Patent Application: US-59993705-A

Abstract:
atherosclerosis can be viewed as a response to injury and it is not atherosclerosis per se that is serious but instead factors leading to rupture of atherosclerotic plaques . in the present invention we have identified such factors . a decreased binding of annexing v to the endothelium was seen in patients with a history of atherothrombosis . the use of native annexin v or an n - terminal fragment as an active component or a subfraction of immunoglobulins to manufacture a pharmaceutical composition is proposed to improve said binding . the use of annexin v , n - terminal fragments or immunoglobulins to increase the annexin v - binding to carotid plaque represents novel mechanism for preventing atherothrombosis .

Description:
the risk of atherothrombosis and plaque rupture is strongly raised when annexin v binding to endothelium is decreased as a consequence of antibodies inhibiting the annexin v - plaque binding . restoring of the annexin v binding by administration of annexin v ( or fragments ) or by administering immunoglobulins , preferably a subfraction of immunoglobulins ( ie a subfraction of a pooled immunoglobulin preparation as discussed above ), that inhibit other antibodies ( for example annexin - v - binding antibodies ) that decrease the plaque - annexin binding provides novel proposed therapies for atherothrombosis and especially plaque rupture , the main cause of cardiovascular disease . a first aspect of the invention provides the use of the annexin v protein or an n - terminal fragment of annexin v , optionally in the form of a salt , in the manufacture of a pharmaceutical composition to prevent atherothrombosis and / or plaque rupture . the term annexin v is well known to those skilled in the art and is used , for example , in documents cited above , for example ep 1 379 266 . as will be apparent to the skilled person , the n - terminal fragment of annexin v is large enough to be recognisable by the skilled person as a fragment of annexin v ( rather than , for example , a fragment of another annexin . the pharmaceutical composition may comprise an effective amount of the annexin v protein or n - terminal fragment of annexin v , optionally in combination with a carrier and additives . suitable carriers and additives which can be used will be well known to those skilled in the art , including the examples used in ep 1 379 266 . the salt can be a pharmaceutically acceptable acid addition salt where the counter ion is , for example , chloride , acetate . the effective amount of the annexin v in the pharmaceutical composition may be determined from a diagnostic status analysis of the annexin v - endothelium binding . thus , the dosage may be determined by assessing the state of annexin v - endothelium binding in the patient . thus , annexin v - endothelium binding may be assessed by assessing the effect of the patient &# 39 ; s serum on binding of annexin v to endothelium , for example using a technique such as that used in the examples to assess the effect of patient plasma on binding of annexin v to cultured endothelial cells ( see the “ culture of endothelial cells binding ; binding of annexin v ” and “ results ” sections ). immunohistochemical staining of biopsy plaque material from the patient ( as described in the “ immunohistochemical staining of human atherosclerotic plaque ” section of the examples ) may also be used . imaging techniques using labelled annexin ( see discussion of reference 16 above ( wo 95 / 34315 )) may also be used . as will be understood by the skilled person , should the patient be found to have very low annexin v binding then a higher dosage of annexin v ( or fragment thereof ) may be required than if the patient is found to have annexin v binding closer to that found in a normal person . a further aspect of the invention provides a method of treating a subject at risk of atherothrombosis and / or plaque rupture , comprising administering to said subject a pharmaceutical composition comprising an effective amount of annexin v or an n - terminal fragment of annexin v , optionally in the form of a salt . the subject at risk may be a systemic lupus erythematosus ( sle ) patient . the sle patient may further have risk factors as discussed above , for example dyslipidemia , increased oxidation of low density lipoprotein ( oxldl ), raised activity in the tumour necrosis factor ( tnf )- system ( closely associated with dyslipidemia ), systemic inflammation as determined by crp , homocystein and anti - phospholipid antibodies ( apl ). the sle patient may further show recurrent thrombosis or frequesnt repeating atherothrombosis , or have survived one or more manifestations of cardiovascular disease , for example thromboembolic , not hemorrhagic or vasculitic stroke , myocardial infarction , angina pectoris or intermittent claudication . the subject at risk may be a patient that has or has had , or is at risk of , a pneumococcal infection ; or may be a patient with vulnerable plaques , as identified based on symptoms and clinical evalutation indicative of imminent cardiovascular disease such as unstable angina , other forms of severe angina , or transient ischemic attacks ( tia ). an annexin v - endothelium binding assessment , as discussed above , may be used in assessing risk . a further aspect of the invention provides a purified subfraction of immunoglobulins ( ie a purified subfraction of a pooled immunoglobulin preparation , as discussed above ) with the capacity to inhibit antibodies binding to annexin v . such a subfraction may be prepared using techniques indicated above , for example using affinity purification . the purified subfraction may be an anti igg antibody subfraction , for example a subfraction selected by affinity binding to igg , in particular to the igg constant domain . a further aspect of the invention provides a purified subfraction of immunoglobulins ( ie a purified subfraction of a pooled immunoglobulin preparation , as discussed above ) with the capacity to promote binding of annexin v to endothelium ( or endothelial cells , for example in culture ). such a subfraction may be prepared using techniques indicated above , for example using affinity purification . the subfraction may be anti ig antibody subfraction , for example a subfraction selected by affinity binding to igg , in particular to the igg constant domain . such a subfraction may contain anti - apaf , anti - alpc , anti - aps or anti - a - pneumococcal vaccine which may reduce levels of apaf , alpc , aps or antibodies to penumococcal vaccine , depletion of which was found to increase annexin v binding ( see the examples ). the capacity of the subfraction to promote binding of annexin v to endothelium may be assessed using an annexin v - endothelium binding assay as referred to above and in the examples . for example , an appropriate subfraction may be one which decreases the inhibitory effect of plasma from sle cases ( high antiphospholipid antibodies ( apls ) titer serum ) on binding of annexin v to endothelial cells . the subfraction may be one prepared by affinity purification based on binding to a phosphorylcholine conjugate , for example pc - bsa or pc - klh , for example as discussed in the examples . such a subfraction may have raised levels of anti phosphorylcholine ( apc ) antibodies eg apc igg and / or apc igm relative to the starting immunoglobulin preparation . as noted in the examples , we have found a statistical correlation between levels of apc - bsa and apc - klh and annexin v binding . it is considered that apc igg and / or igm may bind to some igg ( see , for example , halpern et al ( 1991 ) j clin invest 88 ( 2 ), 476 - 482 ) and are most likely produced by b1 cells , a b cell subtype that may also neutralize b2 cells that produce igg . a further aspect of the invention provides a purified subfraction of the invention for use in medicine , for example for preventing atherothrombosis and / or plaque rupture . a further aspect of the invention provides the use of a purified subfraction according to the preceding aspect of the invention or the use of a commercially available immunoglobulin preparation ( ie a pooled immunoglobulin preparation , as discussed above ) in the manufacture of a medicament to prevent atherothrombosis and / or plaque rupture . a further aspect of the invention provides a method of treating a subject at risk of atherothrombosis and / or plaque rupture , comprising administering to said subject a pharmaceutical composition comprising an effective amount of immunoglobulins ( ie of a pooled immunoglobulin preparation as discussed above ) or a purified subfraction of immunoglogulins ( examples of which are indicated above ). preferences for the subject to be treated ( or for which the medicament is for treating ) are discussed above . the subject may be , for example , a systemic lupus erythematosus ( sle ) patient or a patient who has , has had or is at risk of a pneumococcal infection ; or a patient with vulnerable plaques and / or unstable angina . the invention will now be described in more detail by reference to the following , non - limiting , figures and examples . fig1 : effect of pre - incubation of high antiphospholipid antibodies ( apls ) titer serum with human pooled immunoglobulin gammagard ® on annexin v binding to human umbilical endothelia cells ( huvecs ): flow cytometry analysis after 24 hrs culture . fig2 : effect of pre - incubation of high antiphospholipid antibodies ( apls ) titer serum with pneumococcal capsular polysaccharide on annexin v binding to huvecs : flow cytometry analysis after 24 hrs culture . fig3 : effect of pre - incubation of high antiphospholipid antibodies ( apls ) titer serum with lysopc and paf on annexin v binding to huvecs : flow cytometry analysis after 24 hrs culture fig4 : effect of pre - incubation of high antiphospholipid antibodies ( apls ) titer serum with irrelevant antigen ( tetanus toxoid ) on annexin v binding to huvecs : flow cytometry analysis after 24 hrs culture . fig5 : effect of pre - incubation of high antiphospholipid antibodies ( apls ) titer serum with lysopc on annexin v binding to huvecs : flow cytometry analysis after 24 hrs culture . the study group consisted of 26 women with sle who had survived one or more manifestations of cardiovascular disease , defined as thromboembolic , not hemorrhagic or vasculitic stroke ( n = 15 ), ( confirmed by computed tomography or magnetic resonance imaging ); myocardial infarction ( n = 7 ), ( confirmed by electrocardiography and a rise in creatine kinase ); angina pectoris ( n = 9 ) ( confirmed by exercise stress test ) or intermittent claudication ( n = 4 ) ( peripheral atherosclerosis confirmed by angiogram ), 26 age - matched women with sle and no clinical manifestations of cardiovascular disease and 26 age - matched healthy population - based women . all patients fulfilled the 1982 revised criteria of the american rheumatism association for sle16 . the study was approved by the ethics committee of the karolinska hospital . all participants gave informed consent before entering the study . the right and left carotid arteries were examined with a duplex scanner ( acuson sequoia , mountain view , calif ., usa ) and the degree of atherosclerosis was determined by intima - media thickness ( imt ) was determined . cryopreserved pooled human umbilical venous endothelial cells ( huvecs ) at passage 2 were purchased from cascade biologics , inc . ( portland , oreg ., usa ) the cultures were maintained in egm ™ phenol red - free medium ( clonetics , san diego , calif ., usa ), containing 2 % of fetal bovine serum and supplements . the cells were incubated in 75 cm2 flasks ( tpp , ag , trasadingen , switzerland ) under humidified 5 % co2 in 37 ° c . conditions . all experiments were performed at passage 3 to 4 . huvecs were seeded at 2 × 10 4 cells / ml density in 12 - well plates ( nunc , inc , naperville , ill ., usa ) for flow cytometry analysis ; at density of 1 × 10 4 cells / well / 100 μl in 96 - well plate ( tpp ) for mtt assay ; at 8 × 10 3 cells / ml . density in 24 - well plates ( nunc ) for dna fragmentation elisa . after allowing 12 - 24 hours for attachment and careful washing with serum - free medium ( sfm ), the cells were made quiescent in sfm for at least 12 hrs prior to treatment . heparin - preserved plasma from the study groups was added to the monolayer at concentration of 10 % in sfm . the cells were harvested non - enzymatically with cell dissociation solution ( cds ; sigma - aldrich , st . louis , mo ., usa ). huvecs were carefully pooled with supernatants , to exclude selective loss of detached floating ec , and centrifuged at 1200 rpm for 7 min . after resuspension in 100 μl of annexin v - binding buffer ( molecular probes inc , eugene , oreg ., usa ) samples were stained with 5 mg / ml of annexin v - fitc ( mol . probes ) and incubated for 15 min on ice . shortly before acquisition 1 mg / ml of propidium iodide ( pi ; r & amp ; dsystems europe ltd , abingdon , uk ) was added . analysis was performed on a facscan flow cytometer ( bd biosciences , san jose , calif ., usa ) equipped with cellquest ™ software . during acquisition a gate was set to exclude events smaller than 230 on linear fcs and ssc scale . for each sample 10000 events were collected . immunostaining was performed on human plaques , characterized previously . plaques were collected from 12 patients undergoing carotid endarterectomy after transient ischemic attacks . all specimens contained advanced atherosclerotic lesions . as a control macroscopically healthy mesenteric artery was obtained after unrelated bowel resection . the cryostat sections were fixed for 20 minutes in 2 % paraformaldehyde in pbs ( sigma chemicals ) at 4 ° c . and stored at − 70 ° c . after blocking endogenous peroxidase , the sections were incubated overnight with monoclonal anti - annexin v antibody ( alexis biochemicals , corp ., lausen , switzerland ) of mouse type igg2a , anti - cd68 ( dakocytomation , glostrup , denmark ) or anti - cd31 ( monosan , uden , the netherlands ). irrelevant mouse igg2a ( serotec ltd , oxford , uk ) served as negative control . all antibodies were diluted in 1 % bsa - 0 . 02 % nan3 in pbs . after washing , 1 % normal horse serum in pbs was used . secondary antibody - biotinylated horse anti - mouse immunoglobulin ( vector laboratories , burlingame , calif ., usa ) was added . the abc peroxidase elite ™ kit was used ( vector laboratories ). the staining was revealed with diaminobenzidine ( vector laboratories ) and counterstaining was done with haematoxyline . all sections were analyzed on a leica dmrxa microscope ( leica , wetzlar , germany ) total igm or igg fraction was separated from commercially available pooled human immunoglobulin ( gammagard ®) at 50 mg / ml using hitrap igm or igg columns ( amersham biosciences ). antibodies against phosphorylcholine ( pc ) were eluted after loading igm or igg fraction on nhs - sepharose columns coupled to pc conjugated either to keyhole limpet haemocyanin protein ( klh ) ( 1 or 5 mg / ml ) or to bovine serum albumin ( bsa ) ( 1 mg / ml ) followed by bsa - only column . pc - bsa ( phosphorylcholine - bovine serum albumin ) and pc - klh was purchased from biosearch technologies , inc ( ca , usa ). eluted fractions were buffer - exchanged on pd - 10 columns and concentrated with millipore centricone ® devices . procedures were performed according to instructions given by manufacturers . the concentration of igm apc prepared was typically 50 μg / ml , and the concentration of igg apc was typically 30 μg / ml . heparin - preserved plasma with high capacity to inhibit annexin v binding was added to huvecs monolayer at concentration of 10 % in sfm . after 24 hrs cells were harvested with cell dissociation solution ( cds ; sigma - aldrich , st . louis , mo ., usa ) and carefully pooled with supernatants , to exclude selective loss of detached floating cells , centrifugation at 1200 rpm for 7 min followed . after resuspension in 100 μl of annexin v - binding buffer ( molecular probes inc , eugene , oreg ., usa ) samples were stained with 2 μl of 5 mg / ml annexin v - fitc ( molecular probes ) and incubated for 15 min on ice . shortly before acquisition 1 mg / ml of propidium iodide ( pi ; a vital dye ; r & amp ; dsystems europe ltd , abingdon , uk ) was added . analysis was performed as described above . depletion of igg subclass of immunoglobulins resulted in up to 2 . 7 - 2 . 6 fold increase in fluorescence intensity of annexin v - binding ( complete serum mean fi : 267 . 95 ± vs . 709 . 91 ±; median fi : 222 . 67 ± vs 567 . 42 ±). reconstitution of igg fraction to the depleted sera decreased the fluorescence intensity while the culture with igg eluate resulted in fluorescence intensity of annexin v binding comparable with that of complete sera . binding of annexin v was significantly lower after 24 hrs when plasma from sle cases was used as compared to controls ( sle cases vs population controls : p = 0 . 002 , sle cases vs sle controls p = 0 . 02 ). depletion of total igg from sera with a high capacity to inhibit binding of annexin v restored this binding completely . there was a striking positive association between annexin v - binding and degree of atherosclerosis ( r = 0 . 73 , p & lt ; 0 . 001 ) among sle cases . immunostaining revealed presence of annexin v in 11 / 12 plaques tested . pooled sera with a high ability to inhibit annexin v binding to ec were 0 . 45 μm filtered and diluted with equal volumes of endothelial basal medium . the hitrap protein g hp , 1 ml column with binding capacity 25 mg human igg / ml gel from amersham biosciences ( uppsala , sweden ) was used according to manufacturer &# 39 ; s instructions . the igg fraction was obtained by eluting the column with 0 . 1 m glycine - hcl , ph 2 . 7 . for neutralization 1 m tris - hcl , ph 9 . 0 was used . complete serum , effluate and eluate were used for incubation with huvec on the day of separation at 1 : 10 dilution in sfm . the frequency of huvecs positive for annexin v staining was determined either as percentage of annexin v + / pi − cells on a bivariate dot plot or percentage of annexin v + cells based on a histogram . annexin v - binding to huvecs in the presence of serum known to decrease binding and preincubated with ivig was determined . preincubation with ivig could restore binding of annexin , indicating that antibodies present in ivig could neutralise binding ( fig1 ). apc - bsa and apc - klh were both associated significantly in sle patients with a history of cvd with annexin v binding to ec ( r = 0 . 45 ; p = 0 . 02 and r = 0 . 03 respectively ). apc - bsa and apc - klh levels were assessed using standard techniques , for example using the following reagents . polysorp f96 microtiter immuno - plates were purchased from nunc ( roskilde denmark ), pc - bsa ( phosphorylcholine - bovine serum albumin ) was purchased from biosearch technologies , inc ( usa ). bovine serum albumin ( bsa ), alkaline phosphatase conjugated goat anti - human igg ( r - chain specific ), alkaline phosphatase conjugated goat anti - human igm ( u - chain specific ), pnpp ( alkaline phosphatase substrate ), were obtained from sigma ( st . louis , mo ., usa ). for example , igg and igm antibodies to pc - bsa were determined by enzyme - linked immunosorbent assay ( elisa ). pooled serum from 17 antiphospholipid syndrome patients was used as an internal standard and tested on every plate . the plateau of antibody binding was reached with the antigen concentration of 10 μg / ml . f96 microtiter polysorp plate was therefore coated with pc - bsa ( 10 μg / ml ) 50 μl / well in pbs . coated plates were incubated overnight at 4 ° c . after five washings with pbs , the plates were blocked with 2 % bsa - pbs for 2 h at room temperature and washed as described above . serum samples were diluted ( 1 : 30 ) in 0 . 2 % bsa - pbs and added at 50 μl / well . preincubation with pneumococcal vaccine ( statens serum institute , denmark ), paf , phosphatidylserine or lysophosphatidylcholine of serum with high capacity to induce decreased binding had the effect of causing decreased serum binding to antigen ; and also restored annexin v binding ( fig2 , 3 , 5 ). in contrast , pc did had no significant effect . this suggests that antibodies binding to pneumococcal vaccine , paf , phosphatidylserine , lysophosphatidylcholine may be involved in reducing annexin v binding to endothelial cells . in conclusion , annexin v is present in atherosclerotic lesions at many sites , especially those that are prone to plaque rupture . when annexin v binding is not optimal but instead decreased as a consequence of antibodies interfering with annexin - plaque binding , the risk of atherothrombosis and plaque rupture is strongly raised . the restoring of the annexin v - binding is therefore a possible novel therapy for atherothrombosis and especially plaque rupture , the main cause of cardiovascular disease . two methods are provided by this invention . one is based on the use of an optimal dose of annexin v or a salt of the protein , which preferably should be administered by intravenous injections , the other is based on the use of ready available immunoglobulins , or an affinity purified subfraction of the immunoglobulins , which can also be administered by injections . the effective amount of annexin v ( or immunoglobulins ) in the dose can be determined from a diagnostic status analysis on the current annexin v - plaque binding . the binding was determined using the analysis method given above . the treatment using a pharmaceutical composition comprising the active component is preferably administered to subjects at risk . a subject at risk is an sle patient with frequent repeating atherothrombosis . another subject at risk is a patient who has , or has had , or is at risk of , a pneumococcal infection . 1 . ross r . atherosclerosis — an inflammatory disease . n engl j med . 1999 ; 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