Patent Application: US-31275203-A

Abstract:
the present invention relates to a novel class of inhibitors of angiogenesis and / or metastasis that targets cell adhesion molecules , and more particularly to antisense phosphorothioate oligonucleotides directed to a subunit of an integrin vitronectin receptor to inhibit angiogenesis and / or metastasis . there is provided an antisense phosphorothioate oligonucleotide directed to one of a α v , β 3 and β 5 subunit of an integrin vitronectin receptor . the antisense phosphorothioate oligonucleotide blocks synthesis of the integrin vitronectin receptor on a target cell , thereby inhibiting angiogenesis and / or metastasis . there is provided a method for blocking angiogenesis and / or metastasis in a patient , comprising delivering an efficient amount of such an antisense phosphorothioate oligonucleotide to the target cell of the patient , thereby blocking synthesis of the integrin vitronectin receptor on the target cell and blocking angiogenesis and / or metastasis .

Description:
the effect of a , antisense phosphorothioate oligodeoxynucleotides ( odn ) on α v β 3 expression and on endothelial migration was assessed using human umbilical vein endothelial cells ( huvec ). it was found that α v antisense phosphorothioate odn reduced α v β 3 expression in endothelial cell cultures and this resulted in a dose - dependent decrease in endothelial cell migration . the α v antisense phosphorothioate odn therefore represents a novel class of angiogenesis inhibitors . primary cultures and early passages of human umbilical vein endothelial cells ( huvec ) were used to establish an in vitro angiogenesis assay and to assess the effect of α v antisense odn on two parameters of the angiogenic process , namely cellular migration and proliferation . the results show that huvec cells respond to motogenic and mitogenic signals triggered by vegf and bfgf , respectively and that pre - treatment of the cells with α v antisense odn inhibited these responses . the integrins expressed by huvec include α 2 β 1 , α 3 β 1 , α 5 β 1 and α v β 3 ( luscinskas f . w ., lawler j . faseb j 8 : 919 , 1994 ). α v β 3 was identified as a marker of angiogenesis on vascular cells in various in vivo and in vitro models ( brooks p . c . et al . science 264 : 569 , 1994a ). α v β 3 can serve as an effective target for anti - angiogenic therapy , based on the present investigation into the potential anti - angiogenic effect of α v antisense odn . three eighteen - base antisense phosphorothioate odn , as1 , as2 and as3 as shown in table 1 , selected to have no homology with dna sequences of other known integrin subunits , α 5 in particular , and previously shown to have α v suppressing effects in a melanoma model , were first used . at a concentration of 40 μm , as2 reduced α v expression by 78 % but as1 and as3 had no effect . this inhibitory effect was confirmed with several huvec cultures and was dose - dependent . a sense odn control had no effect , suggesting the reduction in α v expresssion was due to specific effects of as2 . the reason for the lack of effect of as1 and as3 on α v synthesis may be that not all sites on the target mrna are equally accessible to odn . for example , when the effects of a series of synthetic oligonucleotides complementary to the 5 ′ non - coding and coding regions of rabbit beta - globin mrna on endogenous protein synthesis , was tested using a rabbit reticulocyte cell - free translation system , it was found that the sites most sensitive to inhibition are at the start of the 5 ′ noncoding region and a sequence including the initiation codon and several upstream bases . similarly , when antisense pentadecamers complementary to different sequences between the cap and aug initiation codon of the c - myc mrna were tested , it was found that the activity of antisense sequences complementary to cap - region sequences was 2 - 3 fold higher than the activity of an initiation codon antisense sequence . these observations suggest that the efficacy of an antisense odn sequence depends on its site of hybridization on the mrna sequence . as2 is complementary to the sequence spanning bases 31 - 48 of the human α v subunit , which encodes the signal peptide of the α v molecule ( suzuki s . et al . j bio chem 262 : 14080 , 1987 , fitzgerald l . et al . biochem 26 : 8158 , 1987 ). it is possible that the corresponding α v mrna sequence has a higher binding efficiency for as2 than those targeted by as1 and as3 , although this was not the case in human melanoma cells . having determined the optimal conditions for huvec migration , the effect of as2 on migration was analyzed . it was found that the inhibitory effect on huvec migration exhibited by as2 was dose - dependent and the reduction reached up to 95 % at a concentration of 40 μm . an odn sequence complemetary to mouse α v also decreased the cell migration , but the reduction was lower in magnitude than the reduction caused by as2 . dna synthesis in huvec ( an indirect measure of all division ) was reduced by up to 78 % with 40 μm as2 while mouse α v antisense reduced it up to 62 %. it was observed that individual huvec cultures varied in respect to reductions in α v levels resulting from α v antisense odn treatment . this variability may be due to difference in odn uptake by different cell cultures . as odn are negatively charged , their diffusion across the membrane lipid bilayer is highly inefficient and may be as low as 1 - 2 % of the total oligonucleotides . the efficiency of uptake can however be increased by addition of cationic liposomes ( bennett c . f . et al . j . lip . res . 3 : 85 , 1993 ). it was shown that the length of time that vascular endothelial cells are maintained in culture may affect some of their properties including prostacyclin release , angiotensin - converting enzyme activity , gene expression and cell cycle kinetics ( goldsmith j . c ., et al . lab invest 51 : 643 , 1984 ). to standardize assay conditions and increase reproducibility , huvec in the present study were used only between first and fifth in vitro passages , at which time they were discarded . this necessitated the use of fresh endothelial cell cultures prepared from newly obtained umbilical cords throughout this study . the inconsistency observed in the effects of α v antisense odn on α v β 3 expression and function may also have been related to variability between the cords obtained . diverse factors such as the age of the fetus , mother &# 39 ; s age , mother &# 39 ; s health and clinical history , and environmental factors may have contributed to differences in α v β 3 expression , various cell properties including cell permeability , cell doubling time , expression of other relevant adhesion molecules , and growth factor and growth factor receptor levels expressed by the cells . these factors among others may have contributed to the variability both in cell surface α v expression following antisense odn treatment and in the functional impact of this treatment . future in vitro angiogenesis studies may be facilitated by the use of human endothelial cell lines . the factors responsible for the non - specific effects of murine α v antisense odn are not presently clear . the results indicate however that a murine odn sequence is not an optimal control for the study of human cells . taken together , the present results show that α v antisense odn can significantly reduce cell surface α v levels on endothelial cells and inhibit cellular functions essential for angiogenesis . based on these results , the odn disclosed herein as embodiments of the present invention and others of the present invention have potential clinical applications . moreover , the present results coupled with the fact that antagonists of α v β 3 can block angiogenesis show that targeting of integrins provides an effective anti - angiogenic approach . because a large variety of adhesion molecules are expressed with great specificity on different tumor cells and at different stages of the angiogenic process ( e . g . α v β 3 is only expressed upon angiogenic stimulation ), it is possible to selectively interfere with these adhesive processes without blocking normal established adhesive interactions in the same tissue . such “ anti - adhesive therapies ” may represent a major new approach to the treatment of malignant tumors . the present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope . in the following examples , umbilical cords for obtaining the human umbilical vein endothelial cell ( huvec ) cultures were obtained after normal delivery from the surgical pathology laboratory of the royal victoria hospital ( montreal , quebec , canada ). they were stored at 4 ° c . in a saline solution containing 238 mg / ml n - 2 - hydroxyethylpiperazine - n ′- 2 - ethanesulfonic acid ( hepes ) ( gibco , burlington , ontario , canada ), 2 mg / ml anhydrous d - glucose ( sigma , oakville , ontario , canada ), and 0 . 3 mg / ml kcl ( fisher , nepean , ontario , canada ). umbilical cord veins were cannulated and flushed with ringer &# 39 ; s solution to remove all traces of blood and thrombus , and then the luminal surfaces of the veins were filled with 0 . 1 % collagenase ( sigma ) in phosphate - buffered saline ( pbs ) ( gibco ) and incubated for 15 min at 37 ° c . to detach endothelial cells . the cells were collected by flushing the veins with medium - 199 ( gibco ) and centrifuged for 7 min at 1 , 000 rpm . pellets were resuspended in medium - 199 ( basic medium ) supplemented with 20 % fetal calf serum ( fcs ) ( wisent , st - bruno , quebec , canada ), 238 mg / ml hepes ( gibco ), 10 , 000 u / ml penicillin , 10 , 000 μg / ml streptomycin , 29 . 2 mg / ml l - glutamine ( all purchased from gibco ), and with 1 . 5 mg / ml endothelial cell growth supplement ( ecgs ), and 1 , 500 u / ml heparin ( both obtained from sigma ) ( huvec medium ). cells isolated in this manner were previously identified as & gt ; 90 % endothelial in origin as confirmed by morphology ( cobblestone - like ) and positive immunofluorescence staining with an antibody to von willebrand factor ( mcgill s . n . et al . world j surg 22 : 171 , 1998 ). isolated endothelial cells were plated onto 75 - cm 2 tissue culture flasks ( sarstedt , st - leonard , quebec , canada ) which were pre - coated with 0 . 3 % gelatin ( fisher ) to improve cell attachment , and incubated at 37 ° c . in a 5 % co 2 incubator . the next day , cells were gently washed twice with medium - 199 and fresh huvec medium was added . new culture medium was replenished on alternate days . huvec cultures of 80 - 90 % confluency were used between the first and fifth passages . the effects of the following three α v antisense phosphorothioate oligonucleotide sequences on α v expression by huvec were analyzed . initially , these oligonucleotides were used to determine their effect on α v and upar expression in human melanoma cells . the oligonucleotides were synthesized by the sheldon biotechnology center ( montreal , quebec , canada ) and purified three times using ethanol precipitation ( 2 . 5 vol of ethanol and 1 / 4 vol of 10 m ammonium acetate ). subsequently , the following oligonucleotides were obtained from isis pharmaceuticals ( carlsbad , calif ., usa ): isis 15630 ( as 2 ) described above and , as a control , isis 16205 , as mentioned in table 1 . rat mab 69 - 6 - 5 to human integrin subunit α v ( lehmann m . et al . cancer res 54 : 2102 , 1994 , a gift from j . marvaldi , laboratoire de biochimie cellulaire , universite d &# 39 ; aix - marseille , marseille , france ) was used as a primary antibody . a peroxidase - conjugated goat anti - rat immunoglobulin was used as a secondary antibody ( jackson immunoresearch laboratories inc ., west grove , pa ., usa ). student &# 39 ; s t test was used to analyze migration and thymidine incorporation data . identification of an α v antisense oligonucleotide with a potent inhibitory effect on cell surface α v gene expression three antisense oligonucleotides sequences shown to suppress α v expression in human melanoma cells are herein tested for their effect on α v expression in an early passage huvec culture . huvec monolayers of 80 - 90 % confluency were dispersed with 0 . 05 % trypsin - edta ( sigma ) and plated in 0 , 3 % gelatin - coated 25 - cm 2 tissue culture flasks ( sarstedt ). the cells were allowed to spread in huvec medium . after 4 ˜ 5 h , oligonucleotides were added at the desired concentrations and this was repeated 24 h later for a total incubation time of 2 days at which time the oligonucleotides were removed and fresh huvec medium was added . expression of cell surface α v was assessed by the enzyme - linked immunosorbent assay ( elisa ). cells were treated with oligonucleotides for 48 h in 0 . 3 % gelatin - coated 96 - well tissue culture plates ( sarstedt ) in huvec medium . the cells were washed five times with pbs and non - specific protein binding sites were blocked with 1 % bovine serum albumin ( bsa ) ( boehringer mannheim , laval , quebec , canada ) in pbs for 1 h at room temperature . the cells were fixed with 0 . 125 % glutaraldehyde ( fisher ) in pbs for 2 min and rinsed five times with pbs containing 0 . 1 % bsa ( assay buffer ). to each well , 10 μg / ml mab 69 - 6 - 5 diluted in assay buffer were added for a 90 - min incubation at room temperature . unbound antibody was removed by washing the cells five times with assay buffer . a peroxidase - conjugated goat anti - mouse igg diluted 1 : 1000 in assay buffer was used as a secondary antibody and incubated with the cells for 60 min at room temperature . unbound antibody was removed by 5 washings with assay buffer . a calorimetric reaction was initiated with abts ( 2 , 2 ′- azino - bis ( 3 - ethylbenzthiazoline - 6 - sulfonic acid )) ( boehringer mannheim ) as a substrate and stopped 8 - 10 min later by the addition of 0 . 05 % sodium azide ( fisher ). color intensity was measured with a thermomax ™ microplate reader ( molecular devices corporation , menlo park , calif ., usa ) at a wavelength of 405 nm . the program used to analyze the data was softmax ™ 2 . 32 software package for the maxline ™ microplate readers ( molecular devices corporation ). results shown in fig1 demonstrate that as2 had the most potent inhibitory effect on cell surface α v expression . at a concentration of 40 μm , the level of α v decreased by 78 %. neither of the other two odn , i . e . as1 and as3 , reduced α v expression . as2 was therefore selected for all subsequent studies . in fig1 huvec were treated with α v antisense phosphorothioate oligonucleotides as1 , as2 and as3 at a concentration of 40 μm for 48 h at 37 ° c . ; α v β 3 expression was measured by elisa ; cells were washed with pbs and fixed with 0 . 125 % glutaraldehyde ; to each well , 50 μl of 0 . 1 % bsa ( negative control ) or mab 69 - 6 - 5 ( 10 μg / ml ) were added for a 90 min incubation followed by a 60 min incubation with a peroxidase - conjugated goat anti - mouse antibody ( 1 : 1000 ); abts was used as a substrate and the color intensity was measured at 405 nm ; results are expressed as percent expression relative to untreated cells and based on experiments done in triplicates . the inhibitory effect of as2 on α v expression was confirmed with a second huvec culture , as shown in fig2 . with these cells , the inhibition was shown to be dose - responsive and reached a maximum of 50 % at a concentration of 40 μm odn . a sense odn control showed no effect . in fig2 huvec were treated with as2 at concentrations of 5 - 40 μm for 48 h at 37 ° c . ; α v β 3 expression was measured by elisa as described with respect to fig1 ; results are expressed as percent expression relative to untreated cells ; and a phosphorothioate 18 - base sense sequence corresponding to as2 ( sense 2 ) was used as the control odn . in addition , antisense - treated cells lost adhesiveness and became rounded in morphology probably as a consequence of the reduction in α v expression . this loss of adhesiveness of antisense odn - pretreated endothelial cells was further confirmed by the yield of cells harvested after odn treatment relative to untreated cells . variability in response to α v antisense odn between different endothelial cell cultures the effect of as2 on α v expression is herein examined in multiple cultures derived from individual cords , simultaneously . results shown in fig3 demonstrate that the effect of odn treatment differed among different cell cultures and ranged from no reduction to an 80 % reduction in the level of α v expression in cells treated with as2 . a murine α v antisense odn showed no effect . in fig3 huvec were treated with 40 μm odn for 48 h and seeded into gelatin - coated 96 - well plates at a density of 5 × 10 3 cells / well ; α v β 3 expression was determined 24 h later with elisa as described with respect to fig1 . mouse α v antisense odn was used as a control ; results are expressed as percent expression relative to untreated cells ; and endothelial cell cultures a - e were derived from five individual cords . determination of viability of cells treated with α v antisense oligonucleotides to determine cell viability after treatment with the oligonucleotides , the cells were seeded onto 0 . 3 % gelatin - coated 96 - well tissue culture plates ( sarstedt ) at a density of 5 × 10 3 cells per well in 200 μl medium containing 5 - 40 μm antisense odn . at the intervals indicated in the text , 10 μl of a 5 mg / ml mtt ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyl tetrazolium bromide ) ( sigma ) solution were added to each well and the plates were incubated for 4 h at 37 ° c . to each well , 160 μl of dimethyl sulfoxide ( dmso ) ( fisher ) were added and the wells mixed thoroughly to dissolve the dark blue formazan crystals formed . color intensity was measured with a thermomax ™ microplate reader ( molecular devices corporation ) at a wavelength of 550 nm . the program used to analyze the data was softmax ™ 2 . 32 software package for the maxline ™ microplate readers ( molecular devices corporation ). to confirm that the inhibitory effect of the odn was not due to non - specific cytotoxic effects , cell survival was assessed after odn treatment using the mtt assay . following treatment with 5 - 40 μm odn , the cells were plated into gelatin - coated 96 - well plates and cell survival was analyzed under different culture conditions . in the presence of medium containing 1 % fcs , after treatment with as2 , no significant cell death was observed relative to untreated cells under identical conditions . although some cell lifted due to reduction in cell adhesiveness . the effect of α v antisense odn on cell migration is herein analyzed . cell migration was measured using 8 . 0 - μm nucleopore filters ( fisher ) pre - coated with 0 . 3 % gelatin . the filters were placed into 24 - well tissue culture plates ( sarstedt ) and 4 × 10 4 cells in medium - 199 containing 0 . 1 % bsa were evenly loaded onto each filter . vegf at a concentration of 25 ng / ml with 10 μg / ml human fibronectin ( roche , burlington , ontario , canada ) were placed in the lower chamber to induce cell motility . cell migration was measured 8 - 24 h later . cells were fixed with 0 . 125 % glutaraldehyde for 20 min and stained with 0 . 5 % crystal violet ( fisher ). all non - migrating cells were removed from the upper face of the filter with a cotton swab . migrating cells on the lower surface of the filter were enumerated using a nikon diaphot - tmd ™ inverted microscope ( nikon canada , montreal , quebec , canada ) equipped with an ocular square millimeter grid . to assess the effects of a , antisense odn on endothelial cell migration , huvec were treated with 5 - 40 μm odn for 48 h at 37 ° c . prior to loading of the cells onto gelatin - coated nucleopore filters . results shown in fig4 demonstrate that the inhibition of huvec migration was dose - dependent and that migration could be reduced by up to 95 % at 40 μm , relative to untreated controls . in fig4 huvec were treated with 5 - 40 μm odn for 48 h at 37 ° c . ; cells were dispersed and loaded onto gelatin - coated nucleopore filters for a 24 h incubation at 37 ° c . in the presence of vegf and fibronectin ; results are expressed as fold increase in migration relative to cells incubated in the absence of vegf and fibronectin . the results shown in fig5 demonstrate that cells derived from different umbilical cords varied in the magnitude of their response to migration - inducing factors as well as in their sensitivity to the inhibitory effects of both human ( as2 ) and murine ( mouse odn ) α v antisense odn . the difference in the reduction caused by as2 and mouse odn was significant for endothelial cell cultures a ( p & lt ; 0 . 0005 ) and b ( p & lt ; 0 . 005 ). in fig5 huvec were treated with odn and seeded onto nucleopore filters as described with respect to fig4 ; mouse α v antisense odn was used under the same conditions ; endothelial cell cultures a , b and c were isolated from three individual cords ; results are expressed as fold increase in migration relative to cells not induced by migration factors and are based on 2 filters per migration assay : cells were used after three in vitro passages ; the difference in the reduction caused by human and murine α v antisense odn was significant for endothelial cell cultures a ( p & lt ; 0 . 0005 ) and b ( p & lt ; 0 . 005 ). the effect of α v antisense odn on dna synthesis in response to bfgf was next examined . huvec were treated with the oligonucleotides first in complete huvec medium and 24 h later , in medium - 199 supplemented with 1 % fcs . on the following day , the cells were dispersed with 0 . 05 % trypsin - edta and seeded at a density of 5 × 10 3 cells per well in 96 - well tissue culture plates pre - coated with 0 . 3 % gelatin . the cells were incubated for 48 - 72 h at 37 ° c . in medium - 199 containing 1 % fcs and bfgf at a concentration of 20 ng / ml . the cells were pulsed for 18 h with 1 . 0 μci / ml [ 3 h ]- thymidine ( 50 - 90 mci / mmol , mandel scientific company ltd ., guelph , ontario , canada ), lysed by repeated freezing and thawing , harvested using the skatron cell harvester ( skatron instruments inc ., sunnyvale , calif ., usa ) and absorbed onto filtermats filter papers ( skatron instruments inc .). the filtermats were added into scintillation tubes ( skatron instruments inc .) containing 3 ml of cytoscint scintillation cocktail ( icn , costa mesa , calif ., usa ) and radioactivity was measured in a lkb 1217 rackbeta liquid scintillation counter ( lkb , helsinki , finland ). cells incubated in huvec medium or in medium - 199 containing 1 % fcs served as controls for maximal or baseline uptake levels , respectively . to test effect of anisense odn on dna synthesis huvec were pretreated with 20 or 40 μm odn for a total of 48 h and transferred to gelatin - coated 96 - well plates for the 3 h - thymidine uptake assay performed as described above . at a concentration of 40 μm , as2 odn reduced cell proliferation by up to 69 % and 78 % following 48 and 72 h incubations respectively ( see fig6 a and 6b ). these reductions were significantly higher ( p & lt ; 0 . 05 ) than those seen following treatment with control odn ). in fig6 a and 6b , huvec were pretreated with 20 or 40 μm odn for a total of 48 h and transferred to gelatin - coated 96 - well plates . the cells were incubated for 48 ( a ) or 72 ( b ) h in medium containing 1 % serum and 20 ng / ml bfgf . 3 h - thymidine incorporation was measured after an 18 h pulse . results are expressed as fold increase in 3 h - thymidine incorporation relative to cells cultured in the absence of bfgf . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth , and as follows in the scope of the appended claims . 1 . altman k . - 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