Patent Application: US-73106200-A

Abstract:
the present invention relates to the use of α 1a ar - selective and / or α 1a / α 1d - selective antagonists in a method of preventing restenosis after myocardial infarction and re - perfusion . the invention further relates to a method of identifying agents suitable for us in such a method .

Description:
the present invention results , at least in part , from studies designed to characterize a 1 ar subtype distribution in humans across different vascular beds . the results presented in the example that follows demonstrate a 1 ar subtype expression varies according to vessel bed . specifically , a 1a ar mrna / protein predominates in coronary , splanchnic , renal , and pulmonary arteries , whereas central arteries and veins express all 3 a 1 ars . with aging (& lt ; 55 versus ≧ 65 years ), a two - fold increase in overall mammary artery ( but not saphenous vein ) a 1 ar expression occurs ( a 1b & gt ; a 1a ). robust α 1a and α 1b - mediated contraction for all ages studied indicate these findings have functional significance . α 1 ar - mediated smooth muscle contraction is important in determining tonic and reflex changes in arterial and venous diameter . instantaneous changes in vessel tone are responsible for maintenance of blood pressure and venous return to the heart during stress ( e . g . hypovolemia [ hemorrhage ], shock , and sepsis ). 1 at rest , adult splanchnic vessels contain 30 % total circulating blood volume ; 3 acute sympathetically - mediated constriction is a primary mechanism underlying maintenance of blood pressure during shock or hemorrhage . robustness of compensatory mechanisms is illustrated by blood pressure stability until & gt ; 20 % blood volume is lost . 18 vascular α 1 ars have been studied in animals using a variety of techniques ( table 5 ). after initial controversy , it has been generally agreed a 1d ars mediate vasoconstriction in rat aorta ; 15 , 19 in contrast , contraction in dog , rabbit , and mouse aorta occurs via a 1b ars . 20 - 22 a 1 ar subtype - mediated contraction also differs along mesenteric bed ; a 1d ar mediates contraction in rat superior mesenteric artery ( proximal ) whereas a 1b ars function in distal mesenteric arteries . 19 only a few studies in human vessels have been performed to date ; these identify all three a 1 ar subtype mrnas in human mesenteric artery , 23 a 1b and α 1d in human aorta , 6 and a 1a in saphenous vein 24 and vena cava . 6 a 1b - mediated contraction occurs in human superior vesicle and obturator arteries , 25 and a 1a - mediated contraction in human mesenteric artery . 26 the findings further indicate that α 1a ar - mediated contraction accounts for generalized splanchnic vasoconstriction during stress in humans , although this hypothesis must be confirmed by further contraction studies . other findings of clinical relevance include a 1 ars in renal , pulmonary , and coronary vasculature as possible targets for treatment of renal insufficiency , pulmonary hypertension , and angina . since veins contain all three a 1 ar subtypes , pharmacological isolation preload ( venous return ) and afterload ( arterial vascular resistance ) is possible . while the experiments described in the examples that follow utilized “ normal ” vessels , examination of alterations of a 1 ar subtype distribution by disease can be made . sympathetically - mediated vascular responsiveness changes with age , although precise mechanisms underlying this observation remain unknown . 8 while overall aortic a 1 ar density remains unchanged with age in rat , subtype modulation occurs ( increased a 1a , decreased α 1b , unchanged α 1d ); 27 other studies suggest age decreases all a 1 ars in rat , 28 but increases in sheep . 29 age - related changes are vessel specific , with rat renal α 1b ar mrna declining without change in mesenteric / pulmonary a 1 ars . 28 furthermore , age increases functional a 1d ars in resistance vessels compared with a 1a ar predominance in young rats . 30 in humans , age increases in - hospital mortality associated with major surgery ; 31 risks include vascular - associated conditions such as gastrointestinal infarction and limb ischemia . 2 , 32 , 33 the results reveal age - related increases in mammary artery a 1 ar density ( but not saphenous vein ), and a switch from a 1a predominance in younger adults to a 1b & gt ; a 1a in older patients . other arteries need to be tested to determine whether age - induced arterial changes are global , or mammary artery specific . in support of a global interpretation of the present findings , a recent clinical study demonstrates less blood pressure perturbation in elderly patients with tamsulosin ( a 1a / a 1d - selective antagonist ) compared with alfuzosin ( non - selective ), 34 indicating importance of a 1b ars with aging in resistance vessels . the result presented herein demonstrate human vascular α 1 ar subtype distribution differs from animal models , varies with vessel bed . correlates with contraction in mammary artery , and is modulated by aging . this information provides targets for therapeutic intervention in a clinical settings . certain aspects of the present invention are described in greater detail in the example that follows . vessels were obtained after approval from the duke university institutional review board and individual agencies . sources included discarded tissues from surgery ( 0 - 60 minutes from isolation ), duke university rapid autopsy program ( 0 - 3 hours postmortem ), national disease research interchange ( philadelphia , pa . ; 0 - 5 hours postmortem ), and the international institute for the advancement of medicine ( scranton , pa ; within 12 hours postmortem ). except for functional assays , vessels were snap frozen in liquid nitrogen and stored at − 70 ° c . for later use . vessels were weighed , lumen diameter measured , pulverized under liquid nitrogen , and suspended in cold lysis buffer ( 5 mmol / l tris hcl and 5 mmol / l edta , ph 7 . 4 ) with protease inhibitors . 14 after lysate preparation , membranes were resuspended in cold binding buffer ( 150 mmol / l nacl , 50 mmol / l tris · hcl , 5 mmol / l edta , with protease inhibitors , ph 7 . 4 ) as previously described ; 14 protein concentration was determined using the bicinchoninic acid method ( pierce , rockford , ill .). full saturation binding isotherms were performed in selected human vessels ( aorta , mammary artery , saphenous vein ) in 250 μl binding buffer ( 20 - 60 μg vessel membrane protein ) using the α 1 - adrenergic antagonist [ 125 i ] heat ( 2 -[ b -( hydroxy - 3 [ 125 i ] iodophenyl ) ethyl - aminomethyl ]- tetralone ; dupont - nen ; boston , mass .) as previously described . 14 to measure total a 1 ar density in all vessels , a saturating concentration ( 300 pmol / l ) of the [ 125 i ] heat was used . a kd concentration ( 130 pmol / l [ 125 i ] heat ) was used in competition analysis with antagonists 5 - mu wb4101 , and bmy7378 ( 10 − 12 to 10 − 4 mol / l ). rna isolation and human a 1 ar cdna constructs have previously been described . 14 rpas were performed as previously described ; control b - actin consisted of 0 . 104 kb ( hinp1l / taqi ) fragment in pgem - 4z ( genbank # ab004047 ; nucleotide 119 - 222 ). 14 [ 32 p ] actp ( dupont - nen ) was incorporated into rna probes at the time of synthesis . after digestion with rnase a and t1 , rna samples were separated electrophoretically through a 6 % polyacrylamide gel , dried , and exposed to x - omat film ( eastman kodak company ; rochester , n . y .) for 18 - 24 hours , and phosphorimager plates ( molecular dynamics : sunnyvale , calif .) for 72 hours . volume integration of protected fragments was corrected for background using imagequant image analysis software ( molecular dynamics ) and counts were normalized for b - actin signal and 32 p - actp incorporation ( ctps : a 1a — 97 , a 1b — 219 , a 1d — 133 ). final mrna data are scaled + 1 to + 10 , with + 10 ( 100 arbitrary units ) assigned a 1a ar mrna in liver ( human tissue known to contain maximal a 1 ar mrna ); thus phosphorimager counts / 10 , 000 × 1 . 8 defined phosphorimager units . a 1a ar mrna is highest in mesenteric artery ( 26 units ): therefore , + 3 = 20 - 29 units ; + 2 = 10 - 19 units ; + 1 = 4 - 9 units ; (−)= almost undetectable signal (≦ 3 units ) phosphorimager , negative autoradiograph ; −= lack of signal on both . since the presence of receptor protein does not always correlate with functional response , 15 a 1 ar - mediated contractility in mammary artery was tested using phenylephrine dose response curves in the absence / presence of subtype selective / nonselective antagonists . mammary arteries were immersed in cold oxygenated krebs - ringer bicarbonate solution ( 118 . 3 mmol / l nacl , 4 . 7 mmol / l kcl , 1 . 2 mmol / l mgso 4 , 1 . 2 mmol / l khpo 4 , 42 . 5 mmol / l cacl 2 , 25 mmol / l nahco 3 , 16 mmol / l caedta , 1 . 1 mmol / l glucose ), cleaned of loose connective tissue , cut into 4 - 5 mm long rings , and suspended for isometric tension recording in organ chambers . one stirrup was anchored to the chamber and the other connected to a strain gauge ( ft - 102 ) for measurement of isometric force ( maclab , cb sciences ; milford , mass .). all concentration effect curves were performed at optimum resting tone (˜ 3 g in pilot studies ). contractile response to 60 mmol / l kci was performed ; this determined vessel viability and facilitated normalization of phenylephrine response across vessel rings . phenylephrine dose - response curves were generated ( 10 − 4 - 10 − 9 mol / l ) in ½ log order concentrations in the absence / presence of competitive a 1 ar antagonists . contraction assays using vessel rings from an individual patient were performed simultaneously in separate baths for each antagonist ; hence each vessel ring was exposed to three dose response curves . antagonist potency was expressed as the dissociation constant ( k b ) determined from pk b = log [ b ] log ( dr - 1 ), where [ b ] is antagonist concentration and dr the dose ratio produced by antagonist . dose response curves were analyzed using dose response software ( maclab , cb sciences ). data were tested for normal distribution using shapiro - wilke test of normality . overall { grave over ( α )} 1 ar density was compared between vessels using a general linear multivariate model , and where significant differences identified between specific vascular beds , the exact p value was determined using wilke &# 39 ; s - lambda test ; p & lt ; 0 . 05 was considered significant . since determination of α 1 ar subtype expression involved three subtypes ( a 1a , a 1b , a 1d ), critical α was reduced to 0 . 0167 for these studies . similarly , when comparing α 1 ar subtype expression between different vascular beds , pairwise comparisons were made using a wilcoxon 2 - sample rank sum test , and critical α set at 0 . 0167 . competition binding and functional assays were analyzed using least squares regression analysis with prism software ( graphpad ; san diego , calif .). final data were analyzed using sas system , release v . 6 . 12 ( sas institute inc ., cary , n . c . ), and presented as mean ± sem to two significant figures . 500 vessels from 384 patients ( male , n = 257 ; female , n = 127 ; 64 ± 0 . 82 years [ range 12 - 92 ]) were used . the majority ( 83 %) were collected from operating room specimens , 17 % from autopsy ( cause of death : gun shot , automobile accident , myocardial infarction , cancer ). 95 % vessels were obtained ≦ 3 hours from tissue isolation or death ( within 12 hour postmortem mrna / protein stability period in rats / humans ). 16 , 17 vessels were obtained only from patients without co - existing disease ( e . g . no chronic renal failure , congestive heart failure , diabetes , hypertension , thyroid disease ), or potentially confounding drugs ( e . g . no estrogen supplementation , catecholamines , sympathetic stimulants , antidepressants , or aar drugs ); five years was required to collect enough vessels to complete the study . due to limited vessel rna / protein , n = 1 vessel from a single individual whenever possible , but sometimes represents pooled samples from 2 - 6 patients with similar patient characteristics . the “ fight and flight ” ( stress ) response results in redistribution of blood from splanchnic and “ non - essential ” organs toward vital organs . 2 , 3 in order to test the hypothesis that a 1 ar density in splanchnic versus somatic vessels may be responsible for these effects , kd and bmax were determined for 125 i - heat binding in selected human vessels ( nonspecific binding 30 - 70 %). kd is 130 ± 0 . 20 ( aorta ), 130 ± 3 . 1 ( mammary artery ), and 130 ± 0 . 65 ( saphenous vein ) pmol / l ( n = 2 - 4 each , fig1 ), similar to cloned human a 1 ars . 4 overall human vascular a 1 ar expression is 16 ± 2 . 3 fmol / mg total protein ; central ( conduit ) and small somatic arteries express significantly lower a 1 ar density than splanchnic arteries , p & lt ; 0 . 05 , table 1 ). in contrast , venous a 1 ar density does not change with vessel diameter or vascular bed . a 1 ar subtypes were next examined ; due to limited tissue , molecular approaches were utilized . all three a 1 ar mrnas are present in human vessels ( fig2 ), with α 1a ar predominating overall in arteries ( p & lt ; 0 . 001 ); epicardial coronary arteries express a 1a exclusively ( table 2 ). α 1a ar subtype density is significantly higher in splanchnic versus central vessels ( p & lt ; 0 . 05 ; fig3 ). these findings suggest a 1 ar subtype expression varies with vessel type . to ensure mrna and protein expression correlate , competition analysis was performed . selected vessels were chosen for availability and expression of only one or two α 1 ar subtypes ( to facilitate interpretation of results ). since a 1a ar mrna predominates , 5 - mu ( a 1a - selective antagonist ) was utilized . a 1 ar subtype protein expression in 4 representative human vessels was determined by competition analysis with 5 - mu ( a 1a - selective antagonist ) ( n = 3 experiments per vessel , each performed in triplicate ); table 3 summarizes pki values (− logki ; measure of receptor affinity for antagonist ). 5 - mu binds to two sites in mammary , renal , splenic arteries , and vena cava , with the high affinity pki site consistent with interactions at cloned α 1a ars . 4 although designation of the high affinity binding site is straightforward , low affinity α 1 ar site identification was aided by mrna data in table 2 and confirmed in mammary artery ( and aorta ) using bmy7378 ( a 1d - selective antagonist ). only one binding site was detected in aorta , coronary artery , and hepatic artery , with pki values consistent with α 1d , α 1a , and α 1a ars , respectively . these data suggest mrna and protein expression correlate closely in human vessels . phenylephrine dose response curves were completed in 10 mammary arteries ( patient age 60 ± 2 . 2 years [ range 37 - 73 ]), vessels which contain only a 1a and a 1b ars ; isometric contraction occurs with pd 2 6 . 0 ± 0 . 093 . a 1 ar competitive antagonists produce a concentration dependent shift in potency of phenyiephrine contraction without reducing maximum response ( fig4 ). potency in inhibiting mammary artery contraction ( pk b ) is 9 . 2 ± 0 . 046 ( prazosin , non - selective ), 8 . 4 ± 0 . 63 ( 5 - mu . a 1a - selective ), and 8 . 6 ± 0 . 19 ( spiperone , relatively a 1b - selective ), similar to affinities for each antagonist at cloned human a 1 ars . 4 bmy7378 ( α 1d - selective ) does not produce a shift in dose response . these data suggest a 1a and a 1b ars mediate contraction in human mammary artery . mammary artery α 1 ar density increases significantly with age ( 4 . 4 ± 0 . 78 & lt ; 55 years versus 9 . 3 ± 1 . 7 ≧ 65 years , p = 0 . 003 , fmol / mg total protein ) ( table 4 ). in contrast , saphenous vein α 1 ar density does not change with age . competition analysis with 5 - mu and wb4101 reveals α 1a ars are the major subtype in mammary artery in patients & lt ; 55 years of age ( fig5 ). however , with aging , α 1b ar expression significantly increases ( 3 - fold , p = 0 . 0001 ), becoming the major subtype in patients ≧ 65 years ; α 1a ars also significantly increases with age ( 1 . 5 - fold , p ≦ 0 . 001 ). α 1d ar expression is virtually absent in younger and older patients . 1 . ruffolo r r , jr . distribution and function of peripheral a - adrenoceptors in the cardiovascular system . pharmacol biochem behav . 1985 ; 22 : 827 - 833 . 2 . allen k b , salam a a , lumsden a b . acute mesenteric ischemia after cardiopulmonary bypass . j vasc surg . 1992 ; 16 : 391 - 396 . 3 . reilly p m bulkley g b . vasoactive mediators and splanchnic perfusion . crit care med . 1993 ; 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19 : 81 - 87 . 34 . buzelin j m , fonteyne e , kontturi m . witjes w p j , khan a . comparison of tamsulosin with alfuzosin in the treatment of patients with lower urinary tract symptoms suggestive of bladder outlet obstruction ( symptomatic benign prostatic hyperplasia ). br j urol . 1997 ; 80 : 597 - 605 . all documents cited above are hereby incorporated in their entirety by reference . one skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention .