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:
This application claims priority from Provisional Application No. 60/169,294, filed Dec. 7, 1999, the entire content of which is incorporated herein by reference. 
    
    
     This invention was made with Government support under HL49103 awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     TECHNICAL FIELD 
     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. 
     BACKGROUND 
     Alpha 1 -adrenergic receptor (α 1 AR) stimulation mediates sympathetic nervous system responses such as vascular smooth muscle contraction and myocardial hypertrophy. α 1 AR-mediated vasoconstriction contributes to baseline (tonic) vessel tone, modulates systemic vascular resistance/venous capacitance, and is important in cardiovascular responses to shock. 1  in addition, during “fight and flight” responses, elevated catecholamines result in constriction of “nonessential” vascular beds (e.g. splanchnic) while blood flow to vital organs (e.g., brain, heart) remains uncompromised. 2,3  cDNAs encoding three human α 1 AR subtypes (α 1a , α 1b  and α 1d   4,5 ) were recently cloned, each expressed receptor pharmacologically characterized, 4  and species heterogeneity in α 1 AR subtype tissue distribution identified. 6,7  All three α 1 ARs couple predominantly via Gq to phospholipase C-b activation, resulting in formation of inositol trisphosphate (IP 3 ), calcium release from intracellular stores, and ultimately to smooth muscle contraction. 8    
     Although reasons for existence of three α 1 AR subtypes remain elusive, recent findings suggest subtype and tissue specific regulation may be important. 9,10  While all α 1 AR subtypes mediate smooth muscle contraction, hypertrophic pathways demonstrate subtype specific signaling. 11  α 1 AR agonist exposure to neonatal rat myocytes results in α 1a AR mRNA/protein upregulation (doubling) concurrent with α 1b  and α 1d  downregulation, correlating with induction of myocardial hypertrophy. 12  In contrast, insulin and insulin-like growth factor I induces α 1d AR expression in cultured rat vascular smooth muscle cells. 13  Hence agonist exposure, disease states, and drugs alter α 1 AR subtype expression. 
     The present invention results, at least in part, from studies designed to determine the mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (e.g. aging). Human vascular a 1 AR subtype distribution and function were examined. Specifically, two hypotheses were tested: 1) human α 1 AR subtype expression differs with vascular bed, and 2) age influences human vascular α 1 AR subtype expression. The results 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. 
     SUMMARY OF THE INVENTION 
     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. 
     Objects and advantages of the present invention will be clear from the description that follows. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       FIGS.  1 A- 1 C—Representative saturation binding isotherms for human mammary artery (FIG.  1 A), aorta (FIG.  1 B), saphenous vein (FIG.  1 C). 
       FIG.  2 —Representative RNase protection assay. Autoradiograph (24-hour exposure) demonstrating specific hybridization of a 1 AR subtype radiolabeled antisense riboprobes with total RNA isolated from tRNA (negative control), human aorta, vena cava, iliac artery, and renal artery. 
       FIGS.  3 A and  3 B—Phosphorimager counts from RNase protection assays for each a 1 AR subtype. a 1a AR mRNA expression is significantly higher overall in arteries ( FIG. 3A ) compared with a 1b AR and a 1d AR (**p&lt;0.001), particularly splanchnic (SPL) versus central arteries (CEN) (*p&lt;0.05) (FIG.  3 B). 
       FIGS.  4 A- 4 D—Phenylephrine-induced mammary artery contraction. Antagonists prazosin (nonselective) (FIG.  4 A), 5-MU (a 1a -selective) (FIG.  4 B), spiperone (relatively a 1b -selective) ( FIG. 4C ) demonstrate concentration dependent shift in potency without reducing maximum response. BMY7378 (α 1d -selective) ( FIG. 4D ) does not produce a significant shift. Two concentrations of antagonist are shown: ▪ control; ▴ low (prazosin—10 −9  mol/L; 5-MU/spiperone/BMY7378—10 −8  mol/L); ● higher (prazosin—10 −8  mol/L; 5-MU/spiperone/BMY7378—10 −7 mol/L). 
       FIGS.  5 A- 5 D—a 1 AR subtype expression in mammary artery from young (&lt;55 years, n=6) ( FIGS. 5A and 5C ) versus older (&gt;65 years, n=6) ( FIGS. 5B and 5D ) patients. Competition analysis with 5-MU (a 1a &gt;a 1b =a 1d ) ( FIGS. 5A and 5B ) or WB4101 (a 1a =a 1d &gt;a 1b ) (FIGS.  5 C and  5 D). See Table 4 for pKi values. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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. 
     EXAMPLES 
     Methods 
     Human Vessels 
     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. 
     Membrane Preparation and Radioligand Binding 
     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). 
     RNase Protection Assays (RPAs) 
     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. 
     Functional Assays 
     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). 
     Statistical Analysis 
     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. 
     Results 
     Characterization of Human Vessels 
     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. 
     Human Vascular Total a 1 AR Expression 
     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, FIG.  1 ), 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 Subtype mRNA in Human Vessels 
     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 (FIG.  2 ), 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; FIG.  3 ). These findings suggest a 1 AR subtype expression varies with vessel type. 
     a 1 AR Subtype Protein in Human Vessels 
     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. 
     Human Mammary Artery Contraction 
     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 (FIG.  4 ). 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. 
     Regulation of Vascular a 1 AR Subtype Expression by Age 
     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 (FIG.  5 ). 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. 
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     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.