Patent Application: US-49348605-A

Abstract:
an anti - restenosis agent comprises a phosphorothioate - modified oligonucleotide includes at least on hairpin loop and a tg sequence . the hairpin loop preferably has the sequence cag cga agc . especially preferred oligonucleotides are ones which include a sequence selected from tgggg tgggg t ggggt ggggt cag cga agc and ttggg ttggg t gggtt gggtt cag cga agc . the anti - restenosis agent may be used as or in a coating on a device for implantation into the body , for instance a stent .

Description:
anti - restenosis oligonucleotides according to the invention comprise a anti - restenosis core sequence and are modified to include a hairpin loop at the 3 ′ and / or 5 ′ end and a tg sequence at the 3 ′ and / or 5 ′ end . methods of making such modifications to an oligonucleotide will be well known to a person skilled in the field of oligonucleotide chemistry . particularly preferred hairpin loops at either end of the anti - restenosis agent are cag cga agc or cag cga agc tg at the 3 ′ end and cga agc gac or gt cga agc g ac at the 5 ′ end . the core anti - restenosis sequence may or may not include a multi - guanosine sequence . suitable sequences for the core portion comprise those described in the paper of burgess et al ( 5 ). suitable oligonucleotides which do not include a multi - guanosine sequence are described in wo 97 / 12899 . particularly preferred oligonucleotides according to the invention are selected from the group of : tgggg tgggg t ggggt ggggt cag cga agc ; tgggg tgggg t ggggt ggggt cag cga agc tg ; ttggg ttggg t gggtt gggtt cag cga agc ; and ttggg ttggg t gggtt gggtt cag cga agc tg . these oligonucleotides may be synthesised according to conventional techniques in the art , by utilising any of the commercially available , automated nucleic acid synthesizers without any further chemical modifications . prior to attachment to a stent , an anti - restenosis oligonucleotide according to the invention is purified by high performance liquid chromatography ( hplc ) on a reversed phase c18 column using tetraethylammonium acetate / acetonitrile elution buffers . in order to monitor the incorporation of the oligonucleotide on the stent , radioactive labelling may be carried out according to a conventional technique using 32 p as an isotopic label . the labelling may be carried out by transfer of a 32 p radioactive phosphate group from 32 p - gamma - atp to the 5 ′ position of the oligonucleotide by polynucleotide kinase at 37 ° c . in labelling buffer medium . a suitable technique for attaching an oligonucleotide according to the invention to - a stent is described in wo 99 / 03517 in which a polymer matrix is attached to the stent prior to incorporation of the oligonucleotide by the polymer matrix . in a typical example , and referring to fig1 and 3 , the polymer matrix is formed from 3 , 4 - ethylenedioxythiophene . to coat a stent under good conditions , a connection system , as shown in any of fig4 to 9 , is used to increase contact points between a stent and a conventional electrochemical cell without alteration and / or deformation of the medical device . before electro - deposition , a surface pre - treatment of the couple stent / connection system is made with absolute ethanol under sonication . according to fig1 to 3 , electropolymerisation is carried out using a conventional electrochemical cell comprising three electrodes , a counter electrode 1 , a silver wire 2 covered with silver chloride acting as reference electrode providing a constant potential , and a working electrode 3 giving a potential with respect to the counter electrode . a tank 4 holds the solution of monomer to be polymerised , a salt providing electrical conduction and a stent 5 which in this case is formed of stainless steel . the monomer is 3 , 4 - ethylenedioxythiophene , to which polyvinylpyrolidone ( 2 × 10 − 3 m ) is added . the electrolytic solution is pbs ( phosphate buffered saline ) at ph 7 . 4 , 10 mm comprising the following salts na 2 hpo 4 ; nah 2 po 4 . the monomer is added to the pbs solution . the potentiodynamic method ( chronoamperometry ) is used , the potential could range from + 1 . 1v to + 1 . 3v with respect to ag / agcl . the oligonucleotide is added to the electrolytic solution before polymerisation . the potential is maintained for approximately 10 to 40 minutes . this way to prepare the odn - coated stent is also called “ one step polymerization ”. in fig4 to 9 , reference numeral 3 indicates the working electrode , 5 indicates the stent , 6 indicates stainless steel wire and 7 indicates polymer wire . these figures illustrate examples of a connection system involving the stent and the working electrode that may be employed in the electrochemical cell used to deposit a suitable polymer matrix onto the stent such as is illustrated in fig1 to 3 . the oligonucleotide - coated stent formed according to the method described above , may be used in ptca procedures . the invention is not limited to the embodiments herein before described which may be varied in both construction and detail . in vitro and proliferation studies were performed using two different phosphorothioate - modified oligonucleotides c and d ( as defined below ) at four different concentrations ( 5 , 10 , 20 and 60 μmol l − 1 ). the studies were conducted on human coronary artery smooth muscle cells ( hcasmc ) in order to investigate which oligonucleotide presented the best inhibitory effect on cell proliferation with the lowest cytotoxic effect . the protocol was based on an adaptation of the requirements of the c . a . stein ( 29 ) protocol . the hcasmc were transferred and propagated at 37 ° c . in a gaseous environment of 5 % co 2 in an open flask containing medium 231 supplemented with 5 % smooth muscle growth supplement ( smgs ). the cells were used after four passages . verification of hcasmc phenotype was performed via positive staining for α - actin and negative staining for von willebrand factor . the cells were washed in phosphate buffer saline and fixed in methanol at − 20 ° c . the cells were permeabilized with a triton solution . specific antibodies were used ( monoclonal anti - human α - actin and goat igg anti - von willebrand factor ). then , specific solutions with conjugated antibodies marked with fluoresceine iso thio cyanate were added ( antimousse igg or anti goat igg ). the preparations were covered with a mounting medium and examined under a fluorescence microscope . human umbilical vein endothelial cells ( huvec ) pooled from multiple isolates cells were used as control : positive for the von willebrand factor and negative for the α - actin staining . positive staining for von willebrand factor was observed on huvec cells . negative staining for von willebrand factor was observed on hcasmc cells . the cytotox 96 ® non - radioactive cytotoxicity assay ( promega ) was used . the cytotox 96 ® assay quantitatively measures ldh , a stable cytosolic enzyme that was released upon cell lysis using a calorimetric method . released ldh in culture supernatant was measured with an enzymatic assay which results in the conversion of a tetrazolium salt ( int ) into a red formazan product , using diaphorase enzyme . the amount of color formed , detected at the visible wavelength of 490 nm , was proportional to the number of lysed cells . the general chemical reactions of the cytotox 96 ® assay are as follows : this assay can reveal early , low - level damage to cell membrane that may be missed using other methodologies . triplicate 100 μl of basal culture medium ( m199 ) or culture medium ( m199 ) supplemented with 0 . 2 % bovine serum albumin ( bsa ) or 10 % fetal calf serum ( fcs ) or 100 ng / ml platelet derived growth factor - ab ( pdgf ), in the absence of cells and placed in the same conditions as described for the test articles were used as the culture medium background . volume correction control : triplicate 100 μl of basal culture medium ( m199 ) or culture medium ( ml99 ) supplemented with 0 . 2 % bsa or 10 % fcs or 100 ng / ml pdgf in the absence of cells , in which 15 μl of lysis solution ( 10 ×) was added and placed in the same conditions as described for the test articles , were used as the volume correction control . triplicate 100 μl of basal culture medium ( m199 ) or culture medium ( m1 99 ) supplemented with 0 . 2 % bsa or 10 % fcs or 100 ng / ml pdgf , in the presence of cells and placed in the same conditions as described for the test articles , were used as the negative control . triplicate 100 μl of ldh positive control ( bovine heart ldh , 1 : 5000 dilution in phosphate buffered saline ) prepared just before use was used as the positive control . in that way , a preliminary test was allowed to optimise the cell seeding in order to reach a high enough optical density ( od ) to ensure an adequate signal - to - noise ratio . different cell seeding was tested 5 × 10 3 , 1 × 10 4 and 2 × 10 4 cells / 100μl . triplicate cell monolayers were cultured during 48 hours in 96 - well plates in the presence of 100 μl culture medium ( m199 ) supplemented with 0 . 2 % bsa . thereafter , the cells were incubated with 100 μl of basal culture medium ( m199 ) or culture medium supplemented with 0 . 2 % bsa , culture media supplemented with 10 % fcs or 100 ng / ml pdgf during 48 hours . a culture medium background control was prepared . 15 μl of lysis buffer was then added to some culture wells , incubated at 37 ° c . during 45 minutes and centrifuged at 250 g for 4 minutes . 50 μl aliquots from all wells were transferred to an enzymatic assay plate , 50 μl of substrate mix was added and the plates were incubated 30 minutes at room temperature protected from light . then , 50 μl of stop solution was added in each well and the optical densities ( od ) were recorded within 1 hour . the appropriate concentration of cells was determined by od values as at least twice the od of the culture medium background control . the results are shown below . the increase in od after lysis was higher with the 2 × 10 4 cells seeding than with the 5 × 10 3 cells seeding . this cell concentration ( 2 × 10 4 cells ) was therefore selected for further experiments . in the presence of m199 + 10 % fbs medium , an interference was observed , but the results were interpreted according to the formula presented on page 6 . pdgf enhanced only very slightly the hcasmc growth , particularly as compared to fbs . triplicate cell monolayers were cultured in duplicate , using the seeding which was determined in the primary test ( wells for the test before and after cell lysis ), in 96 - well plates for 48 hours in the presence of 100 μl culture medium ( m199 ) supplemented with 0 . 2 % bsa . thereafter , the cells were incubated under the conditions shown in table 5 for 48 hours . a culture medium background control , a volume correction control , negative and positive controls were prepared . 15 μl of lysis buffer was added to one exemplar of the experiment ( maximum ldh release ), incubated at 37 ° c . for 45 minutes and centrifuged at 250 g for 4 minutes . the other exemplar ( experimental ldh release ), in which cell morphology was noted for each condition , was not treated for cell lysis . then , 50 μl aliquots from all wells were transferred to an enzymatic assay plate , 50 μl of substrate mix was added in each well and the plate incubated for 30 minutes at room temperature protected from light . afterwards , 50 μl of stop solution was added to each well and the od was recorded within 1 hour . a mean od was calculated for each condition and the percentage of cytotoxity was calculated as follows : % ⁢ ⁢ cytotoxicity = experimental ⁢ ⁢ mean ⁢ ⁢ ldh ⁢ ⁢ release ⁢ ⁢ ( od ) - ⁢ mean ⁢ ⁢ control ⁢ ⁢ culture ⁢ ⁢ medium ⁢ ⁢ background ⁢ ⁢ ( od ) maximum ⁢ ⁢ ldh ⁢ ⁢ release ⁢ ⁢ mean ⁢ ⁢ ( od ) - volume ⁢ ⁢ correction ⁢ ⁢ control ⁢ ⁢ mean ⁢ ⁢ ( od ) × 100 table 8 results of ldh release expressed as optical density ( od ) at 490 nm : culture reagents + 100 ng / ml pdgf od ( mean of 3 assays ) % at 490 nm mean of % cytotoxicity concentration before after % cytotoxicity related to ( μmol / l ) cell lysis cell lysis cytotoxicity ( 1 ) and ( 2 ) the product * cmbc + ( 1 ) / 0 . 048 / / / / 100 ng / ml ( 2 ) / 0 . 056 / / / / pdgf vcc + ( 1 ) / 0 . 048 / / / 100 ng / ml ( 2 ) / 0 . 059 / / / pdgf negative ( 1 ) / 0 . 331 1 . 330 22 . 1 28 . 7 0 control + ( 2 ) / 0 . 511 1 . 353 35 . 2 100 ng / ml pdgf ( 1 ) first assay ( 2 ) second assay *% of cytotoxicity of assays − % of cytotoxicity of the negative control table 10 results of ldh release expressed as optical density ( od ) at 490 nm : positive and negative controls od ( mean of 3 assays ) concentration at 490 nm ( μmol / l ) before cell lysis after cell lysis positive control ( 1 ) / 0 . 936 / ldh ( 2 ) 1 . 846 / positive control ( 1 ) 6 . 4 g / l / 1 . 495 phenol ( 2 ) 1 . 751 ( 1 ) first assay ( 2 ) second assay *: % of cytotoxicity of assays − % of cytotoxicity of the negative control control plates exhibited a relative cytotoxic effect , ranging from about 14 . 6 % in the case of fbs to about 28 . 7 % in the case of pdgf . this situation was not unlikely related to culture conditions realizing a previous “ starvation ” of the cells prior to exposure to the active components ( 48 hours in contact with m199 + 0 . 2 % bsa , without fbs or pdgf ). pdgf without fbs did not represent optimal growth and survival conditions for hcasmc . both oligonucleotides c and d exhibited a cytotoxic effect which was dose dependent and more marked in the presence of pdgf as compared to fbs . under identical concentrations , oligonucleotide c was regularly more cytotxic than oligonucleotide d . triplicate cell monolayers were grown to 60 - 70 % confluence in 12 - well plates using m231 medium supplemented with smgs ( 0 . 5 ml per well ). the cell monolayers were washed 3 times with m199 and incubated for 48 hours in the presence of 0 . 5 ml of culture medium ( m199 ) supplemented with 0 . 2 % bsa . thereafter , the cells were incubated under the conditions mentioned in table 5 for 48 hours . negative and positive controls were prepared . cells were trypsinized , trypan blue was added and the cells were counted twice using malassez cell . a mean was calculated and the percentage of proliferation assay was calculated as follows : % ⁢ ⁢ proliferation = experimental ⁢ ⁢ condition ⁢ ⁢ mean ⁢ ⁢ ( cells ⁢ / ⁢ cm 2 ) × 100 experimental ⁢ ⁢ condition ⁢ ⁢ corresponding ⁢ ⁢ control ⁢ ⁢ mean ⁢ ⁢ ( cells ⁢ / ⁢ cm 2 ) ⁢ triplicate culture wells containing 0 . 5 ml of basal culture medium ( m199 ) or culture medium ( m199 ) supplemented with 0 . 2 % bsa and 10 % fcs or 100 ng / ml pdgf , in the presence of cells and placed in the same conditions as described for the test articles , were used as the negative control . triplicate culture wells containing 0 . 5 ml of basal culture medium ( m199 ) or culture medium ( m199 ) supplemented with 0 . 2 % bsa or 10 % fcs or 100 ng / ml pdgf , in the presence of cells in which 6 . 4 g / l phenol was added and placed in the same conditions as described for the test articles , were used as the positive control . table 13 results of the proliferation test expressed as a number of cells : oligonucleotide c and oligonucleotide d ( m199 + 100 ng / ml pdgf ) see annex 4 - graph 4 mean % of cell count inhibition ( number as oligonucleotides / concentration of cells ) % compared controls μmol / l (× 10 3 ) proliferation to control negative control / 78 . 8 100 0 ( m199 + 100 ng / ml pdgf ) positive control / 0 0 100 ( m199 + 100 ng / ml pdgf ) + phenol oligonucleotide c 5 17 . 7 22 . 5 77 . 5 ( m199 + 100 10 11 . 4 14 . 5 85 . 5 ng / ml pdgf ) 20 13 . 0 16 . 5 83 . 5 60 3 . 81 4 . 8 95 . 2 oligonucleotide 5 65 . 3 82 . 9 17 . 1 d ( m199 + 100 10 38 . 9 49 . 4 50 . 6 ng / ml pdgf ) 20 38 . 1 48 . 4 51 . 6 60 24 . 6 31 . 2 68 . 8 the results in table 13 are shown graphically in fig1 . these results showed that both oligonucleotides c and d have an inhibitory effect on the proliferation of hcasmc , which was dose dependent . this effect was observed in the presence of fbs 10 % and in the presence of pdgf 100 ng / ml . whichever fbs or pdgf were used , oligonucleotide c always exhibited a greater inhibition than oligonucleotide d . concentrations of 5 μmol / l of oligonucleotide c were already efficient to reduce by 45 % and 78 % respectively hcasmc growth in the presence of fbs or pdgf . the concentration of oligonucleotide c able to inhibit the cell growth at a level of 78 % was of 5 jmol / l in the presence of pdgf , whereas a similar inhibition was obtained with 60 μmol / l in the presence of fbs . this could be explained , either by a relative neutralization of the oligonucleotide c by fbs , and / or by a stronger proliferation effect of fbs on the cells . in vitro and proliferation studies were performed according to the procedures described in example 1 using two different phosphorothioate - modified oligonucleotides cl and c2 ( as defined below ). the studies were conducted on hcasmc . the phosphorothioate - modified oligonucleotides c1 and c2 were as follows : the hcasmc were transferred and propagated at 37 ° c . in a gaseous environment of 5 % co 2 in an open flask containing medium 231 supplemented with 5 % smooth muscle growth supplement ( smgs ). the cells were used after 6 passages . verification of hcasmc phenotype was performed via positive staining for α - actin and negative staining for von willebrand factor as described in example 1 . triplicate cell monolayers were cultured in duplicate , using the seeding which was determined in the primary test ( example 1 ) ( wells for the test before and after cell lysis ) in 96 - well plates for 48 hours in the presence of 100 μl culture medium ( m199 ) supplemented with 0 . 2 % bsa . thereafter , the cells were incubated under the conditions shown in table 14 for 48 hours . a culture medium background control , a volume correction control , negative and positive controls were prepared . 15 μl of lysis buffer was added to one exemplar of the experiment ( maximum ldh release ), incubated at 37 ° c . for 45 minutes and centrifuged at 250 g for 4 minutes . the other exemplar ( experimental ldh release ), in which cell morphology was noted for each condition , was not treated for cell lysis . then , 50 μl aliquots from all wells were transferred to an enzymatic assay plate , 50 μl of substrate mix was added in each well and the plate incubated for 30 minutes at room temperature protected from light . afterwards , 50 μl of stop solution was added to each well and the od was recorded within 1 hour . a mean od was calculated for each condition and the percentage of cytotoxicity was calculated as follows : % ⁢ ⁢ cytotoxicity = experimental ⁢ ⁢ mean ⁢ ⁢ ldh ⁢ ⁢ release ⁢ ⁢ ( od ) - mean ⁢ ⁢ control ⁢ ⁢ culture ⁢ ⁢ medium ⁢ ⁢ background ⁢ ⁢ ( od ) maximum ⁢ ⁢ ldh ⁢ ⁢ release ⁢ ⁢ mean ⁢ ⁢ ( od ) - volume ⁢ ⁢ correction ⁢ ⁢ control ⁢ ⁢ mean ⁢ ⁢ ( od ) × 100 table 19 results of ldh release expressed as optical density ( od ) at 490 nm : positive and negative controls concentration od ( mean of 3 assays ) at 490 nm ( μmol / l ) before cell lysis after cell lysis positive control / 1 . 846 / ldh positive control 6 . 4 g / l / 1 . 751 phenol control plates exhibited a relative cytotoxic effect , about 11 . 5 % in the case of fbs . in the presence of pdgf , the control plates exhibited an important cytotoxicity about 35 . 2 %. this situation was not unlikely related to culture conditions realizing a previous “ starvation ” of the cells prior to exposure to the active components ( 48 hours in contact with m199 + 0 . 2 % bsa , without fbs or pdgf ). pdgf without fbs did not represent optimal growth and survival conditions for hcasmc . the two oligonucleotides exhibited comparative cytotoxic effects in the presence of fbs about 8 %. in the presence of pdgf , the two oligonucleotides exhibited a proliferation effect . this proliferation was more important in the presence of c 1 . this was carried out as described in the procedure ii - 1 ) in example 1 , except that the cells were incubated under the conditions mentioned in table 14 for 48 hours . table 21 results of the proliferation test expressed as a number of cells : oligonucleotide c1 + 10 % fbs and oligonucleotide c2 + 10 % fbs mean cell count % of inhibition oligonucleotides / ( number of cells ) % of as compared controls (× 10 4 ) proliferation to control negative control + 8 . 69 100 0 10 % fbs positive control + 0 0 100 10 % fbs + phenol oligonucleotide 4 . 83 55 . 6 44 . 4 c 1 + 10 % fbs at 10 μmol / l oligonucleotide 5 . 37 61 . 8 38 . 2 c 1 + 10 % fbs at 10 μmol / l these results showed that both oligonucleotides have an inhibitory effect on the proliferation of hcasmc . this effect was observed in the presence of fbs 10 % and in the presence of pdgf 100 ng / ml . whichever fbs or pdgf were used , oligonucleotide c 1 exhibited always a greater inhibition than oligonucleotide c 2 . in the conditions of the study : the phenotype of the cells ( positive staining with anti α - actin antibodies and negative staining with anti - von willebrand factor antibodies ) has been validated . both oligonucleotides exhibited a slight cytotoxic effect in the presence of fbs and a proliferation effect in the presence of pdgf . at 10 μmol / l concentration , oligonucleotide c 2 was regularly more cytotoxic than oligonucleotide c 1 . at 10 μmol / l concentration oligonucleotide c 1 was more potent than oligonucleotide c 2 to inhibit hcasmc growth . 1 . bennett , m . r ., lindner , v ., deblois , d ., reidy , m . a . and schwartz , s . m . “ effects of phosphorothioate oligonucleotides on neointima formation in the rat carotid artery , dissecting the mechanism of action ”. arterioscler . thromb . vasc . biol . 1997 , 17 : 2326 - 32 . 2 . wang , w . z ., chen , h . j ., schwartz , a ., cannon , p . j ., stein , c . a . and rabbani , l . e . “ sequence - independent inhibition of in vitro vascular smooth muscle cells proliferation , migration and in vivo neointimal formation by phosphorothioate oligodeoxynucleotides ”. j . clin . invest . 1996 , 98 : 443 - 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