Patent Application: US-56116205-A

Abstract:
the present invention relates to a novel method to prevent or attenuate atrial fibrillation promotion resulting from atrial tachycardia . in a study conducted with 39 dogs , the hmg - coa reductase inhibitor simvastatin was found to significantly attenuate af promotion . this finding constitutes the basis for an interesting new pharmaceutical approach for preventing the consequences of atrial tachycardia remodeling .

Description:
definitions : unless otherwise specified , the terms used herein have the meanings that would be understand by those of skill in the art . for convenience , the following recurring terms have been defined . “ atrial fibrillation ”: atrial fibrillation ( often termed “ af ”) is a heart rhythm disorder ( arrhythmia ). it usually involves a rapid heart rate , in which the upper heart chambers ( atria ) are stimulated to contract in a very disorganized and abnormal manner . “ atrial fibrillation promotion ”: a process that makes af easier to initiate or maintain . “ atrial tachyarrhythmia ”: a too - rapid , irregular rhythm in the heart &# 39 ; s upper chambers that can impair a person &# 39 ; s quality of life . if left untreated , atrial tachyarrhythmias can lead to a fivefold increase in the risk of stroke . “ atrial tachycardia ”: a sustained , irregular heart rhythm that occurs in the upper chamber of the heart and causes it to beat too rapidly . “ atrial tachycardia - induced remodeling ”, or “ atrial tachycardia remodeling ”: these terms define the changes of atrial electrophysiologic properties taking place in atrial myocytes during atrial fibrillation and / or following periods of sustained atrial fibrillation ( af ). this is also called “ electrophysiological remodeling ”. “ crp ”: c - reactive protein ( crp ), a marker for inflammation , is analyzed as a predictor of cardiovascular disease . crp is a pentameric globulin with mobility near the gamma zone . it is an acute phase reactant which rises rapidly , but nonspecifically in response to tissue injury and inflammation . it is particularly useful in detecting occult infections , acute appendicitis , particularly in leukemia and in postoperative patients . in uncomplicated postoperative recovery , crp peaks on the 3rd post - op day , and returns to pre - op levels by day 7 . it may also be helpful in evaluating extension or reinfarction after myocardial infarction , and in following response to therapy in rheumatic disorders . “ hmg - co a reductase inhibitor ”: 3 - hydroxy - 3 - methyl - glutaryl coenzyme a reductase inhibitors (“ statins ”) are compounds that act by blocking an enzyme that is needed by the body to make cholesterol . sometimes referred to antihyperlipidemic drugs , they thus help to lower cholesterol in the body . members include the following medicaments : atorvastatin ( lipitor ®), cerivastatin ( baycol ®), fluvastatin ( lescol ®), lovastatin ( mevacor ®), altocor ®), pravastatin ( pravachol ®), simvastatin ( zocor ®), epistatin , eptastatin , mevinolin , and synvinolin . they have a common mechanism of action and are thought to behave in a biologically similar fashion . “ r - r interval ”: the time between two consecutive heartbeats measured by the distance from one ecg qrs complex to the next qrs complex . note that the average r - r interval in seconds is obtained by dividing 60 seconds by the heart rate measured . in normal individuals , the r - r interval is somewhat variable . thirty - nine mongrel dogs ( body weight 20 to 37 kg ) were anesthetized with ketamine ( 5 . 3 mg / kg iv ), diazepam ( 0 . 25 mg / kg iv ), and halothane ( 1 . 5 %). unipolar leads were inserted through jugular veins into the right ventricular ( rv ) apex and right atrial ( ra ) appendage under fluoroscopic guidance and connected to pacemakers ( medtronic ) in subcutaneous pockets in the neck . a bipolar electrode was also inserted into the ra for atrial stimulation and recording during serial electrophysiological study ( eps ). av block was created by radiofrequency catheter ablation to control ventricular response during atrial tachypacing ( atp ) and the rv pacemaker was programmed to pace at 80 bpm . after 24 hours for recovery , a baseline closed - chest eps was performed under anesthesia with ketamine , diazepam , and isoflurane , and then atp ( 400 bpm ) was initiated . closed - chest eps was repeated at 2 , 4 and 7 days of atp and a final open - chest eps was performed on day 8 under anesthesia with morphine and α - chloralose . results in 7 atrial tachypaced dogs without any treatment ( atp - only group ) and 9 non - paced control dogs were each compared with those of dogs subjected to atp during oral treatment with : 1 ) simvastatin , 80 mg / day ( n = 6 ), beginning 3 days prior to atp onset ; 2 ) vitamin c , 500 mg twice daily ( n = 6 ); and 3 ) combined vitamin c , 500 mg and vitamin e , 200 iu twice daily ( n = 6 ), beginning 1 day prior to atp onset and continued throughout the study period . in addition , because no clear effect of vitamin c was observed at this dose , we studied an additional group of 5 dogs receiving sustained - release vitamin c 1 . 5 g daily in divided doses beginning 1 day before atp . on each closed - chest eps day , dogs were anesthetized with ketamine ( 5 . 3 mg / kg iv ), diazepam ( 0 . 25 mg / kg iv ), and isoflurane ( 1 . 5 %), and ventilated mechanically . the atrial pacemaker was then deactivated and the ra appendage effective refractory period ( erp ) was measured at basic cycle lengths ( bcls ) of 150 , 200 , 250 , 300 , and 360 ms . erp was measured with 10 basic stimuli ( s1 ) at various bcls followed by a premature extrastimulus ( s2 ) with 5 ms decrements . the longest s1 - s2 failing to capture the atria defined the erp . af was induced with atrial burst pacing at 10 hz and 4 times threshold current . mean af duration was calculated based on 10 inductions for af & lt ; 20 minutes and 5 inductions for af lasting 20 to 30 minutes . af lasting longer than 30 minutes was considered sustained and terminated by dc cardioversion . a 20 - minute rest period was then allowed before continuing measurements . if sustained af was induced twice during an experiment , no further af induction was performed . for open - chest eps , dogs were anesthetized with morphine ( 2 mg / kg sc ) and α - chloralose ( 120 mg / kg iv , followed by 29 . 25 mg / kg / h ), and ventilated mechanically . body temperature was maintained at 37 ° c ., and a femoral artery and both femoral veins were cannulated for pressure monitoring and drug administration . a median sternotomy was performed , and bipolar electrodes were hooked to the ra and left atrial ( la ) appendage for recording and stimulation . a programmable stimulator ( digital cardiovascular instruments ) was used to deliver twice - threshold currents . five silicon sheets containing 240 bipolar electrodes were sutured onto the atrial surfaces as previously described . 7 - 9 atrial erp was measured over a range of bcls in ra and la appendages and at bcl 300 ms in 6 additional sites : ra posterior wall , ra inferior wall , ra bachmann &# 39 ; s bundle , la posteriorwall , la inferior wall , and la bachmann &# 39 ; s bundle . af duration was assessed as described above and af vulnerability was determined as the percentage of atrial sites at which af could be induced by single extrastimuli . blood samples were collected on the final open - chest study day . serum was removed following centrifugation ( 3000 rpm , 20 minutes ) and stored at − 80 ° c . for subsequent c - reactive protein ( crp ) analysis . crp was measured with the phase range ® canine crp elisa kit ( tri - delta diagnostics inc .). at the end of open - chest studies , ra and la tissue samples were fast - frozen in liquid nitrogen and stored at − 80 ° c . to isolate proteins , tissues were homogenized in ripa buffer with a protease inhibitor cocktail ( 5 μg / μl leupeptin , 5 μg / μl soybean trypsin inhibitor and 10 μg / ml benzamidine ; sigma ) added to prevent protein degradation . the suspension was incubated on ice and then centrifuged ( 14000 g , 10 minutes , 4 ° c .). the soluble fraction was stored at − 80 ° c . protein concentrations were measured by bradford assay with bovine albumin as a standard . proteins ( 200 - μg samples ) were denatured in laemmli buffer , electrophoresed on 7 . 5 % sds - polyacrylamide gels and then transferred to polyvinylidene difluoride ( pvdf ) membranes overnight , blocked for 2 hours with 0 . 1 % tween - 80 - tris - buffered saline ( ttbs ) at room temperature ( rt ) and then incubated with primary antibody ( alomone labs , anti - cardiac ca v 1 . 2 , 1 : 100 ) at 4 ° c . overnight . after 3 washes , membranes were re - blocked in 1 % nonfat dry milk in ttbs for 10 minutes and incubated with secondary antibody ( jackson laboratories , goat anti - rabbit ) for 90 minutes at rt . after 3 additional washes in ttbs , antibody detection was performed with western lightning ™ western blot chemiluminescence reagent plus . band densities were quantified by densitometry ( quantity one software ) standardized to gapdh and normalized to the control sample on each gel . data are presented as mean ± sem . multiple - group comparisons were obtained by anova . a t - test with bonferroni correction was used to evaluate differences between individual means . a two - tailed p & lt ; 0 . 05 was considered statistically significant . there were no significant differences among groups in mean body weight or in hemodynamic variables at final open - chest study ( table 1 ). although crp tended to be slightly higher at end - study in atp - only dogs , crp varied widely among dogs and there were no statistically - significant crp differences among groups . changes in erp caused by 7 days of atp in atp - only dogs are shown in fig1 . erp decreased substantially within 2 days and reached steady - state changes at 4 days ( fig1 a ). af duration increased substantially from 10 ± 7 seconds prior to atp to values averaging hundreds of seconds on days 4 and 7 of atrial tachycardia ( fig1 b ). fig2 compares erp changes as measured at cycle lengths of 300 ( panel a ) and 150 ( panel b ) ms in dogs subjected to atp - only with dogs subjected to atrial tachycardia in the presence of each of the drug interventions . under baseline conditions ( day 0 ) there were no significant differences in erp among groups . with the onset of atrial tachycardia , erp decreased rapidly and to a similar extent in atp - only dogs and in dogs subjected to atrial tachycardia in the presence of each of the antioxidant vitamin regimens . in simvastatin - treated dogs subjected to atrial tachycardia , the erp changes were smaller and erp values were larger than in atp - only dogs for both cycle lengths . fig3 shows the progression of mean af duration in dogs subjected to atrial tachycardia in the presence of each of the interventions studied . with vitamin c ( panel a ), vitamins c and e ( panel b ) and sustained - release vitamin c ( panel c ), progressive increases in af duration to means between ˜ 400 to 600 seconds occurred by day 7 , not significantly different from atp - only dogs . in contrast , atrial tachycardia - induced af promotion was virtually abolished in simvastatin - treated dogs ( panel d ). erp values measured as a function of cycle length during the final open - chest study are illustrated in fig4 . dogs subjected to atp without drug intervention had atrial erps averaging & lt ; 80 ms at all basic cycle lengths , and virtually no rate - adaptation of the erp was detectable . no significant differences were observed between atp - only dogs and dogs subjected to atrial tachycardia in the presence of vitamin c ( panel a ), vitamins c and e ( panel b ) or sustained - release vitamin c ( panel c ). dogs subjected to atrial tachycardia in the presence of simvastatin showed erp values that were significantly greater than those subjected to atp without drug intervention ( panel d ). in non - paced control dogs , af always terminated spontaneously within 5 minutes . af requiring cardioversion for termination was induced following atp in 57 % of atp - only dogs , 33 % of vitamin c - treated dogs , 50 % of combined vitamin c and e - treated dogs and 40 % of sustained - release vitamin c - treated dogs . no sustained af requiring cardioversion occurred in atrial tachypaced dogs treated with simvastatin . fig5 summarizes differences in mean af duration and atrial vulnerability at open - chest study among the different groups of dogs . non - paced control dogs had mean af durations averaging 42 seconds ( panel a ), and atp increased mean af duration at open - chest study to over 1000 seconds . dogs subjected to atp in the presence of each of the antioxidant vitamin regimens had mean af durations greater than 500 seconds and not significantly different from atp - only . dogs subjected to atp in the presence of simvastatin had substantial attenuation of the af maintenance - promoting effect of atrial tachycardia , with a mean af duration (˜ 40 seconds ) equivalent to that of non - paced controls . af vulnerability changes are shown in panel b . af was induced by single extrastimuli at a mean of over 50 % of atrial sites in atp - only dogs , significantly greater than the less than 15 % of sites at which af could be induced in non - paced controls . in dogs subjected to atp during therapy with antioxidant vitamins , af was induced at an average of & gt ; 50 % of sites in each group . in simvastatin - treated dogs exposed to atp , atrial vulnerability was significantly reduced compared to atp - only , to an average of ˜ 20 %. fig6 shows values of atrial erp in different atrial regions . erp decreases caused by atp were regionally variable , as previously described , 16 with the largest changes occurring in ra inferior wall , posterior wall and appendage , as well as la appendage . there were no significant differences between erp values in atp - only dogs and dogs in each of the antioxidant vitamin groups ( panels a - c ). simvastatin significantly attenuated atp effects on erp in ra appendage , posterior wall and inferior wall . la erp reductions induced by atp were not significantly altered by simvastatin therapy . reductions in l - type ca 2 + - current , 17 apparently due to transcriptional down - regulation of the α1c pore - forming ca 2 + - channel subunit , ca v 1 . 2 , 18 - 20 are important in mediating electrophysiological changes caused by atrial tachycardia remodeling . the expression of ca v 1 . 2 protein in ra and la appendage was therefore quantified with the use of western blot techniques in non - paced dogs and dogs subjected to atp during treatment with simvastatin , vitamin c and vitamins c and e . a clear signal was obtained at 207 kda , corresponding to the expected molecular mass of ca v 1 . 2 protein ( fig7 a ). atp alone significantly reduced cav1 . 2 protein expression in both ra ( fig7 b ) and la ( fig7 c ) tissue samples . neither vitamin c alone nor vitamins c plus e altered the tachypacing - induced ca v 1 . 2 changes . in contrast , simvastatin significantly attenuated ca v 1 . 2 downregulation . simvastatin was found to prevent af promotion by 1 week of atp in dogs . this action was associated with significant attenuation of ra erp abbreviation and of atrial tachycardia - induced effects on ca v 1 . 2 protein expression . these actions were not shared by the antioxidant vitamin c nor by vitamins c and e in combination . although several articles have demonstrated beneficial effects of l - type ca channel blockers on short - term atrial tachycardia - induced remodeling , 21 - 24 they appear to be ineffective against longer - term (& gt ; 24 - hour ) remodeling . 8 , 25 a variety of other compounds , including na + , h + - exchange blockers and angiotensin converting - enzyme inhibitors , have also been found effective in short - term 25 , 26 but not longer - term 28 af . carnes et al 10 demonstrated effectiveness of vitamin c at doses equivalent to those in the present study in attenuating erp changes caused by 48 - hour atrial tachycardia in the dog ( changes in arrhythmia promotion were not reported ). here , the effectiveness of vitamin c , alone or in combination with vitamin e , in preventing erp or af - promoting effects of 7 - day atrial tachycardia was not observed . the t - type ca 2 + - channel blocker mibefradil 7 , 8 and the broad - spectrum antiarrhythmic amiodarone 9 do prevent the effects of 1 - week atrial tachycardia in the dog . however , mibefradil has been removed from the market because of adverse drug interactions and amiodarone &# 39 ; s value is limited by a range of potentially - serious adverse effects . the present study is believed to be the first to demonstrate the effectiveness of a hmg - coa reductase ( here , simvastatin ) in atrial - tachycardia remodeling and af promotion . statins act as antioxidants by inhibiting superoxide production , 29 as well as by increasing nitric oxide bioavailability . 30 , 31 simvastatin increases catalase and glutathione peroxidase activity . 32 thus , without wishing to be bound by any particular hypothesis , it would appear that simvastatin &# 39 ; s efficacy is due to an antagonism of oxidant pathways involved in atrial tachycardia remodeing . 10 , the antioxidant properties of both vitamin c and e are well - recognized , 33 , 34 however , the ability of exogenous vitamin c and e to increase the body &# 39 ; s already substantial stores of these important endogenous antioxidants may be insufficient to significantly alter atrial antioxidant capacity . an alternative explanation lies in the anti - inflammatory properties of statins 14 , 15 in the context of the potential role of inflammation in af . 12 , 13 although crp concentrations were measured in the dogs used in the experiments described above , no significant changes with atp or simvastatin administration were observed . simvastatin was much more effective in preventing tachycardia - induced ra erp changes than those in the la ( fig6 d ). the basis of this regionally - determined efficacy is unclear , particularly in view of the ability of simvastatin to prevent la ca v 1 . 2 downregulation ( fig7 c ). these observations point to a role for factors other than ca v 1 . 2 downregulation in contributing to atrial - tachycardia induced erp changes , and may be related to the observation that nitric - oxide synthase downregulation with atrial - tachycardia remodeling is more significant in la than in ra . 35 atrial - tachycardia remodeling has significant clinical consequences , particularly for af occurrence and maintenance , and inhibition of such remodeling may be an interesting novel approach to af therapy . 6 to date , the drugs shown to prevent atrial - tachycardia remodeling in dog models have either been unavailable clinically ( mibefradil ) or have a variety of other potent electrophysiological and extra - cardiac actions ( amiodarone ). the doses of simvastatin that were used in the study ( 2 mg / kg daily ) are equal to those used in some experimental dog studies 36 and smaller than in others , 37 but are somewhat higher than those in common clinical use ( about 0 . 1 to 1 mg / kg ). simvastatin prevents the af - promoting actions of atrial tachycardia in dogs , and may open up interesting new approaches to preventing the arrhythmic consequences of atrial - tachycardia remodeling in man . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit , scope and nature of the subject invention , as defined in the appended claims . 1 . dresing t j , and schweikert r a , atrial fibrillation . the cleveland clinic , department of cardiovascular medicine , may 30 , 1992 . 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