin from the aspirin 4 . 2 mg / kg dose was 870 ng · hr / ml . aucs of equimolar doses of st0702 were : 249 mg · hr / ml ( non - micronised , 31 % of aspirin auc ) and 506 ng · hr / ml ( micronized , 58 % of aspirin auc ). these data demonstrate ( i ) that plasma txb2 as a marker of aspirin efficacy is suppressed by isana ( st0702 ); ( ii ) that when micronized , isana is better absorbed and releases more aspirin ; that when micronized , isana provides an aspirin auc that is 58 % of the corresponding aspirin dose in vivo . the apparent txb2 suppression activity of micronized isana is greater than would be predicted by the aspirin auc . taken together , these data suggest that isana ( st0702 ) is a surprisingly good candidate for development as an aspirin prodrug for aspirin - like antiplatelet ( additional antiplatelet effect not attributable to aspirin ) efficacy . the objectives of this study are to determine the comparative pharmacokinetics of st0702 at 9 . 6 and 4 . 8 mg / kg with single oral gavage dose administration to the cynomolgus monkey and to determine dose - response effects . results demonstrate that in terms of aspirin release , micronized st0702 4 . 8 mg / kg has a similar cmax to equimolar doses of aspirin ( 2 . 1 mg / kg , figure b ( ii )). micronised st0702 9 . 6 mg / kg has a twice the cmax of aspirin ( 2 . 1 mg / kg ) and micronised st0702 4 . 8 mg / kg . cmax for st0702 groups occurred consistently at 30 minutes whereas aspirin absorption continued with a cmax at 1 hour . txb2 suppression was 47 % complete with micronized st0702 4 . 8 mg / kg at 24 hours compared to the 70 % complete with 9 . 6 mg / kg . pharmacokinetic data for st0702 4 . 8 mg / kg : cmax 211 ng / ml , tmax 0 . 5 hr , auc 221 ng · hr / ml . corresponding data for st0702 9 . 6 mg / kg : cmax 522 ng / ml , tmax 0 . 5 hr , auc 571 ng · hr / ml . the modeled auc of aspirin from the aspirin 4 . 2 mg / kg dose was 923 ng · hr / ml . aucs of equimolar doses of micronised st0702 was 571 ng · hr / ml ( 62 % of aspirin auc ). together with data from study 2a , these data suggest that micronised st0702 delivers 58 - 62 % of the expected aspirin in - vivo , compared to approximately 40 % in - vitro . these data demonstrate that dose - response effects of micronized st0702 are linear and predictable . accordingly it should be possible to achieve desired aspirin - like effects and txb2 suppression with careful dose selection of st0702 . in the case of the prodrugs , and exemplified by the data on st0702 ( isana —“ nicotinate ”), there are non - aspirin antiplatelet effects of a metabolite . because platelets are activated by multiple pathways , these non - aspirin antiplatelet effects are thought to supplement the aspirin effects to reduce platelet activation , particularly in disease states with high levels of inflammation or in patients with genetic polymorphisms that render them aspirin resistant . isana (“ nicotinate ”, st0702 ) demonstrates an unusual property of greater inhibition of tumour cell induce platelet aggregation ( tcipa ) compared to aspirin and compared to other prodrugs . these demonstrate non - aspirin antiplatelet effects ( discussed in greater detail below ). similar data have been obtained using the breakdown metabolite of st0702 , isosorbide 2 salicylate 5 nicotinate , suggesting that the antiplatelet effects of st0702 in response to tcipa may arise from an identified metabolite . although the half life of the isosorbide 2 salicylate 5 nicotinate metabolite is very short in - vitro , we investigated the serum taken from cynomologus monkeys participating in study 2b above . the comparative pharmacokinetics of isosorbide 2 salicylate 5 nicotinate from monkeys receiving a single oral gavage dose of micronized st0702 at 9 . 6 and 4 . 8 mg / kg were evaluated . the concentrations of the breakdown products of isana were quantified using lcms methods . these data are presented in figure c ( i ) and demonstrate that there are detectable levels of the two metabolites in plasma up until 2 hours post dosing . target age at the initiation of dosing : 2 . 0 to 4 . 0 years target weight at the initiation of dosing : 3 . 8 to 5 kg animals are transferred from pcs - shg in - house colony . a minimum acclimation period of one week is allowed between transfer and the start of treatment in order to accustom the animals to the laboratory environment . before dosing initiation , all animals are weighed and assigned to treatment groups using a computerized randomization procedure . randomization is by stratification using body weight as the parameter . animals in poor health or at extremes of body weight range are not be assigned to groups . the disposition of all animals are documented in the study records . spare animals are maintained with the main study population for the duration of the study . animals are socially housed ( up to 3 animals of same sex and same dosing group together ) in stainless steel cages equipped with a stainless steel mesh floor and an automatic watering valve unless deemed inappropriate by the study director and / or clinical veterinarian . animals are separated during designated procedures / activities . each cage is clearly labeled with a color coded cage card indicating study , group , animal and tattoo number , and sex . light cycle : 12 hours light and 12 hours dark ( except during designated procedures ) temperature and humidity are monitored and recorded continuously in each animal room by an environmental monitoring system . in the event of a system failure , manual recording are performed ( once daily ) as defined in the standard operating procedures . the light cycle interruptions are documented in the study data . a minimum of ten air changes / hour are maintained . all animals will have access to a standard certified pelleted commercial primate food ( certified pmi 5k9c ) twice daily except during designated procedures . maximum allowable concentrations of contaminants in the diet ( e . g ., heavy metals , aflatoxin , organophosphate , chlorinated hydrocarbons , pcbs ) are controlled and routinely analyzed by the manufacturers . the results of the analysis are retained at pcs - shg . it is considered that there are no known contaminants in the dietary materials that could interfere with the objectives of the study municipal tap water which has been softened , purified by reverse osmosis and exposed to ultraviolet light are freely available ( except during designated procedures ). periodic analysis of the water is subcontracted to management authorized analytical laboratories which are audited by the qau of pcs - shg . the analytical results are retained in the archives of pcs - shg . it is considered that there are no known contaminants in the water that could interfere with the objectives of the study . animals are socially housed for psychological / environmental enrichment and are provided with items such as perches , floor toys , foraging devices and / or hanging devices , except during designated activities . additional enrichment , such as music , natural sounds or color videos films is also provided . each animal is offered food supplements ( such as certified treats , fresh fruit and / or prima foraging crumbles ). additional enrichment may be provided if deemed appropriate by the study director , in consultation with the clinical veterinarian . following transfer , each animal is given a general physical examination by a member of the veterinary staff to assess health status . all animals have previously been tested at least three times for tuberculosis by intradermal injection of tuberculin . as required by sop , animals are retested approximately every three to four months thereafter following the last injection . veterinary care is available throughout the course of the study and animals are examined by the veterinary staff as warranted by clinical signs or other changes . all veterinary examinations and recommended therapeutic treatments are documented in the study records . in the event that animals show signs of illness or distress , the responsible veterinarian may make initial recommendations about treatment of the animal ( s ) and / or alteration of study procedures , which must be approved by the study director . animals are acclimated to the oral gavage procedure for at least 3 days prior to the commencement of dose formulation administration . deionized water is administered by oral gavage using a disposable catheter attached to a plastic syringe at a dose volume of 5 ml / kg . animals are treated at approximately the same time on each dosing day . dose formulations are administered by oral gavage using a disposable catheter attached to a plastic syringe . following each daily dose , the gavage tube is rinsed with 6 ml of ultra pure water into the animal &# 39 ; s stomach . each animal will be dosed with a clean gavage tube on each dosing occasion . the dosing volume is 10 ml / kg . each actual volume administered will be based on the most recent practical body weight of each animal . the dose formulations will be stirred continuously during dose administration . blood is collected from an appropriate vein once pre - treatment and during the dosing and recovery phases as specified below . after collection , samples are transferred to the appropriate laboratory for processing . additional blood samples may be obtained if permissible sampling frequency and blood volume are not exceeded . food is removed overnight ( minimum 12 hours ) from animals before blood sampling ( for clinical chemistry and immunochemistry ). samples are collected according to the following table . samples are mixed gently and placed on crushed wet ice until centrifugation , which are carried out as soon as practical . the samples are centrifuged for approximately 10 minutes in a refrigerated centrifuge ( approximately 4 ° c .) at 2700 rpm . the resultant plasma are separated , transferred to uniquely labeled clear polypropylene tubes , and frozen immediately over dry ice and transferred to a freezer set to maintain − 80 ° c . the plasma samples are transferred to the bioanalytical laboratory at the testing facility . plasma samples are analyzed for concentration of aspirin , salicylic acid and nicotinic acid using a qualified analytical procedure . analysis are performed by lc ms / ms under analytical procedure . data collection is performed using analyst from ab sciex . statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations , accuracy and precision are performed using watson laboratory information management system ( lims ) and microsoft excel . toxicokinetic parameters are estimated using winnonlin pharmacokinetic software ( pharsight corp ., mountain view , calif .). all parameters are generated from individual aspirin , salicylic acid and nicotinic acid concentrations in plasma unless otherwise stated . mean concentrations are derived from where possible . parameters are estimated using sampling times relative to the start of each dose administration . isana appears to be more effective ( approximately 60 % of equivalent aspirin release ) in vivo than in vitro ( 40 % of expected release ) from an aspirin delivery point of view . there is a dramatic improvement in kinetics and efficacy with simple micronisation of isana which bodes remarkably well for formulation given the basic nature of the oral administration of isana . txb2 suppression with micronised isana is as effective as equimolar doses of aspirin . there appears to be a more impressive effect of isana on txb2 suppression ( equivalent ) than the pharmacokinetic data would suggest ( aucs approximately 60 %). the metabolite of interest is detectable for & gt ; 2 hours in monkey plasma at both 4 . 8 and 9 . 6 mg / kg which is likely to be significant for the clinically relevance of the metabolite . all reagents were purchased from sigma - aldrich ( dublin , ireland ) unless otherwise stated . collagen and adp were obtained from chronolog ( havertown , pa ., u . s . a .). allophycocyanin ( apc )- conjugated monoclonal antibody against high - affinity gpiib / iiia ( pac - 1 - apc ) and apc - conjugated monoclonal antibody against human platelet p selectin ( cd62p ) were purchased from bd biosciences ( oxford , uk ). human ( monoclonal , polyclonal ) anti - ces 1 and ces 2 antibodies were purchased from sigma - aldrich ( dublin , ireland ). wild type butrylcholinesterase ( buche ) was a kind gift from oksana lockridge , university of nabraska , usa . human liver and intestinal microsomes , bd gentest ™ were obtained from bd biosciences , usa . test compounds were dissolved in dmso , than diluted in prp or wp to give a final concentration not more than 0 . 25 % dmso , which pilot studies had determined not to affect platelet aggregation . no precipitation of test compound was observed following dilution . blood was collected from fully - consented healthy volunteers at the school of pharmacy and pharmaceutical sciences who had not taken any drugs known to affect platelet function for at least 14 days prior to the study . prp and wp ( 2 . 5 × 10 8 platelets ml − 1 ) were prepared from blood . briefly 36 ml whole blood was collected into 4 ml of 3 . 15 % sodium citrate ; this was centrifuged at 250 ¥ g for 20 min ( with gentle acceleration and deceleration ). at this point prp , which had separated from white blood cells and red blood cells , was gently removed . when wp were required this prp was centrifuged at 700 g for 10 min ( with gentle acceleration and deceleration ) in the presence of prostacyclin . the platelet - poor plasma ( ppp ) was removed and the platelet pellet was washed three times with tyrode &# 39 ; s buffer , before being re - suspended in tyrode &# 39 ; s buffer to a concentration of 2 . 5 ¥ 108 platelets / ml . platelet aggregation was measured by light aggregometry . briefly , prp and wp samples ( 2 . 5 × 10 8 / ml ) were placed in an eight channel platelet aggregation profiler ® model pap - 8e and incubated for 10 min at 37 ° c ., with stirring at 900 r . p . m ., prior to the addition of aggregating agents . aggregation was initiated by the addition of agonists , and monitored by aggro - link software for at least 6 min . for experiments using inhibitors , aggregation was initiated after 10 min preincubation with test compounds . to study the aggregatory potency of adp , the concentration - response ( 0 . 3 - 10 μm ) curves were generated . collagen at different concentrations ( 2 - 5 μg / ml ) was also used to induce platelet aggregation . sumbmaximal concentrations of agonists , i . e . the concentrations that gave approximately 95 % of the maximal aggregation were used to study the effects of inhibitors of aggregation . aspirin and aspirin prodrugs was incubated for various intervals prior to the addition of aggregating agents . results were expressed in percent % changes in maximal light transmission , with 100 % representing light transmission of platelet medium alone . to study the involvement of plasma buche in the activation of st0701 , purified buche ( 3 u / ml ) was incubated with wp for 5 min in the aggregometer with stirring at 900 r . p . m . before the addition of the test compounds st0701 which was incubated with the wp for a further 10 min , prior to the addition of collagen ( 5 ug / ml ). eserine ( in dmso , 10 μm , giving a final concentration ≦ 0 . 25 % dmso , which pilot studies had determined not to affect platelet aggregation ) was preincubated with prp or wp for 5 min in the aggregometer with stirring at 900 r . p . m . before the addition of test compounds ( st0701 - 05 ), which were incubated in prp or wp for a further 10 min prior to the addition of an agonist ( 10 μm adp or 5 μg / ml collagen ) to induce platelet aggregation . 1h -[ 1 , 2 , 4 ] oxadiazolo [ 4 , 3 - a ] quinoxalin - 1 - one ( odq , dissolved in 100 % dmso , used at a final concentration of 10 μm , which gave a final concentration not more than 0 . 25 % dmso ) was preincubated with prp or wp for 5 min in the aggregometer with stirring at 900 r . p . m . before the addition of test compounds ( st0701 - 05 ), which were incubated in prp or wp for a further 10 min prior to the addition of an agonist ( 10 μm adp or 5 □ g / ml collagen ) to induce platelet aggregation . phenylmethylsulphonylfluoride ( pmsf ) 20 , 50 and 100 μm dissolved in etoh (≦ 0 . 2 % etoh , which pilot studies had determined not to affect platelet aggregation ) was preincubated in wp in the aggregometer for 5 min with stirring at 900 r . p . m . before the addition of test compound st0702 to the wp , which was further incubated for 10 min prior to the addition of collagen ( 2 μg / ml ) to induce platelet aggregation . prp , wp samples ( 2 . 5 × 10 8 platelets ml 1 ), lysed wp samples and ppp were analyzed for the presence of buche activity , according to the method of ellman , with some modifications . s - butyryltiocholine iodide ( btcl ) at 1 mm in phosphate buffer ph8 . 0 at 37 ° c . was used as substrate to measure the activity of bche in wp , prp , ppp and in wp lysed with triton x - 100 . combined ache and bche activity was measured with 1 mm acetylythiocholine iodide ( atcl ). an aliquot of 4 μl of wp , prp , ppp or lysed wp , ellman &# 39 ; s reagent ( dtnb 10 μm ) and phosphate buffer ph 8 . 0 were incubated in a 96 well plate at 37 ° c . for 30 min . btcl or atcl was added to give a final concentration of 1 mm and the change in absorbance at 405 nm was measured over a period of 10 min on a plate reader . wp , prp , ppp or lysed wp and 50 mm trishcl ph 7 . 4 were incubated on a 96 well plate at 37 ° c . for 30 min . pna at a final concentration of 3 mm was added to the plate and the change in absorbance at 405 nm was measured over a period of 10 min . npa hydrolyse activity could be due to ce ( ec 3 . 1 . 1 . 1 ), buche , acetylcholinesterase ( ache , ec 3 . 1 . 1 . 7 ) or paraoxanase / arylesterase ( pon , ec 3 . 1 . 8 . 1 ). to identify which esterases were involved in the hydrolysis of pna , various inhibitors were incubated with the wp , prp , ppp or lysed wp and 50 mm trishcl ph 7 . 4 for 30 min at 37 ° c . prior to the addition of the pna and the measurement of the change in absorbance at 405 nm . physostigmine ( 100 μm ), a cholinesterase inhibitor was used to check if buche / ache played a role in the hydrolysis of pna . iso - ompa a selective buche inhibitor was also used . pmsf ( 10 - 100 μm ) was used to determine if serine proteases played any part in the hydrolysis of pna by platelets . edta a calcium chelator was used to investigate the role played by pon esterases . bnpp a ce inhibitor of was used to investigate if carboxylesterases played a role in hydrolysis of pna by platelets . control experiments were performed throughout the course of each experiment to establish normal aggregation responses . prior to each experiment , a sample of prp or / and wp was incubated with 100 of dmso for 10 min at 37 ° c . with stirring to ensure the solvent was having no inhibitor effect on the aggregation response . in order to analyze receptor expression on the surface of individual platelets and to minimize platelet activation caused by sample preparation procedures , no stirring or vortexing steps were used . the abundance of activated gpiib / iiia and p - selectin on the surface of platelets in the presence and absence of inhibitors was measured by flow cytometry . platelet samples were first activated with agonists either collagen or adp . when platelet aggregation reached 50 % maximal light transmission the reaction was terminated by 10 - fold dilution with physiologic saline . resting platelets were used as control . in most of the experiments , platelets were preincubated with inhibitors for 10 min prior to the addition of agonists . platelet samples were then incubated in the dark without stirring for 5 min at room temperature in the presence of saturating concentrations ( 10 μg / ml ) of p - selectin ( cd62p - apc ). the activated gpiib / iiia platelet receptors were measured using pac - 1 monoclonal antibody at the same concentration as above . pac - 1 specifically recognizes an epitope on the high - affinity gpiib / iiia complex of activated platelets at or near the platelet ( ref ). following incubation , samples were diluted in facs flow fluid and analyzed within 5 min using a bd facsarray ( bd biosciences , oxford , uk ). flow cytometry was performed on single stained platelet samples as described before ( ref ). the instrument was set up to measure the size ( forward scatter ), granularity ( side scatter ) and cell fluorescence . a two - dimensional analysis gate of forward and side scatter was drawn in order to include single platelets and exclude platelet aggregates and microparticles . antibody binding was measured by analyzing individual platelets for fluorescence . the mean fluorescence intensity was determined after correction for cell autofluorescence . for each sample , the fluorescence was analyzed using a logarithmic scale . fluorescence histograms were obtained for 10 , 000 individual events . data were analyzed using bd facsarray software and expressed as a percentage of control fluorescence in arbitrary units . platelet lysates were prepared by lysing wp with 10 × ripa buffer ( 20 mm tris ph 7 . 4 , 50 mm nacl , 50 mm naf , 5 mm edta , 20 mm pyrophosphate , 1 mm na 3 vo 4 , 10 % triton x , 10 mm pmsf , pics ) for 1 h on ice with intermittent vortexing . a bradford assay was performed to determine the protein concentration of the platelet lysate samples . platelet lysate samples were mixed with 2 × non - reducing loading buffer ( 50 mm tris ph6 . 8 , 2 % sds , 0 . 1 % bromophenol blue , 20 % glycerol ), or reducing loading buffer ( 50 mm tris ph 6 . 8 , 2 % sds , 0 . 1 % bromophenol blue , 20 % glycerol , 0 . 5 % ( 3 - mercaptoethanol ), boiled for 5 min , centrifuged and loaded onto sds - page gels . proteins were electrophoretically separated under non - reducing and reducing conditions according to laemmli . sds - page was performed with 7 % ( wt / vol ) separation gels at a constant voltage of 160 v / h with the use of a mini - protean ii gel system from bio - rad laboratories ( hemel , hempstead , uk ). after electrophoretic separation , proteins were transferred from the gel onto immobilon - nc membranes , according to the method of towbin et al ., using a bio - rad mini - protean ii blotting system ( bio - rad laboratories ). proteins separated by sds page were transferred to nitrocellulose membranes by the method of towbin et al nitrocellulose membranes were blocked for 1 h in 5 % bsa / nt buffer ( 50 mm tris ph 7 . 4 , 170 mm nacl , 0 . 2 % igepal ) and incubated overnight at 4 ° c . with primary antibody anti - ces2 diluted 1 : 1000 , or anti - ces1 diluted ( 1 : 2000 ) subsequently , immunoblots were washed twice for 10 min each with 100 ml of 5 % bsa / nt per blot and incubated for 1 h with peroxidase - conjugated secondary antibodies at a dilution of 1 : 10000 . after the blots had been washed twice for 10 min with 5 % bsa / nt and rinsed twice with nt buffer , nitrocellulose sheets were developed by enhanced chemiluminescence . calibration of molecular masses was based on the sds - page molecular weight standards from bio - rad . liver microsomes were used as a positive control for ces1 and intestinal microsomes were used as a positive control for ces2 . the data were analyzed using one - way analysis of variance ( graphpad prism software 3 . 0 ). the results are expressed as mean ± s . e . m . of at least three independent experiments . tukey - kramer multiple comparisons test , and paired and unpaired student &# 39 ; s t - tests were performed , where appropriate . statistical significance was considered when p & lt ; 0 . 05 . as shown in fig2 , the test compounds and aspirin ( 100 - 300 μm ) caused inhibition of platelet aggregation in prp and wp stimulated by collagen ( 5 μg / ml ). the ic 50 values for inhibition of wp aggregation are presented in table 1 . the prodrugs st0701 - 05 were significantly less potent in wp suspension than in prp ( fig2 a ). in contrast aspirin was more potent in wp ( ic 50 35 μm ) than in prp ( ic 50 92 μm ). this effect may be due to protein binding ( 50 %) or hydrolysis ; aspirin interacts with albumin partly through transacetylation which complicates measurement of free drug . of the prodrug compounds , st0702 was the most efficacious inhibitor of collagen induced aggregation in wp ( ic 50 154 μm ). st0701 was more potent than st0702 in this regard but less efficacious ( 50 % inhibition at 500 μm ( fig2 b )). the lack of effect of the other compounds in the absence of plasma buche strongly suggests that their anti - platelet effect in prp are due to aspirin release . a role for plasma buche in activation of st0701 was supported by the observation that its inhibitory actions were rescued in wp by the addition of human buche purified from plasma ( 3 u / ml ). aspirin and the aspirin prodrugs were tested as inhibitors of adp ( 10 μm ) induced platelet aggregation in prp and wp ( n = 6 ) ( fig3 a ). the inventors have already reported some initial results for st0701 , st0703 - 04 at low concentration of adp ( 3 μm ) 7 . nitrate compounds st0703 - 04 were significantly more potent inhibitors of adp ( 10 μm ) induced aggregation than the st0701 - 02 or aspirin ( fig3 b ). aspirin caused consistently less inhibition of aggregation than st0701 / 02 ( which were similar in effect ) but the difference was not significant at any single concentration . st0701 and st0702 ( 200 μm ) were incubated in prp for several time intervals ( 2 , 5 , 10 , 20 , 30 min ) before addition of collagen ( 5 μg / ml ) to stimulate platelet aggregation . inhibition by st0701 reached its maximum at 5 min ( 40 % at 2 min ). no significant inhibition of platelet aggregation was observed with st0702 following 0 - 5 min preincubation . the inhibitory activity of st0702 increased gradually with time and became maximal following 15 - 20 min precincubation . several products of plasma hydrolysis of st0701 ( fig1 b ) were available from a previous study that identified st0701 as a true aspirin prodrug 6 . these were tested for their effect on collagen ( 5 μg / ml ) induced aggregation in order to see if they contributed to the inhibitory actions of st0701 . isosorbide , salicylic acid , isosorbide - 2 , 5 - disalicylate and isosorbide - 5 - salicylate were incubated in prp in the concentration range 10 - 300 μm before triggering aggregation ( fig1 b ). none of the hydrolysis products significantly inhibited aggregation induced by collagen ( 5 μg / ml ) ( n = 3 at 11 concentration levels ). the compounds were also co - incubated in the presence of aspirin to see if they potentiated its actions ( either pharmacologically or by affecting disposition ). none of the compounds affected aspirin &# 39 ; s activity apart from isosorbide - disalicylate trended non - significantly towards an attenuation of aspirin &# 39 ; s inhibitory effect ( maximally 10 %) when co - incubated in the range 50 - 500 μm . st0703 - 04 were significantly more potent than aspirin and the other aspirin prodrugs ( st10701 - st0702 ) at inhibiting adp ( 10 μm ) induced platelet aggregation . in order to determine if no played a role in this effect , the irreversible guanyl cyclase inhibitor odq was preincubated with the prp in the aggregometer for 5 min before the addition of aspirin or aspirin prodrugs . platelet aggregation was then initiated by the addition of adp ( 10 μm ). odq cause a small but significant attenuation of the inhibitory action of nitrates st0703 / 04 ( fig3 c ). odq caused some inhibition when incubated alone with adp and it increased the inhibition of adp induced platelet aggregation by aspirin , st0701 and st0702 . the test compounds and aspirin were incubated in prp at maximally effective concentration of 300 μm to inhibit collagen - induced aggregation in the presence or absence of 10 μm physostigmine ( eserine ). this concentration level was separately shown to cause complete inhibition of plasma buche . all of the prp experiments were conducted in samples that had normal buche levels ( there are several polymorphisms leading to low buche activity ). eserine addition resulted in an almost complete loss of inhibitory activity of all of the test compounds ( fig4 ) in prp . in contrast , eserine did not exert significant effects on inhibition of aggregation afforded by aspirin . in wp suspension eserine did not affect the inhibitory actions of the prodrugs or aspirin towards collagen - induced aggregation ( fig4 ). the activation of glycoprotein integrin receptor gpiib / iiia is crucial for platelet aggregation to occur . in addition the translocations of p - selectin from α - granules to the platelet surface membrane underlie platelet adhesion . we measured the expression of these receptors following collagen stimulation in the presence of the test compounds at 60 μm . as shown in fig5 a - b , st0701 and nitrate hybrid st0703 significantly suppressed activation of gpiib / iiia and translocation of p - selectin in prp to resting levels but not in wp . st0705 did not cause significant inhibition of either platelet marker in prp or in wp . st0703 was equipotent with aspirin in prp but not in wp . the effects overall correlated with the known extent of aspirin generation in plasma solution . u46619 a thromboxane mimetic was used to investigate if the prodrugs had any inhibitory effects downstream of thromboxane . neither aspirin nor the aspirin prodrugs had any inhibitory effect on platelet aggregation induced by u46619 in prp . plasma and platelet buche activity was measured using the ellman assay with butyrylthiocholine and acetylthiocholine as substrate . npa hydrolase activity was also determined since this substrate can be hydrolysed by ces , which hydrolyse choline esters only slowly . buche activity in plasma and prp was 6 - 9 μm butyrylthiocholine mini ml − 1 . in contrast there was negligible esterase activity detectable in wp or washed platelet homogenate using the buche substrate . weak activity ( 3 - 5 μm min − 1 ml − 1 ) was also detected in prp and ppp using the substrate pnpa . this was probably due to cholinesterase since there is little or no ce in human plasma . there was significant pnpa hydrolysing activity in wp and in platelet lysate . as pnpa hydrolysis activity could be due to ce , buche , ache or ppon esterases a series of inhibitors were used to classify the esterases present in the wp . physostigmine a cholinesterase inhibitor caused a 25 % reduction in pnpa hydrolyse activity of platelets . pmsf a serine protease inhibitor almost completely inhibited npa activity in wp at concentration of 1000m and inhibited npa activity by 50 % at 10 μm ( fig6 ). edta a calcium chelator which inhibits pon had no effect on npa activity of wp at concentrations up to 0 . 5 mm . iso - ompa ( 10 - 100 μm ) a selective butrylcholinesterase inhibitor had no effect on pna activity of wp or lysed platelets . surprisingly , bnpp ( 10 - 100 μm ) a specific ce inhibitor had no significant effect on npa activity of wp or lysed platelets . the npa hydrolyse activity in the platelet suspension was not due to buche or pon esterases . probing platelet lysates run on nonreducing polyacrylamide gels with anti ces1 and anti - ces2 antibodies we observed bands with mwt ranging from 150 kda - 400 kda , not at the predicted mwt of 62 kda ( fig6 ). these bands were similar in mwt to the bands observed by oertel et al by staining for alphana esterases 30 . these bands appear to be a carboxylesterase type because of their cross reactivity with ces - 1 / 2 antibody and ability to process pnpa . when the gels were reduced these high mwt bands disappeared and were replaced by a doublet , with one band at ˜ 50 kda and another at ˜ 65 kda ( fig6 ). wp suspensions were next treated with pmsf , before addition of st0702 and platelet activating agent . pmsf attenuated the ability of st0702 to inhibit platelet aggregation in a concentration dependent manner with maximal effects at 100 mm . of the prodrug compounds , 2 ( st0702 ) was the most efficacious inhibitor of collagen induced aggregation in wp ( ic 50 154 μm ). st0702 had a greater than expected potency in wp where there is an absence of esterases responsible for the hydrolysis of these prodrugs in prp . to determine if any pmsf - sensitive enzyme in platelets play a role in the hydrolysis of st0702 in wp , pmsf was pre - incubated with wp at increasing concentrations up to 100 mm for 5 min , with stirring before the addition of st0702 ( 200 mm ). platelet aggregation was then initiated by addition of collagen ( 2 mg / ml ). pmsf had no significant effect on collagen - induced aggregation . however pmsf ( 100 mm ) significantly attenuated the effect of st0702 ( 200 mm ) on collagen - induced aggregation in wp ( fig7 b ). lower amounts of pmsf ( 50 mm , 20 mm ), also slightly attenuated the effect of st0702 on collagen - induced aggregation in wp , although this attenuation was not significant . this is the first pharmacological study into mechanisms of platelet inhibition by true aspirin prodrugs . the compounds in this study were reported to undergo productive processing generating aspirin in the presence of buche from human plasma 6 , 7 . this is due to a particular fit for the buche active site that overrides the usual acetyl group preference for this enzyme , which prevents other aspirin esters from releasing aspirin . the compounds are rapidly hydrolysed in plasma solution generating aspirin to 30 - 80 % of the starting ester concentration 6 , 7 . st0701 is the most effective aspirin prodrug ever discovered producing slightly less than a stoichiometric equivalent of aspirin . however , it is a significantly more potent inhibitor of platelet aggregation than aspirin . the platelet inhibitory properties of the nicotinate codrug st0702 ( 2 ), which releases & lt ; 40 % aspirin on a stoichiometric basis , have not been reported previously . in this study , st0702 was equipotent with aspirin ( 90 mm ) in the inhibition of aggregation in prp stimulated by collagen ( 5 mg / ml ). the nitrate compounds st0703 and st0704 were reported to be more potent inhibitors of platelet aggregation in prp than aspirin in response to collagen ( 5 μg / ml ) and adp ( 3 μm ) 6 , 7 . in this study we used a higher concentration of adp to obtain amore reliable biphasic response to further probe the effect of the compounds on adp - stimulated aggregation . meanwhile , the nitrate hybrid st0705 releases & lt ; 10 % aspirin in solutions containing human plasma which made it a useful control to probe for the role of aspirin and no release in the effects of in the nitric oxide - aspirin hybrids ( st0703 and st0704 ). several lines of evidence from this study indicate that the platelet inhibitory actions of the prodrugs st0701 - 04 are due to aspirin release : ( i ) the test compounds were significantly more potent or effective in prp than in wp ; ( ii ) their inhibitory actions in prp were abrogated following preincubation with the cholinesterase inhibitor eserine ; ( iii ) inhibition of collagen induced aggregation in prp by 200 μm st0701 and st0702 was time dependent ( whereas aspirin &# 39 ; s inhibition effects were not ); ( iv ) effects of the prodrugs on platelet gpiib / iiia and p - selectin expression were similar qualitatively to aspirin indicating that the inhibitory effects occurred upstream of receptor release / translocation ; ( v ) the products of plasma esterase hydrolysis of st0701 did not exert platelet inhibitory effects or modulate the platelet inhibitory properties of aspirin when co - incubated ; ( v ) ester st0705 , which does not release aspirin , was not active . aspirin &# 39 ; s failure to effectively inhibit adp induced aggregation is a clinically important deficiency of the drug which has prompted others to evaluate aspirin - no hybrids . a number of no - aspirin hybrids types including aspirin - furoxans , - diazeniumdiolates and - nitrates have been studied in vitro as platelet aggregation inhibitors . the furoxans inhibit platelet aggregation in prp mainly through nitric oxide release since this hybrid type is exclusively hydrolysed at the acetyl ester in media containing human plasma . the nitrate ester ncx - 4016 does not inhibit platelet aggregation in prp . it inhibits platelet aggregation in wpbut in an no and cgmp independent manner . ncx - 4016 inhibits purified cox preparations by direct interaction , i . e . direct acyl transfer onto cox . this ability is lost in media containing human plasma because of a more rapid deacetylation by buche than transacetylation of cox . inhibition of platelet aggregation with ncx - 4016 is therefore only observed in wp where esterase levels are low . the use of organic nitrates in alleviating the symptoms of angina has its mechanistic basis in nitric oxide release from endothelial and smooth muscle cells . platelets are able to stimulate no release from organic nitrates but much less effectively than smooth muscle cells . nitric oxide has a well characterised endogenous role in gastric mucosal protection and maintenance of mucosal defence is a general property of organic nitrate drugs . while organic nitrates produce insufficient nitric oxide locally to affect platelet aggregation , they have potential to mimic or augment its endogenous protective role in the intestinal mucosa . whereas collagen - induced aggregation at submaximal concentrations depends predominantly on the production of txa2 , it is highly susceptible to inhibition by cox inhibitors such as aspirin . adp addition to platelet suspension produces a biphasic aggregation profile . the first phase of adp induced aggregation is independent of txa2 production and hence cox inhibition ( and thus resistant to aspirin ). however the second phase of adp - induced aggregation is dependent on the production of txa2 and can be inhibited by cox inhibitors such as aspirin . we were interested to see if the no - aspirin prodrugs retained the ability to inhibit the second ( cox dependent ) phase of adp - induced aggregation while being able to dampen or reverse the cox - independent first phase . this could be useful in a disease situation where platelets are exposed to multiple pathophysiological stimuli . the nitrate substituted compounds st0703 and st0704 were significantly more effective inhibitors of adp ( 10 μm ) induced aggregation than the non - nitrate substituted compounds and aspirin . this effect appears to be due partly to nitric oxide release because it was attenuated in the presence of the soluble guanyl cyclase inhibitor odq which abrogates the platelet inhibitory effects of nitric oxide . the effect of st0703 and st0704 on adp - induced aggregation was attenuated in the presence of the soluble guanyl cyclase inhibitor odq which blocks to some extent the platelet inhibitory effects of no . [ 50 ] in this study , odq itself partially inhibited platelet aggregation in response to collagen and it amplified the platelet inhibitory effects of the aspirin and prodrug st0701 which does not produce no . odq therefore had only a modest effect when co - incubated with the nitrate hybrids st0703 and st0704 , because while it attenuated the effect of no release it amplifed the aspirin effect . overall , the evidence for a contribution from a no - mediated effect from st0703 and st0704 derives from their inhibitory efficacy towards adp - induced aggregation , as well as the observed reversal of the trend towards increased inhibition when aspirin and its prodrugs were co - incubated with odq . interestingly , the inhibitory effect of st0703 and st0704 in adp induced aggregation were abolished in the presence of eserine , which blocks aspirin release in prp ( data not shown ). st0705 , which releases nitric oxide but little aspirin , was not effective as an inhibitor of adp induced aggregation . these observations imply that aspirin and no were acting synergistically in causing the inhibitory effects of st0703 and st0704 . the other outstanding question in this study was the greater than expected potency of the ester compounds and the significant inhibitory actions in wp suspension of st0701 and st0702 where buche was absent . we showed that buche is not relevant to the inhibitory actions of st0702 in wp because the inhibitory effect was not abolished by pretreatment with eserine . a possible explanation for this was that the compounds were taken up by platelets and activated by a platelet esterase , possibly a ce . we therefore profiled the esterase activity in prp , ppp , wp and platelet lysate using a range of substrate and esterase specific inhibitors . as expected , significant buche activity was detected in prp and ppp but weak ce and ache activity . platelets are reported to possess a low level buche activity possibly plasma derived but trapped in the platelet canalicular system . a platelet membrane - associated ache activity has also been reported . these reports are consistent with our observation of weak wp and lysate turnover of the appropriate thiocholine esters . several α - naphthyl acetate ( αna ) hydrolysing esterase has been separated from platelets although platelet αna was reported to be resistant to inhibition by pmsf . our results show that there is a multimeric protein in platelets that produces reduced fragments in the range 50 - 60 kd with high immunoreactivity towards human ces - 1 and ce - 2 . these proteins are presumed to be the enzyme ( s ) catalysing the hydrolysis of ce substrate pnpa . the platelet hydrolytic activity was blocked by the general serine esterase / protease inhibitor pmsf ( 50 - 100 μm ). the inhibitory activity of st0702 ( 200 - 300 μm ) in wp were abolished when the suspension was pretreated with pmsf ( 100 μm ) indicating that a pmsf sensitive enzyme in platelets causes the activation of st0702 in wp . the result has significance for the potential utility of the compound for it shows that i ) the compound could be activated and cause platelet inhibition in a patient with low plasma esterase activity ; ii ) it indicates that the st0702 does not possess intrinsic anti - platelet activity and iii ) it raises the prospect of increased cellular uptake and intracellular activation , potentially increasing potency . despite releasing only 30 - 40 % aspirin in ppp relative to a stoichiometric amount of aspirin , st0702 has similar anti - platelet activity with respect to stimulation with adp and collagen . the ester prodrugs are significantly more lipophilic than aspirin at ph 7 . 4 as reflected in rphplc retention and as expected from abrogation of the carboxylate . a plausible explanation for their high potency in prp is that they partition into the platelet membrane to a greater extent than aspirin from where they undergo activation mediated by esterases at the platelet surface or intracellularly producing locally high concentrations of aspirin . enhanced cellular uptake would be interesting to measure in other pathologically relevant cell types . inhibition of platelet aggregation by aspirin prodrugs st0701 - 04 is primarily due to aspirin release . in the case of nitrate substituted compounds inhibitory effects are due to release of both no and aspirin with promising effects on adp - induced aggregation . st0702 , a nicotinic acid - aspirin codrug is activated by a pmsf sensitive platelet esterase in wp platelet suspensions . its unexpected potency in prp appears to be due to platelet uptake and drug release . thus , aspirin prodrugs effectively inhibit human platelet aggregation and as such may be an alternative to conventional aspirin . tumour cell - induced platelet aggregation ( tcipa ) facilitates cancer cell invasion , angiogenesis and the formation of metastatic foci . tumour cell - induced platelet aggregation can be modulated by pharmacological inhibitors of matrix metalloproteinase - 2 ( mmp - 2 ) and adp , however , the major cyclooxygenase inhibitor aspirin has failed to prevent tcipa . the inventors have tested the pharmacological effects of a new group of isosorbide - based aspirin prodrugs on tcipa . tcipa was induced by human adenocarcinoma and fibrosarcoma cells under no flow and flow conditions . the release of gelatinases and p - selectin expression during tcipa were studied by zymography and flow cytometry , respectively . tumour cells caused platelet aggregation . this aggregation resulted in the release of mmp - 2 and a significant up - regulation of p - selectin on platelets indicative of platelet activation . pharmacological modulation of tcipa revealed that st0702 , one of the aspirin prodrugs , downregulated tcipa while aspirin was ineffective . one of st0702 metabolites , 5 - nicotinate salicylate ( st0702 salicylate ) was also studied . it was found that st0702 salicylate downregulated both adp - stimulated platelet aggregation and tcipa . the results provided and discussed below results show that st0702 is an effective inhibitor of tcipa in vitro . the salicylate metabolite is thought to contribute to the effects of st0702 by inhibiting adp - mediated tcipa . the inventors have developed a new group of isosorbide - based aspirin pro - drugs that were found to be potent inhibitors of adp - and collagen - induced platelet aggregation ( jones et al ., 2009 ). indeed , several pro - drugs were more potent inhibitors of aggregation than regular aspirin . the aim of the present study was to determine if the observed increase in potency in platelet aggregation assays translates into an effect in tcipa . accordingly , a selection of pro - drugs ( fig1 a ) were evaluated as inhibitors of tcipa under conditions where aspirin itself is ineffective . some of these compounds are designed to simultaneously release a second pharmacologically active moiety ( e . g . nitric oxide ) that might make them more suitable for interrupting processes such as tcipa that operate along multiple activation pathways . one of the test compounds , an aspirin - nicotinic acid codrug ( st0702 ), under development as a dual anti - platelet and lipid modifying agent , markedly inhibited tcipa under both no flow and flow conditions . we have been able to determine the mode of action of st0702 as an inhibitor of tcipa by monitoring its activation during platelet - tumour cell interactions . all reagents were purchased from sigma - aldrich ( dublin , ireland ) unless otherwise indicated . collagen and adp was obtained from chronolog ( havertown , pa ., u . s . a .). allophycocyanin ( apc )- conjugated monoclonal antibody against human platelet p selectin ( cd62p ) were purchased from bd biosciences ( oxford , uk ). the compounds were dissolved in dmso , then diluted in prp or wp to give a final concentration not more than 0 . 25 % dmso , which pilot studies had determined not to affect platelet aggregation . no precipitation of any drug was observed following dilution . three human tumor cell lines , 59m ovarian adenocarcinoma , caco - 2 colon adenocarcinoma and ht1080 fibrocarcinoma cells , were obtained from the european cell culture collection . cell lines were cultured as monolayers in 75 ml culture flasks at 37 ° c . in a humidified atmosphere with 5 % co 2 . 59m cells and ht1080 were cultured in dulbecco &# 39 ; s minimum essential medium ( dmem ) supplemented with 10 % fetal bovine serum ( fbs ) and gentamycin ( 0 . 05 mg / ml ), penicillin ( 0 . 06 mg / ml ), and streptomycin ( 0 . 01 mg / ml ). caco - 2 cell line was cultured in minimum essential medium ( mem ) supplemented with 20 % fbs and the same antibiotics as above . the cells were supplied with fresh medium and subcultured three times each week . blood was collected from healthy volunteers who have not taken any drugs known to affect platelet function for at least 14 days prior to the study . washed platelet suspensions ( 2 . 5 × 10 11 platelets l − 1 ) were prepared from blood an mixed with the anticoagulant sodium citrate ( 0 . 315 % final concentration ). the interactions between platelets and tumor cells were measured by light aggregometry ( alonso - escolano et al ., 2004 ; jurasz et al ., 2001b ; radomski et al ., 1991 ). briefly , washed platelet samples ( 2 . 5 × 10 8 / ml ) were placed in an eight channel pap 8 aggregometer ( bio / data corporation , horsham , pa ., u . s . a .) and incubated for 10 min at 37 ° c ., with stirring at 900 r . p . m ., prior to the addition of aggregating agents . for most experiments , collagen at a concentration that resulted in maximal aggregation ( 5 μg / ml ) was used as a control agonist . tcipa was initiated by the addition of cancer cells , and monitored by aggro - link software for at least 30 min . for experiments using inhibitors , aggregation was initiated after 10 min preincubation with these compounds . to study the ability of the aspirin pro - drugs to inhibit tcipa in wp cancer cell lines such as ht1080 cells ( 2 × 105 / ml ), caco2 cells ( 1 . 5 × 103 / ml ) or 59m cells ( 1 × 103 / ml ) were used in the presence or absence of aspirin nicotinate ( st0702 ), isas , orthonitrate and metanitrate at 300 and 500 μm . platelet aggregation was initiated in washed platelets ( wp ) using ht1080 cells ( 2 × 10 5 / ml ), caco2 cells ( 1 . 5 × 10 3 / ml ) or 59m cells ( 1 × 10 3 / ml ). in addition , regular aspirin ( 300 and 500 μm ) was used as control . results were expressed in percent changes in maximal light transmission , with 100 % representing light transmission of platelet medium alone . st0702 is hydrolyzed by plasma esterases along two pathways liberating both aspirin and 5 - nicotinate salicylate ( st0702 salicylate )( metabolite ). the latter is further processed to isosorbide - 5 - nicotinate and eventually to nicotinic acid . the orthonitrate and metanitrate compounds liberate aspirin and nitric oxide ( no ). isas meanwhile is hydrolysed to aspirin and salicylic acid . aspirin ( 300 and 500 μm ) was used in control experiments . results were expressed in percent changes in maximal light transmission , with 100 % representing light transmission of platelet medium alone . further experiments were performed with st0702 salicylate ( 500 μm ). this was obtained by flash chromatography as a hydrolysis product of st0702 during isolation of the parent compound . its purity and identity was confirmed by nmr , hplc and hrms . to study whether or not aspirin prodrugs had any direct effect on tumour cells , nictoinate was preincubated with different cell lines and added to the platelet suspension . tcipa was monitored for at least 30 min . to study also the effect of physostigmine on activity of aspirin prodrugs , physostigmine ( 10 um dissolved in etoh ) was preincubated with wp for 5 min in aggregometer with stirring at 900 r . p . m . before the addition of aspirin or aspirin prodrugs . platelet aggregation was initiated after 10 min incubation with aspirin / aspirin prodrugs by the addition of ht1080 cells ( 2 × 10 5 / ml ), caco2 cells ( 1 . 5 × 10 3 / ml ) or 59m cells ( 1 × 10 3 / ml ). eserine ( 10 μm ) was incubated with prp for 5 min in the aggregometer with stirring at 900 r . p . m . before the addition of test compound st0702 salicylate ( 500 μm ), which was incubated in prp for a further 10 min prior to the addition of the agonist adp ( 10 μm ) to induce platelet aggregation . in order to analyze receptor expression on the surface of individual platelets and to minimize platelet activation caused by sample preparation procedures , no stirring or vortexing steps were used . the abundance of p - selectin on the surface of platelets in the presence and absence of inhibitors was measured by flow cytometry . platelet samples were first activated with 59m cells ( 1 × 10 3 / ml ). when platelet aggregation reached 50 % maximal light transmission the reaction was terminated by 10 - fold dilution with physiologic saline . resting platelets were used as control . in most of the experiments , platelets were preincubated with inhibitors for 10 min prior to the addition of 59m cells ( 1 × 10 3 / ml ). platelet samples were then incubated in the dark without stirring for 5 min at room temperature in the presence of saturating concentrations ( 10 μg / ml ) of p - selectin ( cd62p - apc ). following incubation , samples were diluted in facs flow fluid and analyzed within 5 min using a bd facsarray ( bd biosciences , oxford , uk ). flow cytometry was performed on single stained platelet samples . the instrument was set up to measure the size ( forward scatter ), granularity ( side scatter ) and cell fluorescence . a two - dimensional analysis gate of forward and side scatter was drawn in order to include single platelets and exclude platelet aggregates and microparticles . antibody binding was measured by analyzing individual platelets for fluorescence . the mean fluorescence intensity was determined after correction for cell autofluorescence . for each sample , the fluorescence was analyzed using a logarithmic scale . fluorescence histograms were obtained for 10 , 000 individual events . data were analyzed using bd facs array software and expressed as a percentage of control fluorescence in arbitrary units . the structure of platelet - tumor cell aggregates was studied using phase - contrast microscopy ( alonso - escolano et al ., 2004 ; jurasz et al ., 2001 ). briefly , 59m cells ( 1 × 10 3 / ml ) were added to the platelet suspension ( 2 . 5 × 108 / ml ) in the presence or absence of aspirin and aspirin prodrugs , and aggregation was terminated at 50 % maximal aggregation , as determined using the aggregometer . the samples were fixed by adding 2 % paraformaldehyde in tyrode &# 39 ; s solution , ph 7 . 4 , and then incubated for 30 min at room temperature . aliquots of each sample were then taken for phase - contrast microscopy examination using an olympus ckx41 microscope ( olympus america inc ., melville , n . y ., u . s . a .). photomicrographs were captured using a digital camera and microfire ( olympus america inc .) software ( alonso - escolano et al ., 2004 ; jurasz et al ., 2001 ). sample collection was carried out as previously described ( jurasz et al ., 2001 ; medina et al ., 2006 ). briefly , platelets at a concentration of 2 . 5 × 10 8 / ml were placed into lumi - aggregometer and tumor cells ( 59m ) were added at 1 × 10 3 / ml . when platelet aggregation reached 50 % maximal light transmission the reaction was terminated and samples were collected . the samples were then centrifuged at 900 × g at room temperature for 10 min . after centrifugation , platelet releasates were collected and stored at − 80 c until assayed for the presence of mmp activity by zymography . gelatin zymography was used to detect the activity of mmp - 2 in the releasates as previously described ( alonso - escolano et al ., 2004 ; jurasz et al ., 2001 ; medina et al ., 2006 ). briefly , samples were subjected to 10 % sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) with copolymerized gelatin ( 0 . 2 %; sigma chemical co , st . louis , mo .) incorporated as a substrate for gelatinolytic proteases . after electrophoresis , the gels were washed with 2 . 5 % triton x - 100 ( 3 times , 20 minutes each ), and then incubated for 48 h at 37 c .° in enzyme assay buffer ( 25 mm tris hcl , 0 . 9 % nacl , 5 mm cacl 2 and 0 . 05 % na 3 n , ph = 7 . 5 ). the conditioned medium of ht - 1080 human fibrosarcoma cells ( that contains high amounts of prommp - 2 , mmp - 2 , prommp - 9 and mmp - 9 ), was used as control . after 72 hours development , gels were fixed and stained in 40 % methanol , 10 % acetic acid and 0 . 1 % ( wt / v ) coomassie blue r - 250 ( sigma chemical co , st . louis , mo .) for 1 hour and then de - stained in 4 % methanol with 8 % acetic acid . the gelatinolytic activities were detected as transparent bands against the background of coomassie blue - stained gelatin . the intensities of the separate bands were analyzed and quantified using chemidoc xrs system ( bio - rad , hercules , calif ., u . s . a .). the gelatinolytic activity of each band was expressed as arbitrary units of density / mg protein . the ultrasound trap had three essential features : a transducer ( ferroperm , kvistgard , denmark ) in a housing of radial symmetry , an aqueous phase and a reflector that provided optical access from above . the trap was driven with a function generator ( hewlett packard 33120a , uk ). microscopic observation occurred with a fast , high - resolution xm10 camera ( soft imaging system , sis , gmbh ) mounted on an olympus bx51m reflection epi - fluorescence microscope . images were captured by a standard pc equipped with the cell - d image acquisition software ( soft imaging system , sis , gmbh ). the experimental procedure was as follows : 59m cells were introduced into the trap , the acoustic field ( 2 . 13 mhz , 0 . 85 mpa ) was initiated and cell clusters were allowed to form . clusters remained levitated in suspension for 10 min . washed platelets ( 2 . 5 × 10 8 / ml ) were introduced into the trap at a flow rate of 3 μl / min . perfusion of washed platelets around the cluster proceeded following initial adhesion of platelets to the periphery of the cluster . to study the effect of aspirin and aspirin prodrugs in tcipa , washed platelets were pre - incubated for 10 min at room temperature with the inhibitors : aspirin , nicotinate , isas , ismna , ortho and meta ( all at a concentration of 500 μm ), prior to their introduction into the ultrasound trap . those aspirin pro - drugs which were found to have an inhibitory effect under static conditions were tested ; st0702 , isas , orthonitrate and metanitrate ( 500 μm ); aspirin ( 500 μm ) was used as control . the cell cluster - platelet aggregate remained under microscopic observation for further 10 min ( the upper time limit established in the current study ) under continuous flow conditions . further experiments were performed with st0702 salicylate ( 500 μm ). to study the hydrolysis of the prodrug st0702 in tcipa , hplc analysis of supernatants and lysates was performed . briefly wp ( 2 . 5 × 108 / ml ) were placed in an eight channel pap 8 aggregometer . st0702 ( 500 μm ) was incubated with the platelets with stirring for 10 min , prior to the addition of cancer cells , ht1080 ( 2 × 105 / ml ) or caco2 ( 1 . 5 × 103 / ml ) to induce aggregation . tcipa was monitored for 30 min , supernatants / lysates were collected as soon as control ( ht1080 or caco2 alone ), plateaued and reached maximal aggregation . for collection of supernatant , after the control plateaued and reached maximal aggregation , 100 μm pmsf was added and the washed platelet / st0702 suspension was centrifuged at 13000 × g for 5 min at 4 ° c ., the supernatant was removed and stored at − 20 ° c . until analysis by hplc . for collection of lysate after the control plateaued and reached maximal aggregation , ice cold 10 × ripa lysis buffer ( 20 mm tris ph 7 . 4 , 50 mm nacl , 50 mm naf , 5 mm edta , 20 mm pyrophosphate , 1 mm na 3 vo4 , 10 % triton - x , 10 mm pmsf and 10 × pics ( 5 mm aebsf , 1 . 5 mm aprotinin , 10 um e - 64 protease inhibitor , 10 um leupeptin hemisulfate )) was added to wp / st0702 suspension . samples were stored on ice for 1 h with frequent vortexing to promote lysis . the samples were centrifuged at 13000 × g for 5 min at 4 ° c . and the supernatant lysates stored at − 20 ° c . until analysis by hplc . hplc was performed as described previously but with detection at 260 nm ( jones , 2009 ). the data were analyzed using one - way analysis of variance ( graphpad prism software ). the results were expressed as mean ± s . e . m . of at least three independent experiments . tukey - kramer multiple comparisons test , and paired and unpaired student &# 39 ; s t - tests were performed , where appropriate . statistical significance was considered when p & lt ; 0 . 05 . caco - 2 , ht1080 and 59m cells were tested for their ability to induce platelet aggregation . when platelets were incubated in the aggregometer for 30 min at 37 ° c . without the addition of tumor cells , no platelet aggregation was detected . however , all cell lines were able to induce platelet aggregation ( fig8 a and 8b ). since the concentration of 2 × 10 5 / ml ( ht1080 cells ), 1 . 5 × 10 3 / ml ( caco2 cells ) and 1 × 10 3 / ml ( 59m cells ) were sufficient to induce platelet aggregation , all remaining experiments were performed at this cell density . different aspirin prodrugs and regular aspirin which inhibit the txa 2 pathway were evaluated as inhibitors of tcipa . first , platelet suspensions were preincubated with aspirin and prodrugs . aspirin , nicotinate , isas , ismna , otho and meta at two different concentrations ( 300 and 500 μm ) were used to inhibit tcipa in caco2 , ht1080 and 59m cell lines . aspirin did not exert any significant effect on tcipa ( fig9 ). however , this concentration of aspirin inhibited collagen - induced aggregation by 56 ± 7 % ( n = 4 ) and abolished arachidonic acid ( 100 μm )- induced aggregation ( n = 4 ). in contrast to aspirin , nicotinate consistently inhibited tcipa in all three cell lines , as shown by aggregometry ( fig9 ) and phase contrast microscopy ( fig1 ). interestingly , nicotinate was the only aspirin prodrug that inhibited platelet aggregation induced by ht1080 ( fig9 ). in addition , isas , ortho and meta were able to inhibit platelet aggregation induced by caco - 2 and 59m cells ( fig9 and 10 ). however , ismna only inhibited platelet aggregation induced by caco - 2 cells ( fig9 and 10 ). when tumour cells ( caco2 and 59m ) were preincubated with nicotinate , no effect on tcipa was observed ( p & gt ; 0 . 05 , n = 4 ). effects of isas , st0702 , ortho - and metanitrate or aspirin on tumour cell - induced platelet aggregation as measured by aggregometry st0702 consistently inhibited tcipa in response to all three cell lines , as shown by aggregometry ( fig9 ) and phase contrast microscopy ( fig1 ). only st0702 and the no - releasing orthonitrate inhibited platelet aggregation induced by ht1080 ( fig3 ). in addition , isas , orthonitrate and metanitrate inhibited platelet aggregation induced by caco - 2 and 59m cells ( fig9 and 10 ). in contrast , aspirin ( 300 - 500 μm ) did not exert a significant effect on tcipa ( fig9 ). however , as expected aspirin ( 300 μm ) inhibited collagen - induced aggregation by 56 ± 7 % ( n = 4 ). the general pattern of tcipa inhibition was consistent with the observations of platelet - cancer masses detected by microscopy following treatment with aspirin or the prodrugs ( fig1 ). mmp - 2 release measured by zymography as mmp - 2 is released during tcipa , zymographic analysis was conducted to study whether or not mmp - 2 was involved in our experiments . we have previously shown that the pro - mmp - 2 is the major gelatinase detected during platelet aggregation induced by both ht1080 and caco2 cells ( jurasz et al ., 2001 ; medina et al ., 2006 ). therefore , we studied the release of mmp - 2 in platelet aggregation induced by 59m . in our studies , we found that pro - mmp - 2 was also released during tcipa , as shown by the 72 kda band ( fig1 ). however , aspirin and all prodrugs ( 300 um ) failed to prevent the release of pro - mmp - 2 during platelet aggregation induced by 59m cells ( p & gt ; 0 . 05 ; n = 4 ), indicating that all drugs did not exert any effect on mmp - 2 release ( fig1 ). for these experiments , 59m cells were used . the interactions of platelets with ht1080 and caco - 2 cells have been previously characterized by our group ( jurasz et al ., 2001 ; medina et al ., 2006 ). the interactions of platelets with 59m cells induced a significant ( p & lt ; 0 . 005 ; n = 4 ) increase in the number of copies of p - selectin on platelet surface ( fig1 a ). flow cytometry performed on platelets pre - incubated with aspirin and prodrugs ( 300 μm ) and then activated by 59m cells was analyzed . nicotinate ( st0702 ) significantly ( p & lt ; 0 . 01 , n = 4 ) inhibited 59m - mediated increase in total p - selectin ( fig1 a and 11b ). in addition , ortho ( orthonitrate ) and meta were able to significantly ( p & lt ; 0 . 05 , n = 4 ) reduce the expression of p - selectin on platelet surface ( fig1 a and 11b ). in contrast , aspirin ( asa ) and isas and ismna failed to prevent the increase in platelet surface abundance of p - selectin ( p & gt ; 0 . 05 , n = 4 ) ( fig1 a and 11b ). following levitation of a 59m cell cluster in the trap for 10 min ( fig1 a ), platelet perfusion was initiated . platelets approached the aggregate within 1 min and established contact with its periphery . complete platelet ‘ encapsulation ’ of the aggregate was seen within 2 min ( fig1 b ). platelet activation ( identified as a transition to a gel - like sheet around the cell cluster ) occurred within 4 min from platelets - cell cluster contact and resulted in the cancer aggregate disruption ( fig1 c ). st0702 was the only inhibitor that significantly arrested tcipa ( p & lt ; 0 . 05 , n = 4 ) ( fig1 d ). st0702 releases small amounts of aspirin and its salicylate during the tcipa following the tcipa experiment with ht1080 or caco - 2 cells , supernatants were collected for further analysis along with the cellular pellets which were lysed in the presence of esterase / protease inhibitors . the supernatants and lysis fractions were analysed by hplc ( fig1 ). this indicated that in the presence of cancer cell and platelet esterases , st0702 produces substantial amounts of its deacylated metabolite ( isosorbide - 2 - salicylate - 5 - nicotinate ( st0702 - salicylate ), fig1 ) along with smaller amounts of aspirin , salicylic acid , nicotinic acid and isosorbide - 5 - nicotinate . there were no significant differences between the supernatants or lysates following stimulation with caco - 2 or ht1080 cells . to further study the mechanism of action of st0702 in tcipa , more experiments were carried out with its in vitro metabolites identified in the hplc experiments : nicotinic acid , isosorbide - 5 - nicotinate and the st0702 salicylate . st0702 salicylate inhibit tcipa in response to ht1080 and caco - 2 cells under no flow conditions ( fig1 a ), however , unlike the parent st0702 , the salicylate did not inhibit tcipa under flow conditions ( fig1 d ). unlike its aspirin - releasing parent st0702 , the salicylate did not inhibit collagen - induced platelet aggregation ( fig1 b ). however , the salicylate inhibited adp - induced aggregation in prp ( fig1 c ), an effect that became significant in the presence of the esterase inhibitor eserine , which protected the salicylate from further hydrolysis in response to esterases in prp . unlike st0702 , the salicylate did not inhibit tcipa in the ultrasound trap model , indicating that under flow conditions , its adp inhibitory properties were insufficient to prevent tcipa . the remaining fragments identified in cell lysates by hplc ( nicotinic acid and isosorbide - 5 - nicotinate ) did not inhibit tcipa at up to 3 mm . the main function of platelets is the maintenance of vascular haemostasis . platelets also play crucial roles in the pathogenesis of vascular thrombosis and disease . there is increasing evidence that platelet - cancer cell interactions participate in the complex multi step process of carcinogenesis including blood - borne metastasis . when platelets are activated the arachidonic acid cascade is initiated , leading to txa2 synthesis . this reaction is catalysed by a number of enzymes , the most important being cyclooxygenase ( cox ) which converts arachidonic acid to prostaglandin h2 ( pgh2 ) and thromboxane synthase which converts pgh2 to txa2 . txa2 mediates one of major pathways of platelet aggregation by stimulating platelet thromboxane receptors leading to activation of platelet inositol phosphate pathways and an increase in intracellular ca2 + and release of dense - and α - granules . aspirin reduces the synthesis of txa2 by irreversibly inhibiting platelet cox , blocking pgg2 production . aspirin preferentially inhibits the cox - 1 isoform of the enzyme , but its effects on cox - 2 are an important part of the explanation for its anti - inflammatory and putative anti - cancer effects . numerous studies have shown an inverse relationship between aspirin consumption and cancer incidence ( elwood et al ., 2009 ; rothwell et al ., 2011 ). epidemiological and randomized trial data indicates that aspirin - mediated cancer preventative effects are related to dose , duration of use and length of follow up ( langley et al ., 2011 ). the strongest evidence for an anti - cancer effect of aspirin is from patients with cox - 2 over - expressing tumours , suggesting the effect is dosedependent considering aspirin &# 39 ; s cox - 1 selectivity . the evidence for a therapeutic effect of aspirin treatment in cancer patients is more equivocal . two recent nonrandomized trials have reported a reduction in colorectal and breast cancer specific mortality ( chan et al ., 2009 ; holmes et al ., 2010 ), however several older studies failed to detect an improvement in survival in patients on high dose aspirin ( lebeau b et al ., 1993 ; lipton a et al ., 1982 ). consistent with its limited therapeutic efficacy , aspirin fails to inhibit tcipa in vitro ( medina et al ., 2006 ). the implication of this is that in stimulating platelet activation and recruitment cancer cells can surmount the cox - 1 blockade resulting from pharmacologically relevant levels of aspirin . in this context we assessed aspirin pro - drugs as inhibitors of tcipa because of their greater efficacy and potency in response to classical platelet stimuli such as collagen and adp and ability to produce additional metabolites including no ( ortho and metanitrates ), salicylic acid ( isas ) or niacin ( st0702 ). the isosorbide - based aspirin pro - drugs caused significant inhibition of tcipa in response to ht1080 , 59m and caco - 2 cell lines . of the test compounds , the niacin aspirin co - drug st0702 most consistently inhibited platelet aggregation under static conditions but all of the prodrugs exhibited some activity . it &# 39 ; s worth mentioning that nicotinic acid has been shown to mildly inhibit platelet aggregation , an effect which differs from other anti - platelet drugs such as aspirin suggesting potential opportunities for therapeutic combination in this field , however , in this study , nicotinic acid by itself did not inhibit tcipa up to 3 mm ( serebruany et al , 2010 ) evaluation of tumour cell - platelet interactions , has usually been performed under static conditions . we have recently reported the development of a new method to study tcipa under flow conditions using an ultrasound standing wave trap ( bazou et al ., 2011 ). the approach permits the study of tcipa and assessment of inhibitors under more realistic ( patho ) physiological conditions where flow dynamics play a role in adhesion and tumour mass rupture . initial studies with this method have shown that platelet recruitment and degranulation by tumour cells are followed by rupture of tumour cell mass with consequent evolution of satellite aggregates . aspirin treatment fails to delay the rupture of the tumour cell - platelet aggregates ( bazou et al ., 2011 ). st0702 was the only pro - drug to effectively inhibit flow - induced tcipa . surprisingly the no - releasing pro - drugs ( ortho and metanitrate ) did not interfere with tcipa under flow conditions . nitroaspirins or no donating aspirin compounds ( e . g . ncx4016 ) have been extensively evaluated in vitro and in vivo as chemopreventative agents but not in models of tcipa . interpretation of the biochemical efficacy of ncx4016 is moreover complicated by its metabolic conversion to a quinone methide that irreversibly modifies cellular biomolecules leading to reduced viability . nevertheless there is substantial evidence that no release from nitro - aspirins can augment the anti - platelet effects of the aspirin component . the ortho - and metanitrate pro - drugs evaluated in the present study are among the first to release aspirin and no . these inhibit adp - induced aggregation in a manner that is sensitive to inhibition by the soluble guanylate cyclase inhibitor odq ( jones et al ., 2009 ). one interpretation of the present data is that no amplification of aspirin effects may be insufficient to prevent key steps in the mutual activation of platelets and cancer cells . one of the main pathways involved in tcipa is the mmp - 2 dependent pathway . we have previously shown the requirement for activated mmp - 2 to induce the mmp - 2 dependent pathway both in agonist and platelet aggregation induced by ht1080 and caco2 cells ( jurasz et al ., 2001 ; medina et al ., 2006 ). phenanthroline , a synthetic broad spectrum mmp inhibitor , was able to reduce tcipa and the abundance of receptors on platelet surface . therefore , we studied the effect of aspirin prodrugs on mmp - 2 release during tcipa . we have found that none of the test drugs significantly reduced the release of mmp - 2 . these results clearly indicate that the effect of aspirin prodrugs on tcipa is mmp - independent . tcipa is partly mediated by adp ( alonso - escolano et al ., 2004 ; medina et al ., 2006 ). although adp is a weak agonist it is essential for platelet function . the release of adp from activated platelets and stimulation of p2y2 purinergic receptors accounts for the non - txa2 , non - mmp - 2 - mediated pathway of platelet aggregation . in order to find out why st0702 was more efficacious inhibitor of tcipa than its analogous prodrugs , we analysed platelet - tumour cell supernatants and lysates following tcipa experiments using a hplc method capable of separating and identifying potential metabolites of st0702 . the most prominent byproduct of cellular hydrolysis of st0702 was the corresponding salicylate ester resulting from esterase mediated - deacylation ( st0702 salicylate ). interestingly , st0702 salicylate was able to inhibit adp - stimulated platelet aggregation but not collagen - induced aggregation , which is more aspirin sensitive . furthermore the salicylate caused inhibition of tcipa under no flow conditions implicating adp blockade in the mode of action of st0702 . notably , the salicylate did not inhibit tcipa under flow conditions , unlike st0702 , which can also release aspirin suggesting that adp inhibition by st0702 is not sufficient for the inhibitory effects observed . the present results are consistent with our previous studies where scavenging adp with potato and human apyrase decreased tcipa ( alonso - escolano et al ., 2006 ; jurasz et al ., 2003 ; medina et al ., 2006 ). similar effects to apyrase could be demonstrated using selective inhibitors of the p2y12 receptor such as 2 - methylthio - amp ( alonso - escolano et al ., 2004 ). since platelet receptors mediate tcipa , we next studied the changes in the abundance of p - selectin on platelets induced by 59m cells . in fact , p - selectin and its association with mucin is likely to mediate tcipa in a variety of mucin producing cancers . in our study , we found that 59m cells increased the number of copies of p - selectin , as measured by flow cytometry . these results are in agreement with our previous studies in vitro ( medina et al ., 2006 ). we next studied the effect of aspirin prodrugs on pselectin in tcipa . indeed , we have previously shown that tcipa inhibition is strongly associated with p - selectin down - regulation ( medina et al ., 2006 ). our results showed that st0702 again was the most efficacious inhibitor of p - selectin expression during tcipa . interestingly , orthonitrate and metanitrate but not isas , significantly reduced p - selectin expression but to a lesser extent . this may be due to the fact that ortho - 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