angiogenesis - related diseases are those involving angiogenesis in their onset or progression and include neovascularization - induced ocular diseases , such as diabetic retinopathy , macular degeneration , proliferative vitreoretinopathy , glaucoma , atherosclerotic processes and tumors , such as carcinomas , lymphomas , leukemia , sarcomas , melanomas , gliomas , neuroblastomas and other solid tumors . in a particularly preferred embodiment , the invention provides a method for treating a tumor disease selected from lymphoma and leukemia in a subject in need thereof , comprising administering to said subject an effective amount of adenosine - 5 ′- triphosphate - 2 ′, 3 ′- dialdehyde . for therapeutical use , o - atp can be formulated with pharmaceutically acceptable carriers and excipients , and administered through the oral , topical or parenteral route . pharmaceutical forms suitable for the different administration routes comprise tablets , pills , capsules , granulates , powders , suppositories , syrups , solutions , suspensions , creams , ointments , gels , pastes , lotions , emulsions , sprays . pharmaceutical compositions can be prepared as described in remington &# 39 ; s pharmaceutical sciences handbook , mack pub . co ., ny , usa , xvii ed . the amount of active substance per dose unit ranges from 0 . 01 to 100 mg per kg of body weight , to be administered once a day or more according to the type and severity of the pathology . in general the daily dose will range from 1 to 300 mg , preferably from 10 to 100 mg . in another embodiment , the invention refers to combined preparations of o - atp and other biologically active substances for the treatment of angiogenesis - mediated pathologies . according to a preferred embodiment , o - atp is used in combination with antitumor substances such as alkaloids , antibiotics , cytotoxic or cytostatic compounds , antimetabolites , antihormonal agents , alkylating agents , peptides , biological response modulators , cytokines . alternatively , oatp is used in combination with antiatherosclerotic substances , preferably with lipid lowering drugs or statins . the different active substances can be administered either simultaneously or separately . the choice of the specific combination of active substances , their dosage and way of administration depend on the specific disease , its resistance to pharmacological treatments , patient &# 39 ; s tolerance and other variables to be determined on a case by case basis . human endothelial cells ( huvec ) were isolated from umbilical vein , counted and seeded in a constant number in a 96 - well plate . the cells were cultured as described ( jaffe , e . a . ( 1984 ) biology of endothelial cells , martinus nighoff publisher , boston , usa , pp . 1 - 260 ), with or without ( control ) vegf ( 50 ng / ml ), in the presence of o - atp ( 100 μm ), and o - atp + vegf . after 24 hours cultivation with or without stimulus , the cells were washed and counted with an optical microscope using a burker chamber . the results are reported in fig1 and represent the mean ± sd of 10 experiments . transwell chambers for cell cultures ( polycarbonate filters 0 . 4 μm , costar ) were used . in short , confluent endothelial cells , in monolayer , were exposed to vegf , o - atp , atp ( 300 μm ), atp + o - atp , o - atp + vegf ( at the previously indicated concentrations ) for 1 hr and thoroughly washed . albumin marked with 125 i ( nen , boston , mass .) was added to the upper compartment ; cold albumin ( 1 . 5 mg / ml ) was added to the culture medium to minimize transcytosis . one hour after the addition of 125 i - labelled albumin to each well , samples were taken from the lower compartment . the radioactivity of the samples was measured with a gamma counter ( packard , sterling , va .). the results , reported in fig2 , represent the mean ± sd of 10 independent experiments and are expressed as percentage of migrated endothelial cells . huvec were isolated from human umbilical cord by collagenase treatment and cultured in 1 % gelatine - coated flasks using endotoxin - free medium 199 , containing 20 % heat - inactivated fetal bovine serum , 1 % bovine retinal - derived growth factor , 90 microg / ml heparin , 100 iu / ml penicillin and 100 microg / ml streptomycin . all experiments were carried out with huvec at the passage 1 - 4 . we used oatp at the concentration of 500 microm . huvec were treated with oatp over night , washed and fixed with glutaraldeide 2 % in pbs . the cells were colored with crystal violet 0 . 1 %, washed and dried . the dye solubilization was performed with acetic acid 10 % and the absorbance was measured spectrophotometrically at 595 nm , using a microplate reader . the optical density was proportional to the number of cells . as reported in the fig3 , cell growing number is significantly reduced by 500 microm oatp treatment ( mean ± sem of 7 experiments ). vegf is a prototypic angiogenetic factor which induces endothelial cell proliferation , angiogenesis and capillary permeability . the latter is evidenced by the assay of transendothelial migration . it is known that vegf increases transendothelial migration . migration assay was performed using a transwell double chamber system ( 5 micrometer polycarbonate membrane ). huvec ( 5 × 10 4 cells / well ) were seeded on the filter , in the presence or absence of tnfα ( 200 u / ml ) and oatp 500 microm . freshly obtained pmbc ( peripheral blood mononuclear cells ) by means of fycoll from buffy - coat were added in the upper compartment and allowed to migrate over night to the lower chamber which contained rpmi and 10 % fetal calf serum . at the end of the culture , migrated cells were recovered from the lower chamber and counted fig4 shows the transendothelial migration assay . the measure of migrated pbmc through a huvec monolayer indicates that the addition of tnfα ( 200 u / ml ) significantly increases the number of migrated pbmc , expressed as percentage over basal , whereas the addition of 500 microm oatp significantly reduces the tnfα - induced transendothelial migration . data represent mean ± sem of 7 experiments . in another series of experiments , we tested the possible direct effect of oatp on tumor cell growth . we used , both in vitro and in vivo , the rma cells . rma cells were derived from the rausher leukemia virus - induced mouse t - cell lymphoma rbl - 5 of b6 origin . rma wild type ( wt ) murine lymphoma t cell line ( atcc ) was grown in rpmi 1640 supplemented with 10 % heat inactivated fetal bovine serum , 100 u / ml penicillin , 100 microg / ml streptomycin . lymphocytes were obtained from a buffy - coat by a fycoll gradient and depleted from monocytes by adherence . apoptosis was evaluated by facs analysis , after staining with annexin - v fitc - conjugate to show the exposure of phosphatidyl serine on the external side of plasma membranes , and with propidium iodide ( pi ). two thousand rma wt cells and lymphocytes were treated with or without different concentrations of oatp for 24 and 48 hours . the cells were then washed with pbs with ca ++ and mg ++ and stained . ten thousand cell / sample were analysed and the percentage of annexin v / pi positive cells was calculated with fcs - express software ( de novo software ). rma wt cells were treated with different concentrations of oatp for 24 and 48 hours , then fixed for at least four hours in ice - cold 70 % ethanol . cells were stained with a solution containing np - 40 , rnase and pi and cell cycle distribution was detected using facscan ( becton dickinson , san jose , calif .) and analyzed with fcs - express software . the obtained results indicate that oatp , at high concentrations , is able to induce apoptosis of rma cells and not of lymphocytes . sensitivity of rma wild type cell line to different concentrations of oatp evaluated as percentage of annexinv - pi positive cells at 24 and 48 hours of treatment ( fig5 ). sensitivity of lymphocytes from buffy - coat ( pmbc ), activated or not with pha , at different concentrations of oatp , evaluated as percentage of annexinv - pi positive cells at 24 hours of treatment ( fig6 ). the values of the cell cycle progression are reported in the table . cell cycle progression of rma wt cell line treated with different concentrations of oatp for 24 or 48 hours . at high doses of oatp , there is a high mortality of rma wt cells by apoptotic mechanism , as demonstrated by sub g1 peak , in accordance with the percentage of annexinv / pi positive cells ( fig5 ). concomitantly , surviving cells loss partially the capacity to entry in s and / or g2 / m phases of the cell cycle , mainly at 24 hours of treatment . when the concentration of the oatp decreases , cells display a trend similar with the control one . at high dose of oatp ( 1 mm ) and 48 hours of treatment , there is a paradoxical decrease in the percentage of cell death , assessed by sub g1 peak , at variance with the percentage of annexinv / pi positive cells which strongly increases ( fig5 ). this could depend on the proliferation of cells escaped from the treatment . to note that , at low doses , there is increase of the percentage of cells in the g2 / m phase of the cell cycle . rma cells were derived from the rausher leukemia virus - induced mouse t - cell lymphoma rbl - 5 of b6 origin and maintained in rpmi 1640 medium , supplemented with fetal bovine serum , 1 % penicillin / streptomycin and 1 % glutamine ( complete medium ). c57bl / 6 female mice weighing about 18 - 20 g ( 8 week old ) were used . the cells were washed twice with 0 . 9 % nacl and subcutaneously injected in each mouse in a volume of 100 microliters containing 7 × 10 4 cells . after 10 days from rma cell injection , we treated the mice with oatp . five groups of mice were studied ( each of 7 mice ): 1 ) controls , untreated ; 2 ) locally ( subcutaneously ) ( sc ) treated with 0 . 5 mg of oatp ; 3 ) locally ( sc ) treated with 1 mg oatp ; 4 ) intraperitoneally treated with 1 mg oatp ; 5 ) locally ( sc ) treated with 1 mg oatp for 3 days only . the mice were observed until the 20 th day from the tumor inoculation : they were weighed daily and the size of the tumor mass was daily measured . the tumor mass ( measured by arbitrary units ) did not significantly grow in the groups 3 and 5 for three days from the beginning of the treatment ( e . g . until the 14 th day ). successively , the control of the tumor growing is mainly exerted until the number of the tumor cells is not exponentially expanded . however , at 20 days from rma cell injection , in the group 3 the tumor growth was less evident than that observed in the other groups ( fig7 ); in addition , the tumor mass obtained in 20 days in the mice from groups 3 was less solid than the tumor mass obtained in the other groups . no significant differences were observed between the body weights of the mice measured during the different treatments . our data show that the local continuous treatment with 1 mg oatp is efficient in significantly slowing the tumor growth . it is possible that more elevated doses of oatp necessitate to control the tumor growth in the time . our in vitro data suggest that elevated concentrations of oatp ( 1 mg for about 70 , 000 rma cells ) are able to induce the apoptosis of these cells . we compared the effects of oatp to that of two known compounds able to antagonize the p2x7 receptors : 1 ) the pyridoxal phosphate - 6 - azophenil - 2 ′, 4 ′- disulphonic acid ( ppads ), a non - specific p2x7 antagonist , and 2 ) 1 -( n , o - bis [ 5 - isoquinolinesulphonyl ]- n - methyl - l - tyrosyl )- 4 - phenylpiperazine ( kn62 ), a potent p2x7 antagonist ( especially at the human receptor ). we used atp ( able to increase the p2x7 receptor expression on the cells ), and the p2x7 inhibitors , oatp , ppads and kn62 at the concentrations reported in the literature , e . g . : atp 3 mm , oatp 300 microm , ppads 50 microm and kn62 10 microm . in the first series of experiments we studied the proliferation of human umbilical vein endothelial cells ( huvec ), obtained as previously described ( 1 ). the huvec were treated with each of the compounds for 36 hours , washed and fixed with glutaraldeide 2 % in pbs . the cells were colored with crystal violet 0 . 1 %, washed and dried . the dye solubilization was performed with acetic acid 10 % and the absorbance was measured spectrophotometrically at 595 nm , using a microplate reader . the optical density was proportional to the number of cells . as reported in the fig8 , cell growing number is significantly reduced by all the treatments ( mean ± sem of 7 experiments ), but the maximal reduction of the proliferation has been induced by oatp . it is possible that the inhibition of the atp - dependent pathway can induce the shift to another metabolic activity , as the glycolysis . we studied if the compounds were able to induce apoptosis or necrosis of the huvec , by staining the cells with annexin v and propidium iodide ( see the following description ). the block of the huvec proliferation is further evidenced by the absence of cell apoptosis due to the treatment of the cells with the tested compounds , as reported in the fig9 ( the results are representative of 5 experiments ). only the treatment with atp was able to induce about 4 . 5 % pi positive , e . g . necrotic cells . in addition , oatp was cytofluorometrically assayed for its ability to modulate the expression of tnfalpha receptors on endothelial cells . as reported in the fig9 a , the use of human tnfalpha ( 100 u / ml ) improved the expression of the tnfalpha receptor 1 ( tnfr1 ) on huvec . the addition of oatp alone did not modify such expression . however , oatp was able to downregulate the tnfalpha - induced increased expression of tnfr1 . in the second series of experiments , we studied the differences in the apoptosis induction on human promyelocytic leukemia hl60 cells by atp and by the inhibitors of the p2x7 receptors . apoptotic cells and necrotic cells were analyzed by staining the cells with annexin v and propidium iodide ( pi ) ( bd pharmingen apoptosis kit , san diego , calif .). briefly , an aliquot of 10 5 cells was incubated with annexin v - fluorescein isothiocyanate ( fitc ) and pi for 15 minutes at room temperature in the dark . the cells were immediately analyzed by facscalibur ( becton dickinson , heidelberg , germany ). the emission / excitation wavelengths were 530 / 488 nm for annexin v fitc ( fl1 ) and 640 nm / 488 nm for pi ( fl2 ). the necrotic cells were annexin v - and pi - positive , whereas apoptotic cells were annexin v - positive and pi - negative . the percentage of cells stained in each quadrant was quantified using the cellquest software ( bd bioscience , san josew , calif .). the data , reported in the fig1 , and representative of 5 experiments , show that oatp was able to induce cell apoptosis at better extent with respect to ppads and kn62 and such effect was dependent on the concentration of oatp . the combined use of atp with one of the p2x7 inhibitors increased the number of necrotic cells . fig1 summarizes the effects of the different treatments on the apoptosis / necrosis induction in the hl60 cells .
the use of o - atp as a pharmacological agent useful for the treatment of diseases in whose onset or progression angiogenesis is involved .